US20230008421A1 - Method of forming solid cell cultures - Google Patents
Method of forming solid cell cultures Download PDFInfo
- Publication number
- US20230008421A1 US20230008421A1 US17/511,240 US202117511240A US2023008421A1 US 20230008421 A1 US20230008421 A1 US 20230008421A1 US 202117511240 A US202117511240 A US 202117511240A US 2023008421 A1 US2023008421 A1 US 2023008421A1
- Authority
- US
- United States
- Prior art keywords
- cells
- cell
- cell culture
- cancer
- chip
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 168
- 239000007787 solid Substances 0.000 title claims abstract description 118
- 238000004113 cell culture Methods 0.000 title claims description 320
- 239000000017 hydrogel Substances 0.000 claims abstract description 43
- 239000013610 patient sample Substances 0.000 claims abstract description 32
- 238000010186 staining Methods 0.000 claims abstract description 15
- 210000004027 cell Anatomy 0.000 claims description 480
- 206010028980 Neoplasm Diseases 0.000 claims description 55
- 210000001519 tissue Anatomy 0.000 claims description 28
- 201000011510 cancer Diseases 0.000 claims description 27
- 239000000523 sample Substances 0.000 claims description 23
- 238000012258 culturing Methods 0.000 claims description 20
- 210000002220 organoid Anatomy 0.000 claims description 18
- 238000012545 processing Methods 0.000 claims description 17
- 230000008859 change Effects 0.000 claims description 15
- 210000002865 immune cell Anatomy 0.000 claims description 14
- 238000002271 resection Methods 0.000 claims description 13
- 210000002536 stromal cell Anatomy 0.000 claims description 12
- 238000000799 fluorescence microscopy Methods 0.000 claims description 11
- 210000003470 mitochondria Anatomy 0.000 claims description 10
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 8
- 210000004369 blood Anatomy 0.000 claims description 8
- 239000008280 blood Substances 0.000 claims description 8
- 230000003278 mimic effect Effects 0.000 claims description 7
- 208000026310 Breast neoplasm Diseases 0.000 claims description 6
- 239000008358 core component Substances 0.000 claims description 6
- 201000010099 disease Diseases 0.000 claims description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 6
- 238000013188 needle biopsy Methods 0.000 claims description 6
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 5
- 102000008186 Collagen Human genes 0.000 claims description 5
- 108010035532 Collagen Proteins 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 229920001436 collagen Polymers 0.000 claims description 5
- 230000009089 cytolysis Effects 0.000 claims description 5
- 230000029087 digestion Effects 0.000 claims description 5
- 210000002744 extracellular matrix Anatomy 0.000 claims description 5
- 229920002674 hyaluronan Polymers 0.000 claims description 5
- 229960003160 hyaluronic acid Drugs 0.000 claims description 5
- 208000003174 Brain Neoplasms Diseases 0.000 claims description 4
- 206010006187 Breast cancer Diseases 0.000 claims description 4
- 206010009944 Colon cancer Diseases 0.000 claims description 4
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 4
- 208000008839 Kidney Neoplasms Diseases 0.000 claims description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 4
- 206010033128 Ovarian cancer Diseases 0.000 claims description 4
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 4
- 206010038389 Renal cancer Diseases 0.000 claims description 4
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 4
- 238000003501 co-culture Methods 0.000 claims description 4
- 206010017758 gastric cancer Diseases 0.000 claims description 4
- 201000010982 kidney cancer Diseases 0.000 claims description 4
- 201000005202 lung cancer Diseases 0.000 claims description 4
- 208000020816 lung neoplasm Diseases 0.000 claims description 4
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 4
- 201000002528 pancreatic cancer Diseases 0.000 claims description 4
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 4
- 201000011549 stomach cancer Diseases 0.000 claims description 4
- 230000009028 cell transition Effects 0.000 claims description 3
- 210000000130 stem cell Anatomy 0.000 claims 2
- 230000004044 response Effects 0.000 abstract description 127
- 238000003384 imaging method Methods 0.000 abstract description 25
- 238000011282 treatment Methods 0.000 description 203
- 239000003795 chemical substances by application Substances 0.000 description 140
- 239000001963 growth medium Substances 0.000 description 58
- 239000000975 dye Substances 0.000 description 50
- 238000009826 distribution Methods 0.000 description 38
- 239000012530 fluid Substances 0.000 description 33
- 239000013598 vector Substances 0.000 description 32
- 239000007789 gas Substances 0.000 description 25
- 230000005012 migration Effects 0.000 description 25
- 238000013508 migration Methods 0.000 description 25
- 238000004458 analytical method Methods 0.000 description 24
- 230000012292 cell migration Effects 0.000 description 24
- 230000000875 corresponding effect Effects 0.000 description 22
- 230000008569 process Effects 0.000 description 21
- 230000008878 coupling Effects 0.000 description 20
- 238000010168 coupling process Methods 0.000 description 20
- 238000005859 coupling reaction Methods 0.000 description 20
- 239000000306 component Substances 0.000 description 19
- 230000003833 cell viability Effects 0.000 description 15
- 238000010586 diagram Methods 0.000 description 13
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 12
- 238000005259 measurement Methods 0.000 description 12
- 239000000853 adhesive Substances 0.000 description 10
- 230000001070 adhesive effect Effects 0.000 description 10
- 239000012528 membrane Substances 0.000 description 10
- 239000007788 liquid Substances 0.000 description 9
- 239000002246 antineoplastic agent Substances 0.000 description 8
- 239000007850 fluorescent dye Substances 0.000 description 8
- 230000006870 function Effects 0.000 description 8
- 239000003814 drug Substances 0.000 description 7
- 238000010191 image analysis Methods 0.000 description 7
- 239000003086 colorant Substances 0.000 description 6
- 238000003066 decision tree Methods 0.000 description 6
- 229960004679 doxorubicin Drugs 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 230000033001 locomotion Effects 0.000 description 6
- 238000010801 machine learning Methods 0.000 description 6
- 238000000386 microscopy Methods 0.000 description 6
- 230000035899 viability Effects 0.000 description 6
- 238000000429 assembly Methods 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 230000006399 behavior Effects 0.000 description 5
- 239000000090 biomarker Substances 0.000 description 5
- 238000004891 communication Methods 0.000 description 5
- 239000002131 composite material Substances 0.000 description 5
- 239000000356 contaminant Substances 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 238000003860 storage Methods 0.000 description 5
- 230000000007 visual effect Effects 0.000 description 5
- 230000004663 cell proliferation Effects 0.000 description 4
- 238000004624 confocal microscopy Methods 0.000 description 4
- -1 instrumentation Substances 0.000 description 4
- 230000002572 peristaltic effect Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 230000000712 assembly Effects 0.000 description 3
- 238000013135 deep learning Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 239000000092 prognostic biomarker Substances 0.000 description 3
- 230000007704 transition Effects 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- 238000012800 visualization Methods 0.000 description 3
- 206010061818 Disease progression Diseases 0.000 description 2
- 238000011394 anticancer treatment Methods 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 238000000339 bright-field microscopy Methods 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 239000000104 diagnostic biomarker Substances 0.000 description 2
- 230000005750 disease progression Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000002085 persistent effect Effects 0.000 description 2
- 230000000704 physical effect Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000013517 stratification Methods 0.000 description 2
- 238000012706 support-vector machine Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000011269 treatment regimen Methods 0.000 description 2
- 229920001410 Microfiber Polymers 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009118 appropriate response Effects 0.000 description 1
- 238000013528 artificial neural network Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000004611 cancer cell death Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 210000003040 circulating cell Anatomy 0.000 description 1
- 229940000425 combination drug Drugs 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229910003460 diamond Inorganic materials 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000012632 fluorescent imaging Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000004973 liquid crystal related substance Substances 0.000 description 1
- 238000007477 logistic regression Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000003658 microfiber Substances 0.000 description 1
- FAQDUNYVKQKNLD-UHFFFAOYSA-N olaparib Chemical compound FC1=CC=C(CC2=C3[CH]C=CC=C3C(=O)N=N2)C=C1C(=O)N(CC1)CCN1C(=O)C1CC1 FAQDUNYVKQKNLD-UHFFFAOYSA-N 0.000 description 1
- 229960000572 olaparib Drugs 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000002210 silicon-based material Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 210000000352 storage cell Anatomy 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T7/00—Image analysis
- G06T7/0002—Inspection of images, e.g. flaw detection
- G06T7/0012—Biomedical image inspection
- G06T7/0014—Biomedical image inspection using an image reference approach
- G06T7/0016—Biomedical image inspection using an image reference approach involving temporal comparison
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T7/00—Image analysis
- G06T7/0002—Inspection of images, e.g. flaw detection
- G06T7/0012—Biomedical image inspection
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/80—Hyaluronan
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/10—Image acquisition modality
- G06T2207/10016—Video; Image sequence
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/10—Image acquisition modality
- G06T2207/10024—Color image
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/10—Image acquisition modality
- G06T2207/10056—Microscopic image
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/10—Image acquisition modality
- G06T2207/10064—Fluorescence image
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/20—Special algorithmic details
- G06T2207/20081—Training; Learning
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/20—Special algorithmic details
- G06T2207/20084—Artificial neural networks [ANN]
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/30—Subject of image; Context of image processing
- G06T2207/30004—Biomedical image processing
- G06T2207/30024—Cell structures in vitro; Tissue sections in vitro
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/30—Subject of image; Context of image processing
- G06T2207/30004—Biomedical image processing
- G06T2207/30096—Tumor; Lesion
Definitions
- the described embodiments relate generally to systems and methods for determining the efficacy of a treatment, such as an anti-cancer agent.
- a range of treatment agents may be used to treat cancerous cells, such as those associated with various types of tumors.
- Factors such as tumor type, progression, patient characteristics, anti-cancer agent characteristics, and so on may impact the efficacy of a given treatment. These and other factors may hinder the ability of a medical provider to select the most appropriate anti-cancer agent, such as that with the highest efficacy.
- Anti-cancer treatments may be administered that result in a minimal efficacy and/or detract from overall patient treatment.
- other types of treatments for certain diseases may vary for different patients based on specific patient factors.
- Embodiments of the present invention are directed to treatment efficacy prediction systems and methods.
- a microfluidic chip in one example, includes a body defining a channel and a cell culture chamber fluidly coupled to the flow channel.
- the microfluidic chip further includes a coupling portion attached to the body and defining an inlet and an outlet.
- the channel defines a flow path extending between the inlet and the outlet with the cell culture chamber positioned therebetween.
- the microfluidic chip further includes a gas permeable membrane covering the cell culture chamber.
- the channel is configured to deliver a growth media to the cell culture chamber.
- the cell culture chamber may have a substantially cylindrical shape.
- the cell culture chamber may have a substantially cylindrical shape with a diameter of preferably about 6.75 mm. In other cases, the diameter may be more or less than 6.75 mm based on the particular application, such as being at least about 5.0 mm, at least about 3.0 mm or other appropriate diameter.
- the body defines the cell culture chamber having a closed bottom end and an open top end.
- the gas permeable membrane may cover the open top end of the cell culture chamber. Further, the gas permeable membrane may be attached to the body portion by an adhesive.
- the cell culture chamber may be a first cell culture chamber.
- the body may further define a second cell culture chamber fluidly coupled to the channel along the flow path between the first cell culture chamber and the inlet or the outlet of the coupling portion.
- the first cell culture chamber may have a first volume and the second cell culture chamber may have a second volume that is different from the first volume; however, this is not required.
- the first cell culture chamber may have a first shape and the second cell culture chamber may have a second shape that is different from the first shape; however, this is not required.
- the body includes a multi-layered structure.
- the multi-layered structure includes a first body portion layer defining the cell culture chamber.
- the multi-layered structure further includes a second body portion layer connected to the first body portion layer and defining the channel fluidly coupled to the cell culture chamber.
- the multi-layered structure further includes a third body portion layer connected to the second body portion layer opposite the first body portion layer and defining an opening or hole above the cell culture chamber.
- the coupling portion may be attached to the third body portion layer.
- the third body portion layer further defines a first lumen fluidly coupled to the inlet of the coupling portion and extending to the flow channel of the second body portion layer.
- the third body portion may further define a second lumen fluidly coupled to the outlet of the coupling portion and extending to the flow channel of the second body portion layer.
- the inlet and the outlet define tube barbs.
- the barbs may protrude from a topmost surface of the chip.
- the body may include or be formed fully from an acrylic material or a silicone-based material.
- a microfluidic device may include a dosing bank including a plurality of reservoirs. Each reservoir of the plurality of reservoirs may be configured to hold a growth media.
- the microfluidic device may further include a staging section or “stage” configured to arrange a plurality of microfluidic chips.
- the plurality of reservoirs can correspond to the plurality of microfluidic chips. In some cases, multiple reservoirs may be used for any given microfluidic chip.
- the microfluidic device may further include a pump fluidly coupleable with the plurality of reservoirs and the plurality of microfluidic chips to define fluid circuits between each corresponding pair of the plurality of reservoirs and the plurality of microfluidic chips. The pump further may cause a circulation of a growth media through the fluid circuits for each corresponding pair.
- each reservoir of the plurality of reservoirs are fluidly isolated from one another.
- the fluid circuits may therefore be fluidically isolated from one another.
- the pump may be further configured to selectively cause a circulation of the growth media through an individual fluid circuit of the fluid circuits.
- the growth media may include a treatment agent, such as an anti-cancer agent and/or cells in suspension.
- the cells may be in circulation with the growth media such that the drugs, cells, and/or other agents are included.
- circulating cells can be used to make immune models or test cell therapies. Additionally or alternatively, other circulating agents can help characterize the behavior or properties of the cells in circulation.
- the plurality of reservoirs may be exposable to atmosphere.
- the reservoirs may have lids that can be opened to expose the reservoir to air for the purpose of loading treatments. Once the treatment is loaded, the lid can be closed.
- the microfluidic device may operate as described herein upon the closing of the reservoirs with the lids.
- the plurality of reservoirs may be configured to receive a treatment agent or combination of agents during operation of the pump.
- the dosing bank may include a tray or other structure configured to hold the plurality of reservoirs in a substantially upright position.
- the device may further include tubes fluidly coupling the pump to each reservoir and each microfluidic chip housed in the staging section.
- a method of forming a solid culture includes isolating target cells from a patient sample.
- the method further includes forming stained cells from the isolated cells by staining the isolated cells with a light-responsive dye.
- the method further includes encapsulating the stained cells in a hydrogel.
- the hydrogel may include hyaluronic acid, collagen and/or other elements that are configured to mimic core components of human tissue extracellular matrices and/or disease-specific cell niches.
- the method may further include culturing dissociated cells in the hydrogel.
- the culturing may further include forming two-dimensional cell cultures of the dissociated cells.
- the culturing may further include forming three-dimensional cell cultures of the dissociated cells.
- the culturing may further include forming cell cultures of a single population of dissociated cells.
- the culturing may further include forming cell cultures from multiple cell types.
- the dissociated cells include cancer cells as well as normal/non-transformed cells, stromal cells, or immune cells.
- the culturing further comprises forming co-culture of cancer, normal/non-transformed, stromal, and/or immune cells.
- the dissociated cells may be isolated from a patient-derived tissue or tumor sample.
- the method may further include forming the spheroid or an organoid.
- the method may include culturing the spheroid or the organoid in a hydrogel.
- the spheroid or the organoid may include cancer cells, normal/non-transformed cells, stromal cells, or immune cells.
- the culturing further includes forming co-culture of cancer, normal/non-transformed, stromal, and/or immune cells.
- the isolated cells may be isolated from a patient-derived tissue or tumor sample.
- the method further includes processing the patient sample using a digestion enzyme-based operation, a blood lysis solution, or a selecting operation to isolate target cells of the patient sample.
- Processing may allow for the isolation of many different cell types, such as many different cell types that stay alive from the patient tissue or tumor sample.
- different cells can be co-cultured, including cells in addition to cancer, normal/non-transformed, immune, and/or stromal cells, in either a dissociate cell culture or spheroid/organoid.
- the light-responsive dye may be configured to allow for tracking of the target cells via fluorescence microscopy.
- the light-responsive dye may be configured to stain mitochondria of the target cells for live cell tracking.
- the light-responsive dye may be configured to stain nuclei of the target cells for dead cell tracking.
- forming stained cells further includes staining isolated cells with a first light-responsive dye.
- the first light-responsive dye may be configured to stain mitochondria of the target cells for live cell tracking.
- Forming stained cells may further include staining isolated cells with a second light-responsive dye, the second light-responsive dye being configured to stain nuclei of the target cells for dead cell tracking.
- the light-responsive dye may be configured to cause a color change in the stained cell when the stained cell transitions from a living cell to a dead cell.
- the target cells may be, without limitation, cells of a tumor, the tumor comprising a breast cancer, a colorectal cancer, a lung cancer, a kidney cancer, a pancreatic cancer, an ovarian cancer, a brain cancer, or a gastric cancer.
- the patient sample includes tissue slices, surgical resections and/or xenografts.
- the patient sample may also include biopsy samples, including core needle biopsy samples in certain circumstances.
- the method may further include culturing the tissue slices, cores, surgical resections and/or xenografts in a hydrogel.
- a method of loading a microfluidic chip includes arranging a solid cell culture in a cell culture chamber of the microfluidic chip.
- the microfluidic chip includes a body defining the cell culture chamber and a channel that traverses the cell culture chamber and extends between an inlet and an outlet of the microfluidic chip.
- the method further includes positioning a gas permeable membrane over the cell culture chamber while the inlet and outlet remain exposed for coupling to a circulation system.
- the positioning further includes adhering the gas permeable membrane to the body and covering the cell culture chamber.
- the arranging of the solid cell culture may further include dropping a quantity of the solid cell culture (e.g., the cell culture in a hydrogel) into the cell culture chamber using a pipette.
- the solid cell culture may be a first solid cell culture and the cell culture chamber may be a first cell culture chamber.
- the method may further include arranging a second solid cell culture in a second cell culture chamber of the microfluidic chip.
- the second cell culture chamber may be defined by the body and fluidly coupled to the channel between the inlet and the outlet.
- the first cell culture chamber may have a first volume and the second cell culture chamber may have a second volume that is different from the first volume.
- the first cell culture chamber may have a first shape and the second cell culture chamber may have a second shape that is different from the first shape.
- a method of operating a microfluidic chip includes fluidly coupling the microfluidic chip with a microfluidic device to define a fluid circuit between the microfluidic chip, a flow restrictor, a reservoir, and a pump; the microfluidic chip including a solid cell culture, the reservoir including a growth media.
- the method further includes causing a flow of the growth media through the circuit such that the solid cell culture of the microfluidic chip is exposed to the growth media to form an exposed cell culture.
- a “solid cell culture” refers to the cells in the hydrogel material.
- an “exposed cell culture” refers to a circumstance in which the solid cell culture is exposed to media (e.g., growth media) while the cell culture itself remains solid (e.g., the growth media flowing along the hydrogel with cells.).
- a “liquid cell culture” refers to cells that are in a liquid medium exclusively.
- a liquid cell culture may include immune cells put into a growth media/serum and then circulated using the systems and techniques described herein. The method includes analyzing a response of the solid cell culture to the growth media.
- the growth media includes a treatment agent.
- the fluid coupling further includes fluidly coupling a first tube portion to an inlet of the microfluidic device.
- the first tube portion may be connected to a second tube portion fluidly coupled with a reservoir, the first and second tube portions defining a common tube.
- the fluid coupling may further include fluidly coupling a third tube portion to an outlet of the microfluidic device and the reservoir to define a fluid circuit.
- the microfluidic device may define a flow path therethrough.
- the microfluidic chip may include a cell culture chamber that holds the hydrogel including the target cells.
- the flow path may traverse and/or run adjacent to the cell culture chamber. The method may therefore further include causing a flow of the growth media along the flow path while a hydrogel restrains the target cells from exit from the culture cell culture chamber.
- first tube portion may be fluidly coupled to the pump.
- the second tube portion may be fluidly coupled to the reservoir.
- the first and second tube portions may be portions of the same tube.
- the microfluidic device may further include a third tube portion connected to the reservoir and the microfluidic chip to complete the circuit.
- the circuit may be a closed-circuit.
- the method further includes fluidly coupling a second microfluidic chip with the microfluidic device to define a second fluid circuit between the second microfluidic chip, a second reservoir, and the pump; the second microfluidic chip including a second solid cell culture, the second reservoir including a second growth media.
- the method may further include causing a second flow of the second growth media through the second circuit such that the second solid cell culture of the second microfluidic chip is exposed to the second growth media.
- the method may further include analyzing a response of the second solid cell culture to the second growth media.
- the growth media and the second growth media include different treatment agents, such as different anti-cancer agents or cells.
- the method may further include comparing the response of the first solid cell culture and the response of the second solid cell culture to determine a treatment efficacy.
- the first circuit and the second circuit may be fluidly isolated from one another.
- the pump may be configured to control the first flow and the second flow independently.
- the method may further include, prior to the analyzing, fluidly uncoupling the microfluidic chip from the microfluidic device.
- the method may further include fluidly coupling the microfluidic chip with the microfluidic device to define the fluid circuit between the microfluidic chip, the reservoir, and the pump.
- the method may further include causing another flow of the growth media (e.g., the same growth media or a different growth media) through the circuit such that the solid cell culture of the microfluidic chip is exposed to the growth media.
- the method may further include, subsequent to the causing of another flow, analyzing a subsequent response of the solid cell culture to the growth media.
- the method may therefore further include comparing the response of the solid cell culture to the growth media and a subsequent response of the solid cell culture to the growth media to determine a treatment efficacy.
- the method may further include analyzing the response of the solid cell culture to the growth media to determine a first cell population quantity.
- the method may further include analyzing the subsequent response of the solid cell culture to the growth media to determine a second cell population quantity.
- the method may further include analyzing further subsequent responses of the solid cell culture and determining a third cell population quantity, a fourth cell population quantity, and so one over a course of days or other appropriate interval.
- the method may further include comparing the first cell population quantity and the second cell population quantity to determine a change in cell population quantity indicative of a treatment efficacy.
- the method may further include analyzing the response of the solid cell culture to the growth media to determine a first cell population position, and analyzing the subsequent response of the solid cell culture to the growth media includes determining a second cell population position.
- the method may further include analyzing further responses of the solid cell culture and determining a third cell population position, a fourth cell population position, and so one over a course of days or other appropriate interval.
- the method may further include comparing the first cell population position and the second population position to determine a change in cell population position indicative of a treatment efficacy.
- the analyzing may include conducting a fluorescence microscopy operation on the solid cell culture of the microfluidic chip.
- the analyzing includes collecting three-dimensional images of the solid cell culture of the microfluidic chip.
- the analyzing may further include collecting two-dimensional images in a z-stack of the solid cell culture of the microfluidic chip.
- the analyzing may further include analyzing multiple responses of the solid cell culture to the growth media over time. The analyzing may be conducted daily.
- the analyzing may include conducting a confocal microscopy operation on the solid cell culture of the microfluidic chip. Additionally or alternatively, the analyzing may include conducting a brightfield microscopy operation on the solid cell culture of the microfluidic chip. Additionally or alternatively, the analyzing may include conducting a lattice light sheet microscopy operation on the solid cell culture of the microfluidic chip.
- the analyzing includes executing instructions of a non-transitory computer-readable media, with one or more processing elements of a computer, to determine a treatment efficacy of a treatment agent of the growth media, on the solid cell culture.
- a method of analyzing a solid cell culture over time includes determining a first response of a solid cell culture to a growth media including a treatment agent.
- the solid cell culture is held in a cell culture chamber of a microfluidic chip.
- the method further includes determining a second response of the solid cell culture to the growth media, including a treatment agent.
- the method further includes comparing the first and second responses to determine a treatment efficacy.
- multiple responses of the solid cell culture may be compared to determine treatment efficacy, such as comparing three, four, or more responses for each solid cell culture/chip as appropriate for a given application.
- the computer vision and imaging techniques described herein may be applied to comparing and analyzing any number of responses over any appropriate time period.
- one or both of the first response or the second response includes at least one of a color of the solid cell culture, a pixel intensity of an image of the solid cell culture, a shape of the solid cell culture, a size of the solid cell culture, a position of cells of the solid cell culture, or a quantity of cell of the solid cell culture.
- the treatment efficacy may be indicative of a viability of cells of the solid cell culture in response to the treatment agent.
- determining the first response comprises capturing an image of the solid cell culture.
- the method may further include determining a cell viability from the image and predicting a patient response to treatment agent based on the cell viability.
- the method may further include determining a cell proliferation from the image and predicting a patient response to treatment agent based on the cell proliferation.
- the method may further include determining a cell position from the image and predicting a patient response to treatment agent based on the cell position.
- the image may be a first image.
- determining the second response may include capturing a second image of the solid cell culture.
- the method may further include comparing the first image and the second image (and/or additional images) to determine a cell migration distance of a cell of the solid cell culture over time.
- the method may further include predicting a patient response to the treatment agent using the cell migration distance.
- the method may further include comparing the first image and the second image to determine a cell migration speed of a cell of the solid cell culture over time.
- the method may further include predicting a patient response to the treatment agent using the cell migration speed.
- the method may further include comparing the first image and the second image to determine a migration distance of a plurality of cells that define a subset of the solid cell culture over time.
- the method may further include predicting a patient response to the treatment agent using the migration distance.
- the method may further include comparing the first image and the second image to determine a migration speed of a plurality of cells over time.
- the method may further include predicting a patient response to the treatment agent using the migration speed.
- the subset of cells may include the 5% most aggressive cells of the plurality of cells.
- the plurality of cells may include the 2% most aggressive cells of the plurality of cells.
- the plurality of cells may include the 1% most aggressive cells of the plurality of cells.
- the plurality of cells may include the subset of cells expressing a specific biomarker.
- the method may further include comparing the first image and the second image to determine a cell having a maximum migration vector in the solid cell culture over time.
- the method may further include predicting a patient response to the treatment agent using the maximum migration vector.
- the method may further include comparing the first image and the second image to determine a cell having a maximum migration speed in the solid cell culture over time.
- the method may further include predicting a patient response to the treatment agent using the maximum migration speed.
- determining the first response or determining the second response include determining characteristics of a single cell of the plurality of cells.
- the single cell of the plurality of cells may have characteristics that can be used to predict a response of the plurality of cells (e.g., the cell with longest migration vector may be a predictive biomarker, as one example).
- determining the first response includes determining characteristics of the single cell at a first time.
- Determining the second response includes determining characteristics of the single cell at a second time subsequent to the first time.
- the method further includes predicting a patient response to the treatment agent based on a comparison of the measured characteristics of the single cell at the first time and the second time and/or additional times, as described herein.
- the method may further include predicting a patient response to the treatment agent based on the weighted cell measurement.
- the first image comprises a first weighted cell measurement and the second image comprises a second weighted cell measurement.
- the method further includes predicting a patient response to the treatment agent based on a comparison of the first weighted cell measurement and the second weighted cell measurement.
- the method may further include predicting a patient response to the treatment agent based on the radius or the diameter of a spheroid or an organoid.
- the first image includes a first radius or a first diameter of a spheroid or an organoid and the second image includes a second radius or a second diameter of the spheroid or an organoid.
- the method further includes predicting a patient response to the treatment agent based on a comparison of the first radius or the first diameter with the second radius or the second diameter.
- dissociated/single cells may be analyzed in a similar manner.
- dissociated/single cells may be tracked or measured or otherwise measured according to substantially any of the associated metrics, as described herein.
- a count and/or a position of a dissociated/single cell that leaves the spheroid, a migration distance, a migration speed, and so on may be determined and analyzed according to the techniques described herein for determining treatment efficacy.
- the method further includes predicting a patient response to the treatment agent based on a length, a width, or a height of a surgical resection, tissue slice and/or xenograft.
- the first image includes a first length, a first width, or a first height of an surgical resection, tissue slice and/or xenograft and the second image includes a second length, a second width, or a second height of the surgical resection, tissue slice and/or xenograft.
- the method further includes predicting a patient response to the treatment agent based on a comparison of the first length, the first width, or the first height with the second length, the second width, or the second height.
- comparing further includes executing instructions of a non-transitory computer-readable media, with one or more processing elements of a computer, to determine the treatment efficacy.
- FIG. 1 depicts a functional diagram of a treatment efficacy prediction system.
- FIG. 2 depicts a flow diagram for determining a treatment efficacy.
- FIG. 3 depicts an example patient sample.
- FIG. 4 depicts a user interface including information associated with stained cells.
- FIG. 5 depicts a chart showing the relative concentration and size distribution of living cells and dead cells.
- FIG. 6 depicts a 2D micrograph of an example spheroid.
- FIG. 7 A depicts a first representation of an ovarian tumor indicative of living cells.
- FIG. 7 B depicts a second representation of the ovarian tumor of FIG. 7 A indicative of dead cells.
- FIG. 7 C depicts a third visual representation of the ovarian tumor of FIG. 7 A including a composite of living and dead cells.
- FIG. 8 A depicts an exploded view of an example microfluidic chip.
- FIG. 8 B is a cross-sectional view of the microfluidic chip of FIG. 8 A .
- FIG. 8 C is a cross-section view of a flow restrictor for use with the microfluidic chip in a fluid circuit.
