US20170218321A1 - Cell culture insert - Google Patents
Cell culture insert Download PDFInfo
- Publication number
- US20170218321A1 US20170218321A1 US15/492,730 US201715492730A US2017218321A1 US 20170218321 A1 US20170218321 A1 US 20170218321A1 US 201715492730 A US201715492730 A US 201715492730A US 2017218321 A1 US2017218321 A1 US 2017218321A1
- Authority
- US
- United States
- Prior art keywords
- cell culture
- insert
- cells
- culture insert
- array
- 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.)
- Abandoned
Links
- 238000004113 cell culture Methods 0.000 title claims abstract description 191
- 239000012528 membrane Substances 0.000 claims abstract description 77
- 230000001464 adherent effect Effects 0.000 claims abstract description 36
- 238000000034 method Methods 0.000 claims abstract description 36
- 238000012258 culturing Methods 0.000 claims abstract description 11
- 239000000758 substrate Substances 0.000 claims description 53
- 239000000463 material Substances 0.000 claims description 23
- 239000006143 cell culture medium Substances 0.000 claims description 9
- 238000004891 communication Methods 0.000 claims description 9
- 239000012530 fluid Substances 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 abstract description 7
- 210000004027 cell Anatomy 0.000 description 143
- 210000004379 membrane Anatomy 0.000 description 65
- 102000004169 proteins and genes Human genes 0.000 description 32
- 108090000623 proteins and genes Proteins 0.000 description 32
- 229940127285 new chemical entity Drugs 0.000 description 21
- 239000000306 component Substances 0.000 description 20
- 210000003494 hepatocyte Anatomy 0.000 description 19
- 238000012360 testing method Methods 0.000 description 19
- 229940127286 new biological entity Drugs 0.000 description 13
- 230000006870 function Effects 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 8
- 238000003556 assay Methods 0.000 description 7
- 238000010586 diagram Methods 0.000 description 7
- 210000004185 liver Anatomy 0.000 description 7
- 230000004060 metabolic process Effects 0.000 description 7
- 238000010521 absorption reaction Methods 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- 229940079593 drug Drugs 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 210000001035 gastrointestinal tract Anatomy 0.000 description 5
- 229920000642 polymer Polymers 0.000 description 5
- 241000282414 Homo sapiens Species 0.000 description 4
- 239000004793 Polystyrene Substances 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 230000005484 gravity Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000002503 metabolic effect Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- -1 polyethylene Polymers 0.000 description 4
- 229920002223 polystyrene Polymers 0.000 description 4
- 238000013456 study Methods 0.000 description 4
- 206010013710 Drug interaction Diseases 0.000 description 3
- 238000000423 cell based assay Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000000099 in vitro assay Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 239000002356 single layer Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 2
- 239000004372 Polyvinyl alcohol Substances 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 150000001336 alkenes Chemical class 0.000 description 2
- 238000010868 cell confinement Methods 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 230000036267 drug metabolism Effects 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000010579 first pass effect Methods 0.000 description 2
- 230000004077 genetic alteration Effects 0.000 description 2
- 231100000118 genetic alteration Toxicity 0.000 description 2
- 239000000017 hydrogel Substances 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 210000004347 intestinal mucosa Anatomy 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- DLBFLQKQABVKGT-UHFFFAOYSA-L lucifer yellow dye Chemical compound [Li+].[Li+].[O-]S(=O)(=O)C1=CC(C(N(C(=O)NN)C2=O)=O)=C3C2=CC(S([O-])(=O)=O)=CC3=C1N DLBFLQKQABVKGT-UHFFFAOYSA-L 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 229920000515 polycarbonate Polymers 0.000 description 2
- 239000004417 polycarbonate Substances 0.000 description 2
- 229920000570 polyether Polymers 0.000 description 2
- 229920005862 polyol Polymers 0.000 description 2
- 150000003077 polyols Chemical class 0.000 description 2
- 229920002451 polyvinyl alcohol Polymers 0.000 description 2
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000041 toxicology testing Toxicity 0.000 description 2
- 238000012605 2D cell culture Methods 0.000 description 1
- 102000007350 Bone Morphogenetic Proteins Human genes 0.000 description 1
- 108010007726 Bone Morphogenetic Proteins Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 206010048610 Cardiotoxicity Diseases 0.000 description 1
- 229920000089 Cyclic olefin copolymer Polymers 0.000 description 1
- 239000004713 Cyclic olefin copolymer Substances 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 108091006020 Fc-tagged proteins Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010029155 Nephropathy toxic Diseases 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 241000932156 Proteinus Species 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 206010039020 Rhabdomyolysis Diseases 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- 230000001919 adrenal effect Effects 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000000712 assembly Effects 0.000 description 1
- 238000000429 assembly Methods 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229960000182 blood factors Drugs 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 229940112869 bone morphogenetic protein Drugs 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 231100000259 cardiotoxicity Toxicity 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000010267 cellular communication Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000010226 confocal imaging Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000002059 diagnostic imaging Methods 0.000 description 1
- 239000004205 dimethyl polysiloxane Substances 0.000 description 1
- 235000013870 dimethyl polysiloxane Nutrition 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 230000002357 endometrial effect Effects 0.000 description 1
- 230000034964 establishment of cell polarity Effects 0.000 description 1
- XJRPTMORGOIMMI-UHFFFAOYSA-N ethyl 2-amino-4-(trifluoromethyl)-1,3-thiazole-5-carboxylate Chemical compound CCOC(=O)C=1SC(N)=NC=1C(F)(F)F XJRPTMORGOIMMI-UHFFFAOYSA-N 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 229920002313 fluoropolymer Polymers 0.000 description 1
- 239000004811 fluoropolymer Substances 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 239000005428 food component Substances 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000010224 hepatic metabolism Effects 0.000 description 1
- 231100000304 hepatotoxicity Toxicity 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000003463 hyperproliferative effect Effects 0.000 description 1
- 238000002952 image-based readout Methods 0.000 description 1
- 239000012216 imaging agent Substances 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 231100000386 immunotoxicity Toxicity 0.000 description 1
- 230000007688 immunotoxicity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 230000031891 intestinal absorption Effects 0.000 description 1
- 230000003870 intestinal permeability Effects 0.000 description 1
- 238000010849 ion bombardment Methods 0.000 description 1
- 230000005865 ionizing radiation Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000007056 liver toxicity Effects 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 230000002132 lysosomal effect Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 230000004898 mitochondrial function Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 231100000150 mutagenicity / genotoxicity testing Toxicity 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 210000001087 myotubule Anatomy 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 230000007694 nephrotoxicity Effects 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 description 1
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 1
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 1
- 229920002492 poly(sulfone) Polymers 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 239000004926 polymethyl methacrylate Substances 0.000 description 1
- 229920000306 polymethylpentene Polymers 0.000 description 1
- 239000011116 polymethylpentene Substances 0.000 description 1
- 229920000098 polyolefin Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920005606 polypropylene copolymer Polymers 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 210000000512 proximal kidney tubule Anatomy 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 230000002381 testicular Effects 0.000 description 1
- 229960000103 thrombolytic agent Drugs 0.000 description 1
- 230000002537 thrombolytic effect Effects 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 231100000513 vascular toxicity Toxicity 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- KAKZBPTYRLMSJV-UHFFFAOYSA-N vinyl-ethylene Natural products C=CC=C KAKZBPTYRLMSJV-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- 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
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
-
- 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
- C12M23/12—Well or multiwell plates
-
- 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
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/02—Membranes; Filters
- C12M25/04—Membranes; Filters in combination with well or multiwell plates, i.e. culture inserts
Definitions
- the present disclosure relates to apparatuses, systems and methods for culturing cells.
- the present disclosure relates generally to cell culture inserts for use in culturing cells to promote the formation of spheroids and methods of using these spheroid-promoting cell culture inserts.
- Spheroids are three-dimensional (3D) cell clusters that can provide more in vivo-like functions to the cells than cells cultured as monolayers in 2D cell culture systems.
- 3D three-dimensional
- cell culture inserts for use in culturing cells to promote the formation of spheroids and methods of using these spheroid-promoting cell culture inserts are described.
- a cell culture insert as described herein can be nested in another cell culture insert or another cell culture insert can be nested in a cell culture insert as described herein.
- the disclosure describes a cell culture insert having a body and a porous membrane.
- the body has a first open end, a second end wherein the second end defines an opening having a diametric dimension in a range from 100 ⁇ m to 1000 ⁇ m (e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 and all values and ranges therein between; e.g., 200 to 500, 200 to 700, 400 to 600, etc.), and one or more sidewalls extending from the first open end to the second end.
- 100 ⁇ m to 1000 ⁇ m e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 and all values and ranges therein between; e.g., 200 to 500, 200 to 700, 400 to 600, etc.
- the one or more sidewalls, or a portion thereof, are sloped (e.g., greater than 5 degrees from perpendicular relative to first or second ends; e.g., >10°, >20°, >30°, >40°, >50°, etc.).
- the sidewalls, if sloped, are preferably sloped such that the diameter at the second is less than the diameter at the first open end.
- a porous membrane is disposed over the opening of the second end.
- the disclosure describes a cell culture insert having a body and a porous membrane.
- the body has a first open end, a second end wherein the second end defines an opening, and one or more sidewalls extending from the first open end to the second end.
- the one or more sidewalls, or a portion thereof, are sloped.
- the porous membrane is disposed over the opening of the second end and is non-adherent to cells.
- the disclosure describes a permeable support device configured to be at least partially inserted into a reservoir of a cell culture device.
- the permeable support device comprises a first well having a tapered shape and bottom at least partially defined by a first permeable support.
- the disclosure described a cell culture insert.
- the insert includes a body having a first open end, a second end, and one or more sidewalls extending from the first open end to the second end.
- the second end comprises a substrate having an array of microwells defining wells with a porous membrane at the bottom, wherein at least a portion of a substrate having an array of microwells defining a well is sloped.
- the disclosure describes a nested permeable support device comprising a first well having a tapered shape and a bottom portion at least partially defined by a first permeable support, and a reservoir having a bottom located below the first well.
- the reservoir is made from gas permeable material or has an array of microwells or both.
- the disclosure describes a nested permeable support device comprising a first well, a second well and a third well.
- the first well has a bottom, wherein at least a portion of the bottom is formed by a first porous membrane.
- the second well has a bottom, wherein at least a portion of the bottom is formed by a second porous membrane.
- a portion of either the first or the second permeable support may comprise an arcuate shape.
- the second well and the second permeable support are located below the first well and the first permeable support.
- the third well has a non-liquid permeable, gas permeable bottom which is located below the second well and the second permeable support.
- the disclosure describes a nested permeable support device comprising a first well, a second well, and a third well.
- the first well has a substrate forming a microwell array, the bottom of which is a porous membrane.
- the second well has a substrate forming a microwell array, the bottom of which is a porous membrane.