- FIG. 9 A depicts an operation of the microfluidic chip of FIG. 8 A .
- FIG. 9 B depicts another operation of the microfluidic chip of FIG. 8 A .
- FIG. 9 C depicts another operation of the microfluidic chip of FIG. 8 A .
- FIG. 9 D depicts another operation of the microfluidic chip of FIG. 8 A .
- FIG. 9 E depicts another operation of the microfluidic chip of FIG. 8 A .
- FIG. 10 depicts a top schematic view of a microfluidic device for use with the microfluidic chip of FIG. 8 A .
- FIG. 11 depicts an isometric view of the microfluidic device performing a dosing operation.
- FIG. 12 depicts an example spheroid of isolated and dyed tumor cells after a dosing operation.
- FIG. 13 A depicts a chart showing a first quantity of cells at a first time point.
- FIG. 13 B depicts another chart showing a second quantity of cells at a second time point.
- FIG. 14 A depicts a chart showing a three-dimensional position of a quantity of cells at a first time point.
- FIG. 14 B depicts another chart showing a three-dimensional position of a quantity of cells at a second time point.
- FIG. 15 depicts a chart showing a quiver plot indicative of the three-dimensional movement of cells.
- FIG. 16 depicts an example report including visualizations of different dosing operations performed on a given patient sample.
- FIG. 17 depicts a chart showing a distribution of cancerous cell viability based on one or more of dosing operations described herein.
- FIG. 18 depicts a flow diagram for forming a cell culture.
- FIG. 19 depicts a flow diagram for loading a microfluidic chip.
- FIG. 20 depicts a flow diagram for dosing and imaging a culture of microfluidic chip.
- FIG. 21 depicts a flow diagram for determining a treatment efficacy.
- FIG. 22 depicts a functional block diagram of a system including a computing device.
- the present disclosure relates to systems and methods for predicting a patient response to various agents and/or combinations of agents using ex vivo dosing and imaging.
- the systems and methods may be applicable to oncology, ex vivo monitoring of disease progression, such as cancer progression, testing of treatment agents, such as anti-cancer agents, patient stratification, and other medical treatment efficacy testing.
- the system and methods allow discovery, identification, or validation of therapeutic, diagnostic and/or prognostic biomarkers for the purpose of drug development and treatment decision making.
- an organ-on-a-chip and computer vision system are used to analyze biomarkers for predicting patient response to treatment agents, such as anti-cancer agents.
- tumor and/or healthy tissue samples may be cultured in a hydrogel or other environment and placed in a cell culture chamber of a microfluidic chip. Tumor and/or healthy tissue samples may be split into multiple segments, such as aliquots, to culture in multiple chips or separate cell culture chambers.
- the samples are labelled or stained for tracking, such as via light-responsive dyes, such as fluorescent dyes.
- the stained cells may be tracked by microscopy and processed and analyzed by a variety of computer-implemented techniques, such as the disclosed computer vision techniques of the present disclosure.
- the fluorescent dyes may selectively stain live cells. Other fluorescent dyes selectively stain dead cells. Some dyes may stain all cells, regardless of whether they are alive or dead. In some cases, dyes may be used to stain cells that express certain biomarkers or targets, such as certain proteins. DNA or RNA may also be stained using dyes according to the techniques disclosed herein.
- the microfluidic chip including a cell culture chamber, may be arranged with (e.g. fluidly coupled with) a microfluidic device or other system or pump in order to introduce growth media, treatment agents, and other media to the cell culture of the chip.
- the device may include various treatment agents held in reservoirs with growth media.
- the media is circulated from the cell culture chamber to the microfluidic chip using a pump, such as a peristaltic pump, pneumatic pump, and so on.
- a solid cell culture including target cells and hydrogel may be deposited in the microfluidic chip.
- the solid cell culture may be exposed to the circulating media, which may include the treatment agents, in order to define an exposed solid cell culture within the microfluidic chip.
- the cell culture chamber of the microfluidic chip having the solid cell culture may be imaged using various techniques, such as microscopy techniques, including confocal microscopy, to track the live and dead cells over time.
- the imaging may be based on identification of the cells due to staining or labeling of the cells.
- the live/dead status of the cells may be tracked using the fluorescent dye or other tracking dye.
- Three-dimensional (3D) images, or stacks of two-dimensional (2D) images taken layer-by-layer, are collected for analysis using a computer vision tool.
- the computer vision tool which may be executed by one or more computing devices (e.g., via software executing one or more algorithms or machine learning models) may track cell characteristics (e.g., the shape, size, position (x, y, z) and color) of cells in a given image. As the cells may be stained with selectively activated dyes, the number of live and dead cells in an image may be determined. For example, the computer vision may analyze pixel information, such as hue, intensity, and the like, to determine location of specific cells (e.g., live cells may have a first color and dead cells may have a second color) and the location and number of the different cells may be tracked by analyzing subsequent images or image frames over time.
- cell characteristics e.g., the shape, size, position (x, y, z) and color
- the computer vision may analyze pixel information, such as hue, intensity, and the like, to determine location of specific cells (e.g., live cells may have a first color and dead cells may have a second color) and the location and
- One or more images captured at different points in time may be used to monitor the progression of malicious cells (e.g., cancer cells) ex vivo and can be used to predict the response a patient may have to a given agent. For example, comparing a first image frame having a live cell in a first location, such as a first pixel, may be compared to a second image frame captured at a second point in time, having a live cell in a different location, where the system may estimate that the cell has migrated or moved from the first location to the second location. The distance may then be determined by analyzing the difference in pixel locations in the image. Various metrics may be derived from these images, including cell viability, distance and speed of cell migration over time. These assessments, used alone or in combination, may be used to predict patient outcomes to a given agent.
- malicious cells e.g., cancer cells
- microfluidic chips may be analyzed in parallel to determine treatment efficacy across a range of anti-cancer treatments.
- microfluidic chips exposed to either no agent (baseline) or various single and/or combination agents, such as chemotherapies, a comparison may be made between images to predict patient response.
- the techniques described herein may be used to discover therapeutic, diagnostic and prognostic biomarkers that aid the treating of cancer patients and the development of new therapeutics.
- the treatment efficacy prediction system 100 may broadly be configured to facilitate the ex vivo monitoring of disease progression, such as cancer progression, testing of treatment agents, and patient stratification.
- the system 100 may include modules, devices, assemblies, sub-assemblies, and so on (described in relation to the examples of FIGS. 3 - 17 ) that cooperate with one another to evaluate the efficacy of a selected treatment agent, combination agents and/or other treatments for use in treating a particular patient. It will be appreciated that any of the modules, devices, assemblies, sub-assemblies, and so on may be implemented separately from one another, as described herein.
- Treatment agents and other agents may be more or less effective based on the particularized circumstances of a treatment regimen, patient characteristic, cancer type and progression or stage, in addition to a range of other complex and often interrelated factors.
- Conventional treatment regimens may lack the ability to appropriately assess these factors, which may result in the delivery of treatment agents that are suboptimal.
- various treatment agents may be administered and monitored in vivo, which may hinder the treatment of the patient and overall health outcomes. For example, with conventional approaches, it may be impractical or even impossible to determine if a particular cancer treatment would be effective without administering the treatment to the patient and evaluating the patient over an extended period of time. Further, a patient may have an adverse or undesirable outcome to a particular cancer treatment, once administered. And it may take an exceedingly long period of time for a clinician to assess the efficacy of the treatment, during which time, a patient's overall health conditions may deteriorate.
- the system 100 depicted functionally in FIG. 1 may mitigate these and other hindrances by allowing for ex vivo monitoring of cancer progression in the context of proposed treatment agents.
- the system 100 may permit a clinician or other provider to evaluate a spectrum of various different treatment agents for one or more patient samples, such as a sample including a cancerous tumor.
- the system 100 may allow for the creation of an ex vivo environment for the patient sample that replicates in vivo conditions.
- target cells such as any of the cells described herein, may be cultured and encapsulated in a hydrogel in order to provide structural stability to the cells, mechanical properties and microenvironmental cues to mimic physiological environments in human organs.
- a modular environment may be provided including a range of cell culture conditions, including conditions in which human serum is present, in order to provide conditions that may be similar to the conditions of the human body.
- the clinician may monitor the progression and response of each tissue sample for each of the treatment agents.
- the treatment agents with the most appropriate response or most suitable efficacy may be identified for administration to the patient for treatment.
- FIG. 1 depicts the system 100 broadly including a culture module 104 , a dosing module 108 , and an analysis module 112 .
- the culture module 104 may generally include various mechanical components, instrumentation, solutions, and devices and so on that are used to prepare a patient sample for evaluation and dosing. While many functions are contemplated and described herein, the culture module 104 may broadly be configured to isolate target cells from a patient sample, form stained cells from the isolated cells, and encapsulate the stained cells in a media.
- the target cells may be cells from a patient-derived tissue or tumor sample.
- the target cells may be from tissue slices, cores, surgical resections and/or xenografts.
- Sample target cells may include cells associated with malicious tissues, such as cells from a breast cancer, a colorectal cancer, a lung cancer, a kidney cancer, a pancreatic cancer, an ovarian cancer, a brain cancer, or a gastric cancer, and so on.
- the cell culture module 104 may isolate the target cells and stain the cells with the light-responsive dye, such as a fluorescent dye. Staining, such as with a fluorescent dye may stain or tag the entirety of the cell population or stain individual cell populations, or stain individual components of a given cell population, such as a stain on the mitochondria and a separate stain on a nuclei, to allow for tracking of the cell via fluorescence microscopy or other procedures.
- the light-responsive dye such as a fluorescent dye
- the stained cells may be encapsulated in a media, such as a hydrogel, thus forming the solid cell culture, in the culture module 104 in order to promote replication of the core components of human tissue.
- the stained cells may be encapsulated in a media, such as a hydrogel or may be formed as a spheroid or organoid, thus forming the solid cell culture.
- the dosing module 108 may generally include various mechanical components, instrumentation, solutions, and devices and so on that are used to administer treatment agents to a cell culture and collect data regarding the response of the culture to the treatment agents.
- the dosing module 108 may include a microfluidic chip that is configured to receive a solid cell culture (e.g., cells and hydrogel). Growth media may be put in circulation with the solid cell culture to define an exposed solid cell culture.
- the microfluidic chip may hold the cells in the hydrogel and allow for the circulation of treatment agents alongside growth media in a gas permeable environment (e.g., to provide the cell with oxygen).
- a pump of a microfluidic device of the dosing module 108 may cause circulation of the treatment agents and growth media through the chip.
- a plurality of chips may permit the device to circulate different treatment agents to each chip to deposit the treatments within the cell culture chambers to evaluate the efficacy of different treatments.
- the chips may be analyzed at select intervals, such as daily (for one, two, three, four, five, or more days), and a response to the treatment agent may be determined.
- fluorescence microscopy may be used to determine the concentration of living and dead cells in a given culture, using the light-responsive dye. More generally, and as described herein, any appropriate camera or imaging device may be used. Images may be captured over time, and presented in a two-dimensional and/or three-dimensional format, in order to provide a sufficient data set for analysis of the treatment efficacy.
- the analysis module 112 may generally include various computer vision systems (e.g., computer system 2200 of FIG. 22 ) or image analysis systems, computer implemented techniques, and analysis devices that may be operated to determine the efficacy of treatment of one or more of treatment agents administered to the cells.
- the light-responsive dye may be used to indicate the concentration of dead cells, living cells, as well as the transition of cells from a living to dead state. The concentration of cells living or dead, over time, may provide an indication of the viability of cancerous cells, as one example, over the course of administration of a particular treatment agent.
- Properties of the captured images may be further analyzed to predict a treatment efficacy, including, without limitation, cell characteristics, such as a color of the solid or liquid cell culture, a pixel intensity of an image of the solid or liquid cell culture, a shape of solid or liquid cell culture, a size of the solid or liquid cell culture, a position of cells of the solid or liquid cell culture, or a quantity of cells of the solid or liquid cell culture.
- cell characteristics such as a color of the solid or liquid cell culture, a pixel intensity of an image of the solid or liquid cell culture, a shape of solid or liquid cell culture, a size of the solid or liquid cell culture, a position of cells of the solid or liquid cell culture, or a quantity of cells of the solid or liquid cell culture.
- a two-dimensional or three-dimensional image of the cell culture may be generated at multiple different times over the course of ex vivo administration.
- the images may include particularized information for each cell, including cell position and type.
- the images can be compared to determine various properties, including cell migration, cell migration speed, and cell migration distance.
- the images can be further analyzed to determine a maximum migration speed and/or a maximum migration vector, among other characteristics.
- the collective images of the ex vivo response of the cells to the treatment agent may be indicative of an in vivo response to the treatment agent, and thus used to predict treatment efficacy.
- FIG. 2 depicts a flow diagram 200 for determining a treatment efficacy.
- the flow diagram 200 may implement one or more of the operations of the system 100 described above with respect to FIG. 1 . Accordingly, the operations described with respect to the flow diagram 200 may be implemented by one or more of the culture module 104 , the dosing module 108 , and/or the analysis module 112 .
- a patient sample is collected.
- Patient samples may arrive at a lab, including samples containing a tumor of various cancer types.
- tumor samples may be processed using a digestion enzyme-based cell isolation kit, blood lysis solution and other selecting steps to isolate viable cells while removing blood and contaminants.
- the output may be a viable mixed cell population containing the various cells from the primary tumor, including cancer cells, normal/non-transformed cells, stromal cells, and immune cells.
- cells may be stained using live and/or dead cell labeling dyes. Additionally or alternatively, cell-specific/biomarker-targeting dyes may be used. These dyes may be light-responsive dyes that stain or mark the cells. For example, the dyes may be specifically selected for the ability to track cells without requiring the cells to be fixed.
- MitoViewTM 633 may be used to track live cells by staining the mitochondria of the cells. This dye exhibits a red color when imaged using fluorescence microscopy or other imaging techniques, including confocal and lattice light sheet microscopy. The red color may disappear after cells die.
- NucView® 488 may be used to track dead cells by staining the nucleus of the cells.
- the dye may have a green color when imaged using fluorescence microscopy or other fluorescent imaging techniques; however, it will be appreciated that substantially any color may be used and the foregoing are provided as illustrative example colors. When cells die, the green color may appear. Additionally or alternatively, cells may be stained with both dyes simultaneously to more accurately track cells as they transition from living to dead. It will be appreciated that the red and green colors are described above for purposes of illustration. More generally the light-responsive dyes described herein may have a range of different colors depending in part on the fluorescent dye used. In this regard, various other colors, intensity of colors, and so may be used for staining the cells and performing one or more analysis operations, such as any of the analysis techniques described herein.
- spheroids are optionally formed.
- an ultra-low attachment (ULA) plate may be used to form the spheroid.
- the cells or spheroids may be embedded within hydrogel.
- the hydrogel may contain hyaluronic acid and collagen to mimic core components of human tissue extracellular matrices and/or disease-specific cell niches.
- the spheroids may include the target and/or stained cells in a generally spheroid shape which may facilitate the determining of the response of the cells to a treatment agent, for example, by permitting the tracking of spheroid characteristics, including, but not limited to, a size, shape or other properties of the spheroid over time.
- the spheroid, dissociated cell culture (e.g., without a spheroid architecture), or other cell culture, including a hydrogel and the stained cells, may be set in a microfluidic chip (e.g., the microfluidic chip 800 of FIGS. 8 A and 8 B ).
- the microfluidic chip may include one or more cell culture chambers.
- the solid cell culture may be deposited in a first cell culture chamber with a pipette or other instrument.
- a flow channel is fluidly connected to the one or more cell culture chambers.
- the flow channel may allow the microfluidic chip to fluidly couple to a circulation system, as described herein with reference to FIGS. 10 and 11 .
- the microfluidic chip may be configured to permit circulation through the flow channels to promote interaction between a circulated flow and the hydrogel and cells of the chip.
- the hydrogel and cells are within the microfluidic chip and covered by a gas permeable membrane.
- a gas permeable membrane may be positioned over the cell culture chamber and adhered to a body of the chip. Once the gas permeable membrane is positioned over the chip, the cell culture chambers are covered. This may allow growth media to flow into the chip without leaking or overflowing using tubing connected to the chip's inlet and outlet barbs.
- Each microfluidic device may include reservoirs that are connected to a corresponding microfluidic chip via tubing.
- a peristaltic pump is used to collect growth media in a 15 mL conical tube or reservoir and circulate the growth media to the inlet of the chip. The growth media may then travel through the flow channel and exit through the outlet of the chip. The growth media then returns to the reservoir using another line of tubing.
- chips/reservoirs may be connected using the peristaltic or other pump. (e.g., FIGS. 10 and 11 ).
- One chip may be a control where only growth media is circulated to the dissociated cell culture in the circular chamber.
- Various drugs and combinations may then be circulated to the other chips via their corresponding cell culture chambers, including Olaparib, AC-T and AC-CarboTaxol.
- imaging may commence.
- the imaging may be periodic, such as being at select intervals, such as daily, every twelve hours, and so on as appropriate for a given application.
- the imaging may occur on a daily basis.
- the media is cleared from the microfluidic chips.
- the microfluidic chips may be detached from the microfluidic device so they can be transferred to a microscope for imaging.
- a confocal microscope is used to conduct three-dimensional fluorescence imaging. Each chip has the length and width of a standard microscope slide to ensure compatibility. The chips are placed in the slide holder of the confocal microscope and the laser settings are selected to match the excitation wavelength and detection wavelength of each cell staining dye.
- two-dimensional images of the chips are captured.
- the confocal microscope may take images layer by layer from the bottom to the top of the chamber.
- a step size of 5 micrometers may be used. It will be appreciated that in other cases, other steps sizes may be appropriate, such as a step size of 10 micrometers or more.
- the chamber(s) containing cells may be imaged in its entirety to capture the location of all cells.
- the circular chamber may be imaged on a daily basis. In other instances, imaging is conducted at larger intervals (e.g., day 1 and day 5), and can also be conducted in shorter intervals (e.g., hour 1 and hour 12). Between each imaging of the chip, the chip may be recoupled with the microfluidic device in order to provide additional treatment agents to the chip. Accordingly, the subsequent imaging of the chip may indicate the progression and response of the tumor to the treatment agent over time.
- the two-dimensional images may be stitched together.
- the raw microscope images may be ordered by their z position. Once the images are ordered from the bottom to the top, each layer can be stitched onto each other along the z axis. Cells are detected on each two-dimensional image and assigned an x, y and z position.
- three-dimensional images and other visual representations of the cells may be generated in order to determine a treatment efficacy. For example, once the z-stack is complete, some cells may appear on more than one stack due to the confocal microscopy capturing the same cell in more than one layer.
- the brightest pixel is identified as the true z position of the cell, and optionally may be the only pixel displayed in the final three-dimensional reconstruction.
- the final three-dimensional reconstruction contains every detected cell with an x, y and z coordinate.
- these duplicate cells may be identified through machine learning techniques, such as K-nearest neighbor (KNN) and other techniques. This process can result in the removal of duplicate cells across multiple Z positions in the z-stack.
- KNN K-nearest neighbor
- machine learning techniques may also be configured to match multiple stained mitochondria to the same cell. Deep learning and other associated techniques of the computer vision system described herein may also be used. Accordingly, where the cell has multiple mitochondria, the techniques described herein may be used to account for the multiple mitochondria in order to obtain a more accurate cell count.
- the process 200 may include further analysis operations 252 associated with the analysis of two-dimensional and three-dimensional images and response of the cells to a treatment agent.
- the analysis operations 252 may be performed using the computer vision and associated systems described herein, such as those described in greater detail below with respect to FIGS. 13 A- 16 .
- the operations may include executing instructions of a non-transitory computer-readable media, with one or more processing elements of a computer, such as the computing system 2200 of FIG. 22 .
- a computer vision tool is disclosed herein that is configured to identify the color of each cell based on the light-responsive or fluorescent dye. Since each cell is treated as a unique object, the color of each cell can also be changed (e.g., all red cells can be changed to blue, or a subset can be changed to blue based on their position or size).
- an array may be configured to store data on each cell, such as the coordinates and color of each cell, for analysis between multiple time points.
- Data at a first time point may be indicative of a first response of the cell culture to a treatment agent (e.g., at a first time).
- Data at a second time point may be indicative of a second response of the cell culture to the treatment agent (e.g., as a second time, subsequent the first time).
- cells with red fluorescence from MitoViewTM 633 may be counted to determine the number of live cells on a first day (e.g., day 1), then compared to an image of the same sample on a later day (e.g., day 5) to determine how many cells died between the first day and the later day.
- Attributes of cells such as the average z position of the cells may be calculated on both the first day and the later day to determine, for example, average upward or downward migration of cells between the first day and the later day.
- Various other cell metrics may be analyzed to determine a change over time as cells are exposed to a drug or combination of drugs.
- a computer vision analysis system configured to determine at a single-cell level the behavior of each cell over time.
- the computer vision analysis system may be further configured to analyze the single-cell level behavior to differentiate cancer cells from immune or other cells.
- the computer vision analysis system may be further configured to identify and differentiate among sub-types of cancer cells.
- deep learning including using neural networks, can be used to make predictions regarding cells and groups of cells.
- deep learning can allow for the classification of cells by type (e.g., cancer, stromal or immune).
- machine learning can also be used to support one or more of the analysis functions described herein.
- Further machine learning methods such as K-means and support vector machines (SVMs) may be used to classify single cells and groups of cells.
- K-means and support vector machines (SVMs) may be used to classify single cells and groups of cells.
- SVMs support vector machines
- KNN algorithm is used to match a single cell between the first day and the later day (e.g., day 1 and day 5).
- the migration vector of each cell may then be visualized using a quiver plot (e.g., FIG. 15 herein).
- the sum of the vectors may indicate various trends, such as the total movement of cells in a sample.
- a subset of vectors e.g., the top 10%
- individual vectors can be used to help make predictions of patient response to a given treatment.
- a combination of metrics extracted from the image data may be used to predict patient response to various treatments, including single agents and combination drugs.
- thresholds are set to classify treatment responses using a single metric, such as cell viability, cell migration vector total, or largest single cell migration vector.
- Simple classifiers, receiver operating characteristic (ROC) curves and logistic regression are example methods of correlating single metrics in the invention to patient response.
- the metrics outputted by the computer vision process can be used as input features to perform various predictions.
- decision trees may be used to make predictions about the patient outcome using a complex decision making process correlating multiple patient response features.
- decision trees may be used for complex weighing of multiple inputs to optimize prediction accuracy.
- Training data may be used to correlate inputs to patient response and determine which inputs have the highest impact on predicting patient outcomes.
- the decision tree may have multiple nodes and branches to facilitate the predictions of a patient response.
- the final node of the tree (the prediction) may be either the patient's predicted response likelihood or the optimal predicted treatment or even both.
- the decision tree may be optimized to maximize sensitivity and specificity on a population of patients.
- the decision tree may also be optimized to maximize positive predictive value and negative predictive value. For example, on a per patient basis, for each proposed treatment, a predicted response with the decision tree (e.g., complete, partial or no response) can be outputted.
- FIGS. 3 - 17 example implementations of the system 100 of FIG. 1 and process 200 are presented herein.
- mechanical components, instrumentation, solutions, devices, user interfaces, charts, computing devices, and so on are described for purposes of illustration, which may be used to execute one or more of the functions or techniques described above with respect to FIGS. 1 and 2 .
- FIGS. 3 - 17 rather than limit the disclosure, assist in illustrating various features and functions of the present disclosure.
- other mechanical components, instrumentation, solutions, devices, user interfaces, charts, computing devices, and so on are contemplated herein for implementing the functions and techniques described above in the system 100 and the process 200 .
- the patient sample 304 may be a patient-derived tumor sample that is obtained during the operation 204 described above with respect to FIG. 2 .
- the patient sample 304 may include cells of or associated with a malignant tissue, such as cells of a breast cancer, a colorectal cancer, a lung cancer, a kidney cancer, a pancreatic cancer, an ovarian cancer, a brain cancer, or a gastric cancer, among other cancers.
- the patient sample 304 may include tissue slices, cores, surgical resections, xenografts and/or core needle biopsies.
- the patient sample 304 is shown as being substantially any patient-derived sample collected with respect to the culture module 104 described above.
- the patient sample 304 may include target cells that are arrangeable in a culture for dosing and analysis by the dosing module 108 and the analysis module 112 .
- the patient sample 304 may include dissociated or isolated cells.
- the patient sample 304 shown in FIG. 3 may be representative of a patient sample prior to forming a solid cell culture or otherwise modifying the patient sample.
- the sample 304 may therefore further include cancer cells, normal/non-transformed cells, stromal cells, or immune cells.
- the sample 304 may also include blood and other contaminants, which as described herein, may be selected or filtered or removed using a digestion enzyme-based operation, a blood lysis solution, or a selecting operation among other possibilities.
- a user interface 400 is depicted including information associated with cells tagged with a tracking dye.
- the user interface 400 may be a visual representation that is associated with the stained cells.
- the target cells may be stained with a first light-responsive dye that facilitates the tracking of a live cell count, and a second light-responsive dye that facilitates the tracking of the dead cell count.
- the user interface 400 may allow for visualization of the presence of the first light-responsive dye for live cell tracking and the presence of the second light-responsive dye for dead cell tracking.
- the user interface 400 includes a display region 402 .
- the display region 402 may represent at least a portion of cells from the operation 212 .
- the display region 402 may be indicative of a portion of the dissociated cells that is analyzed in order to facilitate counting of live and dead cells.
- the display region 402 is shown including live cells 406 and dead cells 410 .
- the live cells 406 may be those having or otherwise associated with the first color
- the dead cells 410 may be those having or otherwise associated with the second color.
- the live cells 406 and the dead cells 410 are represented in the display region 402 based on a magnification 414 , which may be modifiable by a user or computer-implemented technique.
- the representative first color and second color of the live cells 406 and the dead cells 410 may allow for the determination of analysis of various properties of the cells.
- the differing colored live cells 406 and dead cells 410 may allow for a quantity of cells per unit volume to be counted or determined.
- the quantity of per unit volume of live cells 406 and the quantity of per unit volume dead cells 410 may be compared in order to determine a relative concentration of living cells to dead cells.
- the display region 402 may be overlaid or associated with a control panel 452 .
- the control panel 452 may display the current determination of a living cell concentration 456 versus a dead cell concentration 470 .
- a determination of the concentration of live cells 406 relative to the dead cells 410 may result in the living cell concentration 456 having a value of 90% and the dead cell concentration 470 having a value of 10%. It will be appreciated that the user interface 400 shows information associated with how many cells were isolated from the patient tumor sample.
- a chart 500 is depicted including additional information corresponding to the living cells 406 and the dead cells 410 shown in FIG. 4 .
- the chart 500 may show a relative concentration or number of living/dead cells with respect to cell size.
- the chart 500 includes a cell size axis 504 , a cell quantity axis 508 , a live cell distribution 512 , a dead cell distribution 516 , live cell size average 506 a , dead cell size average 506 b .
- the cell size axis 504 may be representative of an approximate size of a given target cell. In FIG. 5 , the size is represented in micrometers; however, other appropriate units may also be used.
- the cell quantity axis 508 may include a count of the number of cells for a given sample or portion thereof measured.
- the live cell distribution 512 and the dead cell distribution 516 may be representative of a quantity of cells for a respective cell size. In some cases, the distributions 512 , 516 may be a histogram or other representation in which a quantity of cells are depicted for a given range of cell sizes.
- the live cell average 506 a may represent an average size of the live cells represented by the live cell distribution 512 .
- the dead cell average 506 b may represent an average size of the dead cells represented by the dead cell distribution 516 .
- the culture 600 may represent a solid cell culture formed via the optional spheroid formation operation 216 and hydrogel embedding operation 220 , described above with respect to FIG. 2 .
- the culture 600 is shown in FIG. 6 as including a hydrogel 608 .
- the hydrogel 608 may contain hyaluronic acid and collagen to mimic core components of human tissue extracellular matrices and disease-specific cell niches, like those found in breast tumors.
- the hydrogel 608 is shown encapsulating a spheroid 604 .
- the spheroid 604 may include target cells 612 , such as any of the cells described herein.
- the target cells 612 may be stained with a light-responsive dye.
- the arrangement of the cell culture including the spheroid 604 may facilitate the tracking of tumor properties over time.
- a representative circumference 616 is shown in FIG. 6 .
- the representative circumference 616 may correspond to a size value for the spheroid 604 or other cell or portion of the culture 600 .
- the culture 600 may be exposed to treatment agents as described herein.
- a size of the circumference 616 or other property of the culture 600 may be measured in order to determine a change over time.
- the change may be indicative of a treatment efficacy for the treatment agent when compared against the control/baseline.
- a decrease in a size of the circumference 616 may provide information to a clinician regarding the efficacy of a treatment that was administered to the cell culture 600 .
- the solid cell culture 600 may be stained with one or more of the light-responsive dyes described herein. Properties of the solid cell culture 600 and spheroid 604 may therefore be measured using the light-responsive dyes.
- FIGS. 7 A- 7 C representations of a solid cell culture 700 are shown under different conditions for analyzing the components of the cell culture.
- the example of FIGS. 7 A- 7 C shows a spheroid of an ovarian tumor for purposes of illustrations, in other cases, other tumors may be represented using similar techniques.