- the third well has a substrate forming a microwell array, the bottom of which is a non-liquid permeable, gas permeable support.
- the second well with the microwell array substrate with the porous membrane bottom is located beneath the first well with the microwell array substrate with the first porous membrane bottom and above the third well with the microwell array substrate with the non-liquid permeable, gas permeable bottom.
- FIGS. 1A-C shows schematic diagrams of embodiments of cell culture inserts in which cells can be cultured to form a spheroid.
- FIGS. 2A and 2B shows schematic diagrams of embodiments of a portion of a cell culture insert in which cells can be cultured to form a spheroid.
- FIGS. 3A-D shows A, schematic diagram (side view) of embodiments of a cell culture insert in which cells can be cultured to form one or more spheroids; B, one embodiment of a substrate having an array of microwells at the second end of a cell culture insert; C, schematic diagram (top view) of the second end of a cell culture insert in which cells can be cultured to form one or more spheroids; D, schematic diagram (enlarged side view) of an array of microwells on a substrate with a bottom having a porous membrane forming the second end of a cell culture insert in which cells can be cultured to form one or more spheroids.
- FIGS. 4A-B shows schematic diagrams of some embodiments of nested cell culture inserts in which cells can be cultured to form one or more spheroids.
- FIG. 5A-B shows schematic diagrams of some embodiments of nested cell culture inserts in which cells can be cultured to form one or more spheroids on any or all of the cell culture substrates forming the well bottoms.
- the present disclosure describes, among other things, spheroid-promoting cell culture inserts and methods of using spheroid-promoting cell culture inserts.
- the spheroid-promoting cell culture inserts will be contained in a cell culture apparatus.
- the spheroid-promoting cell culture inserts can be placed in another cell culture insert or another spheroid-promoting cell culture insert.
- the spheroid-promoting cell culture insert can contain another cell culture insert or another spheroid-promoting cell culture insert.
- a method of using spheroid-promoting cell culture inserts includes performing an experiment to test a New Chemical Entity (NCE) or a New Biological Entity (NBE).
- Cell culture devices or apparatuses that include nested permeable support devices can be adapted to have a spheroid-promoting cell culture insert.
- Examples of such cell culture devices or apparatuses include TRANSWELL® Permeable Supports (Corning, Inc.), and MILLICELL® Cell Culture Inserts (EMD Millipore), as well as the cell culture articles and methods described in U.S. Pat. No. 8,163,537, which is incorporated herein by reference in its entirety to the extent that it does not conflict with the disclosure presented herein.
- the apparatuses 500 include spheroid-promoting cell culture inserts 100 that have one or more sidewalls 110 that are non-adherent to cells to cause the cells in the insert 100 to associate with each other and form spheroids 200 .
- the insert fits inside a reservoir or a well 150 of a multiwell cell culture plate.
- the one or more sidewalls 110 can be coated with an ultra-low binding material to make the wall non-adherent to cells.
- non-adherent material include perfluorinated polymers, olefins, or like polymers or mixtures thereof.
- Other examples include agarose, non-ionic hydrogels such as polyacrylamides, or polyethers such as polyethyleneoxide or polyols such as polyvinylalcohol or like materials or mixtures thereof.
- the one or more sidewalls 110 of the spheroid-promoting cell culture inserts 100 include a portion that is sloped 115 .
- the one or more sidewalls can be sloped along their entire length. In other embodiments, only a portion of one or more sidewalls is sloped.
- the one or more sidewalls 110 and other components of the spheroid-promoting cell culture inserts 100 can be formed of any suitable material.
- materials intended to contact cells or culture media are compatible with the cells and the media.
- cell culture components are formed from polymeric material.
- suitable polymeric materials include polystyrene, polymethylmethacrylate, polyvinyl chloride, polycarbonate, polysulfone, polystyrene copolymers, fluoropolymers, polyesters, polyamides, polystyrene butadiene copolymers, fully hydrogenated styrenic polymers, polycarbonate PDMS copolymers, and polyolefins such as polyethylene, polypropylene, polymethyl pentene, polypropylene copolymers and cyclic olefin copolymers, and the like.
- the spheroid-promoting cell culture insert 100 includes a body having a first open end 101 and a second end 102 wherein the end 102 defines an opening.
- a porous membrane 120 can cover the opening of the second end.
- the porous membrane 120 can be adherent to cells.
- the porous membrane 120 , or a portion thereof can be non-adherent to cells 200 .
- the opening of the second end 102 of the body of the spheroid-promoting cell culture insert 100 can have a variety of shapes.
- the opening forms a circle or an oval.
- the opening defines a rectangle or other quadrilateral.
- the opening of the second end has diametric dimension, such as a diameter, a width, a diagonal of a square or rectangle, or the like, d 1 in a range from 100 Lm to 1000 ⁇ m.
- the opening of the second end can have a diametric dimension d 1 of 100 ⁇ m, 150 ⁇ m, 200 ⁇ m, 250 ⁇ m, 300 ⁇ m, 350 ⁇ m, 400 ⁇ m, 450 ⁇ m, 500 ⁇ m, 550 ⁇ m, 600 ⁇ m, 650 ⁇ m, 700 ⁇ m, 750 ⁇ m, 800 ⁇ m, 850 ⁇ m, 900 ⁇ m, 950 ⁇ m, or 1000 ⁇ m, and any dimension encompassed within the range from 100 ⁇ m to 1000 ⁇ m.
- the porous membrane 120 can have a variety of shapes. In some embodiments, the porous membrane 120 completely covers the opening of the second end of the body of the spheroid-promoting cell culture insert 100 . In some embodiments the porous membrane can have an arcuate or curved shape. In some embodiments the second end of the body of the spheroid promoting cell culture insert is comprised of a microwell array with a porous membrane forming the bottom.
- the porous membrane can be made of a variety of different materials including but not limited to track-etched membrane or a woven or non-woven porous material.
- the material of the porous membrane may be treated or coated to make it more adherent or more non-adherent to cells. Treatment may be accomplished by any number of methods known in the art which include plasma discharge, corona discharge, gas plasma discharge, ion bombardment, ionizing radiation, and high intensity UV light. Coatings can be introduced by any suitable method known in the art including printing, spraying, condensation, radiant energy, ionization techniques or dipping. The coatings may then provide either covalent or non-covalent attachment sites.
- Such sites can be used to attach moieties, such as cell culture components (e.g., proteins that facilitate growth or adhesion). Further, the coatings may also be used to enhance the attachment of cells (e.g., polylysine). Alternatively, cell non-adherent coatings as described above can be used to prevent or inhibit cell binding.
- moieties such as cell culture components (e.g., proteins that facilitate growth or adhesion).
- the coatings may also be used to enhance the attachment of cells (e.g., polylysine).
- cell non-adherent coatings as described above can be used to prevent or inhibit cell binding.
- the porous membrane may be a substrate having an array of microwells.
- the spheroid formed by the cells 200 occludes the porous membrane 120 of the spheroid-promoting cell culture insert. (See for example, FIG. 1A ). In some aspects, this occlusion prevents the passage of proteins, small molecules, and/or media from going around the spheroid.
- the combination of, for example, non-adherent sidewalls, geometry, and gravity can define a confinement volume in which growth of cells cultured in the inserts is limited. In embodiments, this combination can promote the formation of spheroids by cells cultured in the inserts.
- the confinement volume can be defined by the portion of the one or more sidewalls proximate the second end and the width or diagonal of the second opening. In some embodiments, the portion of the one or more sidewalls proximate the second end d 2 is 500 ⁇ m, 450 ⁇ m, 400 ⁇ m, 350 ⁇ m, 300 ⁇ m, 250 ⁇ m, 200 ⁇ m, 150 ⁇ m, or 100 ⁇ m, or any length in between.
- the confinement volumes are defined by the wells of the microwell array substrate with the porous membrane that forms the bottom of the second opening.
- the second opening approximates the size of the first opening with the wells of the microwell array substrate comprising the confinement volume and have a diameter of d 1 500 ⁇ m, 450 ⁇ m, 400 ⁇ m, 350 ⁇ m, 300 ⁇ m, 250 ⁇ m, 200 ⁇ m, 150 ⁇ m, or 100 ⁇ m, or any length in between.
- the spheroid-promoting cell culture insert can further include a ledge 130 extending around the perimeter of the first open end where the ledge 130 is sized to support the spheroid-promoting cell culture insert 100 when it is positioned inside a reservoir 150 .
- the reservoir 150 is gas permeable.
- a spheroid 200 can grow in a spheroid-promoting cell culture insert 100 , a portion of which can include a porous membrane 120 .
- One or both of the porous membrane 120 and the lower sidewalls 110 can be non-adherent to cells.
- the slope of the sidewall 110 encourages the seeded cells 200 to aggregate on the porous membrane 120 .
- One or more of gravity, an ultra-low binding material, the sidewall geometry of the cell culture insert, and the arcuate shape of the porous membrane can facilitate the formation of a spheroid.
- the porous membrane may contain an array of microwells structured and arranged to form spheroids.
- a spheroid can grow in a spheroid-promoting cell culture insert 100 , a portion of which is a porous membrane 120 .
- the porous membrane 120 and the lower sidewalls 110 can be non-adherent to cells.
- the slope of the sidewall 110 encourages the seeded cells to aggregate on or occlude the porous membrane 120 .
- One or more of gravity, an ultra-low binding material, the sidewall geometry of the cell culture insert, and the cell confinement volume around the porous membrane 120 can facilitate the formation of a spheroid.
- the size of the spheroid can be limited by the confinement volume.
- the second end of the insert can have a shape that provides a confinement volume that promotes spheroid formation, a spheroid confinement volume.
- a spheroid-promoting cell culture insert 105 can have a first open end 101 and a second end, 102 and one or more sidewalls 110 extending from the first open end to the second end. In some embodiments, the sidewalls are sloped. In one aspect, the second end of the spheroid-promoting cell culture insert 105 contains multiple spheroid-promoting wells 400 , where each spheroid-promoting well may have a sidewall 410 . In some embodiments, the spheroid-promoting cell culture insert 105 can have one or more sidewalls 110 that are non-adherent to cells.
- the substrate having an array of microwells is comprised of hexagonal close-packed well structures.
- An image of an embodiment of such a substrate having an array of microwells 410 is shown in FIG. 3B , showing the hexagonally shaped wells 400 .
- FIG. 3C is a schematic drawing showing a top-down view of an embodiment of a substrate having an array of microwells 410 .
- cells cultured within each well 400 form a single spheroid 200 .
- the wells 400 of the spheroid-promoting cell culture insert 105 have an inner surface that defines an upper aperture and a nadir, or low point or surface. At the upper aperture the wells have a diametric dimension, such as a diameter, a width, a diagonal of a square or rectangle, or the like, d 3 , in a range from 100 ⁇ m to 1000 ⁇ m.