- the cell culture 700 is shown in a condition in which a live cell dye causes a representation 704 to be illuminated or represented.
- the representation 704 may be indicative of the physical properties of live cells, including cell position and size.
- the cell culture 700 is shown in a condition in which a dead cell dye causes a representation 708 to be illuminated or represented.
- the representation 708 may be indicative of the physical properties of dead cells, including cell position and size. In some circumstances, it may be beneficial to overlay the representation 704 , 708 , for example, to more accurately track cells as the cells transition from living to dead.
- FIG. 7 C the cell culture 700 is shown in which the representations 704 , 708 are overlaid to define a representation 712 .
- the representation 712 may be a composite visual representation of the living cell and the dead cell dye.
- the representations 704 , 708 , 712 may change over time, such as changing in response to administration of one or more treatment agents.
- the change in representation 704 , 708 , 712 as explained herein, may be analyzed to determine a treatment efficacy.
- a microfluidic chip 800 is disclosed.
- the microfluidic chip 800 may be used in conjunction with the operation 224 of setting the hydrogel in a chip, as described above with reference to FIG. 2 .
- the microfluidic chip 800 may be configured to mimic an environment of a human organ.
- the microfluidic chip 800 may be configured to replicate an environment of a human organ in order to establish an environment where cells can live ex vivo for a period of time.
- the microfluidic chip 800 may include a compartment or cell culture chamber to hold cells, such as any of cells or cell cultures described herein.
- the microfluidic chip 800 may include layers to cover or shield the cell cultures to block contaminants from the cell culture while also allowing gas, such as oxygen to reach the cell culture.
- the microfluidic chip 800 may also be configured to circulate growth media and treatment agents to the solid cell culture held therein. The growth media may allow the cells to survive for a period of time, ex vivo.
- the microfluidic chip 800 may be a multilayered structure.
- FIG. 8 A shows an exploded view of a stack up of layers and components that may be used to form the microfluidic chip 800 .
- Each component layer of FIG. 8 A is shown in a top view.
- a base layer 802 a first body portion layer 812 , a second body portion layer 822 , a third body portion layer 832 , a coupling portion 842 , and a gas permeable layer 852 is shown.
- the microfluidic chip 800 may include more or fewer layers, such as including one or more sterilization layers, films, adhesives, and so on that may be used to prepare the microfluidic chip 800 for dosing.
- the microfluidic chip 800 may also be associated with one or more flow restrictors 880 ( FIG. 8 C ).
- the base layer 802 may be a substantially solid section of the chip 800 upon which the other layers of the chip 800 are arranged.
- the base layer 802 may have a size corresponding to a size of a standard microscope slide to foster compatibility.
- the first body portion layer 812 , the second body portion layer 822 , and the third body portion layer 832 may be layers of a body 801 that define a channel 862 , a first volume 864 (including a first cell culture chamber 814 ) and a second volume 866 (including a second cell culture chamber 816 ) of the chip 800 .
- the first body portion layer 812 may define a first cell culture chamber 814 and a second cell culture chamber 816 , which may each be defined by openings or through portions through the first body layer 812 .
- the second body portion layer 822 may define a second body portion first hole 824 and a second body portion second hole 826 , which may each define openings or through portions through the second body layer 822 .
- the third body portion layer 832 may define a third body portion first hole 834 and a third body portion second hole 836 , which may each define openings or through portions through the third body layer 832 .
- the first body portion layer 812 , the second body portion layer 822 , and the third body portion layer 832 may be stacked relative to one another such that the first cell culture chamber 814 , second body portion first hole 824 , and third body portion first hole 834 cooperate to define a first volume 864 .
- the first volume 864 including the first cell culture chamber 814 , may have a substantially cylindrical shape with a closed bottom end and an open top end.
- the first cell culture chamber 814 may have a diameter of preferably about 6.75 mm. In other cases, the diameter may be more or less than 6.75 mm such as being less than about 5 mm, less than about 3 mm and/or other appropriate value for a given application.
- first body portion layer 812 , the second body portion layer 822 , and the third body portion layer 832 may be stacked relative to one another such that the second cell culture chamber 816 , second body portion second hole 826 , and third body portion second hole 836 cooperate to define the second volume 866 .
- the second volume 866 including the second cell culture chamber 816 , may have a diamond or rectangular shape with a closed bottom end and an open top end.
- the body 801 may also define the channel 862 .
- the second body portion layer 822 includes an elongated through portion 828 .
- the elongated through portion 828 may extend through a complete thickness of the second body portion layer 822 and through a portion of the second body portion first opening 824 and the second body portion second opening 826 .
- the first body portion layer 812 , the second body portion layer 822 , and the third body portion layer 832 may be stacked relative to one another such that the first body portion layer 812 and the third body portion layer 832 close the bottom and the top of the elongated through portion 828 , respectively, in order to define the channel 862 .
- the elongated through portion 828 may extend between opposing ends of the second body portion layer 822 .
- the third body portion layer 832 may cover the elongated through portion 828 and define a first lumen 833 a and a second lumen 833 b , each extending into the elongated through portion 828 .
- the first lumen 833 a may extend through a thickness of the third body portion layer 832 at a first end and into the elongated through portion 828 .
- the second lumen 833 b may extend through the thickness of the third body portion layer 832 at a second, opposing end and into the elongated through portion 828 .
- the first and second lumens 833 a , 833 b may extend into the elongated through portion 828 in order to facilitate a fluidic coupling of the channel 862 to a fluid circuit.
- the coupling portion 842 may include an inlet feature 843 a and an outlet feature 843 b .
- the inlet feature 843 a and the outlet feature 843 b may be barbs that protrude from a topmost surface of the microfluidic chip 800 .
- the inlet feature 843 a may be alignable with or receivable by the first lumen 833 a and configured to define a fluid connection between a tubing of a circulation system and the channel 862 .
- the outlet feature 843 b may be alignable with or receivable by the second lumen 833 b and configured to define a fluid connection between additional tubing of the circulation system and another end of the channel 862 .
- this configuration may allow the chip 800 to be fluidly coupled to a circulation system in which treatment agents are circulated through the chip 800 alongside growth media.
- fluid or media of a variety of purposes may be introduced to the chip 800 via inlet feature 843 a and caused to flow through the chip 800 via the first lumen 833 a , the channel 862 , the second lumen 833 b , and exiting via the outlet feature 843 b.
- the gas permeable layer 852 may include a membrane, film, or other layer configured to shield and cover the first and second volumes 864 , 866 , containing the first and second cell culture chambers 814 , 816 .
- the gas permeable layer 852 may be configured to shield or cover the volumes 864 , 866 while allowing gas, such as oxygen, ingress and egress between the cell culture chambers 814 , 816 and an external environment. In this manner, cell cultures held therein may be exposed to an oxygen rich environment and/or an oxygen poor environment as needed based on a particular application.
- the third body portion layer 832 is shown as including an adhesive surface 835 .
- the gas permeable layer 852 may be attachable to the body via the adhesive surface 835 subsequent to loading the chip 800 with a cell culture, as described herein.
- the chip 800 may be coupled such that the base layer 802 , the first body portion layer 812 , the second body portion layer 822 , the third body portion layer 832 , the coupling portion 842 , and the gas permeable layer 852 are stacked relative to one another.
- the channel 862 is shown extending through the body 801 between the first lumen 833 a and the second lumen 833 b .
- the first lumen 833 a is fluidly coupled to the inlet feature 843 a .
- the second lumen 833 b is fluidly coupled to the outlet feature 843 b .
- the inlet feature 843 a may be fluidly coupled with a first tube 906 a
- the outlet feature 843 b may be fluidly coupled with a second tube 906 b
- the first and second tubes 906 a , 906 b may be portions of tubing of a circulation system (e.g., such as that provided by the microfluidic device 1000 of FIGS. 10 and 11 ).
- the tubes 906 a , 906 b may be inserted or received by the respective inlet feature 843 a and outlet feature 843 b .
- the inlet and outlet features 843 a , 843 b may be configured to define a sealed connection with the respective tubes 906 a , 906 b.
- the chip 800 may be configured to define a segment of a flow path for the circulation system.
- the first tube 906 a may introduce an inlet flow F i into the chip 800 at the inlet feature 843 a .
- the inlet flow F i may be a flow of a media including treatment agents.
- the flow of the media and/or treatment agents and/or other agents may progress to the channel 862 defined by the body 801 .
- the channel 862 may run adjacent to both the first cell culture chamber 814 and the second cell culture chamber 816 . In this regard, the flow of media and/or treatment agents continue through the channel 862 and alongside and/or into a portion of the first cell culture chamber 814 and/or the second cell culture chamber 816 .
- first cell culture chamber 814 and/or the second cell culture chamber 816 may include cells encapsulated in a hydrogel, such as any of the arrangements of cells and hydrogels described herein.
- the channel 862 may therefore be configured to route the growth media and/or treatment agents for interaction with the cells/hydrogel included in the respective cell culture chambers 814 , 816 .
- a solid cell culture (including cells and hydrogel) may be deposited into the first cell culture chamber 814 and/or the second cell culture chamber 816 .
- the growth media may be circulated through the chip 800 , such as along the channel 862 in order to flow over the cells and hydrogel held therein.
- An exposed solid cell culture may therefore be defined in the chip once the solid cell culture is put in the chip and the growth media is circulated therethrough.
- the interaction of the media and/or the treatment agents with the cells may cause the cells to retain at least a portion of the media and/or treatment agents in the cell culture chamber.
- the flow may continue along the channel 862 and exit the chip at the outlet feature 843 b at a flow F o .
- FIG. 8 C is a cross-section view of a flow restrictor 880 for use with the microfluidic chip 800 in a fluid circuit.
- the flow restrictor 880 may be implemented in order to facilitate sterility in the system, such as by preventing contamination of the chip 800 and cells therein.
- the flow restrictor 880 may therefore include one or more filter elements.
- the flow restrictor 880 may therefore further include one or more reductions or changes in a diameter in the flow path in order to increase or modify a pressure gradient of the flow across the restrictor 880 , among other uses.
- the flow restrictor 880 is shown including a first straight barb 882 .
- the first straight barb 882 defines a first barb channel 883 therethrough.
- the flow restrictor 880 may generally be arranged adjacent the inlet or outlet of the microfluidic chip 800 .
- the first straight barb 882 may be fluidly coupled to one of the inlet or outlet features 843 a , 843 b .
- the flow restrictor 880 may be fluidly coupled to tubing that is received by the first straight barb 882 and one of the barbs of the inlet or outlet features 843 a , 843 b to complete the fluidic coupling.
- the 8 C also includes a second straight barb 896 that defines a second barb channel 897 .
- the second straight barb 896 may be substantially analogous to the first straight barb 882 .
- the second straight barb 896 may be fluidly coupleable with a component of the microfluidic device (e.g., the microfluidic device 1000 of FIG. 10 ) via tubing and/or other connector.
- Multiple flow restrictors can be used. As one example, a first flow restrictor can be used at an inlet of a given microfluidic chip and a second flow restrictor can be used at an outlet of the microfluidic chip.
- the flow restrictor 880 may include multiple layers arranged generally between the first and second straight barbs 882 , 896 to facilitate the foregoing functionalities.
- the flow restrictor 880 is shown including a first layer 884 and a first hole 885 , and an adjacent second layer 886 and a second hole 887 .
- the first straight barb 882 may be at least partially received by the first hole 885 .
- the second hole 887 may generally be aligned with the first hole 885 .
- the second hole 887 has a diameter that is less than a diameter of the first hole 885 .
- the first barb channel 883 , the first hole 885 and the second hole 887 may define a flow path with the second hole 887 operating to reduce or restrict the flow through the flow restrictor 880 .
- the flow restrictor 880 is further shown as including a filter layer 888 with a filter hole 889 generally adjacent the second hole 887 .
- the filter layer 888 may house or hold a filter 890 in the filter hole 889 .
- Multiple constructions of the filter 890 are possible.
- the filter 890 may include a glass microfiber filter. In some cases, multiple separate filters may be used.
- the flow restrictor 880 Adjacent the filter layer 888 , the flow restrictor 880 is shown as including a third layer 892 and third hole 893 , and an adjacent fourth layer 894 and fourth hole 895 .
- the third layer 892 and third hole 893 , and the fourth layer 894 and fourth hole 895 may generally mirror the first layer 884 and first hole 885 , and second layer 886 and second hole 887 .
- the third hole 893 may be generally smaller in diameter than the fourth hole 895 .
- the second straight barb 896 may be at least partially received by the fourth hole 895 .
- FIGS. 9 A- 9 E depict an operation of loading the microfluidic chip 800 with a cell culture.
- an operation 900 a is shown in which the first cell culture chamber 814 of the first volume 864 is loaded with a solid cell culture (e.g., any combination of cells and hydrogel described herein).
- a pipet 902 or other instrument may selectively introduce a solid cell culture 910 into the first cell culture chamber 814 .
- the second cell culture chamber 816 of the second volume 866 may also be loaded with a solid cell culture.
- an operation 900 b is shown in which an adhesive backing 879 is removed to reveal the adhesive surface 835 of the body 801 .
- the adhesive backing 879 may reveal the adhesive surface 835 in order to prepare the body 801 for attachment with the gas permeable layer 852 .
- an operation 900 c is shown in which the gas permeable layer 852 may be positioned over the body 801 .
- the gas permeable layer 852 may cover the top of both the first and second volumes 864 , 866 , containing the first cell culture chamber 814 and the second cell culture chamber 816 , as shown in FIG. 9 D at operation 900 d .
- the gas permeable layer 852 may be attached to the body 801 via the adhesive surface 835 .
- an operation 900 e is shown in which the first tube 906 a is fluidly coupled with the inlet feature or barb 843 a and the second tube 906 b is fluidly coupled with the outlet feature or barb 843 b.
- a microfluidic device 1000 is shown.
- the microfluidic device 1000 may be configured to implement the operation 232 of FIG. 2 , in which the solid cell culture (e.g., the cells and hydrogel desired herein) is exposed to treatment agents.
- the microfluidic device 1000 may be configured to provide treatment agents alongside growth media to multiple microfluidic chips, in parallel.
- the microfluidic device 1000 may define closed circuits for each chip that are generally fluidly isolated and separated from the circuits of the other chips.
- the microfluidic device 1000 may be configured to provide different treatment agents, growth media, or other solutions to the chips, so that a response of the solid cell cultures may be determined over time.
- the microfluidic device 1000 may include a housing 1002 , a platform 1004 , a staging section 1010 , a dosing bank 1020 , and a pump 1030 .
- the housing 1002 may provide a structural platform for the various components of the microfluidic device 1000 , including the pump 1030 and the dosing bank 1020 .
- the housing 1002 may also provide a structure upon which to arrange and temporarily store chips during dosing.
- the housing 1002 may have one or more open sides, as shown in FIG. 11 . In other cases, the housing 1002 may enclose one or more components of the microfluidic device 1000 . In yet other cases, the housing 1002 may be omitted entirely.
- the staging section 1010 may be configured to arrange a plurality of microfluidic chips in the microfluidic device 1000 .
- the staging section 1010 may be a portion of the housing 1002 .
- the staging section 1010 may include a raised platform, slots, or other features for receiving and securing chips in a particular position in the staging section 1010 .
- the staging section 1010 may include a chip tray 1012 .
- the chip tray 1012 is shown as holding six microfluidic chips: a first microfluidic chip 800 a , a second microfluidic chip 800 b , a third microfluidic chip 800 c , a fourth microfluidic chip 800 d , a fifth microfluidic chip 800 e , and a sixth microfluidic chip 800 f .
- Each of chips 800 a - 800 f may be substantially analogous to the microfluidic chip 800 described above with respect to FIGS. 8 A- 9 E ; redundant explanation of which is omitted for clarity. While various configurations are possible, the chip tray 1012 may include a recess or groove that is configured to accommodate a single chip.
- a given chip may be received in the groove such that at least side to side movement of the chip is mitigated on the tray 1012 .
- FIGS. 10 and 11 show the chip tray 1012 as including a grooves for six chips, other configurations are possible, including trays that hold more or fewer chips.
- the dosing bank 1020 may include a plurality of reservoirs.
- the dosing bank 1020 may include a plurality of reservoirs corresponding to the plurality of chips arranged at the staging section 1010 .
- the dosing bank may include a first reservoir 1022 a , a second reservoir 1022 b , a third reservoir 1022 c , a fourth reservoir 1022 d , a fifth reservoir 1022 e , and a sixth reservoir 1022 f .
- the reservoirs 1022 a - 1022 f may be configured to hold a media, such as any of the growth media described herein.
- the reservoirs 1022 a - 1022 f may further be configured to hold treatment agents, or substantially any other fluid for introduction to respective ones of the chips 800 a - 800 f .
- the reservoirs 1022 a - 1022 f may be exposed or exposable such that additional media and/or treatment agents may be added over time.
- the reservoirs 1022 a - 1022 f may also be associated optionally with covers to shield the media held therein from contaminants during dosing.
- one or more or all of the reservoirs 1022 a - 1022 f may include a cap, such as an aluminum cap, to hermetically seal the respective reservoir 1022 a - 1022 f.
- the pump 1030 may be configured to complete a fluid circuit between respective ones of the chips 800 a - 800 f and the reservoirs 1022 a - 1022 f .
- the pump 1030 may be a peristaltic pump, pneumatic pump and/or any other appropriate pump or pumping device.
- the pump 1030 may be configured to define separate fluid circuits between a given one of the chips 800 a - 800 f and the reservoirs 1022 a - 1022 f .
- the pump 1030 may be configured to cause circulation of fluid between respective ones of the reservoirs 1022 a - 1022 f and the chips 800 a - 800 f without fluid of one of the reservoirs crossing over or contaminating another reservoir.
- each chip may be dosed separately such that a reading or measurement of each chip may be taken in order to determine the impact from a particular solution held in the corresponding reservoir.
- the pump 1030 is shown as a single assembly, in other cases, the pump 1030 may represent multiple pumps. Further, the pump 1030 is shown as including six circulation paths. In other cases, the pump 1030 may define more or fewer paths.
- the pump 1030 , the chips 800 a - 800 f , and the reservoirs 1022 a - 1022 f are coupled to one another to define six circulation paths.
- the pump 1030 , the first microfluidic chip 800 a , and the first reservoir 1022 a are coupled to one another to define a first fluid circulation path 1002 a .
- the pump 1030 , the second microfluidic chip 800 b , and the second reservoir 1022 b are coupled to one another to define a second fluid circulation path 1002 b .
- the pump 1030 , the third microfluidic chip 800 c , and the third reservoir 1022 c are coupled to one another to define a third fluid circulation path 1002 c .
- the pump 1030 , the fourth microfluidic chip 800 d , and the fourth reservoir 1022 d are coupled to one another to define a fourth fluid circulation path 1002 d .
- the pump 1030 , the fifth microfluidic chip 800 e , and the fifth reservoir 1022 e are coupled to one another to define a fifth fluid circulation path 1002 e .
- the pump 1030 , the sixth microfluidic chip 800 f , and the sixth reservoir 1022 f are coupled to one another to define a sixth fluid circulation path 1002 f .
- the circulation paths 1002 a - 1002 f may be separate fluid paths that are fluidly isolated from one another.
- the pump 1030 may cause a circulation along circulation paths 1002 a - 1002 f independent from one another.
- the pump 1030 may cause media, treatment agents and the like to circulate along the first circulation path between the first reservoir 1022 a and the first chip 800 a , the second reservoir 1022 b and the second chip 800 b , the third reservoir 1022 c and the third chip 800 c , the fourth reservoir 1022 d and the fourth chip 800 d , the fifth reservoir 1022 e and the fifth chip 800 e , and the sixth reservoir 1022 f and the sixth chip 800 f.
- the chips 800 a - 800 f may be removed individually from the microfluidic device 1000 .
- individual ones of the chips 800 a - 800 f may be removed from the microfluidic device 1000 for imagining and analysis.
- an individual chip may be removed and imaged using fluorescence microscopy or other techniques in which properties of the solid cell culture are determined for a single point in time, including, but not limited to, cell culture size color, position, and density.
- the chip may be recoupled to the microfluidic device 1000 .
- the microfluidic device 1000 may proceed to provide additional treatment agents, media, and so one to the respective microfluidic chip.
- the chip may be removed again from the microfluidic device 1000 in order to conduct further imaging of the solid cell culture for a second, later point in time.
- the cell culture color, size, position, and density can be detected at the second point in time and/or a variety of other cell characteristics.
- the characteristics of the solid cell culture at the second point in time can be compared with the characteristics of the solid cell culture at the first point in time to determine other characteristics, including cell migration distance, cell migration speed, maximum migration vector, maximum migration speed, and so on, as described herein.
- the chip may be removed repeatedly, such as a third, fourth, fifth, or more times in order to conduct further imaging of the solid cell culture for a third, fourth, fifth, or more later points in time.
- the width or diameter of a spheroid is measured and compared at different time points.
- Single cells surrounding the spheroid are tracked individually or in clusters. Movement of single cells can be tracked to determine if the cells are entering or exiting the spheroid.
- the change in spheroid size and/or the behavior of single cells can be used individually or together to predict a patient response to a given treatment.
- FIG. 12 shows an example report 1200 including images of representative cell cultures after a period of dosing. A spheroid exposed to a treatment that stays stable or grows may indicate a poor response, while a spheroid that shrinks may indicate a good response.
- the given image or other representation of a cell culture may be compared with an image of the culture prior to dosing in order to determine a change in size of the spheroid.
- the example report 1200 includes a first cell culture column 1208 a , a second cell culture column 1208 b , and a third cell culture column 1208 c .
- each of columns 1208 a - 1208 c may be indicative of a different cell culture held by a different microfluidic chip (e.g., one of the microfluidic chips 800 a - 800 f of FIGS. 10 and 11 ).
- Each of the cell cultures in the respective chips may be dosed with a different treatment agent, different strength or concentration of treatment agent, and/or no treatment agent in order to evaluate the response of the cell culture to a variety of conditions over time (e.g., as may be depicted in various three-dimensional images, as shown herein with respect to FIG. 16 ).
- a different treatment agent different strength or concentration of treatment agent, and/or no treatment agent in order to evaluate the response of the cell culture to a variety of conditions over time (e.g., as may be depicted in various three-dimensional images, as shown herein with respect to FIG. 16 ).
- FIG. 16 In the example of FIG.
- the first cell culture column 1208 a corresponds to a cell culture for administration of a first treatment agent at a first dose (e.g., 40 nM doxorubicin)
- the second cell culture column 1208 b corresponds to a cell culture for administration of a second treatment agent at a second dose (e.g., 50 nM doxorubicin)
- the third cell culture column 1208 c corresponds to a cell culture for administration of a third treatment agent at a third dose (e.g., 100 nM doxorubicin).
- the report 1200 includes a first time point row 1204 a , and a subsequent time point row 1204 b .
- the first time point row 1204 a may correspond to a period of time before dosing, such as at day zero.
- the subsequent time point row 1204 b may correspond to a later time point after some period of dosing.
- the subsequent time point row 1204 b may be after one day of dosing.
- the subsequent time point row 1204 b may correspond to another period of dosing, such as after two days, three days, four days, five day, and so on.
- the cell cultures may be evaluated at each of the time points (and other time points) in order to evaluate treatment efficacy.
- the cell culture associated with the first cell culture column 1208 a may be imaged, according to any of the techniques described herein, at a time associated with the first time point row 1204 a (e.g., a time prior to dosing) in order to produce a first representation 1212 a of the cell culture.
- the cell culture associated with the first cell culture column 1208 a may subsequently be dosed (e.g., with 40 nM doxorubicin) and imaged at a time associated with the second time point row 1204 b (e.g., after one day or so of dosing) in order to produce a second representation 1212 b of the cell culture.
- the cell culture associated with the second cell culture column 1208 b may be imaged, according to any of the techniques described herein, at a time associated with the first time point row 1204 a (e.g., a time prior to dosing) in order to produce a first representation 1216 a of the cell culture.
- the cell culture associated with the second cell culture column 1208 b may subsequently be dosed (e.g., with 50 nM doxorubicin) and imaged at a time associated with the second time point row 1204 b (e.g., after one day or so of dosing) in order to produce a second representation 1216 b of the cell culture.
- the cell culture associated with the third cell culture column 1208 c may be imaged, according to any of the techniques described herein, at a time associated with the first time point row 1204 a (e.g., a time prior to dosing) in order to produce a first representation 1220 a of the cell culture.
- the cell culture associated with the third cell culture column 1208 c may subsequently be dosed (e.g., with 100 nM doxorubicin) and imaged at a time associated with the second time point row 1204 b (e.g., after one day or so of dosing) in order to produce a second representation 1220 b of the cell culture.
- the respective second representations 1212 b , 1216 b , 1220 b may be evaluated using the computer vision tools described herein, including producing various charts, reports, graphs and so on, as described below in relation to FIGS. 13 A- 17 .
- FIGS. 13 A- 17 various charts, reports, and graphs are depicted that are representative of the imaging and analyzing operations of the computer vision system described above with reference to FIG. 2 . It will be appreciated that the following charts, reports, and graphs are depicted as examples. In other cases, additional or alternative charts, reports, and graphs may be used.
- a chart 1300 a is shown including a grid 1304 a and a first representation 1308 a .
- the grid 1304 a may be a three-dimensional image representative of a three-dimensional space associated with the solid cell culture.
- the first representation 1308 a may be representative of a three-dimensional position of particular items of interest, such as living cells in a three-dimensional space.
- the chart 1300 a may be produced, in one example, via two-dimensional images captured of the solid cell culture, in which the size and location of cells of interest are determined using the light-responsive dye. The two-dimensional images may be stitched together in order to produce a three-dimensional image.
- the first representation 1308 a may represent a composite of multiple two-dimensional images taken at a first time point. In the present example, the first representation 1308 a may be taken at a first time point, which may occur prior to any dosing with the treatment agents.
- the first representation 1308 a may also provide information regarding the quantity of the cells of interest, as shown in FIG. 13 A .
- a chart 1300 b is shown including a grid 1304 b and a second representation 1308 b .
- the grid 1304 b may be a three-dimensional image that is representative of a three-dimensional space associated with the solid cell culture represented by the chart 1300 a after a period of time (e.g., a second time point).
- the second representation 1308 b may be representative of a three-dimensional position of particular items of interest, such as living cells described above.
- the second representation 1308 b may represent a composite of multiple two-dimensional images taken at a second time point.
- the second representation 1308 b may be taken at a second time point, which may occur after a series of doses of the treatment agents or no treatment (e.g., with respect to a baseline/negative control).
- the second representation 1308 b may also provide information regarding the quantity of the cells of interest, as shown in FIG. 13 B .
- FIG. 14 A depicts a chart 1400 a showing a first distribution 1420 a in a three-dimensional coordinate system defined by an axis 1404 a , an axis 1408 a , and an axis 1412 a .
- the first distribution 1420 a may be representative of a solid cell culture at a first time point, which may be prior to dosing.
- the first distribution may be produced by stitching as described herein.
- the chart 1400 a provides information regarding the three-dimensional position of target cells in the first distribution 1420 a , including distribution density.
- the first distribution 1420 a has a higher density of cells for lower values along the axis 1412 a.
- FIG. 14 B shows a chart 1400 b having a second distribution 1420 b in a three-dimensional coordinate system defined by an axis 1404 b , an axis 1408 b , and an axis 1412 b .
- the chart 1400 b provides information regarding the three-dimensional position of target cells in the second distribution 1420 b , including distribution density.
- the second distribution 1420 b has a lower density of cells in the three-dimensional coordinate system as compared with the first distribution shown in FIG. 14 A .
- FIG. 15 depicts a chart 1500 showing a quiver or vector diagram indicative of the three-dimensional movement of the quantity of cell from the three-dimensional position of FIG. 14 A to the three dimensional position of FIG. 14 B .
- the three-dimensional position of a cell from the second distribution 1420 b may be compared with a corresponding cell from the first distribution 1420 a to determine a vector for the cell.
- the vector may correspond to a path of travel and/or speed for the cell between the first time point represented by the chart 1400 a and the second time point represented by the chart 1400 b .
- a given cell from the first distribution 1420 a may be a tail of the vector, and the corresponding cell from the second distribution 1420 b may be an arrowhead of the vector.
- the chart 1500 may be a three-dimensional quiver plot defined by an axis 1504 , an axis 1508 , and an axis 1512 .
- a vector distribution 1520 may be plotted in the chart 1500 including vectors for one or more target cells.
- the vector distribution 1520 may provide information regarding the behavior of cells during the administration of the treatment agent or no treatment (e.g., with respect to a baseline/negative control).
- the vector distribution 1520 may include a first region 1522 having a maximum migration vector.
- the maximum migration vector may correspond to a cell of interest that moved the greatest amount, in speed or position, between the measured first and second time points.
- the vector distribution 1520 may further include a second region 1524 that may correspond to a cluster of vectors that are generally larger than other of the vector distribution 1520 .
- the second region 1524 may correspond to a cluster of cells that generally moved the farthest or the fastest during the administration of the treatment agents.
- the vector distribution 1520 may further include a third region 1526 that may correspond to a cluster of vectors that are generally smaller than other vectors of the vector distribution 1520 .
- the third region 1526 may correspond to a cluster of cells that generally moved the least or the slowest during the administration of the treatment.
- a solid cell culture may be prepared using any of the techniques described herein. A portion of the solid cell culture may be deposited into different microfluidic chips, such as in one or more of the six microfluidic chips shown in FIGS. 10 and 11 .
- the microfluidic device 1000 may operate to circulate a flow of a different solution (e.g., different treatment agents) to each of the cell cultures of the microfluidic chips.
- a solid cell culture may be defined for each chip using the different solutions or treatment agents for the cells/hydrogel initially deposited in the chip.
- the microfluidic device 1000 may circulate a flow of different treatment agents to each chip.
- one of the chips may receive no treatment agents, and just receive growth media as a control case.
- the response of the exposed cell cultures to the various different treatment agents may be compared to determine a treatment efficacy. For example, the determined cell characteristics for each cell culture can be compared and analyzed to determine which treatment agent was the most effective in treating the target cells.
- FIG. 16 depicts an example report 1600 including visualizations of different dosing operations performed on a given patient sample.
- the report 1600 may include information regarding the impact of a particular treatment agent for a patient sample over a period of time.
- the chart 1600 may include a first chip column 1608 a , a second chip column 1608 b , a third chip column 1608 c , a fourth chip column 1608 d , a fifth chip column 1608 e , and a sixth chip column 1608 f .
- each of the columns 1608 a - 1608 f may be indicative of a different microfluidic chip (e.g., one of the microfluidic chips 800 a - 800 f of FIGS. 10 and 11 ).
- the microfluidic chips may initially include a solid cell culture, such as any of cell cultures herein, which may include target tumor cells.
- the microfluidic chips may be dosed or treated with treatment agents to define an exposed cell culture in each chip.
- each of the microfluidic chips may be dosed with a different treatment agent in order to determine the efficacy of a particular treatment.
- the response of exposed cell culture of the microfluidic chips may be measured over a period of time.
- the chart 1600 may include a first time point row 1604 a , a second time point row 1604 b , and a final time point row 1604 c .
- the first time point row 1604 a may correspond to a first day
- the second time point row 1604 b may correspond to a second day
- the final time point row 1604 c may correspond to a subsequent day, such as a third, fourth, or fifth day.
- Each microfluidic chip represented by the chip column may be imaged and analyzed for each time point represented by the time point row.
- the microfluidic chip may be imaged to determine a color, size, position, density, and/or other property at a first given time point corresponding to the first time point row 1604 a .
- the imaging may be a two-dimensional image or a three-dimensional image, which may be produced by stitching together the two-dimensional images.
- the imaging may be used to produce a representative chart 1612 a , as shown in FIG. 16 , which includes information for the characteristics of solid cell culture at the first time point.
- the chart 1612 a may be substantially analogous to the charts 1300 a , 1300 b described above for FIGS. 13 A and 13 B .
- the microfluidic chip may be subsequently imaged at a second given time point, such as subsequent to a round of dosing via the treatment agents, and which may correspond to the second time point row 1604 b . Accordingly, the imaging may be used to produce a representative chart 1612 b , which includes information for the characteristics of the solid cell culture at the second time point. Further, the microfluidic chip may be subsequently imaged at a third given time point, such as subsequent to another round of dosing via the treatment agents, and which may correspond to the final time point row 1604 c . Accordingly, the imaging may be used to produce a representative chart 1612 c , which includes information for the characteristics of the solid cell culture at the final time point.
- a response of the solid cell culture of the first microfluidic chip may be analyzed over time to determine a treatment efficacy for the treatment agents administered to the first microfluidic chip, as described herein.
- the chart 1600 and corresponding analysis may allow for the comparison of the treatment efficacy across multiple different treatment agents, including combinations of agents dosed simultaneously and/or sequentially, for the cell culture.
- the second chip column 1608 b may include a representative chart 1618 a at the first time point row 1604 a , a representative chart 1618 b at the second time point row 1604 b , and a representative chart 1618 c at the final time point row 1604 c .
- the third chip column 1608 c may include a representative chart 1622 a at the first time point row 1604 a , a representative chart 1622 b at the second time point row 1604 b , and a representative chart 1622 c at the final time point row 1604 c .
- the fourth chip column 1608 d may include a representative chart 1626 a at the first time point row 1604 a , a representative chart 1626 b at the second time point row 1604 b , and a representative chart 1626 c at the final time point row 1604 c .
- the fifth chip column 1608 e may include a representative chart 1630 a at the first time point row 1604 a , a representative chart 1630 b at the second time point row 1604 b , and a representative chart 1630 c at the final time point row 1604 c .
- the sixth chip column 1608 f may include a representative chart 1634 a at the first time point row 1604 a , a representative chart 1634 b at the second time point row 1604 b , and a representative chart 1634 c at the final time point row 1604 c.
- the report 1600 includes representative charts 1612 c , 1618 c , 1622 c , 1626 c , 1630 c , 1634 c for each of the microfluidic chips at the final time point.
- the final time point may be representative of a conclusion of the dosing of the various treatment agents.
- the information of each of the representative charts 1612 c , 1618 c , 1622 c , 1626 c , 1630 c , 1634 c may be compared to determine which cell culture exhibits the best or most effective response to a given treatment agent with respect to the control/baseline.
- the representative chart 1612 c may show information corresponding to a response of the cell culture to a first treatment agent at the final time point
- the representative chart 1618 c may show information corresponding to a response of the cell culture to a second treatment agent at the final time point
- the representative chart 1622 c may show information corresponding to a response of the cell culture to a third treatment agent at the final time point
- the representative chart 1626 c may show information corresponding to a response of the cell culture to a fourth treatment agent at the final time point
- the representative chart 1630 c may show information corresponding to a response of the cell culture to a fifth treatment agent at the final time point
- the representative chart 1634 c may show information corresponding to a response of the cell culture to a sixth treatment agent (or a control with no treatment agent, which could apply to any single or multiple microfluidic chips) at the final time point.
- characteristics such as target cell color, size, density, count, and so on, may be compared across each of the representations shown in the final time point row 1604 c to determine a treatment efficacy.
- the treatment agent that was used to for the treatment of the microfluidic chip that resulted in the representation may be determined to have the high treatment efficacy among the treatment agents.
- the response of a cell culture to a given treatment agent may be used to determine cell viability.
- the viability of both responders and non-responders may be determined and plotted in order to determine an efficacy of treatment.
- a chart 1700 is depicted showing cell viability with respect to responders, such as a BRCA mutated, and non-responders, such as BRCA wild-type.
- the chart 1700 includes a data set axis 1704 and a viability axis 1708 .
- a first distribution 1710 having a first spread 1724 is shown alongside a second distribution 1720 having a second spread 1722 .
- the viability of the responders of the first distribution 1710 may be compared to the viability of the non-responders of the second distribution 1720 with the chart 1700 in order to provide information to assist in determining treatment efficacy.
- FIGS. 18 - 21 illustrates process 1800 , 1900 , 2000 , 2100 , respectively. While specific steps (and orders of steps) of the methods presented herein have been illustrated and will be discussed, other methods (including more, fewer, or different steps than those illustrated) consistent with the teachings presented herein are also envisioned and encompassed with the present disclosure.
- a process 1800 is disclosed.
- the process 1800 is directed to a method of forming a solid cell culture, such as that described with respect to FIG. 2 .
- target cells are isolated from a patient sample.
- a patient sample 304 may be obtained.
- the sample 304 may arrive at a lab, and include samples containing a tumor of various cancer types.
- a digestion enzyme-based cell isolation kit, blood lysis solution and other selecting or filtering steps may be used to isolate viable cells while removing blood and contaminants.
- the output may be a viable mixed cell population containing the various cells from the primary tumor, including cancer cells, normal/non-transformed cells, stromal cells and immune cells.
- stained cells are formed from the isolated cells by staining the target cells with a light-responsive dye.
- cells may be stained with one or both of a live cell dye and a dead cell staining dye.
- the dyes may be light-responsive dyes.
- the cells may be stained with a first light-responsive dye to facilitate live cell tracking.
- the cells may be further stained with a second light-responsive dye to facilitate dead cell tracking.
- the light-responsive dye is configured to cause a color change in the stained cell when the stained cell transitions from a living cell to a dead cell.
- a spheroid may optionally be formed from the stained cells.
- the spheroid or organoid may also include cancer cells, normal/non-transformed cells, stromal cells, and/or immune cells, which are formed from a patient-derived tissue or tumor sample. Tissue slices, cores, surgical resections, xenografts, and/or biopsies may also be used.
- the stained cells are encapsulated.
- the stained cells such as isolated cells (or spheroids formed from operation 1810 ) may be included in the hydrogel 608 .
- the hydrogel 608 may be configured to replicate components of human tissue, which may facilitate cell growth.
- hyaluronic acid and collagen may be used to mimic core components of human tissue extracellular matrices and disease-specific cell niches, like those found in breast tumors.
- the culturing of process 1800 may include culturing dissociated cells in the hydrogel.
- the dissociated cells may be formed via two-dimensional or three-dimensional cell cultures.
- the dissociated cells may be a single population of cells, whereas in other cases, multiple cell types may be used.
- the dissociated cells may be cancer, normal/non-transformed cells, stromal, and/or immune cells, as one example, derived from a tissue or tumor sample.
- a process 1900 is disclosed.
- the process 1900 is directed to a method of loading a microfluidic chip, such as that shown above with respect to FIGS. 9 A- 9 F .
- a solid cell culture is arranged in a cell culture chamber of a microfluidic chip.
- the microfluidic chip includes a body defining a cell culture chamber and a channel that traverses the cell culture chamber and extends between an inlet and an outlet of the microfluidic chip.
- the solid cell culture 910 is arranged in the first cell culture chamber 814 of the microfluidic chip 800 .
- the microfluidic chip 800 includes the body 801 that defines the first cell culture chamber 814 and the channel 862 that runs along or adjacent the first cell culture chamber 814 and extends between the inlet feature 843 a and the outlet feature 843 b.
- a gas permeable membrane is positioned over a volume containing the cell culture chamber while the inlet and outlet remain exposed for coupling to a circulation system.
- the gas permeable layer 852 is positioned on to the body 801 .
- the gas permeable layer 852 may be adhered to the body 801 via the adhesive surface 835 . As shown in FIG.
- the gas permeable layer 852 may cover both the first volume 864 and the second volume 866 , containing the first cell culture chamber 814 and the second cell culture chamber 816 , while allowing the first cell culture chamber 814 and the second cell culture chamber 816 to be exposed to gas in a surrounding atmosphere of the microfluidic chip 800 .
- a process 2000 is disclosed.
- the process 2000 is directed to a method of operating a microfluidic chip.
- a microfluidic chip is fluidly coupled with a microfluidic device to define a fluid circuit between the microfluidic chip, a flow restrictor, a reservoir, and a pump.
- the microfluidic chip including a cell culture, the reservoir including a media.
- microfluidic chips 800 a - 800 f may be fluidly coupled with the microfluidic device 1000 .
- the microfluidic device 1000 may operate to define fluid circuits 1002 a - 1002 f between each respective microfluidic chip 800 a - 800 f and a corresponding one of the reservoirs 1022 a - 1022 f.
- a flow of the media is caused through the circuit such that the media interacts with the solid cell culture of the microfluidic chip to define an exposed cell culture in the microfluidic chip.
- the microfluidic device 1000 may operate the pump 1030 to cause a flow of media through each of the fluid circuits 1002 a - 1002 f .
- the pump 1030 may be configured to control a flow of circulation through each of the fluid circuits 1002 a - 100 f separately. This may allow the microfluidic device 1000 to administer separate treatment agents to each of the microfluidic chips 800 a - 800 f such a response to each of the different treatment agents may be measured.
- a response of the solid cell culture to the media is analyzed.
- a given microfluidic chip may be removed and fluidly uncoupled from the microfluidic device 1000 .
- the microfluidic chip may be imaged or otherwise analyzed for the given time point at which the microfluidic chip is fluidly uncoupled.
- the microfluidic chip may undergo a process of fluorescence microscopy. Fluorescence microscopy may capture images of the cell culture of the given microfluidic chip under conditions in which the light-responsive dye allows for detection of target cells, e.g., the detection of dead cells and/or living cells.
- the images may be two-dimensional images. As described herein, the two-dimensional images may be captured in a manner that is representative of layers of the cell culture. The layers may be stacked on one another or stitched together to form a three-dimensional image.
- the given microfluidic chip may be fluidly coupled to the microfluidic device again to administer further media, including additional treatment agents.
- the method 2000 may further include causing another flow of the media through the circuit, at a second time point, such that the media interacts with the cell culture of the microfluidic chip, and analyzing a subsequent response of the cell culture to the media. Analyzing of the subsequent response of the cell culture may include imaging, as described, which is used to generate a two-dimensional or three-dimensional image of the cell culture. The imaging of the cell culture at the first and second time points may be analyzed to determine a treatment efficacy. As described herein, additional time points may also be analyzed.
- the method 2000 may include analyzing the image at the first time point to determine a first live/dead cell population quantity.
- the method 2000 may further include analyzing the image at the second time point to determine a second live/dead cell population quantity.
- the first cell population quantity and the second cell population quantity may, in turn, be compared to determine change in cell population quantity indicative of a treatment efficacy.
- the method 2000 may include analyzing the image at the first time point to determine a first cell population position and analyzing the image at the second time point to determine a second cell population position.
- the first cell population position and the second cell population positon may, in turn, be compared to determine change in cell population position indicative of a treatment efficacy.
- a process 2100 is disclosed.
- the process 2100 is directed to analyzing a solid cell culture over time.
- the process 2100 may be executed fully, or in part, using the computing device 2200 described below with reference to FIG. 22 .
- various operations may be performed after the computing device 2200 (or multiple computing devices 2200 ) receive data from the system (e.g., microfluidic system and chips) and utilize this data to make assessments regarding treatment efficacy and patient response.
- a first response of a solid cell culture to a growth media, including a treatment agent is determined.
- the solid cell culture is held in a cell culture chamber of a microfluidic chip.
- a first response of the cell culture 910 may be determined at a first time point.
- the first response may be determined by executing instructions of a non-transitory computer-readable media with one or more processing elements of the computing device 2200 .
- the cell culture 910 may be analyzed, using the computing device 2200 , to determine a cell color, quantity, size, density and/or other characteristics at a first point in time, which may be represented by the first representation 1308 a of FIG. 13 A .
- the first response may be a response of the cell culture 910 prior to a dosing with a treatment agent.
- the computing device 2200 may receive data corresponding to images captured by one or more of the components described herein, where the images may be analyzed to determine cell color, quantity, size, and density, e.g., by utilizing image analysis or computer vision algorithms that can identify pixel colors and distribution corresponding to the cells.
- additional responses of the cell culture to the media is determined.
- additional responses of the cell culture 910 may be determined at a second time point, third time point, fourth time point, fifth time point, and so on.
- the additional responses may be determined by executing instructions of a non-transitory computer-readable media with one or more processing elements of the computing device 2200 .
- the cell culture 910 may be analyzed, using the computing device 2200 , to determine a cell quantity, color, size, density and/or other characteristics at substantially any subsequent time point, which may be represented by one or more of the distributions of FIG. 16 .
- the additional responses may be a response of the cell culture 910 subsequent to a dosing with a treatment agent.
- the computing device 2200 may utilize computer vision and/image analysis algorithms that can identify the cells within one or more images captured and can extract information, such as hue, intensity, or the like, that can be utilized to determine the response information.
- the first and the additional responses are compared to determined treatment efficacy.
- the first and the additional responses may be compared by executing instructions of a non-transitory computer-readable media with one or more processing elements of the computing device 2200 , such as one or more image analysis or computer vision algorithms as described above.
- the color, size, quantity, density and/or other characteristics of the cell cultures may be compared, using the computing device 2200 , between the first time point and any of the subsequently measured time points.
- a relative increase or decrease in one or more of these characteristics may be indicative of an efficacy of the treatment provided for by the corresponding treatment agent.
- Other properties may also be measured and compared with respect to the first and second responses, including, but not limited to, a color of the cell culture, a pixel intensity of an image of the cell culture, a shape of cell culture, a size of cell culture, a position of cells of the cell culture, or a quantity of cell of the cell culture.
- the first and/or second response may include capturing an image, such as a two-dimensional or three-dimensional image of the cell.
- the method 2100 may further include determining one or more of a cell viability, a cell proliferation, a cell position from the image.
- the method 2100 may further include determining a cell viability using a color and/or a color ratio to predict treatment response.
- the image analysis may extract hue and/or intensity information to correlate the same to cell viability, based on a color mapping, machine learning, look up table, or the like.
- the method for determining the cell viability may vary based on the type of image analysis or computer vision algorithms utilized.
- the first color indicative of live cells e.g., a red color
- a second color indicative of dead cells e.g., a green color
- the system may analyze hue information at various pixel locations over time, which can be correlated to cell death within the spheroid. Such information may be used as a predictive metric (e.g., ratio of live and dead cells). In other cases, other techniques may be used to determine a cell viability.
- the method 2100 may further include predicting a patient response to the treatment agent based on one or more of cell viability, cell proliferation, or the cell position, for example, as described herein with respect to operation 252 of FIG. 2 .
- an image indicative of the first response may be compared, using the computing device 2200 , to an image indicative of the second response to facilitate the prediction of the patient response.
- data from the two images may be analyzed by the computer device 2200 to determine via image analysis distinctions in the responses that indicate one response may be better or more improved in terms of cancer cell death as compared to the other.
- the method 2100 may further include comparing the images to determine one or more of a cell migration speed or a cell migration distance for one or more cells over time.
- pixel analysis may be used to generate distance vectors that may then be compared in length or dimension to determine cell migration distances.
- the cell migration speed and/or the cell migration distance over time may be indicative of a patient response to the treatment agent. Maximum or minimum of migration vectors or speeds may also be computed in this regard.
- the cell migration speed and/or the cell migration distance may be determined with respect to a subset of the most aggressive cells, including but not limited to, the 5% most aggressive cells of the cell culture, the 2% most aggressive cells of the cell culture, the 1% most aggressive cells of the cell culture, among other subsets. Additionally or alternatively, the cell migration speed and/or the cell migration distance may be computed with respect to a subset of cells expressing a specific bio marker.
- the analysis of operation 2112 may be performed with respect to a single cell.
- the change in position of a single cell may be tracked between first time point and second time point.
- a cell may be tagged or otherwise identified in one or more images and then tracked over time in determines of movement between different image frames.
- the operation 2112 may be performed with respect to a weighted cell measurement.
- weighting factors may be used based on an association of how much each characteristic of the cell culture affects a treatment response prediction.
- the likelihood of a patient responding to treatment A may be based on a patient having a minimum score of X.
- the score X is calculated by having three quarters of the score coming from the cell viability of the culture and one quarter of the score coming from the cell migration speed of that culture.
- the weighted cell measurement may be a composite score of multiple individual measurements (e.g., cell viability, migration distance, and so on).
- the images of the first and second response described above may be images of spheroid or organoids.
- a radius, diameter or circumference of the spheroid or organoid may be measured or determined using the images and compared across multiple time points.
- the computing device may analyze the image to determine an approximate perimeter for the spheroid or organoid and then calculate a circumference, diameter, or other measurements with respect to an estimated area and/or volume of the spheroid or organoid.
- dissociated/single cell analysis may be accomplished using similar techniques. For example, dissociated/single cells may be tracked and measured using substantially any of the associated metrics, as described herein.
- a count and/or a position of a single cells that leave the spheroid, a migration distance, a migration speed, and so on may be determined and analyzed according to the techniques described herein for determining treatment efficacy.
- the method 2100 may include predicting a patient response to the treatment agent based on a comparison of the first radius or the first diameter with the second radius or the second diameter of the respective first and second images. Additionally or alternatively, surgical resections, tissue slices, xenografts, and/or core needle biopsies may be used, which may have a length, width, and height.
- the length, width, or height of the tissue slices, surgical resections, xenografts, and/or core needle biopsies may be measured using the images and compared across multiple time points.
- the method 2100 may include predicting a patient response to the treatment agent based on a comparison of the first length, the first width, or the first height with the second length, the second width, or the second height of the respective first and second images.
- FIG. 22 presents an example computing device 2200 .
- the schematic representation in FIG. 22 of the computing device 2200 may include systems, components, modules, assemblies, and subassemblies configured to implement or execute any of the techniques and processes described herein.
- the computing device 2200 may include any appropriate hardware (e.g., computing devices, data centers, switches), software (e.g., applications, system programs, engines), network components (e.g., communication paths, interfaces, routers) and the like (not necessarily shown in the interest of clarity) for use in facilitating any appropriate operations disclosed herein.
- the computing device 2200 may include a processing unit or element 2208 a operatively connected to computer memory 2212 and computer-readable media 2216 .
- the processing unit 2208 a may be operatively connected to the memory 2212 and computer-readable media 2216 components via an electronic bus or bridge (e.g., such as system bus 2210 ).
- the processing unit 2208 a may include one or more computer processors or microcontrollers that are configured to perform operations in response to computer-readable instructions.
- the processing element 2208 a may be a central processing unit of the computing device 2200 . Additionally or alternatively, the processing unit 2208 a may be other processors within the device including application specific integrated chips (ASIC) and other microcontroller devices.
- ASIC application specific integrated chips
- the memory 2212 may include a variety of types of non-transitory computer-readable storage media, including, for example, random access memory (RAM), read-only memory (ROM), erasable programmable memory (e.g., EPROM and EEPROM), or flash memory.
- RAM random access memory
- ROM read-only memory
- EPROM erasable programmable memory
- flash memory flash memory
- the memory 2212 is configured to store computer-readable instructions, sensor values, and other persistent software elements.
- Computer-readable media 2216 may also include a variety of types of non-transitory computer-readable storage media including, for example, a hard-drive storage device, a solid state storage device, a portable magnetic storage device, or other similar device.
- the computer-readable media 2216 may also be configured to store computer-readable instructions, sensor values, and other persistent software elements.
- the processing unit 2208 a is operable to read computer-readable instructions stored on the memory 2212 and/or computer-readable media 2216 .
- the computer-readable instructions may adapt the processing unit 2208 a to perform the operations or functions described above with respect to FIGS. 1 - 21 .
- the computer-readable instructions may be provided as a computer-program product, software application, or the like.
- the computing device 2200 may also include a display 2218 .
- the display 2218 may include a liquid-crystal display (LCD), organic light emitting diode (OLED) display, light emitting diode (LED) display, or the like.
- the computing device 2200 may also include a battery 2224 that is configured to provide electrical power to the components of the computing device 2200 .
- the battery 2224 may include one or more power storage cells that are linked together to provide an internal supply of electrical power.
- the battery 2224 may be a component of a power source 2228 (e.g., including a charging system or other circuitry that supplies electrical power to components of the computing device 2200 ).
- the computing device 2200 may also include one or more sensors 2240 that may be used to detect a touch and/or force input, environmental condition, orientation, position, or some other aspect of the computing device 2200 .
- the computing device 2200 may also include a camera 2232 that is configured to capture a digital image or other optical data.
- the computing device 2200 may also include a communication port 2244 that is configured to transmit and/or receive signals or electrical communication from an external or separate device.
- the communication port 2244 may be configured to couple to an external device via a cable, adaptor, or other type of electrical connector. In some embodiments, the communication port 2244 may be used to couple the computing device 2200 with a computing device and/or other appropriate accessories configured to send and/or receive electrical signals.
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Pathology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Genetics & Genomics (AREA)
- Theoretical Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medical Informatics (AREA)
- Computer Vision & Pattern Recognition (AREA)
- Physiology (AREA)
- Quality & Reliability (AREA)
- Radiology & Medical Imaging (AREA)
- General Engineering & Computer Science (AREA)
- Toxicology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Biophysics (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Systems and methods for predicting a patient response to various agents and/or combinations of agents using ex vivo dosing and imaging are disclosed. In one example, a method of forming a solid culture that includes isolating target cells from a patient sample, forming stained cells from the isolated cells by staining the isolated cells with a light-responsive dye; and encapsulating the stained cells in a hydrogel
Description
- This application claims priority to PCT Application No. PCT/US2021/054411, filed Oct. 11, 2021, entitled “Treatment Efficacy Prediction Systems and Methods,” which claims priority to U.S. Provisional Application No. 63/219,697, filed Jul. 8, 2021, entitled “Treatment Efficacy Prediction Systems and Methods,” both of which are incorporated herein in their entirety for all purposes.
- The described embodiments relate generally to systems and methods for determining the efficacy of a treatment, such as an anti-cancer agent.
- A range of treatment agents, such as anti-cancer agents, may be used to treat cancerous cells, such as those associated with various types of tumors. Factors such as tumor type, progression, patient characteristics, anti-cancer agent characteristics, and so on may impact the efficacy of a given treatment. These and other factors may hinder the ability of a medical provider to select the most appropriate anti-cancer agent, such as that with the highest efficacy. Anti-cancer treatments may be administered that result in a minimal efficacy and/or detract from overall patient treatment. Similarly, other types of treatments for certain diseases may vary for different patients based on specific patient factors. As such, there is a need for systems and techniques to facilitate individualized biomarker discovery, diagnostics and/or prognostics associated with the efficacy of particular medical treatments, such as, treatment with anti-cancer and other treatment agents.
- Embodiments of the present invention are directed to treatment efficacy prediction systems and methods.
- In one example, a microfluidic chip is disclosed. The microfluidic chip includes a body defining a channel and a cell culture chamber fluidly coupled to the flow channel. The microfluidic chip further includes a coupling portion attached to the body and defining an inlet and an outlet. The channel defines a flow path extending between the inlet and the outlet with the cell culture chamber positioned therebetween. The microfluidic chip further includes a gas permeable membrane covering the cell culture chamber.
- In another example, the channel is configured to deliver a growth media to the cell culture chamber. The cell culture chamber may have a substantially cylindrical shape. For example, the cell culture chamber may have a substantially cylindrical shape with a diameter of preferably about 6.75 mm. In other cases, the diameter may be more or less than 6.75 mm based on the particular application, such as being at least about 5.0 mm, at least about 3.0 mm or other appropriate diameter.
- In another example, the body defines the cell culture chamber having a closed bottom end and an open top end. The gas permeable membrane may cover the open top end of the cell culture chamber. Further, the gas permeable membrane may be attached to the body portion by an adhesive.
- In another example, the cell culture chamber may be a first cell culture chamber. In this regard, the body may further define a second cell culture chamber fluidly coupled to the channel along the flow path between the first cell culture chamber and the inlet or the outlet of the coupling portion. The first cell culture chamber may have a first volume and the second cell culture chamber may have a second volume that is different from the first volume; however, this is not required. Further, the first cell culture chamber may have a first shape and the second cell culture chamber may have a second shape that is different from the first shape; however, this is not required.
- In another example, the body includes a multi-layered structure. In this regard, the multi-layered structure includes a first body portion layer defining the cell culture chamber. The multi-layered structure further includes a second body portion layer connected to the first body portion layer and defining the channel fluidly coupled to the cell culture chamber. The multi-layered structure further includes a third body portion layer connected to the second body portion layer opposite the first body portion layer and defining an opening or hole above the cell culture chamber. In some cases, the coupling portion may be attached to the third body portion layer. As such, the third body portion layer further defines a first lumen fluidly coupled to the inlet of the coupling portion and extending to the flow channel of the second body portion layer. The third body portion may further define a second lumen fluidly coupled to the outlet of the coupling portion and extending to the flow channel of the second body portion layer.
- In another example, the inlet and the outlet define tube barbs. The barbs may protrude from a topmost surface of the chip. While many material constructions are possible, the body may include or be formed fully from an acrylic material or a silicone-based material.
- In another example, a microfluidic device is disclosed. The microfluidic device may include a dosing bank including a plurality of reservoirs. Each reservoir of the plurality of reservoirs may be configured to hold a growth media. The microfluidic device may further include a staging section or “stage” configured to arrange a plurality of microfluidic chips. The plurality of reservoirs can correspond to the plurality of microfluidic chips. In some cases, multiple reservoirs may be used for any given microfluidic chip. The microfluidic device may further include a pump fluidly coupleable with the plurality of reservoirs and the plurality of microfluidic chips to define fluid circuits between each corresponding pair of the plurality of reservoirs and the plurality of microfluidic chips. The pump further may cause a circulation of a growth media through the fluid circuits for each corresponding pair.
- In another example, each reservoir of the plurality of reservoirs are fluidly isolated from one another. The fluid circuits may therefore be fluidically isolated from one another. The pump may be further configured to selectively cause a circulation of the growth media through an individual fluid circuit of the fluid circuits. The growth media may include a treatment agent, such as an anti-cancer agent and/or cells in suspension. In this regard, the cells may be in circulation with the growth media such that the drugs, cells, and/or other agents are included. In some cases, circulating cells can be used to make immune models or test cell therapies. Additionally or alternatively, other circulating agents can help characterize the behavior or properties of the cells in circulation.
- In another example, the plurality of reservoirs may be exposable to atmosphere. For example, the reservoirs may have lids that can be opened to expose the reservoir to air for the purpose of loading treatments. Once the treatment is loaded, the lid can be closed. The microfluidic device may operate as described herein upon the closing of the reservoirs with the lids. In this regard, the plurality of reservoirs may be configured to receive a treatment agent or combination of agents during operation of the pump.