- the well can have a diametric dimension d 3 of 100 ⁇ m, 150 ⁇ m, 200 ⁇ m, 250 ⁇ m, 300 ⁇ m, 350 ⁇ m, 400 ⁇ m, 450 ⁇ m, 500 ⁇ m, 550 ⁇ m, 600 ⁇ m, 650 ⁇ m, 700 ⁇ m, 750 ⁇ m, 800 ⁇ m, 850 ⁇ m, 900 ⁇ m, 950 ⁇ m, or 1000 ⁇ m, and any dimension encompassed within the range from 100 ⁇ m to 1000 ⁇ m.
- the depth of the wells 400 d 4 is 1000 ⁇ m, 500 ⁇ m, 450 ⁇ m, 400 ⁇ m, 350 ⁇ m, 300 ⁇ m, 250 ⁇ m, 200 ⁇ m, 150 ⁇ m, or 100 ⁇ m, or any dimension encompassed within the range from 100 ⁇ m to 1000 ⁇ m.
- a substrate having an array of microwells with a porous support forming the bottom of the microwells 410 covers the second end of the spheroid-promoting cell culture insert 105 .
- at least a portion of the substrate having an array of microwells 410 is non-adherent to cells.
- at least a portion of the substrate having an array of microwells 410 is adherent to cells.
- a portion of the substrate having an array of microwells 410 is porous.
- a portion of the substrate having an array of microwells 410 forming the wells 400 includes openings.
- the substrate having an array of microwells 410 can be adhered to, affixed to, or juxtaposed with a porous membrane 420 .
- the second end of the spheroid-promoting cell culture insert 105 is covered by a porous membrane 420 , and the porous membrane defines the substrate having an array of microwells 410 .
- the substrate having an array of microwells 410 forming the wells 400 of the spheroid-promoting cell culture insert 105 are sloped. In some embodiments, the substrate having an array of microwells 410 forming the wells 400 can be sloped along the entire depth of the well.
- a structured bottom surface as described herein can be formed in any suitable manner.
- a substrate can be coined, injection molded or embossed to form the substrate having an array of microwells 410 .
- a porous material or a gas permeable material can be coined, injection molded or embossed to form a substrate having an array of microwells.
- a spheroid-promoting cell culture insert 100 can be used in a nested permeable support device 600 .
- a spheroid-promoting cell culture insert 100 can be placed in another cell culture insert or device or another spheroid-promoting cell culture insert.
- two, or three, or more spheroid-promoting cell culture inserts 100 can be nested.
- a spheroid-promoting cell culture insert 100 can be placed in another cell culture insert or can have another cell culture insert placed in it.
- the spheroid-promoting cell culture insert can further include a ledge extending around the perimeter of the first open end where the ledge is sized to support the spheroid-promoting cell culture insert when it is positioned inside a reservoir or another cell culture insert.
- a ledge extending around the perimeter of the first open end where the ledge is sized to support the spheroid-promoting cell culture insert when it is positioned inside a reservoir or another cell culture insert.
- Each cell culture insert can have a porous membrane 120 or can be gas permeable. In one embodiment, the lowest cell culture insert or reservoir is gas-permeable and the upper cell inserts have porous membranes 120 .
- a nested device can include an upper 100 and a middle 101 spheroid-promoting cell culture insert.
- the upper 100 and middle 101 spheroid-promoting cell culture inserts can have a porous membrane 120 at the nadir.
- the porous membrane is a substrate having an array of microwells.
- the nested device can further include a spheroid-promoting cell culture reservoir 102 that does not have a permeable support.
- the spheroid-promoting cell culture reservoir 102 can be made of or include a portion of a gas-permeable material.
- the gas permeable material is a substrate having an array of microwells.
- the middle cell culture insert 101 can be a spheroid-promoting cell culture insert, but both the uppermost cell culture insert 300 and middle spheroid-promoting cell culture insert 101 have porous membranes 120 .
- the lowest device can be a reservoir 151 .
- the reservoir 151 can be gas-permeable.
- the middle cell culture insert can be a spheroid-promoting cell culture insert 105 that contains multiple spheroid-promoting porous wells 400 .
- the uppermost cell culture insert 300 can have a porous membrane 120 .
- the spheroid-promoting cell culture insert 105 can be inserted in or nested in a reservoir 151 .
- the reservoir 151 can be gas-permeable.
- FIG. 5B shows an embodiment of an apparatus with first 105 and second 106 spheroid-promoting cell culture inserts having porous membrane bottoms 120 in a spheroid-promoting reservoir 151 having a non-liquid permeable, gas permeable bottom.
- first 105 and second 106 spheroid-promoting cell culture inserts having porous membrane bottoms 120 in a spheroid-promoting reservoir 151 having a non-liquid permeable, gas permeable bottom.
- any combination of cell culture supports each having porous membranes or not, having substrates having an array of microwells for spheroid promotion or not, or being gas permeable or not, are possible depending on the desired cell culture environment.
- the spheroid-promoting cell culture inserts can be used in a method to determine whether a compound or molecule known as a NCE has a desired biological activity.
- a method to determine whether a compound or molecule known as a NCE has a desired biological activity Such methods are described in, for example, U.S. Pat. No. 8,163,537. These methods often entail examining the Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADME-Tox) of the NCE, as well as determining the NCE's level of effectiveness for the targeted therapeutic indication including pharmacokinetic parameters.
- One type of assessment examines the “first pass effect.” This assessment involves experimental determination of the bioavailabilty of the NCE following its absorption through the digestive tract and then its metabolism by the liver.
- the assessment of the “first pass effect” requires two separate in vitro assays to be conducted, and the data combined, to determine the intestinal permeability and the hepatic metabolism. If desired, additional studies may be conducted to determine target selectivity, efficacy and dosage (Lau et al., Drug Metabolism and Disposition, Vol. 32, No. 9, pp. 937-942, 2004).
- a well-known method used today to examine the intestinal absorption of a NCE is known as the Caco 2 cell-based assay which is typically conducted on permeable supports such as the ones sold under the brand name of TranswellTM and manufactured by Corning Inc. (“Transwell® Permeable Supports: Including SnapwellTM and NetwellTM Inserts-Instructions for Use” Corning Inc., September 2007.)
- TranswellTM permeable support facilitates the development of Caco 2 cell polarization to create more in vivo-like test conditions.
- researchers from the Schering-Plough Research Institute have expanded the utility of the Caco 2 cell-based assay by adding hepatocytes in the nutrient medium to a TranswellTM receiver plate which receives the TranswellTM permeable support.
- hepatocytes or Hep G2 cells used in a Caco 2 cell-based assay are cultured in a spheroid-promoting cell culture insert.
- the present disclosure relates to a nested permeable support device and methods for using the nested permeable support device to perform various experiments to test new therapeutic compounds, NBEs, or NCEs.
- the spheroid-promoting cell culture inserts can be used to assess system-like communication information. In some embodiments, the spheroid-promoting cell culture inserts can be used to create cell models that represent a series of human organs in vertical orientation.
- the nested permeable support device is used to form a first pass assay to determine the bioavailability of a NCE or NBE following absorption from the digestive tract and metabolism by the liver.
- the nested permeable support device 600 can be used to perform a first pass assay to determine the bioavailability of a NCE following absorption through the digestive tract and metabolism by the liver. In another embodiment, the nested permeable support device 600 can be used to perform a first pass assay to determine the bioavailability of a NBE following absorption through the digestive tract and metabolism by the liver.
- a researcher using the embodiment shown in FIG. 5A can place a media in a growing reservoir and then place the upper insert 300 in the growing reservoir.
- the upper insert 300 is then filled with a volume of Caco 2 cells in media.
- the upper insert 300 and growing reservoir are in communication until a confluent monolayer of Caco 2 cells is formed across the permeable support of the upper insert 300 . It usually takes about a month for Caco 2 cells to form across the permeable support of the upper insert 300 .
- the Caco 2 cells can be tested electronically to determine how tightly the Caco 2 cells adhere to one another by performing a Trans Epithelial Electrical Resistance (TEER) test, where a probe is inserted into the upper insert 300 and then the probe initiates a pulse that is detected by another probe located in the growing reservoir below the permeable support.
- TEER Trans Epithelial Electrical Resistance
- Another test that can be performed uses a dye called Lucifer yellow, which can pass through gaps in the Caco 2 cell monolayer. The more Lucifer yellow that shows up in the growing reservoir after being introduced in the upper insert 300 , the less mature (or confluent) the monolayer of Caco 2 cells. Tests such as these can be performed to make sure the Caco 2 cell culture is functioning as expected.
- the researcher can place a media in another growing reservoir and then place the middle insert 105 in this growing reservoir.
- the middle insert 105 is then filled with a volume of hepatocytes in media.
- the middle insert 105 and reservoir are in communication until a spheroid of hepatocytes is formed across the second permeable support. Tests could also be conducted to assure that the hepatocytes are functioning appropriately.
- the upper insert 300 and middle insert 105 can be placed in a reservoir 151 to grow the Caco 2 cells and the hepatocytes.
- the upper and middle inserts 300 and 105 would be lifted out of their respective growing reservoirs.
- the middle insert 105 would be placed (nested) in the reservoir 151 which contains a media. Some media would then be placed above the layer of hepatocytes located within the middle insert 105 .
- the upper insert 300 would be placed (nested) in the middle insert 105 .
- the NCE/NBE and media would be dispensed above the layer of Caco 2 cells located within the upper insert 300 .
- the upper insert 300 could be removed and the media in the middle insert 105 could be tested (i.e., LS/MS) to determine if the NCE/NBE passed through the intestinal epithelium (Caco 2 cells). If the NCE/NBE did pass through the Caco 2 cells, then the middle insert 105 could be removed and the media in the reservoir 151 could be tested (i.e., LC/MS) to check the bioavailability of the NCE and/or how the NCE/NBE is metabolized by the liver (hepatocytes) to form metabolic products. The hepatocytes could also show if the NCE/NBE is toxic at the dosage applied.
- the media in the middle insert 105 could be tested (i.e., LS/MS) to determine if the NCE/NBE passed through the intestinal epithelium (Caco 2 cells). If the NCE/NBE did pass through the Caco 2 cells, then the middle insert 105 could be removed and the media in the reservoir 151 could be tested (i.e
- target cells or molecules
- these could be examined (i.e., LC/MS) to determine the drug effects either microscopically, or by using an assay that is separate from the Caco 2 cells and hepatocytes by pulling out the inserts 300 and 105 .
- the target cells (or molecules) on the bottom of the reservoir 151 could be examined using an interrogation system to assess function and viability as described, for example, in U.S. Pat. No. 8,163,537.
- an assay such as this will enable understanding of whether 1) an NCE/NBE can pass through the intestinal epithelium; 2) whether the liver metabolizes or is damaged by an NCE/NBE; and 3) the effect on the target cells of unmodified or liver-metabolized NCE/NBE.