- In another example, the dosing bank may include a tray or other structure configured to hold the plurality of reservoirs in a substantially upright position. The device may further include tubes fluidly coupling the pump to each reservoir and each microfluidic chip housed in the staging section.
- In another example, a method of forming a solid culture is disclosed. The method includes isolating target cells from a patient sample. The method further includes forming stained cells from the isolated cells by staining the isolated cells with a light-responsive dye. The method further includes encapsulating the stained cells in a hydrogel. In one example, the hydrogel may include hyaluronic acid, collagen and/or other elements that are configured to mimic core components of human tissue extracellular matrices and/or disease-specific cell niches.
- In another example, the method may further include culturing dissociated cells in the hydrogel. The culturing may further include forming two-dimensional cell cultures of the dissociated cells. The culturing may further include forming three-dimensional cell cultures of the dissociated cells. The culturing may further include forming cell cultures of a single population of dissociated cells. The culturing may further include forming cell cultures from multiple cell types.
- In another example, the dissociated cells include cancer cells as well as normal/non-transformed cells, stromal cells, or immune cells. In this regard, the culturing further comprises forming co-culture of cancer, normal/non-transformed, stromal, and/or immune cells. The dissociated cells may be isolated from a patient-derived tissue or tumor sample.
- In another example, the method may further include forming the spheroid or an organoid. In some cases the method may include culturing the spheroid or the organoid in a hydrogel. The spheroid or the organoid may include cancer cells, normal/non-transformed cells, stromal cells, or immune cells. In some cases, the culturing further includes forming co-culture of cancer, normal/non-transformed, stromal, and/or immune cells. The isolated cells may be isolated from a patient-derived tissue or tumor sample.
- In another example, the method further includes processing the patient sample using a digestion enzyme-based operation, a blood lysis solution, or a selecting operation to isolate target cells of the patient sample. Processing may allow for the isolation of many different cell types, such as many different cell types that stay alive from the patient tissue or tumor sample. As such, different cells can be co-cultured, including cells in addition to cancer, normal/non-transformed, immune, and/or stromal cells, in either a dissociate cell culture or spheroid/organoid.
- In another example, the light-responsive dye may be configured to allow for tracking of the target cells via fluorescence microscopy. The light-responsive dye may be configured to stain mitochondria of the target cells for live cell tracking. The light-responsive dye may be configured to stain nuclei of the target cells for dead cell tracking.
- In another example, forming stained cells further includes staining isolated cells with a first light-responsive dye. The first light-responsive dye may be configured to stain mitochondria of the target cells for live cell tracking. Forming stained cells may further include staining isolated cells with a second light-responsive dye, the second light-responsive dye being configured to stain nuclei of the target cells for dead cell tracking.
- In another example, the light-responsive dye may be configured to cause a color change in the stained cell when the stained cell transitions from a living cell to a dead cell. The target cells may be, without limitation, cells of a tumor, the tumor comprising a breast cancer, a colorectal cancer, a lung cancer, a kidney cancer, a pancreatic cancer, an ovarian cancer, a brain cancer, or a gastric cancer.
- In another example, the patient sample includes tissue slices, surgical resections and/or xenografts. The patient sample may also include biopsy samples, including core needle biopsy samples in certain circumstances. In this regard, the method may further include culturing the tissue slices, cores, surgical resections and/or xenografts in a hydrogel.
- In another example, a method of loading a microfluidic chip is disclosed. The method includes arranging a solid cell culture in a cell culture chamber of the microfluidic chip. The microfluidic chip includes a body defining the cell culture chamber and a channel that traverses the cell culture chamber and extends between an inlet and an outlet of the microfluidic chip. The method further includes positioning a gas permeable membrane over the cell culture chamber while the inlet and outlet remain exposed for coupling to a circulation system.
- In another example, the positioning further includes adhering the gas permeable membrane to the body and covering the cell culture chamber. The arranging of the solid cell culture may further include dropping a quantity of the solid cell culture (e.g., the cell culture in a hydrogel) into the cell culture chamber using a pipette.
- In another example, the solid cell culture may be a first solid cell culture and the cell culture chamber may be a first cell culture chamber. In this regard, the method may further include arranging a second solid cell culture in a second cell culture chamber of the microfluidic chip. The second cell culture chamber may be defined by the body and fluidly coupled to the channel between the inlet and the outlet. The first cell culture chamber may have a first volume and the second cell culture chamber may have a second volume that is different from the first volume. The first cell culture chamber may have a first shape and the second cell culture chamber may have a second shape that is different from the first shape.
- In another example, a method of operating a microfluidic chip is disclosed. The method includes fluidly coupling the microfluidic chip with a microfluidic device to define a fluid circuit between the microfluidic chip, a flow restrictor, a reservoir, and a pump; the microfluidic chip including a solid cell culture, the reservoir including a growth media. The method further includes causing a flow of the growth media through the circuit such that the solid cell culture of the microfluidic chip is exposed to the growth media to form an exposed cell culture. As used herein, a “solid cell culture” refers to the cells in the hydrogel material. An “exposed cell culture” refers to a circumstance in which the solid cell culture is exposed to media (e.g., growth media) while the cell culture itself remains solid (e.g., the growth media flowing along the hydrogel with cells.). As further used herein, a “liquid cell culture” refers to cells that are in a liquid medium exclusively. For example, a liquid cell culture may include immune cells put into a growth media/serum and then circulated using the systems and techniques described herein. The method includes analyzing a response of the solid cell culture to the growth media.
- In another example, the growth media includes a treatment agent. In some cases, the fluid coupling further includes fluidly coupling a first tube portion to an inlet of the microfluidic device. The first tube portion may be connected to a second tube portion fluidly coupled with a reservoir, the first and second tube portions defining a common tube. The fluid coupling may further include fluidly coupling a third tube portion to an outlet of the microfluidic device and the reservoir to define a fluid circuit. The microfluidic device may define a flow path therethrough.
- In another example, the microfluidic chip may include a cell culture chamber that holds the hydrogel including the target cells. The flow path may traverse and/or run adjacent to the cell culture chamber. The method may therefore further include causing a flow of the growth media along the flow path while a hydrogel restrains the target cells from exit from the culture cell culture chamber.
- In another example, the first tube portion may be fluidly coupled to the pump. The second tube portion may be fluidly coupled to the reservoir. The first and second tube portions may be portions of the same tube. The microfluidic device may further include a third tube portion connected to the reservoir and the microfluidic chip to complete the circuit. The circuit may be a closed-circuit.
- In another example, the method further includes fluidly coupling a second microfluidic chip with the microfluidic device to define a second fluid circuit between the second microfluidic chip, a second reservoir, and the pump; the second microfluidic chip including a second solid cell culture, the second reservoir including a second growth media. The method may further include causing a second flow of the second growth media through the second circuit such that the second solid cell culture of the second microfluidic chip is exposed to the second growth media. The method may further include analyzing a response of the second solid cell culture to the second growth media.
- In another example, the growth media and the second growth media include different treatment agents, such as different anti-cancer agents or cells. The method may further include comparing the response of the first solid cell culture and the response of the second solid cell culture to determine a treatment efficacy. The first circuit and the second circuit may be fluidly isolated from one another. In this regard, the pump may be configured to control the first flow and the second flow independently.
- In another example, the method may further include, prior to the analyzing, fluidly uncoupling the microfluidic chip from the microfluidic device. In this regard, subsequent to the analyzing, the method may further include fluidly coupling the microfluidic chip with the microfluidic device to define the fluid circuit between the microfluidic chip, the reservoir, and the pump. The method may further include causing another flow of the growth media (e.g., the same growth media or a different growth media) through the circuit such that the solid cell culture of the microfluidic chip is exposed to the growth media. The method may further include, subsequent to the causing of another flow, analyzing a subsequent response of the solid cell culture to the growth media.
- In another example, the method may therefore further include comparing the response of the solid cell culture to the growth media and a subsequent response of the solid cell culture to the growth media to determine a treatment efficacy. The method may further include analyzing the response of the solid cell culture to the growth media to determine a first cell population quantity. The method may further include analyzing the subsequent response of the solid cell culture to the growth media to determine a second cell population quantity. As described herein, the method may further include analyzing further subsequent responses of the solid cell culture and determining a third cell population quantity, a fourth cell population quantity, and so one over a course of days or other appropriate interval. The method may further include comparing the first cell population quantity and the second cell population quantity to determine a change in cell population quantity indicative of a treatment efficacy. In this regard, the method may further include analyzing the response of the solid cell culture to the growth media to determine a first cell population position, and analyzing the subsequent response of the solid cell culture to the growth media includes determining a second cell population position. As described herein, the method may further include analyzing further responses of the solid cell culture and determining a third cell population position, a fourth cell population position, and so one over a course of days or other appropriate interval. The method may further include comparing the first cell population position and the second population position to determine a change in cell population position indicative of a treatment efficacy.
- In another example, the analyzing may include conducting a fluorescence microscopy operation on the solid cell culture of the microfluidic chip. In some cases, the analyzing includes collecting three-dimensional images of the solid cell culture of the microfluidic chip. The analyzing may further include collecting two-dimensional images in a z-stack of the solid cell culture of the microfluidic chip. The analyzing may further include analyzing multiple responses of the solid cell culture to the growth media over time. The analyzing may be conducted daily.
- In another example, the analyzing may include conducting a confocal microscopy operation on the solid cell culture of the microfluidic chip. Additionally or alternatively, the analyzing may include conducting a brightfield microscopy operation on the solid cell culture of the microfluidic chip. Additionally or alternatively, the analyzing may include conducting a lattice light sheet microscopy operation on the solid cell culture of the microfluidic chip.
- In another example, the analyzing includes executing instructions of a non-transitory computer-readable media, with one or more processing elements of a computer, to determine a treatment efficacy of a treatment agent of the growth media, on the solid cell culture.
- In another example, a method of analyzing a solid cell culture over time is disclosed. The method includes determining a first response of a solid cell culture to a growth media including a treatment agent. The solid cell culture is held in a cell culture chamber of a microfluidic chip. The method further includes determining a second response of the solid cell culture to the growth media, including a treatment agent. The method further includes comparing the first and second responses to determine a treatment efficacy. In some cases, and as described herein, multiple responses of the solid cell culture may be compared to determine treatment efficacy, such as comparing three, four, or more responses for each solid cell culture/chip as appropriate for a given application. In this regard, while the example of comparing a first and second response is presented for purposes of illustration, it will be appreciated that the computer vision and imaging techniques described herein may be applied to comparing and analyzing any number of responses over any appropriate time period.
- In another example, one or both of the first response or the second response includes at least one of a color of the solid cell culture, a pixel intensity of an image of the solid cell culture, a shape of the solid cell culture, a size of the solid cell culture, a position of cells of the solid cell culture, or a quantity of cell of the solid cell culture. The treatment efficacy may be indicative of a viability of cells of the solid cell culture in response to the treatment agent.
- In another example, determining the first response comprises capturing an image of the solid cell culture. In this regard, the method may further include determining a cell viability from the image and predicting a patient response to treatment agent based on the cell viability. The method may further include determining a cell proliferation from the image and predicting a patient response to treatment agent based on the cell proliferation. The method may further include determining a cell position from the image and predicting a patient response to treatment agent based on the cell position.
- In another example, the image may be a first image. In this regard, determining the second response may include capturing a second image of the solid cell culture. The method may further include comparing the first image and the second image (and/or additional images) to determine a cell migration distance of a cell of the solid cell culture over time. The method may further include predicting a patient response to the treatment agent using the cell migration distance.
- In another example, the method may further include comparing the first image and the second image to determine a cell migration speed of a cell of the solid cell culture over time. In turn, the method may further include predicting a patient response to the treatment agent using the cell migration speed.
- In another example, the method may further include comparing the first image and the second image to determine a migration distance of a plurality of cells that define a subset of the solid cell culture over time. In turn, the method may further include predicting a patient response to the treatment agent using the migration distance.
- In another example, the method may further include comparing the first image and the second image to determine a migration speed of a plurality of cells over time. In turn, the method may further include predicting a patient response to the treatment agent using the migration speed.
- In another example, with reference to determining a migration distance and/or a migration speed, the subset of cells may include the 5% most aggressive cells of the plurality of cells. In other cases, the plurality of cells may include the 2% most aggressive cells of the plurality of cells. In other cases, the plurality of cells may include the 1% most aggressive cells of the plurality of cells. Additionally or alternatively, the plurality of cells may include the subset of cells expressing a specific biomarker.
- In another example, the method may further include comparing the first image and the second image to determine a cell having a maximum migration vector in the solid cell culture over time. In turn, the method may further include predicting a patient response to the treatment agent using the maximum migration vector.
- In another example, the method may further include comparing the first image and the second image to determine a cell having a maximum migration speed in the solid cell culture over time. In turn, the method may further include predicting a patient response to the treatment agent using the maximum migration speed.
- In another example, one or both of determining the first response or determining the second response include determining characteristics of a single cell of the plurality of cells. The single cell of the plurality of cells may have characteristics that can be used to predict a response of the plurality of cells (e.g., the cell with longest migration vector may be a predictive biomarker, as one example). In this regard, determining the first response includes determining characteristics of the single cell at a first time. Determining the second response includes determining characteristics of the single cell at a second time subsequent to the first time. In turn, the method further includes predicting a patient response to the treatment agent based on a comparison of the measured characteristics of the single cell at the first time and the second time and/or additional times, as described herein.
- In another example, one or both of the first image or the second image include a weighted cell measurement of a single cell or a plurality of cells. In turn, the method may further include predicting a patient response to the treatment agent based on the weighted cell measurement. In some cases, the first image comprises a first weighted cell measurement and the second image comprises a second weighted cell measurement. In turn, the method further includes predicting a patient response to the treatment agent based on a comparison of the first weighted cell measurement and the second weighted cell measurement.
- In another example, one or both of the first image or the second image includes information associated with a radius or a diameter of a spheroid or an organoid. The method may further include predicting a patient response to the treatment agent based on the radius or the diameter of a spheroid or an organoid. In some cases, the first image includes a first radius or a first diameter of a spheroid or an organoid and the second image includes a second radius or a second diameter of the spheroid or an organoid. In turn, the method further includes predicting a patient response to the treatment agent based on a comparison of the first radius or the first diameter with the second radius or the second diameter. In some cases, dissociated/single cells may be analyzed in a similar manner. For example, dissociated/single cells may be tracked or measured or otherwise measured according to substantially any of the associated metrics, as described herein. As illustrative examples, a count and/or a position of a dissociated/single cell that leaves the spheroid, a migration distance, a migration speed, and so on may be determined and analyzed according to the techniques described herein for determining treatment efficacy.
- In another example, one or both of the first image or the second image includes information associated with one or more of a length, a width, or a height of a surgical resection, tissue slice and/or xenograft. The method further includes predicting a patient response to the treatment agent based on a length, a width, or a height of a surgical resection, tissue slice and/or xenograft. In some cases, the first image includes a first length, a first width, or a first height of an surgical resection, tissue slice and/or xenograft and the second image includes a second length, a second width, or a second height of the surgical resection, tissue slice and/or xenograft. In turn, the method further includes predicting a patient response to the treatment agent based on a comparison of the first length, the first width, or the first height with the second length, the second width, or the second height.
- In another example, comparing further includes executing instructions of a non-transitory computer-readable media, with one or more processing elements of a computer, to determine the treatment efficacy.
- In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following description.
-
FIG. 1 depicts a functional diagram of a treatment efficacy prediction system. -
FIG. 2 depicts a flow diagram for determining a treatment efficacy. -
FIG. 3 depicts an example patient sample. -
FIG. 4 depicts a user interface including information associated with stained cells. -
FIG. 5 depicts a chart showing the relative concentration and size distribution of living cells and dead cells. -
FIG. 6 depicts a 2D micrograph of an example spheroid. -
FIG. 7A depicts a first representation of an ovarian tumor indicative of living cells. -
FIG. 7B depicts a second representation of the ovarian tumor ofFIG. 7A indicative of dead cells. -
FIG. 7C depicts a third visual representation of the ovarian tumor ofFIG. 7A including a composite of living and dead cells. -
FIG. 8A depicts an exploded view of an example microfluidic chip. -
FIG. 8B is a cross-sectional view of the microfluidic chip ofFIG. 8A . -
FIG. 8C is a cross-section view of a flow restrictor for use with the microfluidic chip in a fluid circuit. -
FIG. 9A depicts an operation of the microfluidic chip ofFIG. 8A . -
FIG. 9B depicts another operation of the microfluidic chip ofFIG. 8A . -
FIG. 9C depicts another operation of the microfluidic chip ofFIG. 8A . -
FIG. 9D depicts another operation of the microfluidic chip ofFIG. 8A . -
FIG. 9E depicts another operation of the microfluidic chip ofFIG. 8A . -
FIG. 10 depicts a top schematic view of a microfluidic device for use with the microfluidic chip ofFIG. 8A . -
FIG. 11 depicts an isometric view of the microfluidic device performing a dosing operation. -
FIG. 12 depicts an example spheroid of isolated and dyed tumor cells after a dosing operation. -
FIG. 13A depicts a chart showing a first quantity of cells at a first time point. -
FIG. 13B depicts another chart showing a second quantity of cells at a second time point. -
FIG. 14A depicts a chart showing a three-dimensional position of a quantity of cells at a first time point. -
FIG. 14B depicts another chart showing a three-dimensional position of a quantity of cells at a second time point. -
FIG. 15 depicts a chart showing a quiver plot indicative of the three-dimensional movement of cells. -
FIG. 16 depicts an example report including visualizations of different dosing operations performed on a given patient sample. -
FIG. 17 depicts a chart showing a distribution of cancerous cell viability based on one or more of dosing operations described herein. -
FIG. 18 depicts a flow diagram for forming a cell culture. -
FIG. 19 depicts a flow diagram for loading a microfluidic chip. -
FIG. 20 depicts a flow diagram for dosing and imaging a culture of microfluidic chip. -
FIG. 21 depicts a flow diagram for determining a treatment efficacy. -
FIG. 22 depicts a functional block diagram of a system including a computing device. - The description that follows includes sample systems, methods, and apparatuses that embody various elements of the present disclosure. However, it should be understood that the described disclosure may be practiced in a variety of forms in addition to those described herein.
- The present disclosure relates to systems and methods for predicting a patient response to various agents and/or combinations of agents using ex vivo dosing and imaging. In some examples, the systems and methods may be applicable to oncology, ex vivo monitoring of disease progression, such as cancer progression, testing of treatment agents, such as anti-cancer agents, patient stratification, and other medical treatment efficacy testing. The system and methods allow discovery, identification, or validation of therapeutic, diagnostic and/or prognostic biomarkers for the purpose of drug development and treatment decision making.
- In one embodiment, an organ-on-a-chip and computer vision system are used to analyze biomarkers for predicting patient response to treatment agents, such as anti-cancer agents. In an example implementation, tumor and/or healthy tissue samples may be cultured in a hydrogel or other environment and placed in a cell culture chamber of a microfluidic chip. Tumor and/or healthy tissue samples may be split into multiple segments, such as aliquots, to culture in multiple chips or separate cell culture chambers. The samples are labelled or stained for tracking, such as via light-responsive dyes, such as fluorescent dyes. As described herein, the stained cells may be tracked by microscopy and processed and analyzed by a variety of computer-implemented techniques, such as the disclosed computer vision techniques of the present disclosure. The fluorescent dyes may selectively stain live cells. Other fluorescent dyes selectively stain dead cells. Some dyes may stain all cells, regardless of whether they are alive or dead. In some cases, dyes may be used to stain cells that express certain biomarkers or targets, such as certain proteins. DNA or RNA may also be stained using dyes according to the techniques disclosed herein.
- The microfluidic chip, including a cell culture chamber, may be arranged with (e.g. fluidly coupled with) a microfluidic device or other system or pump in order to introduce growth media, treatment agents, and other media to the cell culture of the chip. For example, the device may include various treatment agents held in reservoirs with growth media. The media is circulated from the cell culture chamber to the microfluidic chip using a pump, such as a peristaltic pump, pneumatic pump, and so on. A solid cell culture including target cells and hydrogel may be deposited in the microfluidic chip. The solid cell culture may be exposed to the circulating media, which may include the treatment agents, in order to define an exposed solid cell culture within the microfluidic chip. The cell culture chamber of the microfluidic chip having the solid cell culture may be imaged using various techniques, such as microscopy techniques, including confocal microscopy, to track the live and dead cells over time. The imaging may be based on identification of the cells due to staining or labeling of the cells. For example, the live/dead status of the cells may be tracked using the fluorescent dye or other tracking dye. Three-dimensional (3D) images, or stacks of two-dimensional (2D) images taken layer-by-layer, are collected for analysis using a computer vision tool.
- The computer vision tool, which may be executed by one or more computing devices (e.g., via software executing one or more algorithms or machine learning models) may track cell characteristics (e.g., the shape, size, position (x, y, z) and color) of cells in a given image. As the cells may be stained with selectively activated dyes, the number of live and dead cells in an image may be determined. For example, the computer vision may analyze pixel information, such as hue, intensity, and the like, to determine location of specific cells (e.g., live cells may have a first color and dead cells may have a second color) and the location and number of the different cells may be tracked by analyzing subsequent images or image frames over time. One or more images captured at different points in time may be used to monitor the progression of malicious cells (e.g., cancer cells) ex vivo and can be used to predict the response a patient may have to a given agent. For example, comparing a first image frame having a live cell in a first location, such as a first pixel, may be compared to a second image frame captured at a second point in time, having a live cell in a different location, where the system may estimate that the cell has migrated or moved from the first location to the second location. The distance may then be determined by analyzing the difference in pixel locations in the image. Various metrics may be derived from these images, including cell viability, distance and speed of cell migration over time. These assessments, used alone or in combination, may be used to predict patient outcomes to a given agent.
- Multiple microfluidic chips may be analyzed in parallel to determine treatment efficacy across a range of anti-cancer treatments. In this regard, microfluidic chips exposed to either no agent (baseline) or various single and/or combination agents, such as chemotherapies, a comparison may be made between images to predict patient response. By comparing various agents and/or combinations of agents, the techniques described herein may be used to discover therapeutic, diagnostic and prognostic biomarkers that aid the treating of cancer patients and the development of new therapeutics.
- Reference will now be made to the accompanying drawings, which assist in illustrating various features of the present disclosure. The following description is presented for purposes of illustration and description. Furthermore, the description is not intended to limit the inventive aspects to the forms disclosed herein. Consequently, variations and modifications commensurate with the following teachings, and skill and knowledge of the relevant art, are within the scope of the present inventive aspects.
- With reference to
FIG. 1 , a functional diagram of a treatmentefficacy prediction system 100 is depicted. The treatmentefficacy prediction system 100 may broadly be configured to facilitate the ex vivo monitoring of disease progression, such as cancer progression, testing of treatment agents, and patient stratification. Broadly, thesystem 100 may include modules, devices, assemblies, sub-assemblies, and so on (described in relation to the examples ofFIGS. 3-17 ) that cooperate with one another to evaluate the efficacy of a selected treatment agent, combination agents and/or other treatments for use in treating a particular patient. It will be appreciated that any of the modules, devices, assemblies, sub-assemblies, and so on may be implemented separately from one another, as described herein. Treatment agents and other agents, more generally, may be more or less effective based on the particularized circumstances of a treatment regimen, patient characteristic, cancer type and progression or stage, in addition to a range of other complex and often interrelated factors. Conventional treatment regimens may lack the ability to appropriately assess these factors, which may result in the delivery of treatment agents that are suboptimal. Often in conventional systems, various treatment agents may be administered and monitored in vivo, which may hinder the treatment of the patient and overall health outcomes. For example, with conventional approaches, it may be impractical or even impossible to determine if a particular cancer treatment would be effective without administering the treatment to the patient and evaluating the patient over an extended period of time. Further, a patient may have an adverse or undesirable outcome to a particular cancer treatment, once administered. And it may take an exceedingly long period of time for a clinician to assess the efficacy of the treatment, during which time, a patient's overall health conditions may deteriorate. - The
system 100 depicted functionally inFIG. 1 may mitigate these and other hindrances by allowing for ex vivo monitoring of cancer progression in the context of proposed treatment agents. Thesystem 100 may permit a clinician or other provider to evaluate a spectrum of various different treatment agents for one or more patient samples, such as a sample including a cancerous tumor. Thesystem 100 may allow for the creation of an ex vivo environment for the patient sample that replicates in vivo conditions. For example, target cells, such as any of the cells described herein, may be cultured and encapsulated in a hydrogel in order to provide structural stability to the cells, mechanical properties and microenvironmental cues to mimic physiological environments in human organs. More generally, a modular environment may be provided including a range of cell culture conditions, including conditions in which human serum is present, in order to provide conditions that may be similar to the conditions of the human body. Over a span of time, such as a series of days, the clinician may monitor the progression and response of each tissue sample for each of the treatment agents. As described in greater detail below, the treatment agents with the most appropriate response or most suitable efficacy may be identified for administration to the patient for treatment. - To facilitate the foregoing,
FIG. 1 depicts thesystem 100 broadly including aculture module 104, adosing module 108, and ananalysis module 112. Theculture module 104 may generally include various mechanical components, instrumentation, solutions, and devices and so on that are used to prepare a patient sample for evaluation and dosing. While many functions are contemplated and described herein, theculture module 104 may broadly be configured to isolate target cells from a patient sample, form stained cells from the isolated cells, and encapsulate the stained cells in a media. The target cells may be cells from a patient-derived tissue or tumor sample. The target cells may be from tissue slices, cores, surgical resections and/or xenografts. Sample target cells, without limitation, may include cells associated with malicious tissues, such as cells from a breast cancer, a colorectal cancer, a lung cancer, a kidney cancer, a pancreatic cancer, an ovarian cancer, a brain cancer, or a gastric cancer, and so on. Thecell culture module 104 may isolate the target cells and stain the cells with the light-responsive dye, such as a fluorescent dye. Staining, such as with a fluorescent dye may stain or tag the entirety of the cell population or stain individual cell populations, or stain individual components of a given cell population, such as a stain on the mitochondria and a separate stain on a nuclei, to allow for tracking of the cell via fluorescence microscopy or other procedures. The stained cells may be encapsulated in a media, such as a hydrogel, thus forming the solid cell culture, in theculture module 104 in order to promote replication of the core components of human tissue. The stained cells may be encapsulated in a media, such as a hydrogel or may be formed as a spheroid or organoid, thus forming the solid cell culture. - The
dosing module 108 may generally include various mechanical components, instrumentation, solutions, and devices and so on that are used to administer treatment agents to a cell culture and collect data regarding the response of the culture to the treatment agents. In this regard, thedosing module 108 may include a microfluidic chip that is configured to receive a solid cell culture (e.g., cells and hydrogel). Growth media may be put in circulation with the solid cell culture to define an exposed solid cell culture. The microfluidic chip may hold the cells in the hydrogel and allow for the circulation of treatment agents alongside growth media in a gas permeable environment (e.g., to provide the cell with oxygen). A pump of a microfluidic device of thedosing module 108 may cause circulation of the treatment agents and growth media through the chip. A plurality of chips, each fluidly coupled in a separate closed circuit, may permit the device to circulate different treatment agents to each chip to deposit the treatments within the cell culture chambers to evaluate the efficacy of different treatments. The chips may be analyzed at select intervals, such as daily (for one, two, three, four, five, or more days), and a response to the treatment agent may be determined. As one example, and as described in greater detail below, fluorescence microscopy may be used to determine the concentration of living and dead cells in a given culture, using the light-responsive dye. More generally, and as described herein, any appropriate camera or imaging device may be used. Images may be captured over time, and presented in a two-dimensional and/or three-dimensional format, in order to provide a sufficient data set for analysis of the treatment efficacy. - The
analysis module 112 may generally include various computer vision systems (e.g.,computer system 2200 ofFIG. 22 ) or image analysis systems, computer implemented techniques, and analysis devices that may be operated to determine the efficacy of treatment of one or more of treatment agents administered to the cells. The light-responsive dye may be used to indicate the concentration of dead cells, living cells, as well as the transition of cells from a living to dead state. The concentration of cells living or dead, over time, may provide an indication of the viability of cancerous cells, as one example, over the course of administration of a particular treatment agent. Properties of the captured images may be further analyzed to predict a treatment efficacy, including, without limitation, cell characteristics, such as a color of the solid or liquid cell culture, a pixel intensity of an image of the solid or liquid cell culture, a shape of solid or liquid cell culture, a size of the solid or liquid cell culture, a position of cells of the solid or liquid cell culture, or a quantity of cells of the solid or liquid cell culture. In some cases, a two-dimensional or three-dimensional image of the cell culture may be generated at multiple different times over the course of ex vivo administration. Using the light-responsive dyes, the images may include particularized information for each cell, including cell position and type. By capturing multiple images over time, the images can be compared to determine various properties, including cell migration, cell migration speed, and cell migration distance. The images can be further analyzed to determine a maximum migration speed and/or a maximum migration vector, among other characteristics. In this manner, the collective images of the ex vivo response of the cells to the treatment agent may be indicative of an in vivo response to the treatment agent, and thus used to predict treatment efficacy. -
FIG. 2 depicts a flow diagram 200 for determining a treatment efficacy. The flow diagram 200 may implement one or more of the operations of thesystem 100 described above with respect toFIG. 1 . Accordingly, the operations described with respect to the flow diagram 200 may be implemented by one or more of theculture module 104, thedosing module 108, and/or theanalysis module 112. - At
operation 204, a patient sample is collected. Patient samples may arrive at a lab, including samples containing a tumor of various cancer types. Atoperation 208, tumor samples may be processed using a digestion enzyme-based cell isolation kit, blood lysis solution and other selecting steps to isolate viable cells while removing blood and contaminants. The output may be a viable mixed cell population containing the various cells from the primary tumor, including cancer cells, normal/non-transformed cells, stromal cells, and immune cells. - At
operation 212, cells may be stained using live and/or dead cell labeling dyes. Additionally or alternatively, cell-specific/biomarker-targeting dyes may be used. These dyes may be light-responsive dyes that stain or mark the cells. For example, the dyes may be specifically selected for the ability to track cells without requiring the cells to be fixed. In one example, MitoView™ 633 may be used to track live cells by staining the mitochondria of the cells. This dye exhibits a red color when imaged using fluorescence microscopy or other imaging techniques, including confocal and lattice light sheet microscopy. The red color may disappear after cells die. In another example, NucView® 488 may be used to track dead cells by staining the nucleus of the cells. The dye may have a green color when imaged using fluorescence microscopy or other fluorescent imaging techniques; however, it will be appreciated that substantially any color may be used and the foregoing are provided as illustrative example colors. When cells die, the green color may appear. Additionally or alternatively, cells may be stained with both dyes simultaneously to more accurately track cells as they transition from living to dead. It will be appreciated that the red and green colors are described above for purposes of illustration. More generally the light-responsive dyes described herein may have a range of different colors depending in part on the fluorescent dye used. In this regard, various other colors, intensity of colors, and so may be used for staining the cells and performing one or more analysis operations, such as any of the analysis techniques described herein. - At
operation 216, spheroids are optionally formed. As one example, an ultra-low attachment (ULA) plate may be used to form the spheroid. As shown inoperation 220, the cells or spheroids may be embedded within hydrogel. The hydrogel may contain hyaluronic acid and collagen to mimic core components of human tissue extracellular matrices and/or disease-specific cell niches. The spheroids may include the target and/or stained cells in a generally spheroid shape which may facilitate the determining of the response of the cells to a treatment agent, for example, by permitting the tracking of spheroid characteristics, including, but not limited to, a size, shape or other properties of the spheroid over time. - At
operation 224, the spheroid, dissociated cell culture (e.g., without a spheroid architecture), or other cell culture, including a hydrogel and the stained cells, may be set in a microfluidic chip (e.g., themicrofluidic chip 800 ofFIGS. 8A and 8B ). For example, the microfluidic chip may include one or more cell culture chambers. The solid cell culture may be deposited in a first cell culture chamber with a pipette or other instrument. A flow channel is fluidly connected to the one or more cell culture chambers. The flow channel may allow the microfluidic chip to fluidly couple to a circulation system, as described herein with reference toFIGS. 10 and 11 . The microfluidic chip may be configured to permit circulation through the flow channels to promote interaction between a circulated flow and the hydrogel and cells of the chip. Atoperation 228, the hydrogel and cells are within the microfluidic chip and covered by a gas permeable membrane. For example, to seal the chip, a gas permeable membrane may be positioned over the cell culture chamber and adhered to a body of the chip. Once the gas permeable membrane is positioned over the chip, the cell culture chambers are covered. This may allow growth media to flow into the chip without leaking or overflowing using tubing connected to the chip's inlet and outlet barbs. - At
operation 232, drug dosing may commence. Each microfluidic device may include reservoirs that are connected to a corresponding microfluidic chip via tubing. In one instance, a peristaltic pump is used to collect growth media in a 15 mL conical tube or reservoir and circulate the growth media to the inlet of the chip. The growth media may then travel through the flow channel and exit through the outlet of the chip. The growth media then returns to the reservoir using another line of tubing. There may be a hermetic seal placed on the reservoir lid and filters placed in the chip inlet and/or outlet to ensure sterility against bacterial and/or microbial contamination. - By running multiple chips/cell culture chambers in parallel, various chemical agents, including chemotherapies, can be tested in parallel. In one instance, six chips/reservoirs may be connected using the peristaltic or other pump. (e.g.,
FIGS. 10 and 11 ). One chip may be a control where only growth media is circulated to the dissociated cell culture in the circular chamber. Various drugs and combinations may then be circulated to the other chips via their corresponding cell culture chambers, including Olaparib, AC-T and AC-CarboTaxol. In the case of sequential combination therapies like AC-T and AC-CarboTaxol, a clearing process is in place to initially dose AC, clear out the drug from the cell culture chamber, then add in fresh media containing the Taxol or CarboTaxol for the next round of dosing. - At
operation 236, imaging may commence. The imaging may be periodic, such as being at select intervals, such as daily, every twelve hours, and so on as appropriate for a given application. In the present example, the imaging may occur on a daily basis. In this regard, on a daily basis, the media is cleared from the microfluidic chips. The microfluidic chips may be detached from the microfluidic device so they can be transferred to a microscope for imaging. In one instance, a confocal microscope is used to conduct three-dimensional fluorescence imaging. Each chip has the length and width of a standard microscope slide to ensure compatibility. The chips are placed in the slide holder of the confocal microscope and the laser settings are selected to match the excitation wavelength and detection wavelength of each cell staining dye. - In
operation 240, two-dimensional images of the chips are captured. For example, the confocal microscope may take images layer by layer from the bottom to the top of the chamber. In one instance, a step size of 5 micrometers may be used. It will be appreciated that in other cases, other steps sizes may be appropriate, such as a step size of 10 micrometers or more. The chamber(s) containing cells may be imaged in its entirety to capture the location of all cells. For example, the circular chamber may be imaged on a daily basis. In other instances, imaging is conducted at larger intervals (e.g.,day 1 and day 5), and can also be conducted in shorter intervals (e.g.,hour 1 and hour 12). Between each imaging of the chip, the chip may be recoupled with the microfluidic device in order to provide additional treatment agents to the chip. Accordingly, the subsequent imaging of the chip may indicate the progression and response of the tumor to the treatment agent over time. - At
operation 244, the two-dimensional images may be stitched together. For example, the raw microscope images may be ordered by their z position. Once the images are ordered from the bottom to the top, each layer can be stitched onto each other along the z axis. Cells are detected on each two-dimensional image and assigned an x, y and z position. In this regard, and as reflected byoperation 248, three-dimensional images and other visual representations of the cells may be generated in order to determine a treatment efficacy. For example, once the z-stack is complete, some cells may appear on more than one stack due to the confocal microscopy capturing the same cell in more than one layer. When cells have the same x and y position and appear more than once in adjacent layers on the z axis, the brightest pixel is identified as the true z position of the cell, and optionally may be the only pixel displayed in the final three-dimensional reconstruction. The final three-dimensional reconstruction contains every detected cell with an x, y and z coordinate. In one example, these duplicate cells may be identified through machine learning techniques, such as K-nearest neighbor (KNN) and other techniques. This process can result in the removal of duplicate cells across multiple Z positions in the z-stack. In some cases, such machine learning techniques may also be configured to match multiple stained mitochondria to the same cell. Deep learning and other associated techniques of the computer vision system described herein may also be used. Accordingly, where the cell has multiple mitochondria, the techniques described herein may be used to account for the multiple mitochondria in order to obtain a more accurate cell count. - More broadly, the
process 200 may includefurther analysis operations 252 associated with the analysis of two-dimensional and three-dimensional images and response of the cells to a treatment agent. Theanalysis operations 252 may be performed using the computer vision and associated systems described herein, such as those described in greater detail below with respect toFIGS. 13A-16 . In some cases, the operations may include executing instructions of a non-transitory computer-readable media, with one or more processing elements of a computer, such as thecomputing system 2200 ofFIG. 22 . For example, a computer vision tool is disclosed herein that is configured to identify the color of each cell based on the light-responsive or fluorescent dye. Since each cell is treated as a unique object, the color of each cell can also be changed (e.g., all red cells can be changed to blue, or a subset can be changed to blue based on their position or size). - In some cases, an array may be configured to store data on each cell, such as the coordinates and color of each cell, for analysis between multiple time points. Data at a first time point may be indicative of a first response of the cell culture to a treatment agent (e.g., at a first time). Data at a second time point may be indicative of a second response of the cell culture to the treatment agent (e.g., as a second time, subsequent the first time). For example, cells with red fluorescence from MitoView™ 633 may be counted to determine the number of live cells on a first day (e.g., day 1), then compared to an image of the same sample on a later day (e.g., day 5) to determine how many cells died between the first day and the later day. Attributes of cells, such as the average z position of the cells may be calculated on both the first day and the later day to determine, for example, average upward or downward migration of cells between the first day and the later day. Various other cell metrics may be analyzed to determine a change over time as cells are exposed to a drug or combination of drugs.
- In another example, disclosed herein is a computer vision analysis system configured to determine at a single-cell level the behavior of each cell over time. The computer vision analysis system may be further configured to analyze the single-cell level behavior to differentiate cancer cells from immune or other cells. In some cases, the computer vision analysis system may be further configured to identify and differentiate among sub-types of cancer cells.
- To facilitate the foregoing, deep learning, including using neural networks, can be used to make predictions regarding cells and groups of cells. For example, deep learning can allow for the classification of cells by type (e.g., cancer, stromal or immune). More generally, machine learning can also be used to support one or more of the analysis functions described herein. Further machine learning methods such as K-means and support vector machines (SVMs) may be used to classify single cells and groups of cells. With high resolution and magnification microscopy, cells can be differentiated by their size and shape, and/or various other cell characteristics. As one illustration, a KNN algorithm is used to match a single cell between the first day and the later day (e.g.,
day 1 and day 5). In this regard, the migration vector of each cell may then be visualized using a quiver plot (e.g.,FIG. 15 herein). The sum of the vectors may indicate various trends, such as the total movement of cells in a sample. A subset of vectors (e.g., the top 10%) or individual vectors can be used to help make predictions of patient response to a given treatment. - A combination of metrics extracted from the image data may be used to predict patient response to various treatments, including single agents and combination drugs. In its simplest form, thresholds are set to classify treatment responses using a single metric, such as cell viability, cell migration vector total, or largest single cell migration vector. Simple classifiers, receiver operating characteristic (ROC) curves and logistic regression are example methods of correlating single metrics in the invention to patient response.
- The metrics outputted by the computer vision process can be used as input features to perform various predictions. As one example, decision trees may be used to make predictions about the patient outcome using a complex decision making process correlating multiple patient response features. In some cases, decision trees may be used for complex weighing of multiple inputs to optimize prediction accuracy. Training data may be used to correlate inputs to patient response and determine which inputs have the highest impact on predicting patient outcomes. The decision tree may have multiple nodes and branches to facilitate the predictions of a patient response. The final node of the tree (the prediction) may be either the patient's predicted response likelihood or the optimal predicted treatment or even both. Further, the decision tree may be optimized to maximize sensitivity and specificity on a population of patients. The decision tree may also be optimized to maximize positive predictive value and negative predictive value. For example, on a per patient basis, for each proposed treatment, a predicted response with the decision tree (e.g., complete, partial or no response) can be outputted.
- With reference to
FIGS. 3-17 , example implementations of thesystem 100 ofFIG. 1 andprocess 200 are presented herein. For example, mechanical components, instrumentation, solutions, devices, user interfaces, charts, computing devices, and so on are described for purposes of illustration, which may be used to execute one or more of the functions or techniques described above with respect toFIGS. 1 and 2 . It will be appreciated, therefore, that the examples ofFIGS. 3-17 , rather than limit the disclosure, assist in illustrating various features and functions of the present disclosure. In this regard, other mechanical components, instrumentation, solutions, devices, user interfaces, charts, computing devices, and so on are contemplated herein for implementing the functions and techniques described above in thesystem 100 and theprocess 200. - With reference to
FIG. 3 , anexample patient sample 304 is shown relative to ameasurement device 308. Thepatient sample 304 may be a patient-derived tumor sample that is obtained during theoperation 204 described above with respect toFIG. 2 . Thepatient sample 304 may include cells of or associated with a malignant tissue, such as cells of a breast cancer, a colorectal cancer, a lung cancer, a kidney cancer, a pancreatic cancer, an ovarian cancer, a brain cancer, or a gastric cancer, among other cancers. Thepatient sample 304 may include tissue slices, cores, surgical resections, xenografts and/or core needle biopsies. More broadly, thepatient sample 304 is shown as being substantially any patient-derived sample collected with respect to theculture module 104 described above. In this regard, thepatient sample 304 may include target cells that are arrangeable in a culture for dosing and analysis by thedosing module 108 and theanalysis module 112. Accordingly, thepatient sample 304 may include dissociated or isolated cells. Thepatient sample 304 shown inFIG. 3 may be representative of a patient sample prior to forming a solid cell culture or otherwise modifying the patient sample. Thesample 304 may therefore further include cancer cells, normal/non-transformed cells, stromal cells, or immune cells. Thesample 304 may also include blood and other contaminants, which as described herein, may be selected or filtered or removed using a digestion enzyme-based operation, a blood lysis solution, or a selecting operation among other possibilities. - With reference to
FIG. 4 , auser interface 400 is depicted including information associated with cells tagged with a tracking dye. Theuser interface 400 may be a visual representation that is associated with the stained cells. In the present example, the target cells may be stained with a first light-responsive dye that facilitates the tracking of a live cell count, and a second light-responsive dye that facilitates the tracking of the dead cell count. Theuser interface 400 may allow for visualization of the presence of the first light-responsive dye for live cell tracking and the presence of the second light-responsive dye for dead cell tracking. - As shown in
FIG. 4 , theuser interface 400 includes adisplay region 402. Thedisplay region 402 may represent at least a portion of cells from theoperation 212. For example, thedisplay region 402 may be indicative of a portion of the dissociated cells that is analyzed in order to facilitate counting of live and dead cells. Accordingly, for purposes of illustration, thedisplay region 402 is shown includinglive cells 406 anddead cells 410. Thelive cells 406 may be those having or otherwise associated with the first color, and thedead cells 410 may be those having or otherwise associated with the second color. Thelive cells 406 and thedead cells 410 are represented in thedisplay region 402 based on amagnification 414, which may be modifiable by a user or computer-implemented technique. - The representative first color and second color of the
live cells 406 and thedead cells 410, respectively, may allow for the determination of analysis of various properties of the cells. As one example, the differing coloredlive cells 406 anddead cells 410 may allow for a quantity of cells per unit volume to be counted or determined. The quantity of per unit volume oflive cells 406 and the quantity of per unit volumedead cells 410 may be compared in order to determine a relative concentration of living cells to dead cells. In the example ofFIG. 4 , thedisplay region 402 may be overlaid or associated with acontrol panel 452. Thecontrol panel 452 may display the current determination of a livingcell concentration 456 versus adead cell concentration 470. For example, a determination of the concentration oflive cells 406 relative to thedead cells 410 may result in the livingcell concentration 456 having a value of 90% and thedead cell concentration 470 having a value of 10%. It will be appreciated that theuser interface 400 shows information associated with how many cells were isolated from the patient tumor sample. - For example, and with reference to
FIG. 5 achart 500 is depicted including additional information corresponding to the livingcells 406 and thedead cells 410 shown inFIG. 4 . For example, thechart 500 may show a relative concentration or number of living/dead cells with respect to cell size. As shown inFIG. 5 , thechart 500 includes acell size axis 504, acell quantity axis 508, alive cell distribution 512, adead cell distribution 516, live cell size average 506 a, deadcell size average 506 b. Thecell size axis 504 may be representative of an approximate size of a given target cell. InFIG. 5 , the size is represented in micrometers; however, other appropriate units may also be used. Thecell quantity axis 508 may include a count of the number of cells for a given sample or portion thereof measured. - The
live cell distribution 512 and thedead cell distribution 516 may be representative of a quantity of cells for a respective cell size. In some cases, thedistributions live cell distribution 512. Thedead cell average 506 b may represent an average size of the dead cells represented by thedead cell distribution 516. - With reference to
FIG. 6 , anexample culture 600 is depicted. Theculture 600 may represent a solid cell culture formed via the optionalspheroid formation operation 216 andhydrogel embedding operation 220, described above with respect toFIG. 2 . For example, theculture 600 is shown inFIG. 6 as including ahydrogel 608. Thehydrogel 608 may contain hyaluronic acid and collagen to mimic core components of human tissue extracellular matrices and disease-specific cell niches, like those found in breast tumors. Thehydrogel 608 is shown encapsulating aspheroid 604. Thespheroid 604 may includetarget cells 612, such as any of the cells described herein. Thetarget cells 612 may be stained with a light-responsive dye. - The arrangement of the cell culture including the
spheroid 604 may facilitate the tracking of tumor properties over time. As one example, arepresentative circumference 616 is shown inFIG. 6 . Therepresentative circumference 616 may correspond to a size value for thespheroid 604 or other cell or portion of theculture 600. Theculture 600 may be exposed to treatment agents as described herein. Subsequently, a size of thecircumference 616 or other property of theculture 600 may be measured in order to determine a change over time. The change may be indicative of a treatment efficacy for the treatment agent when compared against the control/baseline. As one example, a decrease in a size of thecircumference 616 may provide information to a clinician regarding the efficacy of a treatment that was administered to thecell culture 600. - The
solid cell culture 600 may be stained with one or more of the light-responsive dyes described herein. Properties of thesolid cell culture 600 andspheroid 604 may therefore be measured using the light-responsive dyes. As an illustration, with reference toFIGS. 7A-7C , representations of asolid cell culture 700 are shown under different conditions for analyzing the components of the cell culture. The example ofFIGS. 7A-7C shows a spheroid of an ovarian tumor for purposes of illustrations, in other cases, other tumors may be represented using similar techniques. With reference toFIG. 7A , thecell culture 700 is shown in a condition in which a live cell dye causes arepresentation 704 to be illuminated or represented. Therepresentation 704 may be indicative of the physical properties of live cells, including cell position and size. With reference toFIG. 7B , thecell culture 700 is shown in a condition in which a dead cell dye causes arepresentation 708 to be illuminated or represented. Therepresentation 708 may be indicative of the physical properties of dead cells, including cell position and size. In some circumstances, it may be beneficial to overlay therepresentation FIG. 7C , thecell culture 700 is shown in which therepresentations representation 712. Therepresentation 712 may be a composite visual representation of the living cell and the dead cell dye. Therepresentations representation - With reference to
FIGS. 8A and 8B , amicrofluidic chip 800 is disclosed. Themicrofluidic chip 800 may be used in conjunction with theoperation 224 of setting the hydrogel in a chip, as described above with reference toFIG. 2 . Themicrofluidic chip 800 may be configured to mimic an environment of a human organ. For example, themicrofluidic chip 800 may be configured to replicate an environment of a human organ in order to establish an environment where cells can live ex vivo for a period of time. In this regard, themicrofluidic chip 800 may include a compartment or cell culture chamber to hold cells, such as any of cells or cell cultures described herein. Themicrofluidic chip 800 may include layers to cover or shield the cell cultures to block contaminants from the cell culture while also allowing gas, such as oxygen to reach the cell culture. Themicrofluidic chip 800 may also be configured to circulate growth media and treatment agents to the solid cell culture held therein. The growth media may allow the cells to survive for a period of time, ex vivo. - The
microfluidic chip 800 may be a multilayered structure.FIG. 8A shows an exploded view of a stack up of layers and components that may be used to form themicrofluidic chip 800. Each component layer ofFIG. 8A is shown in a top view. With reference toFIG. 8A , abase layer 802, a firstbody portion layer 812, a secondbody portion layer 822, a thirdbody portion layer 832, acoupling portion 842, and a gaspermeable layer 852 is shown. In other examples, themicrofluidic chip 800 may include more or fewer layers, such as including one or more sterilization layers, films, adhesives, and so on that may be used to prepare themicrofluidic chip 800 for dosing. Themicrofluidic chip 800 may also be associated with one or more flow restrictors 880 (FIG. 8C ). In the example ofFIG. 8A , thebase layer 802 may be a substantially solid section of thechip 800 upon which the other layers of thechip 800 are arranged. Thebase layer 802 may have a size corresponding to a size of a standard microscope slide to foster compatibility. - The first
body portion layer 812, the secondbody portion layer 822, and the thirdbody portion layer 832 may be layers of abody 801 that define achannel 862, a first volume 864 (including a first cell culture chamber 814) and a second volume 866 (including a second cell culture chamber 816) of thechip 800. For example, the firstbody portion layer 812 may define a firstcell culture chamber 814 and a secondcell culture chamber 816, which may each be defined by openings or through portions through thefirst body layer 812. The secondbody portion layer 822 may define a second body portionfirst hole 824 and a second body portionsecond hole 826, which may each define openings or through portions through thesecond body layer 822. Further, the thirdbody portion layer 832 may define a third body portionfirst hole 834 and a third body portionsecond hole 836, which may each define openings or through portions through thethird body layer 832. - As shown in cross-section view of
FIG. 8A , the firstbody portion layer 812, the secondbody portion layer 822, and the thirdbody portion layer 832 may be stacked relative to one another such that the firstcell culture chamber 814, second body portionfirst hole 824, and third body portionfirst hole 834 cooperate to define afirst volume 864. Thefirst volume 864, including the firstcell culture chamber 814, may have a substantially cylindrical shape with a closed bottom end and an open top end. In one example, the firstcell culture chamber 814 may have a diameter of preferably about 6.75 mm. In other cases, the diameter may be more or less than 6.75 mm such as being less than about 5 mm, less than about 3 mm and/or other appropriate value for a given application. Further, the firstbody portion layer 812, the secondbody portion layer 822, and the thirdbody portion layer 832 may be stacked relative to one another such that the secondcell culture chamber 816, second body portionsecond hole 826, and third body portionsecond hole 836 cooperate to define thesecond volume 866. Thesecond volume 866, including the secondcell culture chamber 816, may have a diamond or rectangular shape with a closed bottom end and an open top end. - The
body 801 may also define thechannel 862. For example, and as shown inFIG. 8A , the secondbody portion layer 822 includes an elongated throughportion 828. The elongated throughportion 828 may extend through a complete thickness of the secondbody portion layer 822 and through a portion of the second body portion first opening 824 and the second body portionsecond opening 826. The firstbody portion layer 812, the secondbody portion layer 822, and the thirdbody portion layer 832 may be stacked relative to one another such that the firstbody portion layer 812 and the thirdbody portion layer 832 close the bottom and the top of the elongated throughportion 828, respectively, in order to define thechannel 862. - The elongated through