- the nested permeable support device 100 can be used to test a NCE which would not pass through the digestive tract but instead would enter the body via inhalation in which case the Caco 2 cells would be replaced with nasal mucosal cells, bronchial cells or lung epithelial cells, etc.
- the researcher would typically select the actual cells used in the nested permeable support device 600 .
- multiple spheroids may be grown.
- the spheroids are all the same.
- two or more different types of spheroids are used (e.g., a co-culture system to, for example, simulate or reconstitute the multicellular functionality of an organ).
- a co-culture system to, for example, simulate or reconstitute the multicellular functionality of an organ.
- Cells cultured in three dimensions can exhibit more in vivo like functionality than their counterparts cultured in two dimensions as monolayers.
- cells can attach to a substrate on which they are cultured.
- the cells interact with each other rather than attaching to the substrate.
- Cells cultured in three dimensions more closely resemble in vivo tissue in terms of cellular communication and the development of extracellular matrices.
- Spheroids thus provide a superior model for cell migration, differentiation, survival, and growth and therefore provide better systems for research, diagnostics, and drug efficacy, pharmacology, and toxicity testing.
- the devices are configured such that cells cultured in the devices form spheroids.
- the wells in which cells are grown can be non-adherent to cells to cause the cells in the wells to associate with each other and form spheres.
- the spheroids expand to size limits imposed by the geometry of the wells.
- the wells are coated with an ultra-low binding material to make the wells non-adherent to cells.
- non-adherent material examples include perfluorinated polymers, olefins, or like polymers or mixtures thereof.
- Other examples include agarose, non-ionic hydrogels such as polyacrylamides, polyethers such as polyethylene oxide and polyols such as polyvinyl alcohol, or like materials or mixtures thereof.
- the combination of, for example, non-adherent wells, well geometry (e.g., size and shape), and/or gravity induce cells cultured in the wells to self-assemble into spheroids. Some spheroids maintain differentiated cell function indicative of a more in vivo-like, response relative to cells grown in a monolayer. Other cells types, such as mesenchymal stromal cells, when cultured as spheroids retain their pluripotency.
- the systems, devices, and methods herein comprise one or more cells.
- the cells are cryopreserved.
- the cells are in three dimensional culture.
- the systems, devices, and methods comprise one or more spheroids.
- one or more of the cells are actively dividing.
- the systems, devices, and methods comprise culture media (e.g., comprising nutrients (e.g., proteins, peptides, amino acids), energy (e.g., carbohydrates), essential metals and minerals (e.g., calcium, magnesium, iron, phosphates, sulphates), buffering agents (e.g., phosphates, acetates), indicators for pH change (e.g., phenol red, bromo-cresol purple), selective agents (e.g., chemicals, antimicrobial agents), etc.).
- one or more test compounds e.g., drug
- a spheroid contains a single cell type. In some embodiments, a spheroid contains more than one cell type. In some embodiments, where more than one spheroid is grown, each spheroid is of the same type, while in other embodiments, two or more different types of spheroids are grown. Cells grown in spheroids may be natural cells or altered cells (e.g., cell comprising one or more non-natural genetic alterations). In some embodiments, the cell is a somatic cell.
- the cell is a stem cell or progenitor cell (e.g., embryonic stem cell, induced pluripotent stem cell) in any desired state of differentiation (e.g., pluripotent, multi-potent, fate determined, immortalized, etc.).
- the cell is a disease cell or disease model cell.
- the spheroid comprises one or more types of cancer cells or cells that can be induced into a hyper-proliferative state (e.g., transformed cells).
- Cells may be from or derived from any desired tissue or organ type, including but not limited to, adrenal, bladder, blood vessel, bone, bone marrow, brain, cartilage, cervical, corneal, endometrial, esophageal, gastrointestinal, immune system (e.g., T lymphocytes, B lymphocytes, leukocytes, macrophages, and dendritic cells), liver, lung, lymphatic, muscle (e.g., cardiac muscle), neural, ovarian, pancreatic (e.g., islet cells), pituitary, prostate, renal, salivary, skin, tendon, testicular, and thyroid.
- the cells are mammalian cells (e.g., human, mice, rat, rabbit, dog, cat, cow, pig, chicken, goat, horse, etc.).
- the cultured cells find use in a wide variety of research, diagnostic, drug screening and testing, therapeutic, and industrial applications.
- the cells are used for production of proteins or viruses. Systems, devices, and methods that culture large numbers of spheroids in parallel are particularly effective for protein production. Three-dimensional culture allows for increased cell density, and higher protein yield per square centimeter of cell growth surface area. Any desired protein or viruses for vaccine production may be grown in the cells and isolated or purified for use as desired.
- the protein is a native protein to the cells.
- the protein is non-native.
- the protein is expressed recombinantly.
- the protein is overexpressed using a non-native promoter.
- the protein may be expressed as a fusion protein.
- a purification or detection tag is expressed as a fusion partner to a protein of interest to facilitate its purification and/or detection.
- fusions are expressed with a cleavable linker to allow separation of the fusion partners after purification.
- the protein is a therapeutic protein.
- proteins include, but are not limited to, proteins and peptides that replace a protein that is deficient or abnormal (e.g., insulin), augment an existing pathway (e.g., inhibitors or agonists), provide a novel function or activity, interfere with a molecule or organism, or deliver other compounds or proteins (e.g., radionuclides, cytotoxic drugs, effector proteins, etc.).
- the protein is an immunoglobulin such as an antibody (e.g., monoclonal antibody) of any type (e.g., humanized, bi-specific, multi-specific, etc.).
- Therapeutic protein categories include, but are not limited to, antibody-based drugs, Fc fusion proteins, anticoagulants, antigens, blood factor, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. Therapeutic proteins may be used to prevent or treat cancers, immune disorders, metabolic disorders, inherited genetic disorders, infections, and other diseases and conditions.
- the protein is a diagnostic protein. Diagnostic proteins include, but are not limited to, antibodies, affinity binding partners (e.g., receptor-binding ligands), inhibitors, antagonists, and the like. In some embodiments, the diagnostic protein is expressed with or is a detectable moiety (e.g., fluorescent moiety, luminescent moiety (e.g., luciferase), colorimetric moiety, etc.).
- a detectable moiety e.g., fluorescent moiety, luminescent moiety (e.g., luciferase), colorimetric moiety, etc.
- the protein is an industrial protein.
- Industrial proteins include, but are not limited to, food components, industrial enzymes, agricultural proteins, analytical enzymes, etc.
- the cells are used drug discovery, characterization, efficacy testing, and toxicity testing.
- Such testing includes, but is not limited to, pharmacological effect assessment, carcinogenicity assessment, medical imaging agent characteristic assessment, half-life assessment, radiation safety assessment, genotoxicity testing, immunotoxicity testing, reproductive and developmental testing, drug interaction assessment, dose assessment, adsorption assessment, disposition assessment, metabolism assessment, elimination studies, etc.
- Specific cells types may be employed for specific tests (e.g., hepatocytes for liver toxicity, renal proximal tubule epithelial cells for nephrotoxicity, vascular endothelial cells for vascular toxicity, neuronal and glial cells for neurotoxicity, cardiomyocytes for cardiotoxicity, skeletal myocytes for rhabdomyolysis, etc.).
- Treated cells may be assessed for any number of desired parameters including, but not limited to, membrane integrity, cellular metabolite content, mitochondrial functions, lysosomal functions, apoptosis, genetic alterations, gene expression differences, and the like.
- the cell culture devices are a component of a larger system.
- the system comprises a plurality (e.g., 2, 3, 4, 5, . . . , 10, . . . , 20, . . . , 50, . . . , 100, . . . , 1000, etc.) of such cell culture devices.
- the system comprises an incubator for maintaining the culture devices at optimal culture conditions (e.g., temperature, atmosphere, humidity, etc.).
- the system comprises detectors for imaging or otherwise analyzing cells.
- Such detectors include, but are not limited to, fluorimeters, luminometers, cameras, microscopes, plate readers (e.g., PERKIN ELMER ENVISION plate reader; PERKIN ELMER VIEWLUX plate reader), cell analyzers (e.g., GE IN Cell Analyzer 2000 and 2200; THERMO/CELLOMICS CELLNSIGHT High Content Screening Platform), and confocal imaging systems (e.g., PERKIN ELMER OPERAPHENIX high throughput content screening system; GE INCELL 6000 Cell Imaging System).
- the system comprises perfusion systems or other components for supplying, re-supplying, and circulating culture media or other components to cultured cells.
- the system comprises robotic components (e.g., pipettes, arms, plate movers, etc.) for automating the handing, use, and/or analysis of culture devices.
- a cell culture insert comprises (i) a body having a first open end, a second end wherein the second end defines an opening having a diametric dimension in a range from 100 ⁇ m to 1000 ⁇ m, and one or more sidewalls extending from the first open end to the second end; wherein the one or more sidewalls are sloped; and (ii) a porous membrane disposed over the opening of the second end.
- a second aspect is a cell culture insert according the first aspect wherein at least a portion of the one or more sidewalls are non-adherent to cells.
- a third aspect is a cell culture insert according to aspect 1 or aspect 2, wherein at least a portion of the porous membrane is non-adherent to cells.
- a fourth aspect is a cell culture insert according to any one of aspects 1 to 3, wherein at least a portion of the porous membrane is adherent to cells.
- a fifth aspect is a cell culture insert according to any one of aspects 1 to 4, wherein a portion of the one or more sidewalls proximate the second end at least partially define a cell confinement volume.
- a sixth aspect is a cell culture insert according to aspect 5 wherein a depth of the confinement volume is in a range from 100 ⁇ m to 1000 ⁇ m.
- a seventh aspect is a cell culture insert according to any one of aspects 1 to 6, wherein the insert is configured such that cells cultured in the insert form a spheroid.
- An eighth aspect is a method for culturing a spheroid comprising (i) placing cell culture insert according to aspect 7 in a reservoir, the reservoir having a bottom, wherein insert is placed in the reservoir such that the second end of body of the insert is positioned above the bottom of the insert; (ii) introducing cells into the insert; (iii) introducing a cell culture medium into the insert; and (iv) culturing the cells in the cell culture medium in the insert to form the spheroid.
- a ninth aspect is a cell culture assembly, comprising (i) a reservoir defining an interior and having a bottom; and (ii) a first cell culture insert according to any one of aspects 1 to 7 configured to be positioned in the interior of the reservoir such that the second end of the body is above the bottom or the reservoir, wherein the body of the first insert defines an interior of the first insert.
- a tenth aspect is a cell culture assembly according to aspect 9, wherein the interior of the first insert, when the first insert is positioned in the interior of the reservoir, is in fluid communication with the interior of the reservoir only through the porous membrane disposed over the opening of the second end of the body of the first insert.
- An eleventh aspect is a cell culture assembly according to aspect 9 or aspect 10, further comprising a second insert having a body defining an interior, the body comprising a first open end, a second end defining an opening, and one or more sidewalls extending from the first open end to the second end, wherein the second insert is configured to be positioned in the interior of the first insert such that the second end of the body of the second insert above the second end of the body of the first insert.
- a twelfth aspect is a cell culture assembly according to aspect 11, wherein the interior of the second insert, when the second insert is positioned in the interior of the first insert, is in fluid communication with the interior of the first insert only through the porous membrane disposed over the opening of the second end of the body of the second insert.
- a thirteenth aspect is a method comprising (i) introducing target cells and a cell culture medium to an interior of a reservoir of a cell culture assembly according to aspect 12 such that the target cells grow on the bottom of the reservoir; (ii) positioning a first cell culture insert according to aspect 12 in the interior of the reservoir; (iii) introducing a plurality of a first type of cells and a cell culture medium into the interior of the first cell culture insert such that cells of the first type grow as a spheroid in proximity to the porous membrane of the first insert; (iv) positioning a second cell culture insert according to aspect 12 in the interior of the first insert; and (v) introducing a plurality of a second type of cells and a cell culture medium into the interior of the second cell culture insert such that the cells of the second type grow in proximity to the porous membrane of the second insert.
- a fourteenth aspect is a method according to aspect 13, wherein the cells of the second type cover the porous membrane of the second insert such that compounds or metabolic derivatives thereof that move from the interior of the second insert to the interior of the first insert pass through the cells of the second type.
- a fifteenth aspect is a method according to aspect 14, wherein the cells of the first type attach to the porous membrane of the first insert such that compounds or metabolic derivatives thereof that move from the interior of the first insert to the interior of the reservoir pass through the cells of the first type.
- a sixteenth aspect is a method according to aspect 15, further comprising: (i) introducing a test compound to the interior of the second insert; and (ii) identifying an effect of the test compound or a metabolic derivative thereof on the target cells.
- a seventeenth aspect is a method according to any of aspects 13 to 16, wherein the cells of the first type are hepatocytes.
- An eighteenth aspect is a method according to any of aspects 13 to 17, wherein the cells of the second type are Caco 2 cells.
- a nineteenth aspect is a cell culture insert comprising: (i) a body having a first open end, a second end wherein the second end defines an opening, and one or more sidewalls extending from the first open end to the second end; wherein the one or more sidewalls are sloped; and (ii) a porous membrane disposed over the opening of the second end, wherein the porous membrane is non-adherent to cells.
- a twentieth aspect is a permeable support device configured to be at least partially inserted into a reservoir of a cell culture device, the permeable support device comprising a first well having a tapered shape and bottom at least partially defined by a first permeable support.
- a twenty-first aspect is a permeable support device of aspect 20 wherein the well is configured such that cells cultured in the well form a spheroid.
- a twenty-second aspect is a permeable support device of either of aspects 20 or 21 wherein at least a portion of the first well is coated with an ultra-low binding material.
- a twenty-third aspect is a permeable support device of any of aspect 20 to 22, wherein at least a portion of the permeable support is configured to attach to cells cultured in the first well.
- a twenty-fourth aspect is a permeable support device of any of aspects 20 to 23 wherein at least a portion of the first well comprises an arcuate shape
- a twenty-fifth aspect is a permeable support device of any of aspects 20 to 24 wherein at least a portion of the first well comprises a conical shape.
- a twenty-sixth aspect is a permeable support device of any of aspects 20 to 25 wherein a portion of the well defines a confinement volume.
- a twenty-seventh aspect is a permeable support device of aspect 26 wherein a diametric dimension of the confinement volume is in a range from 200 ⁇ m to 500 ⁇ m.
- a twenty-eighth aspect The permeable support device of either of aspects 26 or 27 wherein the depth of the confinement volume is in a range from 100 ⁇ m to 500 ⁇ m.
- a twenty-ninth aspect is a permeable support device of aspect any of aspects 20 to 28 wherein the first well is configured and sized to receive a second well having a bottom, wherein the second well is located above the first well.
- a thirtieth aspect is a nested permeable support device comprising: (i) a first well having a tapered shape and a bottom portion at least partially defined by a first permeable support; and (ii) a reservoir having a bottom located below the first well.
- a thirty-first aspect is a nested permeable support device of aspect 30 wherein the well is configured such that cells cultured in the well form a spheroid.
- a thirty-second aspect is a nested permeable support device of either of aspects 30 or 31 wherein at least a portion of the first well is coated with an ultra-low binding material.
- a thirty-third aspect is a nested permeable support device of any of aspects 29 to 32, wherein at least a portion of the permeable support is configured to attach to cells cultured in the first well.
- a thirty-fourth aspect is a nested permeable support device of any of aspects 30 to 33 wherein at least a portion of the first well comprises an arcuate shape.
- a thirty-fifth aspect is a nested permeable support device of any of aspects 30 to 34 wherein at least a portion of the first well comprises a conical shape.
- a thirty-sixth aspect is a nested permeable support device of any of aspects 30 to 35 wherein a portion of the well defines a confinement volume.
- a thirty-seventh aspect is a nested permeable support device of aspect 36 wherein a diametric dimension of the confinement volume is in a range from 100 ⁇ m to 1000 ⁇ m, e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 ⁇ m, including ranges between any of the foregoing.
- a thirty eighth aspect is a nested permeable support device of either of aspects 36 or 37 wherein the depth of the confinement volume is in a range from 100 ⁇ m to 1000 ⁇ m, e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 ⁇ m, including ranges between any of the foregoing.
- a thirty-ninth aspect is a nested permeable support device of any of aspects 30 to 38 further comprising a second well having a bottom at least partially defined by a second permeable support, and wherein the first well and the first permeable support are located below the second well and the second permeable support.
- a fortieth aspect is a nested permeable support device of aspect 39 wherein the second well comprises a tapered shape.
- a forty-first aspect is a nested permeable support device of any of aspect 30 to 40 wherein the bottom of the reservoir comprises a gas permeable material.
- a forty-second aspect is a nested permeable support device comprising: (i) a first well having a bottom, wherein at least a portion of the bottom is formed by a first permeable support; (ii) a second well having a bottom, wherein the bottom of the second well comprises an arcuate shape and at least a portion of the bottom is formed by a second permeable support, and wherein the second well and the second permeable support are located below the first well and the first permeable support; and (ii) a third well having a bottom which is located below the second well and the second permeable support.
- a forty-third aspect is a nested permeable support device of aspect 42 wherein at least a portion of the second well is coated with an ultra-low binding material.
- a forty-fourth aspect is a cell culture insert comprising a body having a first open end, a second end, and one or more sidewalls extending from the first open end to the second end; and wherein the second end comprises a substrate having an array of microwells defining wells, wherein at least a portion of a substrate having an array of microwells defining a well is sloped.
- a forty-fifth aspect is a cell culture insert of aspect 44 wherein the sidewalls are sloped.
- a forty-sixth aspect is a cell culture insert of either of aspect 44 or 45 wherein the sidewalls are non-adherent to cells.
- a forty-seventh aspect is a cell culture insert of any of aspects 44 to 46 wherein at least a portion of the substrate having an array of microwells is non-adherent to cells.
- a forty-eighth aspect is a cell culture insert of any of aspects 44 to 47 wherein the wells have an inner surface defining an upper aperture and wherein the wells have a diametric dimension at the upper aperture in a range from 100 ⁇ m to 1000 ⁇ m.
- a forty-ninth aspect is a cell culture insert of any of aspects 44 to 48 wherein the wells have a depth in a range from 100 ⁇ m to 100 ⁇ m.
- a fiftieth aspect is a cell culture insert of any of aspects 44 to 49 wherein at least a portion of the substrate having an array of microwells is non-adherent to cells.
- a fifty-first aspect is a cell culture insert of any of aspects 44 to 49 wherein at least a portion of the substrate having an array of microwells is adherent to cells.
- a fifty-second aspect is a cell culture insert of any of aspects 44 to 51 wherein at least a portion of the substrate having an array of microwells is porous.
- a fifty-third aspect is a cell culture insert of any of aspects 44 to 52 wherein the substrate having an array of microwells comprises openings.
- a fifty-fourth aspect is a cell culture insert of any of aspects 44 to 53 wherein the substrate having an array of microwells is adhered to, affixed to, or juxtaposed with a porous membrane.
- a fifty-fifth aspect is a cell culture insert of any of aspects 44 to 54 wherein the second end is covered by a porous membrane.
- a fifty-sixth aspect is a cell culture insert of any of aspects 44 to 55 wherein the substrate having an array of microwells comprises a sloped surface.
- a fifty-seventh aspect is a cell culture insert of any of aspects 44 to 56 wherein the substrate having an array of microwells comprises an array of hexagonal structures.
- a fifty-eighth aspect is a cell culture assembly, comprising: (i) a reservoir defining an interior and having a bottom; and (ii) a first cell culture insert according to any one of aspects 44-57 configured to be positioned in the interior of the reservoir such that the second end of the body is above the bottom of the reservoir, wherein the body of the first insert defines an interior of the first insert.
- a fifty-ninth aspect is a cell culture assembly according to aspect 58, wherein the interior of the first insert, when the first insert is positioned in the interior of the reservoir, is in fluid communication with the interior of the reservoir only through the porous membrane disposed over the opening of the second end of the body of the first insert.
- a sixtieth aspect is a cell culture assembly according to aspect 58 or aspect 59, further comprising a second insert having a body defining an interior, the body comprising a first open end, a second end defining an opening, and one or more sidewalls extending from the first open end to the second end, wherein the second insert is configured to be positioned in the interior of the first insert such that the second end of the body of the second insert above the second end of the body of the first insert.
- a sixty first aspect is a cell culture assembly according to aspect 60, wherein the interior of the second insert, when the second insert is positioned in the interior of the first insert, is in fluid communication with the interior of the first insert only through the porous membrane disposed over the opening of the second end of the body of the second insert.
- a sixty second aspect is a cell culture insert comprising: a body sized for insert into a reservoir of a cell culture device, said body having a first open end, a second end having a porous membrane, and one or more sidewalls extending from the first open end to the second end; wherein the one or more sidewalls are sloped; and wherein said second end has an upper surface defining a plurality of microwells sized for spheroid growth.
- a sixty third aspect is the cell culture insert of aspect 62, wherein said microwells each have a diameter in a range from 100 ⁇ m to 1000 ⁇ m.
- references herein refer to a component being “configured” or “adapted to” function in a particular way.
- such a component is “configured” or “adapted to” embody a particular property, or function in a particular manner, where such recitations are structural recitations as opposed to recitations of intended use.
- the references herein to the manner in which a component is “configured” or “adapted to” denotes an existing physical condition of the component and, as such, is to be taken as a definite recitation of the structural characteristics of the component.
- any direction referred to herein, such as “top,” “bottom,” “left,” “right,” “upper,” “lower,” “above,” below,” and other directions and orientations are described herein for clarity in reference to the figures and are not to be limiting of an actual device or system or use of the device or system. Many of the devices, articles or systems described herein may be used in a number of directions and orientations.
- Directional descriptors used herein with regard to cell culture apparatuses often refer to directions when the apparatus is oriented for purposes of culturing cells in the apparatus.
- a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.
- the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
- ADME-Tox Absorption, Distribution, Metabolism, Excretion, and Toxicity
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Sustainable Development (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Clinical Laboratory Science (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
- This is a continuation application of International Patent Application Serial No. PCT/US15/58053 filed on Oct. 29, 2015, which claims the benefit of priority to U.S. Provisional Application Ser. No. 62/069,996, filed on Oct. 29, 2014, the contents of which are relied upon and incorporated herein by reference in their entirety, and the benefit of priority under 35 U.S.C. §120 is hereby claimed.
- The present disclosure relates to apparatuses, systems and methods for culturing cells.
- The present disclosure relates generally to cell culture inserts for use in culturing cells to promote the formation of spheroids and methods of using these spheroid-promoting cell culture inserts. Spheroids are three-dimensional (3D) cell clusters that can provide more in vivo-like functions to the cells than cells cultured as monolayers in 2D cell culture systems. For certain cell types, such as hepatocytes, spheroids can attain and retain better in vivo-like functionality than their 2D cultured counterparts.
- In accordance with various embodiments of the present disclosure, cell culture inserts for use in culturing cells to promote the formation of spheroids and methods of using these spheroid-promoting cell culture inserts are described. In some embodiments, a cell culture insert as described herein can be nested in another cell culture insert or another cell culture insert can be nested in a cell culture insert as described herein.
- In various embodiments, the disclosure describes a cell culture insert having a body and a porous membrane. The body has a first open end, a second end wherein the second end defines an opening having a diametric dimension in a range from 100 μm to 1000 μm (e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 and all values and ranges therein between; e.g., 200 to 500, 200 to 700, 400 to 600, etc.), and one or more sidewalls extending from the first open end to the second end. The one or more sidewalls, or a portion thereof, are sloped (e.g., greater than 5 degrees from perpendicular relative to first or second ends; e.g., >10°, >20°, >30°, >40°, >50°, etc.). The sidewalls, if sloped, are preferably sloped such that the diameter at the second is less than the diameter at the first open end. A porous membrane is disposed over the opening of the second end.
- In various embodiments, the disclosure describes a cell culture insert having a body and a porous membrane. The body has a first open end, a second end wherein the second end defines an opening, and one or more sidewalls extending from the first open end to the second end. The one or more sidewalls, or a portion thereof, are sloped. The porous membrane is disposed over the opening of the second end and is non-adherent to cells.
- In various embodiments, the disclosure describes a permeable support device configured to be at least partially inserted into a reservoir of a cell culture device. The permeable support device comprises a first well having a tapered shape and bottom at least partially defined by a first permeable support.
- In various embodiments, the disclosure described a cell culture insert. The insert includes a body having a first open end, a second end, and one or more sidewalls extending from the first open end to the second end. The second end comprises a substrate having an array of microwells defining wells with a porous membrane at the bottom, wherein at least a portion of a substrate having an array of microwells defining a well is sloped.
- In various embodiments, the disclosure describes a nested permeable support device comprising a first well having a tapered shape and a bottom portion at least partially defined by a first permeable support, and a reservoir having a bottom located below the first well. In embodiments the reservoir is made from gas permeable material or has an array of microwells or both.
- In various embodiments, the disclosure describes a nested permeable support device comprising a first well, a second well and a third well. The first well has a bottom, wherein at least a portion of the bottom is formed by a first porous membrane. The second well has a bottom, wherein at least a portion of the bottom is formed by a second porous membrane. A portion of either the first or the second permeable support may comprise an arcuate shape. The second well and the second permeable support are located below the first well and the first permeable support. The third well has a non-liquid permeable, gas permeable bottom which is located below the second well and the second permeable support.
- In another embodiment, the disclosure describes a nested permeable support device comprising a first well, a second well, and a third well. The first well has a substrate forming a microwell array, the bottom of which is a porous membrane. The second well has a substrate forming a microwell array, the bottom of which is a porous membrane. The third well has a substrate forming a microwell array, the bottom of which is a non-liquid permeable, gas permeable support. The second well with the microwell array substrate with the porous membrane bottom is located beneath the first well with the microwell array substrate with the first porous membrane bottom and above the third well with the microwell array substrate with the non-liquid permeable, gas permeable bottom.
- Additional features and advantages of the subject matter of the present disclosure will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the subject matter of the present disclosure as described herein, including the detailed description which follows, the claims, as well as the appended drawings.
- It is to be understood that both the foregoing general description and the following detailed description present embodiments of the subject matter of the present disclosure, and are intended to provide an overview or framework for understanding the nature and character of the subject matter of the present disclosure as it is claimed. The accompanying drawings are included to provide a further understanding of the subject matter of the present disclosure, and are incorporated into and constitute a part of this specification. The drawings illustrate various embodiments of the subject matter of the present disclosure and together with the description serve to explain the principles and operations of the subject matter of the present disclosure. Additionally, the drawings and descriptions are meant to be merely illustrative, and are not intended to limit the scope of the claims in any manner.
- The following detailed description of specific embodiments of the present disclosure can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which:
-
FIGS. 1A-C shows schematic diagrams of embodiments of cell culture inserts in which cells can be cultured to form a spheroid. -
FIGS. 2A and 2B shows schematic diagrams of embodiments of a portion of a cell culture insert in which cells can be cultured to form a spheroid. -
FIGS. 3A-D shows A, schematic diagram (side view) of embodiments of a cell culture insert in which cells can be cultured to form one or more spheroids; B, one embodiment of a substrate having an array of microwells at the second end of a cell culture insert; C, schematic diagram (top view) of the second end of a cell culture insert in which cells can be cultured to form one or more spheroids; D, schematic diagram (enlarged side view) of an array of microwells on a substrate with a bottom having a porous membrane forming the second end of a cell culture insert in which cells can be cultured to form one or more spheroids. -
FIGS. 4A-B shows schematic diagrams of some embodiments of nested cell culture inserts in which cells can be cultured to form one or more spheroids. -
FIG. 5A-B shows schematic diagrams of some embodiments of nested cell culture inserts in which cells can be cultured to form one or more spheroids on any or all of the cell culture substrates forming the well bottoms. - Reference will now be made in greater detail to various embodiments of the subject matter of the present disclosure, some embodiments of which are illustrated in the accompanying drawings. The same reference numerals will be used throughout the drawings to refer to the same or similar parts. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure labeled with the same number. In addition, the use of different numbers to refer to components is not intended to indicate that the different numbered components cannot be the same or similar to other numbered components.
- The present disclosure describes, among other things, spheroid-promoting cell culture inserts and methods of using spheroid-promoting cell culture inserts. In some embodiments, the spheroid-promoting cell culture inserts will be contained in a cell culture apparatus. In some embodiments, the spheroid-promoting cell culture inserts can be placed in another cell culture insert or another spheroid-promoting cell culture insert. In some embodiments, the spheroid-promoting cell culture insert can contain another cell culture insert or another spheroid-promoting cell culture insert. In various embodiments, a method of using spheroid-promoting cell culture inserts includes performing an experiment to test a New Chemical Entity (NCE) or a New Biological Entity (NBE).
- Cell culture devices or apparatuses that include nested permeable support devices can be adapted to have a spheroid-promoting cell culture insert. Examples of such cell culture devices or apparatuses include TRANSWELL® Permeable Supports (Corning, Inc.), and MILLICELL® Cell Culture Inserts (EMD Millipore), as well as the cell culture articles and methods described in U.S. Pat. No. 8,163,537, which is incorporated herein by reference in its entirety to the extent that it does not conflict with the disclosure presented herein.
- Referring now to
FIGS. 1A-C , schematic side views of embodiments ofcell culture apparatuses 500 are shown. Theapparatuses 500 include spheroid-promoting cell culture inserts 100 that have one or more sidewalls 110 that are non-adherent to cells to cause the cells in theinsert 100 to associate with each other and formspheroids 200. In embodiments, the insert fits inside a reservoir or a well 150 of a multiwell cell culture plate. In some embodiments, the one or more sidewalls 110 can be coated with an ultra-low binding material to make the wall non-adherent to cells. Examples of non-adherent material include perfluorinated polymers, olefins, or like polymers or mixtures thereof. Other examples include agarose, non-ionic hydrogels such as polyacrylamides, or polyethers such as polyethyleneoxide or polyols such as polyvinylalcohol or like materials or mixtures thereof. - The one or more sidewalls 110 of the spheroid-promoting cell culture inserts 100 include a portion that is sloped 115. In some embodiments, the one or more sidewalls can be sloped along their entire length. In other embodiments, only a portion of one or more sidewalls is sloped.
- The one or more sidewalls 110 and other components of the spheroid-promoting cell culture inserts 100 can be formed of any suitable material. Preferably, materials intended to contact cells or culture media are compatible with the cells and the media. Typically, cell culture components are formed from polymeric material. Examples of suitable polymeric materials include polystyrene, polymethylmethacrylate, polyvinyl chloride, polycarbonate, polysulfone, polystyrene copolymers, fluoropolymers, polyesters, polyamides, polystyrene butadiene copolymers, fully hydrogenated styrenic polymers, polycarbonate PDMS copolymers, and polyolefins such as polyethylene, polypropylene, polymethyl pentene, polypropylene copolymers and cyclic olefin copolymers, and the like.
- The spheroid-promoting
cell culture insert 100 includes a body having a firstopen end 101 and asecond end 102 wherein theend 102 defines an opening. Aporous membrane 120 can cover the opening of the second end. In some embodiments, theporous membrane 120 can be adherent to cells. In other embodiments, theporous membrane 120, or a portion thereof, can be non-adherent tocells 200. - Referring now to
FIG. 2A andFIG. 2B , the opening of thesecond end 102 of the body of the spheroid-promotingcell culture insert 100 can have a variety of shapes. In some embodiments, the opening forms a circle or an oval. In other embodiments, the opening defines a rectangle or other quadrilateral. In some embodiments, the opening of the second end has diametric dimension, such as a diameter, a width, a diagonal of a square or rectangle, or the like, d1 in a range from 100 Lm to 1000 μm. Specifically, the opening of the second end can have a diametric dimension d1 of 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, or 1000 μm, and any dimension encompassed within the range from 100 μm to 1000 μm. - The
porous membrane 120 can have a variety of shapes. In some embodiments, theporous membrane 120 completely covers the opening of the second end of the body of the spheroid-promotingcell culture insert 100. In some embodiments the porous membrane can have an arcuate or curved shape. In some embodiments the second end of the body of the spheroid promoting cell culture insert is comprised of a microwell array with a porous membrane forming the bottom. - The porous membrane can be made of a variety of different materials including but not limited to track-etched membrane or a woven or non-woven porous material. The material of the porous membrane may be treated or coated to make it more adherent or more non-adherent to cells. Treatment may be accomplished by any number of methods known in the art which include plasma discharge, corona discharge, gas plasma discharge, ion bombardment, ionizing radiation, and high intensity UV light. Coatings can be introduced by any suitable method known in the art including printing, spraying, condensation, radiant energy, ionization techniques or dipping. The coatings may then provide either covalent or non-covalent attachment sites. Such sites can be used to attach moieties, such as cell culture components (e.g., proteins that facilitate growth or adhesion). Further, the coatings may also be used to enhance the attachment of cells (e.g., polylysine). Alternatively, cell non-adherent coatings as described above can be used to prevent or inhibit cell binding.
- In embodiments, the porous membrane may be a substrate having an array of microwells.
- In some embodiments, the spheroid formed by the
cells 200 occludes theporous membrane 120 of the spheroid-promoting cell culture insert. (See for example,FIG. 1A ). In some aspects, this occlusion prevents the passage of proteins, small molecules, and/or media from going around the spheroid. - The combination of, for example, non-adherent sidewalls, geometry, and gravity can define a confinement volume in which growth of cells cultured in the inserts is limited. In embodiments, this combination can promote the formation of spheroids by cells cultured in the inserts. The confinement volume can be defined by the portion of the one or more sidewalls proximate the second end and the width or diagonal of the second opening. In some embodiments, the portion of the one or more sidewalls proximate the second end d2 is 500 μm, 450 μm, 400 μm, 350 μm, 300 μm, 250 μm, 200 μm, 150 μm, or 100 μm, or any length in between. In some embodiments the confinement volumes are defined by the wells of the microwell array substrate with the porous membrane that forms the bottom of the second opening. In which case, the second opening approximates the size of the first opening with the wells of the microwell array substrate comprising the confinement volume and have a diameter of
d 1 500 μm, 450 μm, 400 μm, 350 μm, 300 μm, 250 μm, 200 μm, 150 μm, or 100 μm, or any length in between. - Referring back to
FIG. 1C , in some embodiments, the spheroid-promoting cell culture insert can further include aledge 130 extending around the perimeter of the first open end where theledge 130 is sized to support the spheroid-promotingcell culture insert 100 when it is positioned inside areservoir 150. In some embodiments, at least a portion of thereservoir 150 is gas permeable. - As shown in
FIG. 1A , aspheroid 200 can grow in a spheroid-promotingcell culture insert 100, a portion of which can include aporous membrane 120. One or both of theporous membrane 120 and thelower sidewalls 110 can be non-adherent to cells. The slope of thesidewall 110 encourages theseeded cells 200 to aggregate on theporous membrane 120. One or more of gravity, an ultra-low binding material, the sidewall geometry of the cell culture insert, and the arcuate shape of the porous membrane can facilitate the formation of a spheroid. In addition, in embodiments, the porous membrane may contain an array of microwells structured and arranged to form spheroids. - As shown in
FIG. 1B , a spheroid can grow in a spheroid-promotingcell culture insert 100, a portion of which is aporous membrane 120. Theporous membrane 120 and thelower sidewalls 110 can be non-adherent to cells. The slope of thesidewall 110 encourages the seeded cells to aggregate on or occlude theporous membrane 120. One or more of gravity, an ultra-low binding material, the sidewall geometry of the cell culture insert, and the cell confinement volume around theporous membrane 120 can facilitate the formation of a spheroid. The size of the spheroid can be limited by the confinement volume. In embodiments, the second end of the insert can have a shape that provides a confinement volume that promotes spheroid formation, a spheroid confinement volume. - As shown in
FIG. 3A , in some embodiments, a spheroid-promotingcell culture insert 105 can have a firstopen end 101 and a second end, 102 and one or more sidewalls 110 extending from the first open end to the second end. In some embodiments, the sidewalls are sloped. In one aspect, the second end of the spheroid-promotingcell culture insert 105 contains multiple spheroid-promotingwells 400, where each spheroid-promoting well may have asidewall 410. In some embodiments, the spheroid-promotingcell culture insert 105 can have one or more sidewalls 110 that are non-adherent to cells. - In some embodiments, the substrate having an array of microwells is comprised of hexagonal close-packed well structures. An image of an embodiment of such a substrate having an array of
microwells 410 is shown inFIG. 3B , showing the hexagonally shapedwells 400.FIG. 3C is a schematic drawing showing a top-down view of an embodiment of a substrate having an array ofmicrowells 410. In some preferred embodiments, cells cultured within each well 400 form asingle spheroid 200. - As shown in
FIG. 3D , in some embodiments, thewells 400 of the spheroid-promotingcell culture insert 105 have an inner surface that defines an upper aperture and a nadir, or low point or surface. At the upper aperture the wells have a diametric dimension, such as a diameter, a width, a diagonal of a square or rectangle, or the like, d3, in a range from 100 μm to 1000 μm. Specifically, the well can have a diametric dimension d3 of 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, or 1000 μm, and any dimension encompassed within the range from 100 μm to 1000 μm. In some embodiments, the depth of the wells 400 d4 is 1000 μm, 500 μm, 450 μm, 400 μm, 350 μm, 300 μm, 250 μm, 200 μm, 150 μm, or 100 μm, or any dimension encompassed within the range from 100 μm to 1000 μm. - In some embodiments, a substrate having an array of microwells with a porous support forming the bottom of the
microwells 410 covers the second end of the spheroid-promotingcell culture insert 105. In some embodiments, at least a portion of the substrate having an array ofmicrowells 410 is non-adherent to cells. In some embodiments, at least a portion of the substrate having an array ofmicrowells 410 is adherent to cells. In some embodiments, a portion of the substrate having an array ofmicrowells 410 is porous. In further embodiments, a portion of the substrate having an array ofmicrowells 410 forming thewells 400 includes openings. In some embodiments, the substrate having an array ofmicrowells 410 can be adhered to, affixed to, or juxtaposed with aporous membrane 420. In one embodiment, the second end of the spheroid-promotingcell culture insert 105 is covered by aporous membrane 420, and the porous membrane defines the substrate having an array ofmicrowells 410. - In some embodiments, at least a portion of the substrate having an array of
microwells 410 forming thewells 400 of the spheroid-promotingcell culture insert 105 are sloped. In some embodiments, the substrate having an array ofmicrowells 410 forming thewells 400 can be sloped along the entire depth of the well. - A structured bottom surface as described herein can be formed in any suitable manner. For example, a substrate can be coined, injection molded or embossed to form the substrate having an array of
microwells 410. A porous material or a gas permeable material can be coined, injection molded or embossed to form a substrate having an array of microwells. - Referring now to
FIGS. 4A-B , in some embodiments, a spheroid-promotingcell culture insert 100 can be used in a nestedpermeable support device 600. In some embodiments, a spheroid-promotingcell culture insert 100 can be placed in another cell culture insert or device or another spheroid-promoting cell culture insert. In one aspect two, or three, or more spheroid-promoting cell culture inserts 100 can be nested. In another aspect, a spheroid-promotingcell culture insert 100 can be placed in another cell culture insert or can have another cell culture insert placed in it. In some embodiments, the spheroid-promoting cell culture insert can further include a ledge extending around the perimeter of the first open end where the ledge is sized to support the spheroid-promoting cell culture insert when it is positioned inside a reservoir or another cell culture insert. One having ordinary skill in the art of cell culture would recognize that any combination of spheroid-promoting cell culture inserts and other cell culture inserts could be constructed. Each cell culture insert can have aporous membrane 120 or can be gas permeable. In one embodiment, the lowest cell culture insert or reservoir is gas-permeable and the upper cell inserts haveporous membranes 120. - For example, as shown in
FIG. 4A , a nested device can include an upper 100 and a middle 101 spheroid-promoting cell culture insert. The upper 100 and middle 101 spheroid-promoting cell culture inserts can have aporous membrane 120 at the nadir. In embodiments, the porous membrane is a substrate having an array of microwells. The nested device can further include a spheroid-promotingcell culture reservoir 102 that does not have a permeable support. In some embodiments, the spheroid-promotingcell culture reservoir 102 can be made of or include a portion of a gas-permeable material. In embodiments the gas permeable material is a substrate having an array of microwells. - As shown in the embodiment shown is
FIG. 4B , only the middlecell culture insert 101 can be a spheroid-promoting cell culture insert, but both the uppermostcell culture insert 300 and middle spheroid-promotingcell culture insert 101 haveporous membranes 120. The lowest device can be areservoir 151. In one embodiment, thereservoir 151 can be gas-permeable. - As shown in
FIG. 5A , the middle cell culture insert can be a spheroid-promotingcell culture insert 105 that contains multiple spheroid-promotingporous wells 400. The uppermostcell culture insert 300 can have aporous membrane 120. The spheroid-promotingcell culture insert 105 can be inserted in or nested in areservoir 151. In one embodiment, thereservoir 151 can be gas-permeable. Using a spheroid-promotingcell culture insert 105 that contains multiple spheroid-promotingwells 400 permits tighter nesting with flat bottomed inserts that culture 2D cell sheets (such as those depicted inFIGS. 4 A&B and would also provide more spheroids for amplifying signal and the cell processing rate, preventing bottlenecks in a testing system.FIG. 5B shows an embodiment of an apparatus with first 105 and second 106 spheroid-promoting cell culture inserts havingporous membrane bottoms 120 in a spheroid-promotingreservoir 151 having a non-liquid permeable, gas permeable bottom. In addition to providing greater signal amplification the more physiologic functionality of the spheroids can better approximate a replacement for animal testing. As will be understood by those of ordinary skill in the art, any combination of cell culture supports, each having porous membranes or not, having substrates having an array of microwells for spheroid promotion or not, or being gas permeable or not, are possible depending on the desired cell culture environment. - In some embodiments, the spheroid-promoting cell culture inserts can be used in a method to determine whether a compound or molecule known as a NCE has a desired biological activity. Such methods are described in, for example, U.S. Pat. No. 8,163,537. These methods often entail examining the Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADME-Tox) of the NCE, as well as determining the NCE's level of effectiveness for the targeted therapeutic indication including pharmacokinetic parameters. One type of assessment examines the “first pass effect.” This assessment involves experimental determination of the bioavailabilty of the NCE following its absorption through the digestive tract and then its metabolism by the liver. Commonly, the assessment of the “first pass effect” requires two separate in vitro assays to be conducted, and the data combined, to determine the intestinal permeability and the hepatic metabolism. If desired, additional studies may be conducted to determine target selectivity, efficacy and dosage (Lau et al., Drug Metabolism and Disposition, Vol. 32, No. 9, pp. 937-942, 2004).
- A well-known method used today to examine the intestinal absorption of a NCE is known as the Caco 2 cell-based assay which is typically conducted on permeable supports such as the ones sold under the brand name of Transwell™ and manufactured by Corning Inc. (“Transwell® Permeable Supports: Including Snapwell™ and Netwell™ Inserts-Instructions for Use” Corning Inc., September 2007.) The design of the Transwell™ permeable support facilitates the development of Caco 2 cell polarization to create more in vivo-like test conditions. Researchers from the Schering-Plough Research Institute have expanded the utility of the Caco 2 cell-based assay by adding hepatocytes in the nutrient medium to a Transwell™ receiver plate which receives the Transwell™ permeable support. In this way, the researchers were able to more accurately predict the oral bioavailability of NCE's. However, the hepatic cell viability under these conditions during a 3 hour incubation period was only 50-70%, limiting the potential of this method (Lau et al., Drug Metabolism and Disposition, Vol. 32, No. 9, pp. 937-942, 2004). Another group of researchers from the University of Tokyo co-cultured Caco-2 cells on the Transwell™ permeable support with monolayers of Hep G2 cells growing on the inner surface of the Transwell™ receiver plate. While useful for some assays, the Hep G2 cells did not maintain the functions that are representative of in vivo hepatocytes (Choi et al., Toxicology in Vitro, vol. 18, pages 393-402, 2004).
- The current United States Food and Drug Administration Guidance regarding drug interaction studies like the first pass assay recommends the use of in vitro assays with fresh or cryopreserved human hepatocytes due to species specific responses. (U.S. Department of Health and Human Services et al. “Guidance for Industry: Drug Interaction Studies-Study, Design, Data Analysis, and Implications for Dosing and Labeling”, Clinical Pharmacology, September 2006.) However, it is well known that primary hepatocytes loose differentiated function rapidly in standard cell culture conditions on tissue culture treated polystyrene. The loss of normal differentiated hepatocyte function decreases the in vivo-like conditions and hence also decreases the relevance of experimental data in ADME-Tox and pharmacokinetic in vitro assays.
- In some embodiments of the methods described herein, hepatocytes or Hep G2 cells used in a Caco 2 cell-based assay are cultured in a spheroid-promoting cell culture insert.
- In some embodiments, the present disclosure relates to a nested permeable support device and methods for using the nested permeable support device to perform various experiments to test new therapeutic compounds, NBEs, or NCEs.
- In some embodiments, the spheroid-promoting cell culture inserts can be used to assess system-like communication information. In some embodiments, the spheroid-promoting cell culture inserts can be used to create cell models that represent a series of human organs in vertical orientation.
- In one aspect, the nested permeable support device is used to form a first pass assay to determine the bioavailability of a NCE or NBE following absorption from the digestive tract and metabolism by the liver.
- In one embodiment, the nested
permeable support device 600 can be used to perform a first pass assay to determine the bioavailability of a NCE following absorption through the digestive tract and metabolism by the liver. In another embodiment, the nestedpermeable support device 600 can be used to perform a first pass assay to determine the bioavailability of a NBE following absorption through the digestive tract and metabolism by the liver. - For instance, a researcher using the embodiment shown in
FIG. 5A can place a media in a growing reservoir and then place theupper insert 300 in the growing reservoir. Theupper insert 300 is then filled with a volume of Caco 2 cells in media. Theupper insert 300 and growing reservoir are in communication until a confluent monolayer of Caco 2 cells is formed across the permeable support of theupper insert 300. It usually takes about a month for Caco 2 cells to form across the permeable support of theupper insert 300. The Caco 2 cells can be tested electronically to determine how tightly the Caco 2 cells adhere to one another by performing a Trans Epithelial Electrical Resistance (TEER) test, where a probe is inserted into theupper insert 300 and then the probe initiates a pulse that is detected by another probe located in the growing reservoir below the permeable support. Another test that can be performed uses a dye called Lucifer yellow, which can pass through gaps in the Caco 2 cell monolayer. The more Lucifer yellow that shows up in the growing reservoir after being introduced in theupper insert 300, the less mature (or confluent) the monolayer of Caco 2 cells. Tests such as these can be performed to make sure the Caco 2 cell culture is functioning as expected. - In parallel, the researcher can place a media in another growing reservoir and then place the
middle insert 105 in this growing reservoir. Themiddle insert 105 is then filled with a volume of hepatocytes in media. Themiddle insert 105 and reservoir are in communication until a spheroid of hepatocytes is formed across the second permeable support. Tests could also be conducted to assure that the hepatocytes are functioning appropriately. Alternatively, theupper insert 300 andmiddle insert 105 can be placed in areservoir 151 to grow the Caco 2 cells and the hepatocytes. - Once the Caco 2 cells and the hepatocytes have been cultured, the upper and
middle inserts middle insert 105 would be placed (nested) in thereservoir 151 which contains a media. Some media would then be placed above the layer of hepatocytes located within themiddle insert 105. Then, theupper insert 300 would be placed (nested) in themiddle insert 105. The NCE/NBE and media would be dispensed above the layer of Caco 2 cells located within theupper insert 300. After a period of incubation, theupper insert 300 could be removed and the media in themiddle insert 105 could be tested (i.e., LS/MS) to determine if the NCE/NBE passed through the intestinal epithelium (Caco 2 cells). If the NCE/NBE did pass through the Caco 2 cells, then themiddle insert 105 could be removed and the media in thereservoir 151 could be tested (i.e., LC/MS) to check the bioavailability of the NCE and/or how the NCE/NBE is metabolized by the liver (hepatocytes) to form metabolic products. The hepatocytes could also show if the NCE/NBE is toxic at the dosage applied. If there are target cells (or molecules) on the bottom of thereservoir 151, then these could be examined (i.e., LC/MS) to determine the drug effects either microscopically, or by using an assay that is separate from the Caco 2 cells and hepatocytes by pulling out theinserts reservoir 151 could be examined using an interrogation system to assess function and viability as described, for example, in U.S. Pat. No. 8,163,537. An assay such as this will enable understanding of whether 1) an NCE/NBE can pass through the intestinal epithelium; 2) whether the liver metabolizes or is damaged by an NCE/NBE; and 3) the effect on the target cells of unmodified or liver-metabolized NCE/NBE. - In another embodiment, the nested
permeable support device 100 can be used to test a NCE which would not pass through the digestive tract but instead would enter the body via inhalation in which case the Caco 2 cells would be replaced with nasal mucosal cells, bronchial cells or lung epithelial cells, etc. In practice, the researcher would typically select the actual cells used in the nestedpermeable support device 600. - In some embodiments, particularly where multiple wells are provided on a second end or where nested supports are employed, multiple spheroids may be grown. In some embodiments, the spheroids are all the same. In other embodiments, two or more different types of spheroids are used (e.g., a co-culture system to, for example, simulate or reconstitute the multicellular functionality of an organ). Where cells are imaged or signal generated from the cells is detected, the response from multiple cells may simultaneously be analyzed or the results from individual cells or groups of cells pooled, as desired.
- Cells cultured in three dimensions, such as spheroids, can exhibit more in vivo like functionality than their counterparts cultured in two dimensions as monolayers. In two dimensional cell culture systems, cells can attach to a substrate on which they are cultured. However, when cells are grown in three dimensions, such as spheroids, the cells interact with each other rather than attaching to the substrate. Cells cultured in three dimensions more closely resemble in vivo tissue in terms of cellular communication and the development of extracellular matrices. Spheroids thus provide a superior model for cell migration, differentiation, survival, and growth and therefore provide better systems for research, diagnostics, and drug efficacy, pharmacology, and toxicity testing.
- In some embodiments, the devices are configured such that cells cultured in the devices form spheroids. For example, the wells in which cells are grown can be non-adherent to cells to cause the cells in the wells to associate with each other and form spheres. The spheroids expand to size limits imposed by the geometry of the wells. In some embodiments, the wells are coated with an ultra-low binding material to make the wells non-adherent to cells.
- Examples of non-adherent material include perfluorinated polymers, olefins, or like polymers or mixtures thereof. Other examples include agarose, non-ionic hydrogels such as polyacrylamides, polyethers such as polyethylene oxide and polyols such as polyvinyl alcohol, or like materials or mixtures thereof. The combination of, for example, non-adherent wells, well geometry (e.g., size and shape), and/or gravity induce cells cultured in the wells to self-assemble into spheroids. Some spheroids maintain differentiated cell function indicative of a more in vivo-like, response relative to cells grown in a monolayer. Other cells types, such as mesenchymal stromal cells, when cultured as spheroids retain their pluripotency.
- In some embodiments, the systems, devices, and methods herein comprise one or more cells. In some embodiments, the cells are cryopreserved. In some embodiments, the cells are in three dimensional culture. In some such embodiments, the systems, devices, and methods comprise one or more spheroids. In some embodiments, one or more of the cells are actively dividing. In some embodiments, the systems, devices, and methods comprise culture media (e.g., comprising nutrients (e.g., proteins, peptides, amino acids), energy (e.g., carbohydrates), essential metals and minerals (e.g., calcium, magnesium, iron, phosphates, sulphates), buffering agents (e.g., phosphates, acetates), indicators for pH change (e.g., phenol red, bromo-cresol purple), selective agents (e.g., chemicals, antimicrobial agents), etc.). In some embodiments, one or more test compounds (e.g., drug) are included in the systems, devices, and methods.
- A wide variety of cell types may be cultured. In some embodiments, a spheroid contains a single cell type. In some embodiments, a spheroid contains more than one cell type. In some embodiments, where more than one spheroid is grown, each spheroid is of the same type, while in other embodiments, two or more different types of spheroids are grown. Cells grown in spheroids may be natural cells or altered cells (e.g., cell comprising one or more non-natural genetic alterations). In some embodiments, the cell is a somatic cell. In some embodiments, the cell is a stem cell or progenitor cell (e.g., embryonic stem cell, induced pluripotent stem cell) in any desired state of differentiation (e.g., pluripotent, multi-potent, fate determined, immortalized, etc.). In some embodiments, the cell is a disease cell or disease model cell. For example, in some embodiments, the spheroid comprises one or more types of cancer cells or cells that can be induced into a hyper-proliferative state (e.g., transformed cells). Cells may be from or derived from any desired tissue or organ type, including but not limited to, adrenal, bladder, blood vessel, bone, bone marrow, brain, cartilage, cervical, corneal, endometrial, esophageal, gastrointestinal, immune system (e.g., T lymphocytes, B lymphocytes, leukocytes, macrophages, and dendritic cells), liver, lung, lymphatic, muscle (e.g., cardiac muscle), neural, ovarian, pancreatic (e.g., islet cells), pituitary, prostate, renal, salivary, skin, tendon, testicular, and thyroid. In some embodiments, the cells are mammalian cells (e.g., human, mice, rat, rabbit, dog, cat, cow, pig, chicken, goat, horse, etc.).
- The cultured cells find use in a wide variety of research, diagnostic, drug screening and testing, therapeutic, and industrial applications.
- In some embodiments, the cells are used for production of proteins or viruses. Systems, devices, and methods