portion 828 may extend between opposing ends of the secondbody portion layer 822. The thirdbody portion layer 832 may cover the elongated throughportion 828 and define afirst lumen 833 a and asecond lumen 833 b, each extending into the elongated throughportion 828. For example, thefirst lumen 833 a may extend through a thickness of the thirdbody portion layer 832 at a first end and into the elongated throughportion 828. Thesecond lumen 833 b may extend through the thickness of the thirdbody portion layer 832 at a second, opposing end and into the elongated throughportion 828. The first andsecond lumens portion 828 in order to facilitate a fluidic coupling of thechannel 862 to a fluid circuit. - For example, and as shown in
FIG. 8A , thecoupling portion 842 may include aninlet feature 843 a and anoutlet feature 843 b. As shown inFIG. 8B , the inlet feature 843 a and theoutlet feature 843 b may be barbs that protrude from a topmost surface of themicrofluidic chip 800. The inlet feature 843 a may be alignable with or receivable by thefirst lumen 833 a and configured to define a fluid connection between a tubing of a circulation system and thechannel 862. Theoutlet feature 843 b may be alignable with or receivable by thesecond lumen 833 b and configured to define a fluid connection between additional tubing of the circulation system and another end of thechannel 862. As described herein, this configuration may allow thechip 800 to be fluidly coupled to a circulation system in which treatment agents are circulated through thechip 800 alongside growth media. For example, fluid or media of a variety of purposes may be introduced to thechip 800 viainlet feature 843 a and caused to flow through thechip 800 via thefirst lumen 833 a, thechannel 862, thesecond lumen 833 b, and exiting via theoutlet feature 843 b. - With further reference to
FIG. 8A , the gaspermeable layer 852 may include a membrane, film, or other layer configured to shield and cover the first andsecond volumes cell culture chambers permeable layer 852 may be configured to shield or cover thevolumes cell culture chambers body portion layer 832 is shown as including anadhesive surface 835. The gaspermeable layer 852 may be attachable to the body via theadhesive surface 835 subsequent to loading thechip 800 with a cell culture, as described herein. - With reference to the cross-sectional view of
FIG. 8B , thechip 800 may be coupled such that thebase layer 802, the firstbody portion layer 812, the secondbody portion layer 822, the thirdbody portion layer 832, thecoupling portion 842, and the gaspermeable layer 852 are stacked relative to one another. In the assembled configuration, thechannel 862 is shown extending through thebody 801 between thefirst lumen 833 a and thesecond lumen 833 b. Thefirst lumen 833 a is fluidly coupled to the inlet feature 843 a. Thesecond lumen 833 b is fluidly coupled to theoutlet feature 843 b. As shown inFIG. 8B , the inlet feature 843 a may be fluidly coupled with afirst tube 906 a, and theoutlet feature 843 b may be fluidly coupled with asecond tube 906 b. The first andsecond tubes microfluidic device 1000 ofFIGS. 10 and 11 ). Thetubes respective tubes - As shown in
FIG. 8B , thechip 800 may be configured to define a segment of a flow path for the circulation system. For example, thefirst tube 906 a may introduce an inlet flow Fi into thechip 800 at the inlet feature 843 a. The inlet flow Fi may be a flow of a media including treatment agents. The flow of the media and/or treatment agents and/or other agents may progress to thechannel 862 defined by thebody 801. Thechannel 862 may run adjacent to both the firstcell culture chamber 814 and the secondcell culture chamber 816. In this regard, the flow of media and/or treatment agents continue through thechannel 862 and alongside and/or into a portion of the firstcell culture chamber 814 and/or the secondcell culture chamber 816. As described herein, one or both of the firstcell culture chamber 814 and/or the secondcell culture chamber 816 may include cells encapsulated in a hydrogel, such as any of the arrangements of cells and hydrogels described herein. Thechannel 862 may therefore be configured to route the growth media and/or treatment agents for interaction with the cells/hydrogel included in the respectivecell culture chambers cell culture chamber 814 and/or the secondcell culture chamber 816. The growth media may be circulated through thechip 800, such as along thechannel 862 in order to flow over the cells and hydrogel held therein. An exposed solid cell culture may therefore be defined in the chip once the solid cell culture is put in the chip and the growth media is circulated therethrough. The interaction of the media and/or the treatment agents with the cells may cause the cells to retain at least a portion of the media and/or treatment agents in the cell culture chamber. The flow may continue along thechannel 862 and exit the chip at theoutlet feature 843 b at a flow Fo. -
FIG. 8C is a cross-section view of aflow restrictor 880 for use with themicrofluidic chip 800 in a fluid circuit. The flow restrictor 880 may be implemented in order to facilitate sterility in the system, such as by preventing contamination of thechip 800 and cells therein. The flow restrictor 880 may therefore include one or more filter elements. The flow restrictor 880 may therefore further include one or more reductions or changes in a diameter in the flow path in order to increase or modify a pressure gradient of the flow across therestrictor 880, among other uses. - In the example of
FIG. 8C , theflow restrictor 880 is shown including a firststraight barb 882. The firststraight barb 882 defines afirst barb channel 883 therethrough. The flow restrictor 880 may generally be arranged adjacent the inlet or outlet of themicrofluidic chip 800. In this regard, the firststraight barb 882 may be fluidly coupled to one of the inlet or outlet features 843 a, 843 b. For example, theflow restrictor 880 may be fluidly coupled to tubing that is received by the firststraight barb 882 and one of the barbs of the inlet or outlet features 843 a, 843 b to complete the fluidic coupling. The flow restrictor 880 shown inFIG. 8C also includes a secondstraight barb 896 that defines asecond barb channel 897. The secondstraight barb 896 may be substantially analogous to the firststraight barb 882. The secondstraight barb 896 may be fluidly coupleable with a component of the microfluidic device (e.g., themicrofluidic device 1000 ofFIG. 10 ) via tubing and/or other connector. Multiple flow restrictors can be used. As one example, a first flow restrictor can be used at an inlet of a given microfluidic chip and a second flow restrictor can be used at an outlet of the microfluidic chip. - The flow restrictor 880 may include multiple layers arranged generally between the first and second
straight barbs flow restrictor 880 is shown including afirst layer 884 and afirst hole 885, and an adjacentsecond layer 886 and asecond hole 887. The firststraight barb 882 may be at least partially received by thefirst hole 885. Thesecond hole 887 may generally be aligned with thefirst hole 885. Thesecond hole 887 has a diameter that is less than a diameter of thefirst hole 885. In this regard, thefirst barb channel 883, thefirst hole 885 and thesecond hole 887 may define a flow path with thesecond hole 887 operating to reduce or restrict the flow through theflow restrictor 880. The flow restrictor 880 is further shown as including afilter layer 888 with afilter hole 889 generally adjacent thesecond hole 887. Thefilter layer 888 may house or hold afilter 890 in thefilter hole 889. Multiple constructions of thefilter 890 are possible. In one example, thefilter 890 may include a glass microfiber filter. In some cases, multiple separate filters may be used. - Adjacent the
filter layer 888, theflow restrictor 880 is shown as including athird layer 892 andthird hole 893, and an adjacentfourth layer 894 andfourth hole 895. Thethird layer 892 andthird hole 893, and thefourth layer 894 andfourth hole 895 may generally mirror thefirst layer 884 andfirst hole 885, andsecond layer 886 andsecond hole 887. In this regard, thethird hole 893 may be generally smaller in diameter than thefourth hole 895. The secondstraight barb 896 may be at least partially received by thefourth hole 895. -
FIGS. 9A-9E depict an operation of loading themicrofluidic chip 800 with a cell culture. - With reference to
FIG. 9A , anoperation 900 a is shown in which the firstcell culture chamber 814 of thefirst volume 864 is loaded with a solid cell culture (e.g., any combination of cells and hydrogel described herein). For example, apipet 902 or other instrument may selectively introduce asolid cell culture 910 into the firstcell culture chamber 814. In some cases, the secondcell culture chamber 816 of thesecond volume 866 may also be loaded with a solid cell culture. With reference toFIG. 9B , anoperation 900 b is shown in which anadhesive backing 879 is removed to reveal theadhesive surface 835 of thebody 801. Theadhesive backing 879 may reveal theadhesive surface 835 in order to prepare thebody 801 for attachment with the gaspermeable layer 852. For example, and with reference toFIG. 9C , anoperation 900 c is shown in which the gaspermeable layer 852 may be positioned over thebody 801. In some cases, the gaspermeable layer 852 may cover the top of both the first andsecond volumes cell culture chamber 814 and the secondcell culture chamber 816, as shown inFIG. 9D atoperation 900 d. The gaspermeable layer 852 may be attached to thebody 801 via theadhesive surface 835. With reference toFIG. 9E , anoperation 900 e is shown in which thefirst tube 906 a is fluidly coupled with the inlet feature orbarb 843 a and thesecond tube 906 b is fluidly coupled with the outlet feature orbarb 843 b. - With reference to
FIGS. 10 and 11 , amicrofluidic device 1000 is shown. Themicrofluidic device 1000 may be configured to implement theoperation 232 ofFIG. 2 , in which the solid cell culture (e.g., the cells and hydrogel desired herein) is exposed to treatment agents. Broadly, themicrofluidic device 1000 may be configured to provide treatment agents alongside growth media to multiple microfluidic chips, in parallel. Themicrofluidic device 1000 may define closed circuits for each chip that are generally fluidly isolated and separated from the circuits of the other chips. Themicrofluidic device 1000 may be configured to provide different treatment agents, growth media, or other solutions to the chips, so that a response of the solid cell cultures may be determined over time. - To facilitate the foregoing, the
microfluidic device 1000 may include ahousing 1002, aplatform 1004, astaging section 1010, adosing bank 1020, and apump 1030. Thehousing 1002 may provide a structural platform for the various components of themicrofluidic device 1000, including thepump 1030 and thedosing bank 1020. Thehousing 1002 may also provide a structure upon which to arrange and temporarily store chips during dosing. Thehousing 1002 may have one or more open sides, as shown inFIG. 11 . In other cases, thehousing 1002 may enclose one or more components of themicrofluidic device 1000. In yet other cases, thehousing 1002 may be omitted entirely. - The
staging section 1010 may be configured to arrange a plurality of microfluidic chips in themicrofluidic device 1000. Thestaging section 1010 may be a portion of thehousing 1002. In other cases, thestaging section 1010 may include a raised platform, slots, or other features for receiving and securing chips in a particular position in thestaging section 1010. In some cases, and as shown in the example ofFIGS. 10 and 11 , thestaging section 1010 may include achip tray 1012. Thechip tray 1012 is shown as holding six microfluidic chips: a firstmicrofluidic chip 800 a, a secondmicrofluidic chip 800 b, a thirdmicrofluidic chip 800 c, a fourthmicrofluidic chip 800 d, a fifthmicrofluidic chip 800 e, and a sixthmicrofluidic chip 800 f. Each ofchips 800 a-800 f may be substantially analogous to themicrofluidic chip 800 described above with respect toFIGS. 8A-9E ; redundant explanation of which is omitted for clarity. While various configurations are possible, thechip tray 1012 may include a recess or groove that is configured to accommodate a single chip. For example, a given chip may be received in the groove such that at least side to side movement of the chip is mitigated on thetray 1012. While the examples ofFIGS. 10 and 11 show thechip tray 1012 as including a grooves for six chips, other configurations are possible, including trays that hold more or fewer chips. - The
dosing bank 1020 may include a plurality of reservoirs. Thedosing bank 1020 may include a plurality of reservoirs corresponding to the plurality of chips arranged at thestaging section 1010. For example, and as shown inFIG. 11 , the dosing bank may include afirst reservoir 1022 a, asecond reservoir 1022 b, athird reservoir 1022 c, afourth reservoir 1022 d, afifth reservoir 1022 e, and asixth reservoir 1022 f. The reservoirs 1022 a-1022 f may be configured to hold a media, such as any of the growth media described herein. The reservoirs 1022 a-1022 f may further be configured to hold treatment agents, or substantially any other fluid for introduction to respective ones of thechips 800 a-800 f. The reservoirs 1022 a-1022 f may be exposed or exposable such that additional media and/or treatment agents may be added over time. The reservoirs 1022 a-1022 f may also be associated optionally with covers to shield the media held therein from contaminants during dosing. For example, one or more or all of the reservoirs 1022 a-1022 f may include a cap, such as an aluminum cap, to hermetically seal the respective reservoir 1022 a-1022 f. - The
pump 1030 may be configured to complete a fluid circuit between respective ones of thechips 800 a-800 f and the reservoirs 1022 a-1022 f. Thepump 1030 may be a peristaltic pump, pneumatic pump and/or any other appropriate pump or pumping device. Thepump 1030 may be configured to define separate fluid circuits between a given one of thechips 800 a-800 f and the reservoirs 1022 a-1022 f. For example, thepump 1030 may be configured to cause circulation of fluid between respective ones of the reservoirs 1022 a-1022 f and thechips 800 a-800 f without fluid of one of the reservoirs crossing over or contaminating another reservoir. This may allow each chip to be dosed separately such that a reading or measurement of each chip may be taken in order to determine the impact from a particular solution held in the corresponding reservoir. While thepump 1030 is shown as a single assembly, in other cases, thepump 1030 may represent multiple pumps. Further, thepump 1030 is shown as including six circulation paths. In other cases, thepump 1030 may define more or fewer paths. - In the example of
FIG. 11 , thepump 1030, thechips 800 a-800 f, and the reservoirs 1022 a-1022 f are coupled to one another to define six circulation paths. For example, thepump 1030, the firstmicrofluidic chip 800 a, and thefirst reservoir 1022 a are coupled to one another to define a firstfluid circulation path 1002 a. Further, thepump 1030, the secondmicrofluidic chip 800 b, and thesecond reservoir 1022 b are coupled to one another to define a secondfluid circulation path 1002 b. Further, thepump 1030, the thirdmicrofluidic chip 800 c, and thethird reservoir 1022 c are coupled to one another to define a thirdfluid circulation path 1002 c. Further, thepump 1030, the fourthmicrofluidic chip 800 d, and thefourth reservoir 1022 d are coupled to one another to define a fourthfluid circulation path 1002 d. Further, thepump 1030, the fifthmicrofluidic chip 800 e, and thefifth reservoir 1022 e are coupled to one another to define a fifthfluid circulation path 1002 e. Further, thepump 1030, the sixthmicrofluidic chip 800 f, and thesixth reservoir 1022 f are coupled to one another to define a sixthfluid circulation path 1002 f. Thecirculation paths 1002 a-1002 f may be separate fluid paths that are fluidly isolated from one another. Thepump 1030 may cause a circulation alongcirculation paths 1002 a-1002 f independent from one another. For example, thepump 1030 may cause media, treatment agents and the like to circulate along the first circulation path between thefirst reservoir 1022 a and thefirst chip 800 a, thesecond reservoir 1022 b and thesecond chip 800 b, thethird reservoir 1022 c and thethird chip 800 c, thefourth reservoir 1022 d and thefourth chip 800 d, thefifth reservoir 1022 e and thefifth chip 800 e, and thesixth reservoir 1022 f and thesixth chip 800 f. - The
chips 800 a-800 f may be removed individually from themicrofluidic device 1000. For example, individual ones of thechips 800 a-800 f may be removed from themicrofluidic device 1000 for imagining and analysis. As described above with respect toFIG. 2 , an individual chip may be removed and imaged using fluorescence microscopy or other techniques in which properties of the solid cell culture are determined for a single point in time, including, but not limited to, cell culture size color, position, and density. Subsequent to the imaging, the chip may be recoupled to themicrofluidic device 1000. Themicrofluidic device 1000 may proceed to provide additional treatment agents, media, and so one to the respective microfluidic chip. The chip may be removed again from themicrofluidic device 1000 in order to conduct further imaging of the solid cell culture for a second, later point in time. For example, the cell culture color, size, position, and density can be detected at the second point in time and/or a variety of other cell characteristics. The characteristics of the solid cell culture at the second point in time can be compared with the characteristics of the solid cell culture at the first point in time to determine other characteristics, including cell migration distance, cell migration speed, maximum migration vector, maximum migration speed, and so on, as described herein. The chip may be removed repeatedly, such as a third, fourth, fifth, or more times in order to conduct further imaging of the solid cell culture for a third, fourth, fifth, or more later points in time. - In one example, the width or diameter of a spheroid is measured and compared at different time points. Single cells surrounding the spheroid are tracked individually or in clusters. Movement of single cells can be tracked to determine if the cells are entering or exiting the spheroid. The change in spheroid size and/or the behavior of single cells can be used individually or together to predict a patient response to a given treatment.
FIG. 12 shows anexample report 1200 including images of representative cell cultures after a period of dosing. A spheroid exposed to a treatment that stays stable or grows may indicate a poor response, while a spheroid that shrinks may indicate a good response. In this regard, the given image or other representation of a cell culture may be compared with an image of the culture prior to dosing in order to determine a change in size of the spheroid. - With reference to
FIG. 12 , theexample report 1200 includes a firstcell culture column 1208 a, a secondcell culture column 1208 b, and a thirdcell culture column 1208 c. In one example, each of columns 1208 a-1208 c may be indicative of a different cell culture held by a different microfluidic chip (e.g., one of themicrofluidic chips 800 a-800 f ofFIGS. 10 and 11 ). Each of the cell cultures in the respective chips may be dosed with a different treatment agent, different strength or concentration of treatment agent, and/or no treatment agent in order to evaluate the response of the cell culture to a variety of conditions over time (e.g., as may be depicted in various three-dimensional images, as shown herein with respect toFIG. 16 ). In the example ofFIG. 12 , the firstcell culture column 1208 a corresponds to a cell culture for administration of a first treatment agent at a first dose (e.g., 40 nM doxorubicin), the secondcell culture column 1208 b corresponds to a cell culture for administration of a second treatment agent at a second dose (e.g., 50 nM doxorubicin), and the thirdcell culture column 1208 c corresponds to a cell culture for administration of a third treatment agent at a third dose (e.g., 100 nM doxorubicin). It will be appreciated that the particular treatment agents and doses are presented for purposes of illustration only, and that substantially any other treatment agent and dose may be used as appropriate for evaluating treatment efficacy. - In this regard, the
report 1200 includes a firsttime point row 1204 a, and a subsequenttime point row 1204 b. In the example ofFIG. 12 , the firsttime point row 1204 a may correspond to a period of time before dosing, such as at day zero. The subsequenttime point row 1204 b may correspond to a later time point after some period of dosing. As shown inFIG. 12 , the subsequenttime point row 1204 b may be after one day of dosing. In other cases, the subsequenttime point row 1204 b may correspond to another period of dosing, such as after two days, three days, four days, five day, and so on. The cell cultures may be evaluated at each of the time points (and other time points) in order to evaluate treatment efficacy. For example, the cell culture associated with the firstcell culture column 1208 a may be imaged, according to any of the techniques described herein, at a time associated with the firsttime point row 1204 a (e.g., a time prior to dosing) in order to produce afirst representation 1212 a of the cell culture. The cell culture associated with the firstcell culture column 1208 a may subsequently be dosed (e.g., with 40 nM doxorubicin) and imaged at a time associated with the secondtime point row 1204 b (e.g., after one day or so of dosing) in order to produce asecond representation 1212 b of the cell culture. Further, the cell culture associated with the secondcell culture column 1208 b may be imaged, according to any of the techniques described herein, at a time associated with the firsttime point row 1204 a (e.g., a time prior to dosing) in order to produce afirst representation 1216 a of the cell culture. The cell culture associated with the secondcell culture column 1208 b may subsequently be dosed (e.g., with 50 nM doxorubicin) and imaged at a time associated with the secondtime point row 1204 b (e.g., after one day or so of dosing) in order to produce asecond representation 1216 b of the cell culture. Further, the cell culture associated with the thirdcell culture column 1208 c may be imaged, according to any of the techniques described herein, at a time associated with the firsttime point row 1204 a (e.g., a time prior to dosing) in order to produce afirst representation 1220 a of the cell culture. The cell culture associated with the thirdcell culture column 1208 c may subsequently be dosed (e.g., with 100 nM doxorubicin) and imaged at a time associated with the secondtime point row 1204 b (e.g., after one day or so of dosing) in order to produce asecond representation 1220 b of the cell culture. The respectivesecond representations FIGS. 13A-17 . - In this regard, with reference to
FIGS. 13A-17 , various charts, reports, and graphs are depicted that are representative of the imaging and analyzing operations of the computer vision system described above with reference toFIG. 2 . It will be appreciated that the following charts, reports, and graphs are depicted as examples. In other cases, additional or alternative charts, reports, and graphs may be used. For example, and with reference toFIG. 13A , achart 1300 a is shown including agrid 1304 a and afirst representation 1308 a. Thegrid 1304 a may be a three-dimensional image representative of a three-dimensional space associated with the solid cell culture. Thefirst representation 1308 a may be representative of a three-dimensional position of particular items of interest, such as living cells in a three-dimensional space. Thechart 1300 a may be produced, in one example, via two-dimensional images captured of the solid cell culture, in which the size and location of cells of interest are determined using the light-responsive dye. The two-dimensional images may be stitched together in order to produce a three-dimensional image. In this regard, thefirst representation 1308 a may represent a composite of multiple two-dimensional images taken at a first time point. In the present example, thefirst representation 1308 a may be taken at a first time point, which may occur prior to any dosing with the treatment agents. Thefirst representation 1308 a may also provide information regarding the quantity of the cells of interest, as shown inFIG. 13A . - With reference to
FIG. 13B , achart 1300 b is shown including agrid 1304 b and asecond representation 1308 b. Thegrid 1304 b may be a three-dimensional image that is representative of a three-dimensional space associated with the solid cell culture represented by thechart 1300 a after a period of time (e.g., a second time point). For example, thesecond representation 1308 b may be representative of a three-dimensional position of particular items of interest, such as living cells described above. In this regard, thesecond representation 1308 b may represent a composite of multiple two-dimensional images taken at a second time point. In the present example, thesecond representation 1308 b may be taken at a second time point, which may occur after a series of doses of the treatment agents or no treatment (e.g., with respect to a baseline/negative control). Thesecond representation 1308 b may also provide information regarding the quantity of the cells of interest, as shown inFIG. 13B . - In some cases, it may be desirable to more precisely determine a three-dimensional position of a cell of interest at different points in time. For example, a three-dimensional position of a cell of interest may be compared between a first time and a second time to determine, among other characteristics, a cell migration vector and cell migration speed. In this regard,
FIG. 14A depicts achart 1400 a showing afirst distribution 1420 a in a three-dimensional coordinate system defined by anaxis 1404 a, anaxis 1408 a, and anaxis 1412 a. Thefirst distribution 1420 a may be representative of a solid cell culture at a first time point, which may be prior to dosing. The first distribution may be produced by stitching as described herein. Thechart 1400 a provides information regarding the three-dimensional position of target cells in thefirst distribution 1420 a, including distribution density. In the example ofFIG. 14A , thefirst distribution 1420 a has a higher density of cells for lower values along theaxis 1412 a. - The solid cell culture associated with the
first distribution 1420 a may undergo one or more dosing producers, as described herein. The solid cell culture may be measured at a second time point subsequent to the dosing procedure. In this regard,FIG. 14B shows achart 1400 b having asecond distribution 1420 b in a three-dimensional coordinate system defined by anaxis 1404 b, anaxis 1408 b, and anaxis 1412 b. Thechart 1400 b provides information regarding the three-dimensional position of target cells in thesecond distribution 1420 b, including distribution density. In the example ofFIG. 14B , thesecond distribution 1420 b has a lower density of cells in the three-dimensional coordinate system as compared with the first distribution shown inFIG. 14A . -
FIG. 15 depicts achart 1500 showing a quiver or vector diagram indicative of the three-dimensional movement of the quantity of cell from the three-dimensional position ofFIG. 14A to the three dimensional position ofFIG. 14B . For example, the three-dimensional position of a cell from thesecond distribution 1420 b may be compared with a corresponding cell from thefirst distribution 1420 a to determine a vector for the cell. The vector may correspond to a path of travel and/or speed for the cell between the first time point represented by thechart 1400 a and the second time point represented by thechart 1400 b. As such, in one example, a given cell from thefirst distribution 1420 a may be a tail of the vector, and the corresponding cell from thesecond distribution 1420 b may be an arrowhead of the vector. In this regard, thechart 1500 may be a three-dimensional quiver plot defined by anaxis 1504, anaxis 1508, and anaxis 1512. Avector distribution 1520 may be plotted in thechart 1500 including vectors for one or more target cells. - The
vector distribution 1520, as plotted in thechart 1500, may provide information regarding the behavior of cells during the administration of the treatment agent or no treatment (e.g., with respect to a baseline/negative control). For example, thevector distribution 1520 may include afirst region 1522 having a maximum migration vector. The maximum migration vector may correspond to a cell of interest that moved the greatest amount, in speed or position, between the measured first and second time points. Thevector distribution 1520 may further include asecond region 1524 that may correspond to a cluster of vectors that are generally larger than other of thevector distribution 1520. In this regard, thesecond region 1524 may correspond to a cluster of cells that generally moved the farthest or the fastest during the administration of the treatment agents. As another example, thevector distribution 1520 may further include athird region 1526 that may correspond to a cluster of vectors that are generally smaller than other vectors of thevector distribution 1520. In this regard, thethird region 1526 may correspond to a cluster of cells that generally moved the least or the slowest during the administration of the treatment. These and other trends may be identified in thechart 1500 and analyzed to determine treatment efficacy for the treatment agents. - The systems and techniques of the present disclosure may be used to monitor the response to multiple different treatment agents. For example, a solid cell culture may be prepared using any of the techniques described herein. A portion of the solid cell culture may be deposited into different microfluidic chips, such as in one or more of the six microfluidic chips shown in
FIGS. 10 and 11 . Themicrofluidic device 1000 may operate to circulate a flow of a different solution (e.g., different treatment agents) to each of the cell cultures of the microfluidic chips. As such, a solid cell culture may be defined for each chip using the different solutions or treatment agents for the cells/hydrogel initially deposited in the chip. For example, themicrofluidic device 1000 may circulate a flow of different treatment agents to each chip. In some cases, one of the chips may receive no treatment agents, and just receive growth media as a control case. The response of the exposed cell cultures to the various different treatment agents may be compared to determine a treatment efficacy. For example, the determined cell characteristics for each cell culture can be compared and analyzed to determine which treatment agent was the most effective in treating the target cells. - A visual representation of this comparison is presented in
FIG. 16 . For example,FIG. 16 depicts anexample report 1600 including visualizations of different dosing operations performed on a given patient sample. Thereport 1600 may include information regarding the impact of a particular treatment agent for a patient sample over a period of time. InFIG. 16 , thechart 1600 may include afirst chip column 1608 a, asecond chip column 1608 b, athird chip column 1608 c, afourth chip column 1608 d, afifth chip column 1608 e, and asixth chip column 1608 f. In one example, each of the columns 1608 a-1608 f may be indicative of a different microfluidic chip (e.g., one of themicrofluidic chips 800 a-800 f ofFIGS. 10 and 11 ). The microfluidic chips may initially include a solid cell culture, such as any of cell cultures herein, which may include target tumor cells. The microfluidic chips may be dosed or treated with treatment agents to define an exposed cell culture in each chip. In one example, each of the microfluidic chips may be dosed with a different treatment agent in order to determine the efficacy of a particular treatment. The response of exposed cell culture of the microfluidic chips may be measured over a period of time. In this regard, thechart 1600 may include a firsttime point row 1604 a, a secondtime point row 1604 b, and a finaltime point row 1604 c. In the example, ofFIG. 16 , the firsttime point row 1604 a may correspond to a first day, the secondtime point row 1604 b may correspond to a second day, and the finaltime point row 1604 c may correspond to a subsequent day, such as a third, fourth, or fifth day. - Each microfluidic chip represented by the chip column may be imaged and analyzed for each time point represented by the time point row. For example, the microfluidic chip may be imaged to determine a color, size, position, density, and/or other property at a first given time point corresponding to the first
time point row 1604 a. The imaging may be a two-dimensional image or a three-dimensional image, which may be produced by stitching together the two-dimensional images. The imaging may be used to produce arepresentative chart 1612 a, as shown inFIG. 16 , which includes information for the characteristics of solid cell culture at the first time point. Thechart 1612 a may be substantially analogous to thecharts FIGS. 13A and 13B . Further, the microfluidic chip may be subsequently imaged at a second given time point, such as subsequent to a round of dosing via the treatment agents, and which may correspond to the secondtime point row 1604 b. Accordingly, the imaging may be used to produce arepresentative chart 1612 b, which includes information for the characteristics of the solid cell culture at the second time point. Further, the microfluidic chip may be subsequently imaged at a third given time point, such as subsequent to another round of dosing via the treatment agents, and which may correspond to the finaltime point row 1604 c. Accordingly, the imaging may be used to produce arepresentative chart 1612 c, which includes information for the characteristics of the solid cell culture at the final time point. - A response of the solid cell culture of the first microfluidic chip may be analyzed over time to determine a treatment efficacy for the treatment agents administered to the first microfluidic chip, as described herein. The
chart 1600 and corresponding analysis may allow for the comparison of the treatment efficacy across multiple different treatment agents, including combinations of agents dosed simultaneously and/or sequentially, for the cell culture. For example, and substantially analogous to thefirst chip column 1608 a, thesecond chip column 1608 b may include arepresentative chart 1618 a at the firsttime point row 1604 a, arepresentative chart 1618 b at the secondtime point row 1604 b, and arepresentative chart 1618 c at the finaltime point row 1604 c. Further, and substantially analogous to thefirst chip column 1608 a, thethird chip column 1608 c may include arepresentative chart 1622 a at the firsttime point row 1604 a, arepresentative chart 1622 b at the secondtime point row 1604 b, and arepresentative chart 1622 c at the finaltime point row 1604 c. Further, and substantially analogous to thefirst chip column 1608 a, thefourth chip column 1608 d may include arepresentative chart 1626 a at the firsttime point row 1604 a, arepresentative chart 1626 b at the secondtime point row 1604 b, and arepresentative chart 1626 c at the finaltime point row 1604 c. Further, and substantially analogous to thefirst chip column 1608 a, thefifth chip column 1608 e may include arepresentative chart 1630 a at the firsttime point row 1604 a, arepresentative chart 1630 b at the secondtime point row 1604 b, and arepresentative chart 1630 c at the finaltime point row 1604 c. Further, and substantially analogous to thefirst chip column 1608 a, thesixth chip column 1608 f may include arepresentative chart 1634 a at the firsttime point row 1604 a, arepresentative chart 1634 b at the secondtime point row 1604 b, and arepresentative chart 1634 c at the finaltime point row 1604 c. - With respect to the final
time point row 1604 c, thereport 1600 includesrepresentative charts representative charts representative chart 1612 c may show information corresponding to a response of the cell culture to a first treatment agent at the final time point, therepresentative chart 1618 c may show information corresponding to a response of the cell culture to a second treatment agent at the final time point, therepresentative chart 1622 c may show information corresponding to a response of the cell culture to a third treatment agent at the final time point, therepresentative chart 1626 c may show information corresponding to a response of the cell culture to a fourth treatment agent at the final time point, therepresentative chart 1630 c may show information corresponding to a response of the cell culture to a fifth treatment agent at the final time point, therepresentative chart 1634 c may show information corresponding to a response of the cell culture to a sixth treatment agent (or a control with no treatment agent, which could apply to any single or multiple microfluidic chips) at the final time point. In this regard, characteristics such as target cell color, size, density, count, and so on, may be compared across each of the representations shown in the finaltime point row 1604 c to determine a treatment efficacy. As one example, where the representation shows a lower tumor cell density or live cell count for the finaltime point row 1604 c, the treatment agent that was used to for the treatment of the microfluidic chip that resulted in the representation may be determined to have the high treatment efficacy among the treatment agents. - For example, the response of a cell culture to a given treatment agent may be used to determine cell viability. For example, the viability of both responders and non-responders may be determined and plotted in order to determine an efficacy of treatment. With reference to
FIG. 17 , achart 1700 is depicted showing cell viability with respect to responders, such as a BRCA mutated, and non-responders, such as BRCA wild-type. With reference toFIG. 17 , thechart 1700 includes adata set axis 1704 and aviability axis 1708. Afirst distribution 1710 having afirst spread 1724 is shown alongside asecond distribution 1720 having asecond spread 1722. The viability of the responders of thefirst distribution 1710 may be compared to the viability of the non-responders of thesecond distribution 1720 with thechart 1700 in order to provide information to assist in determining treatment efficacy. - To facilitate the reader's understanding of the various functionalities of the embodiments discussed herein, reference is now made to the flow diagram in
FIGS. 18-21 , which illustratesprocess - With reference to
FIG. 18 , aprocess 1800 is disclosed. Theprocess 1800 is directed to a method of forming a solid cell culture, such as that described with respect toFIG. 2 . Atoperation 1804, target cells are isolated from a patient sample. For example, and with reference toFIGS. 2 and 3 , apatient sample 304 may be obtained. Thesample 304 may arrive at a lab, and include samples containing a tumor of various cancer types. A digestion enzyme-based cell isolation kit, blood lysis solution and other selecting or filtering steps may be used to isolate viable cells while removing blood and contaminants. The output may be a viable mixed cell population containing the various cells from the primary tumor, including cancer cells, normal/non-transformed cells, stromal cells and immune cells. - At
operation 1808, stained cells are formed from the isolated cells by staining the target cells with a light-responsive dye. For example, and with reference toFIGS. 2, 4, and 7 , cells may be stained with one or both of a live cell dye and a dead cell staining dye. The dyes may be light-responsive dyes. For example, the cells may be stained with a first light-responsive dye to facilitate live cell tracking. The cells may be further stained with a second light-responsive dye to facilitate dead cell tracking. In some cases, the light-responsive dye is configured to cause a color change in the stained cell when the stained cell transitions from a living cell to a dead cell. - At
operation 1810, a spheroid may optionally be formed from the stained cells. The spheroid or organoid may also include cancer cells, normal/non-transformed cells, stromal cells, and/or immune cells, which are formed from a patient-derived tissue or tumor sample. Tissue slices, cores, surgical resections, xenografts, and/or biopsies may also be used. - At
operation 1812, the stained cells are encapsulated. For example, and with reference toFIGS. 2 and 6 , the stained cells, such as isolated cells (or spheroids formed from operation 1810) may be included in thehydrogel 608. Thehydrogel 608 may be configured to replicate components of human tissue, which may facilitate cell growth. For example, and as described herein, hyaluronic acid and collagen may be used to mimic core components of human tissue extracellular matrices and disease-specific cell niches, like those found in breast tumors. - It will be appreciated that various types of cell cultures may be used and/or formed in conjunction with the
process 1800 ofFIG. 18 . For example, the culturing ofprocess 1800 may include culturing dissociated cells in the hydrogel. The dissociated cells may be formed via two-dimensional or three-dimensional cell cultures. In some cases, the dissociated cells may be a single population of cells, whereas in other cases, multiple cell types may be used. For example, the dissociated cells may be cancer, normal/non-transformed cells, stromal, and/or immune cells, as one example, derived from a tissue or tumor sample. - With reference to
FIG. 19 , aprocess 1900 is disclosed. Theprocess 1900 is directed to a method of loading a microfluidic chip, such as that shown above with respect toFIGS. 9A-9F . Atoperation 1904, a solid cell culture is arranged in a cell culture chamber of a microfluidic chip. The microfluidic chip includes a body defining a cell culture chamber and a channel that traverses the cell culture chamber and extends between an inlet and an outlet of the microfluidic chip. For example, and with reference toFIGS. 9B and 9C , thesolid cell culture 910 is arranged in the firstcell culture chamber 814 of themicrofluidic chip 800. Themicrofluidic chip 800 includes thebody 801 that defines the firstcell culture chamber 814 and thechannel 862 that runs along or adjacent the firstcell culture chamber 814 and extends between the inlet feature 843 a and theoutlet feature 843 b. - At
operation 1908, a gas permeable membrane is positioned over a volume containing the cell culture chamber while the inlet and outlet remain exposed for coupling to a circulation system. For example, and with reference toFIGS. 9D and 9E , the gaspermeable layer 852 is positioned on to thebody 801. The gaspermeable layer 852 may be adhered to thebody 801 via theadhesive surface 835. As shown inFIG. 9E , the gaspermeable layer 852 may cover both thefirst volume 864 and thesecond volume 866, containing the firstcell culture chamber 814 and the secondcell culture chamber 816, while allowing the firstcell culture chamber 814 and the secondcell culture chamber 816 to be exposed to gas in a surrounding atmosphere of themicrofluidic chip 800. - With reference to
FIG. 20 , aprocess 2000 is disclosed. Theprocess 2000 is directed to a method of operating a microfluidic chip. Atoperation 2004, a microfluidic chip is fluidly coupled with a microfluidic device to define a fluid circuit between the microfluidic chip, a flow restrictor, a reservoir, and a pump. The microfluidic chip including a cell culture, the reservoir including a media. For example, and with reference toFIGS. 10 and 11 ,microfluidic chips 800 a-800 f may be fluidly coupled with themicrofluidic device 1000. Themicrofluidic device 1000 may operate to definefluid circuits 1002 a-1002 f between each respectivemicrofluidic chip 800 a-800 f and a corresponding one of the reservoirs 1022 a-1022 f. - At
operation 2008, a flow of the media is caused through the circuit such that the media interacts with the solid cell culture of the microfluidic chip to define an exposed cell culture in the microfluidic chip. For example, and with reference toFIGS. 10 and 11 , themicrofluidic device 1000 may operate thepump 1030 to cause a flow of media through each of thefluid circuits 1002 a-1002 f. Thepump 1030 may be configured to control a flow of circulation through each of thefluid circuits 1002 a-100 f separately. This may allow themicrofluidic device 1000 to administer separate treatment agents to each of themicrofluidic chips 800 a-800 f such a response to each of the different treatment agents may be measured. - For example, at operation 2012, a response of the solid cell culture to the media is analyzed. For example, and with reference to
FIGS. 11, 13A, 13B and 16 , a given microfluidic chip may be removed and fluidly uncoupled from themicrofluidic device 1000. The microfluidic chip may be imaged or otherwise analyzed for the given time point at which the microfluidic chip is fluidly uncoupled. As one example, the microfluidic chip may undergo a process of fluorescence microscopy. Fluorescence microscopy may capture images of the cell culture of the given microfluidic chip under conditions in which the light-responsive dye allows for detection of target cells, e.g., the detection of dead cells and/or living cells. Confocal microscopy, brightfield microscopy, and lattice sheet microscopy may all be used, in addition to other imaging techniques. In one example, the images may be two-dimensional images. As described herein, the two-dimensional images may be captured in a manner that is representative of layers of the cell culture. The layers may be stacked on one another or stitched together to form a three-dimensional image. - In another example, the given microfluidic chip may be fluidly coupled to the microfluidic device again to administer further media, including additional treatment agents. In this regard, the
method 2000 may further include causing another flow of the media through the circuit, at a second time point, such that the media interacts with the cell culture of the microfluidic chip, and analyzing a subsequent response of the cell culture to the media. Analyzing of the subsequent response of the cell culture may include imaging, as described, which is used to generate a two-dimensional or three-dimensional image of the cell culture. The imaging of the cell culture at the first and second time points may be analyzed to determine a treatment efficacy. As described herein, additional time points may also be analyzed. As one example, themethod 2000 may include analyzing the image at the first time point to determine a first live/dead cell population quantity. Themethod 2000 may further include analyzing the image at the second time point to determine a second live/dead cell population quantity. The first cell population quantity and the second cell population quantity may, in turn, be compared to determine change in cell population quantity indicative of a treatment efficacy. As another example, themethod 2000 may include analyzing the image at the first time point to determine a first cell population position and analyzing the image at the second time point to determine a second cell population position. The first cell population position and the second cell population positon may, in turn, be compared to determine change in cell population position indicative of a treatment efficacy. - With reference to
FIG. 21 , aprocess 2100 is disclosed. Theprocess 2100 is directed to analyzing a solid cell culture over time. Theprocess 2100 may be executed fully, or in part, using thecomputing device 2200 described below with reference toFIG. 22 . For example, various operations may be performed after the computing device 2200 (or multiple computing devices 2200) receive data from the system (e.g., microfluidic system and chips) and utilize this data to make assessments regarding treatment efficacy and patient response. Atoperation 2104, a first response of a solid cell culture to a growth media, including a treatment agent, is determined. The solid cell culture is held in a cell culture chamber of a microfluidic chip. For example, and with reference toFIGS. 9B and 13A , a first response of thecell culture 910 may be determined at a first time point. The first response may be determined by executing instructions of a non-transitory computer-readable media with one or more processing elements of thecomputing device 2200. For example, thecell culture 910 may be analyzed, using thecomputing device 2200, to determine a cell color, quantity, size, density and/or other characteristics at a first point in time, which may be represented by thefirst representation 1308 a ofFIG. 13A . The first response may be a response of thecell culture 910 prior to a dosing with a treatment agent. More specifically, in one implementation, thecomputing device 2200 may receive data corresponding to images captured by one or more of the components described herein, where the images may be analyzed to determine cell color, quantity, size, and density, e.g., by utilizing image analysis or computer vision algorithms that can identify pixel colors and distribution corresponding to the cells. - At
operation 2108, additional responses of the cell culture to the media is determined. For example, and with reference toFIGS. 9B and 13B , additional responses of thecell culture 910 may be determined at a second time point, third time point, fourth time point, fifth time point, and so on. The additional responses may be determined by executing instructions of a non-transitory computer-readable media with one or more processing elements of thecomputing device 2200. For example, thecell culture 910 may be analyzed, using thecomputing device 2200, to determine a cell quantity, color, size, density and/or other characteristics at substantially any subsequent time point, which may be represented by one or more of the distributions ofFIG. 16 . The additional responses may be a response of thecell culture 910 subsequent to a dosing with a treatment agent. In some implementations, thecomputing device 2200 may utilize computer vision and/image analysis algorithms that can identify the cells within one or more images captured and can extract information, such as hue, intensity, or the like, that can be utilized to determine the response information. - At
operation 2112, the first and the additional responses (e.g., a second response, a third response, a fourth response, a fifth response, and so on) are compared to determined treatment efficacy. The first and the additional responses may be compared by executing instructions of a non-transitory computer-readable media with one or more processing elements of thecomputing device 2200, such as one or more image analysis or computer vision algorithms as described above. For example, and with reference toFIG. 16 , as one example, the color, size, quantity, density and/or other characteristics of the cell cultures may be compared, using thecomputing device 2200, between the first time point and any of the subsequently measured time points. A relative increase or decrease in one or more of these characteristics may be indicative of an efficacy of the treatment provided for by the corresponding treatment agent. Other properties may also be measured and compared with respect to the first and second responses, including, but not limited to, a color of the cell culture, a pixel intensity of an image of the cell culture, a shape of cell culture, a size of cell culture, a position of cells of the cell culture, or a quantity of cell of the cell culture. - In this regard, the first and/or second response may include capturing an image, such as a two-dimensional or three-dimensional image of the cell. The
method 2100 may further include determining one or more of a cell viability, a cell proliferation, a cell position from the image. With respect to spheroids/organoids, themethod 2100 may further include determining a cell viability using a color and/or a color ratio to predict treatment response. For example, the image analysis may extract hue and/or intensity information to correlate the same to cell viability, based on a color mapping, machine learning, look up table, or the like. However, the method for determining the cell viability may vary based on the type of image analysis or computer vision algorithms utilized. - The first color indicative of live cells (e.g., a red color) and a second color indicative of dead cells (e.g., a green color) can be compared at one, two, or more time points to determine, for example, a cell death within a spheroid. In other words, the system may analyze hue information at various pixel locations over time, which can be correlated to cell death within the spheroid. Such information may be used as a predictive metric (e.g., ratio of live and dead cells). In other cases, other techniques may be used to determine a cell viability. The
method 2100 may further include predicting a patient response to the treatment agent based on one or more of cell viability, cell proliferation, or the cell position, for example, as described herein with respect tooperation 252 ofFIG. 2 . In some cases, as described herein, an image indicative of the first response may be compared, using thecomputing device 2200, to an image indicative of the second response to facilitate the prediction of the patient response. For example, data from the two images may be analyzed by thecomputer device 2200 to determine via image analysis distinctions in the responses that indicate one response may be better or more improved in terms of cancer cell death as compared to the other. In this regard, themethod 2100 may further include comparing the images to determine one or more of a cell migration speed or a cell migration distance for one or more cells over time. For example, pixel analysis may be used to generate distance vectors that may then be compared in length or dimension to determine cell migration distances. The cell migration speed and/or the cell migration distance over time may be indicative of a patient response to the treatment agent. Maximum or minimum of migration vectors or speeds may also be computed in this regard. In some cases, the cell migration speed and/or the cell migration distance may be determined with respect to a subset of the most aggressive cells, including but not limited to, the 5% most aggressive cells of the cell culture, the 2% most aggressive cells of the cell culture, the 1% most aggressive cells of the cell culture, among other subsets. Additionally or alternatively, the cell migration speed and/or the cell migration distance may be computed with respect to a subset of cells expressing a specific bio marker. - The analysis of
operation 2112 may be performed with respect to a single cell. For example, the change in position of a single cell may be tracked between first time point and second time point. In this example, a cell may be tagged or otherwise identified in one or more images and then tracked over time in determines of movement between different image frames. In other cases, theoperation 2112 may be performed with respect to a weighted cell measurement. For example, weighting factors may be used based on an association of how much each characteristic of the cell culture affects a treatment response prediction. As an illustration, the likelihood of a patient responding to treatment A may be based on a patient having a minimum score of X. The score X is calculated by having three quarters of the score coming from the cell viability of the culture and one quarter of the score coming from the cell migration speed of that culture. The weighted cell measurement may be a composite score of multiple individual measurements (e.g., cell viability, migration distance, and so on). - In some cases, the images of the first and second response described above may be images of spheroid or organoids. In this regard, a radius, diameter or circumference of the spheroid or organoid may be measured or determined using the images and compared across multiple time points. For example, the computing device may analyze the image to determine an approximate perimeter for the spheroid or organoid and then calculate a circumference, diameter, or other measurements with respect to an estimated area and/or volume of the spheroid or organoid. Additionally or alternatively, dissociated/single cell analysis may be accomplished using similar techniques. For example, dissociated/single cells may be tracked and measured using substantially any of the associated metrics, as described herein. As illustrative examples, a count and/or a position of a single cells that leave the spheroid, a migration distance, a migration speed, and so on may be determined and analyzed according to the techniques described herein for determining treatment efficacy. In this regard, the
method 2100 may include predicting a patient response to the treatment agent based on a comparison of the first radius or the first diameter with the second radius or the second diameter of the respective first and second images. Additionally or alternatively, surgical resections, tissue slices, xenografts, and/or core needle biopsies may be used, which may have a length, width, and height. In this regard, the length, width, or height of the tissue slices, surgical resections, xenografts, and/or core needle biopsies may be measured using the images and compared across multiple time points. In this regard, themethod 2100 may include predicting a patient response to the treatment agent based on a comparison of the first length, the first width, or the first height with the second length, the second width, or the second height of the respective first and second images. -
FIG. 22 presents anexample computing device 2200. The schematic representation inFIG. 22 of thecomputing device 2200 may include systems, components, modules, assemblies, and subassemblies configured to implement or execute any of the techniques and processes described herein. In this regard, thecomputing device 2200 may include any appropriate hardware (e.g., computing devices, data centers, switches), software (e.g., applications, system programs, engines), network components (e.g., communication paths, interfaces, routers) and the like (not necessarily shown in the interest of clarity) for use in facilitating any appropriate operations disclosed herein. - As shown in
FIG. 22 , thecomputing device 2200 may include a processing unit orelement 2208 a operatively connected tocomputer memory 2212 and computer-readable media 2216. Theprocessing unit 2208 a may be operatively connected to thememory 2212 and computer-readable media 2216 components via an electronic bus or bridge (e.g., such as system bus 2210). Theprocessing unit 2208 a may include one or more computer processors or microcontrollers that are configured to perform operations in response to computer-readable instructions. Theprocessing element 2208 a may be a central processing unit of thecomputing device 2200. Additionally or alternatively, theprocessing unit 2208 a may be other processors within the device including application specific integrated chips (ASIC) and other microcontroller devices. - The
memory 2212 may include a variety of types of non-transitory computer-readable storage media, including, for example, random access memory (RAM), read-only memory (ROM), erasable programmable memory (e.g., EPROM and EEPROM), or flash memory. Thememory 2212 is configured to store computer-readable instructions, sensor values, and other persistent software elements. Computer-readable media 2216 may also include a variety of types of non-transitory computer-readable storage media including, for example, a hard-drive storage device, a solid state storage device, a portable magnetic storage device, or other similar device. The computer-readable media 2216 may also be configured to store computer-readable instructions, sensor values, and other persistent software elements. - In this example, the
processing unit 2208 a is operable to read computer-readable instructions stored on thememory 2212 and/or computer-readable media 2216. The computer-readable instructions may adapt theprocessing unit 2208 a to perform the operations or functions described above with respect toFIGS. 1-21 . The computer-readable instructions may be provided as a computer-program product, software application, or the like. - As shown in
FIG. 22 , thecomputing device 2200 may also include adisplay 2218. Thedisplay 2218 may include a liquid-crystal display (LCD), organic light emitting diode (OLED) display, light emitting diode (LED) display, or the like. Thecomputing device 2200 may also include abattery 2224 that is configured to provide electrical power to the components of thecomputing device 2200. Thebattery 2224 may include one or more power storage cells that are linked together to provide an internal supply of electrical power. In this regard, thebattery 2224 may be a component of a power source 2228 (e.g., including a charging system or other circuitry that supplies electrical power to components of the computing device 2200). - The
computing device 2200 may also include one ormore sensors 2240 that may be used to detect a touch and/or force input, environmental condition, orientation, position, or some other aspect of thecomputing device 2200. Thecomputing device 2200 may also include acamera 2232 that is configured to capture a digital image or other optical data. Thecomputing device 2200 may also include acommunication port 2244 that is configured to transmit and/or receive signals or electrical communication from an external or separate device. Thecommunication port 2244 may be configured to couple to an external device via a cable, adaptor, or other type of electrical connector. In some embodiments, thecommunication port 2244 may be used to couple thecomputing device 2200 with a computing device and/or other appropriate accessories configured to send and/or receive electrical signals. - Other examples and implementations are within the scope and spirit of the disclosure and appended claims. For example, features implementing functions may also be physically located at various positions, including being distributed such that portions of functions are implemented at different physical locations. Also, as used herein, including in the claims, “or” as used in a list of items prefaced by “at least one of” indicates a disjunctive list such that, for example, a list of “at least one of A, B, or C” means A or B or C or AB or AC or BC or ABC (i.e., A and B and C). Further, the term “exemplary” does not mean that the described example is preferred or better than other examples.
- The foregoing description, for purposes of explanation, uses specific nomenclature to provide a thorough understanding of the described embodiments. However, it will be apparent to one skilled in the art that the specific details are not required in order to practice the described embodiments. Thus, the foregoing descriptions of the specific embodiments described herein are presented for purposes of illustration and description. They are not targeted to be exhaustive or to limit the embodiments to the precise forms disclosed. It will be apparent to one of ordinary skill in the art that many modifications and variations are possible in view of the above teachings.
Claims (26)
1. A method of forming a solid culture, the method comprising:
isolating target cells from a patient sample;
directly after isolating, staining a plurality of individual isolated dissociated cells with a light-responsive dye to form stained cells, wherein at least one of a mitochondria or a nucleus per individual isolated dissociated cell is stained;
encapsulating the stained cells in a hydrogel; and
culturing the encapsulated stained cells.
2. The method of claim 1 , wherein the hydrogel comprises hyaluronic acid and collagen configured to mimic core components of human tissue extracellular matrices and disease-specific cell niches.
3. (canceled)
4. The method of claim 1 , wherein the culturing further comprising forming two-dimensional cell cultures of the dissociated cells.
5. The method of claim 1 , wherein the culturing further comprises forming three-dimensional cell cultures of the dissociated cells.
6. The method of claim 1 , wherein the culturing further comprises forming cell cultures of a single population of cells.
7. The method of claim 1 , wherein the culturing further comprising forming cell cultures from multiple cell types.
8. The method of claim 1 , wherein the dissociated cells comprise cancer cells, stromal cells, immune cells, or normal or non-transformed cells.
9. The method of claim 1 , wherein the culturing further comprises forming co-culture of cancer, normal or non-transformed, stromal, and immune cells.
10. The method of claim 1 , wherein the dissociated cells are isolated from a patient-derived tissue sample.
11. The method of claim 1 , wherein the dissociated cells are isolated from a patient-derived tumor sample.
12. The method of claim 1 , further comprising forming a spheroid or an organoid.
13. The method of claim 12 , further comprising culturing the spheroid or the organoid in a hydrogel.
14. The method of claim 12 , wherein the spheroid or organoid comprises cancer cells, stromal cells, immune cells, normal or non-transformed cells, or stem cells.
15. The method of claim 13 , wherein the culturing further comprises forming co-culture of cancer, normal or non-transformed, stem, stromal, and immune cells.
16. The method of claim 12 , wherein the isolated cells are isolated from a patient-derived tissue sample.
17. The method of claim 12 , wherein the isolated cells are isolated from a patient-derived tumor sample.
18. The method of claim 1 , further comprising processing the patient sample using a digestion enzyme-based operation, a blood lysis solution, or a selecting operation to isolate target cells of the patient sample.
19. The method of claim 1 , wherein the light-responsive dye is configured to allow for tracking of the target cells via fluorescence microscopy.
20. The method of claim 1 , wherein the light-responsive dye is configured to stain mitochondria of the target cells for live cell tracking.
21. The method of claim 1 , wherein the light-responsive dye is configured to stain nuclei of the target cells for dead cell tracking.
22. The method of claim 1 , wherein forming stained cells further comprises
staining isolated cells with a first light-responsive dye, the first light-responsive dye being configured to stain mitochondria of the target cells for live cell tracking, and
staining isolated cells with a second light-responsive dye, the second light-responsive dye being configured to stain nuclei of the target cells for dead cell tracking.
23. The method of claim 1 , wherein the light-responsive dye is configured to cause a color change in the stained cell when the stained cell transitions from a living cell to a dead cell.
24. The method of claim 1 , wherein the target cells are cells of a tumor, the tumor comprising a breast cancer, a colorectal cancer, a lung cancer, a kidney cancer, a pancreatic cancer, an ovarian cancer, a brain cancer, or a gastric cancer.
25. The method of claim 1 , wherein the patient sample comprises tissue slices, surgical resections, xenografts and/or core needle biopsies.
26. The method of claim 25 , further comprising culturing the tissue slices, surgical resections, xenografts and/or core needle biopsies in a hydrogel.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/511,240 US20230008421A1 (en) | 2021-07-08 | 2021-10-26 | Method of forming solid cell cultures |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202163219697P | 2021-07-08 | 2021-07-08 | |
PCT/US2021/054411 WO2023282925A1 (en) | 2021-07-08 | 2021-10-11 | Treatment efficacy prediction systems and methods |
US17/511,240 US20230008421A1 (en) | 2021-07-08 | 2021-10-26 | Method of forming solid cell cultures |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2021/054411 Continuation WO2023282925A1 (en) | 2021-07-08 | 2021-10-11 | Treatment efficacy prediction systems and methods |
Publications (1)
Publication Number | Publication Date |
---|---|
US20230008421A1 true US20230008421A1 (en) | 2023-01-12 |
Family
ID=84799019
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/511,125 Active US11893733B2 (en) | 2021-07-08 | 2021-10-26 | Treatment efficacy prediction systems and methods |
US17/511,240 Pending US20230008421A1 (en) | 2021-07-08 | 2021-10-26 | Method of forming solid cell cultures |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/511,125 Active US11893733B2 (en) | 2021-07-08 | 2021-10-26 | Treatment efficacy prediction systems and methods |
Country Status (3)
Country | Link |
---|---|
US (2) | US11893733B2 (en) |
EP (1) | EP4366873A1 (en) |
CN (1) | CN117940214A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20230047056A1 (en) * | 2021-08-10 | 2023-02-16 | Palantir Technologies Inc. | Framework for live data migration |
US12050567B2 (en) * | 2022-08-08 | 2024-07-30 | Palantir Technologies Inc. | Framework for live data migration |
Family Cites Families (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7312085B2 (en) | 2002-04-01 | 2007-12-25 | Fluidigm Corporation | Microfluidic particle-analysis systems |
EP1976972B1 (en) * | 2006-01-24 | 2014-03-12 | Brown University | Cell aggregation and encapsulation device and method |
WO2011014674A2 (en) * | 2009-07-29 | 2011-02-03 | Cornell University | Microfluidic device for pharmacokinetic-pharmacodynamic study of drugs and uses thereof |
EP2682187B1 (en) * | 2012-07-02 | 2017-09-13 | Molecular Devices, LLC | Selection method and apparatus |
EP2888349B1 (en) * | 2012-08-24 | 2022-02-09 | Yale University | System, device and method for high-throughput multi-plexed detection |
JP2017502983A (en) * | 2013-10-02 | 2017-01-26 | スリ テクノロジーズ エルティーディー. | Patient-specific immunotherapy to treat heterogeneous tumors |
US11567063B2 (en) * | 2013-11-15 | 2023-01-31 | H. Lee Moffitt Cancer Center And Research Institute, Inc. | Methods for assessing cell viability or predicting cell response to a treatment using cell movement |
WO2016164861A1 (en) * | 2015-04-10 | 2016-10-13 | The Regents Of The University Of California | Microfluidic assisted perfusion devices |
EP3384010B1 (en) * | 2015-12-02 | 2020-11-25 | The Johns Hopkins University | Use of an integrated microfluidic chip for analysis of cell motility and prediction and prognosis of patient survival |
CA3225255A1 (en) | 2016-03-30 | 2017-10-05 | EMULATE, Inc. | Methods of determining tumor marker expression in human tumor cells |
EP3469095A4 (en) | 2016-06-14 | 2020-03-04 | Beth Israel Deaconess Medical Center, Inc. | Automated, digital dispensing platform for microdilution antimicrobial susceptibility testing |
JP6861408B2 (en) * | 2016-12-27 | 2021-04-21 | 東京応化工業株式会社 | Method for manufacturing cell culture chips |
US11846630B2 (en) * | 2019-03-06 | 2023-12-19 | Northeastern University | Coordinately-ordered single cells with individual identities for high-throughput assay |
US11760966B2 (en) * | 2019-06-14 | 2023-09-19 | University Of Connecticut | Multigel tumor-on-a-chip system |
US11412350B2 (en) * | 2019-09-19 | 2022-08-09 | Apple Inc. | Mobile device navigation system |
-
2021
- 2021-10-11 CN CN202180102219.0A patent/CN117940214A/en active Pending
- 2021-10-11 EP EP21949508.2A patent/EP4366873A1/en active Pending
- 2021-10-26 US US17/511,125 patent/US11893733B2/en active Active
- 2021-10-26 US US17/511,240 patent/US20230008421A1/en active Pending
Non-Patent Citations (10)
Title |
---|
Anonymous. EBIOSCIENCE DRAQ5; Invitrogen, pp. 1-2, downloaded from: https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2F65-0880.pdf on 03/29/2023. (Year: 2017) * |
Enderle et al. DYNAMIC IMAGING OF IEL-IEC CO-CULTURES ALLOWS FOR QUANTIFICATION OF CD103-DEPENDENT T CELL MIGRATION; International Journal of Molecular Sciences, Vol. 22, pp. 1-15. (Year: 2021) * |
Fang et al. DEVELOPMENT AND DYNAMIC REGULATION OF MITOCHONDRIAL NETWORK IN HUMAN MIDBRAIN DOPAMINERGIC NEURONS DIFFERENTIATED FROM IPSCS; Stem Cell Reports, Vol. 7, pp. 678-692. (Year: 2016) * |
Fiorini et al. MODELING CELL COMMUNICATION IN CANCER WITH ORGANOIDS: MAKING THE COMPLEX SIMPLE; Frontiers in Cell and Developmental Biology, Vol. 8, pp. 1-12. (Year: 2020) * |
Hof et al. LONG-TERM LIVE IMAGING AND MULTISCALE ANALYSIS IDENTIFY HETEROGENEITY AND CORE PRINCIPLES OF EPITHELIAL ORGANOID MORPHOGENESIS; BMC Biology, Vol. 19, No. 37, pp. 1-22. (Year: 2021) * |
Roberson et al. IMAGING AND ANALYSIS OF 3D TUMOR SPHEROIDS ENRICHED FOR A CANCER STEM CELL PHENOTYPE; Journal of Biomolecular Screening, Vol. 15, No. 7, pp. 820-829. (Year: 2010) * |
Robinson et al. THE SELECTIVE DETECTION OF MITOCHONDRIAL SUPEROXIDE BY LIVE CELL IMAGING; Nature Protocols, Vol. 3, No. 6, pp. 941-947. (Year: 2008) * |
Sokol et al. GROWTH OF HUMAN BREAST TISSUES FROM PATIENT CELLS IN 3D HYDROGEL SCAFFOLDS; Breast Cancer Research; Vol. 18, No. 19, pp. 1-13. (Year: 2016) * |
Wash et al. FUNCTIONAL OPTICAL IMAGING OF PRIMARY HUMAN TUMOR ORGANOIDS: DEVELOPMENT OF A PERSONALIZED DRUG SCREEN; The Journal of Nuclear Medicine, Vol. 58, No. 9, pp. 1367-1372. (Year: 2017) * |
Wlodkovic et al. CYTOMETRY IN CELL NECROBIOLOGY REVISITED. RECENT ADVANCES AND NEW VISTAS; Cytometry A, Vol. 77, No. 7, pp. 1-29. (Year: 2010) * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20230047056A1 (en) * | 2021-08-10 | 2023-02-16 | Palantir Technologies Inc. | Framework for live data migration |
US12050567B2 (en) * | 2022-08-08 | 2024-07-30 | Palantir Technologies Inc. | Framework for live data migration |
Also Published As
Publication number | Publication date |
---|---|
EP4366873A1 (en) | 2024-05-15 |
US20230010963A1 (en) | 2023-01-12 |
CN117940214A (en) | 2024-04-26 |
US11893733B2 (en) | 2024-02-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10843190B2 (en) | Apparatus and method for analyzing a bodily sample | |
US20230145084A1 (en) | Artificial immunohistochemical image systems and methods | |
US8320655B2 (en) | Process and system for analyzing the expression of biomarkers in cells | |
CN107748256B (en) | Liquid biopsy detection method for circulating tumor cells | |
CN111699510A (en) | Transformation of digital pathology images | |
CN103592262B (en) | cell analysis apparatus and cell analysis method | |
Sharma et al. | High-throughput screening in primary neurons | |
US20060234332A1 (en) | Predicting hepatotoxicity using cell based assays | |
CN110446803A (en) | Automatically the cell specified number is collected | |
JP5580117B2 (en) | Cell analyzer | |
CN101784895A (en) | Method for predicting biological systems responses in hepatocytes | |
CN108319815A (en) | A kind of method and its system virtually dyed for cell | |
US7235353B2 (en) | Predicting hepatotoxicity using cell based assays | |
Wanner et al. | Methods for mapping neuronal activity to synaptic connectivity: lessons from larval zebrafish | |
US11893733B2 (en) | Treatment efficacy prediction systems and methods | |
US20210018441A1 (en) | Quantitative liquid biopsy diagnostic system and methods | |
WO2023282925A1 (en) | Treatment efficacy prediction systems and methods | |
JP2024527371A (en) | Treatment Efficacy Prediction System and Method | |
CN116590144A (en) | Lung chip, lung model, construction method of lung model and compound detection method | |
WO2023278692A1 (en) | Plaque detection method and apparatus for imaging of cells | |
US20200101455A1 (en) | Orthogonal polybiosensing and imaging systems | |
JP2022523394A (en) | Automatic microfluidic system for life expectancy and healthy life expectancy analysis in C. elegans | |
US11908130B2 (en) | Apparatuses and methods for digital pathology | |
Zhang | Cancer Drug Screening Scale-up: Combining Biomimetic Microfluidic Platforms and Deep Learning Image Analysis | |
US20240026263A1 (en) | Modeling, monitoring and manufacturing of multi-organ systems for human physiology and pathology |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: OUROTECH, INC., DELAWARE Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HU, SACHA;PEERANI, ELEONORA;PRIMO, GASTON AGUSTIN;AND OTHERS;SIGNING DATES FROM 20211007 TO 20211009;REEL/FRAME:057920/0053 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: ADVISORY ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |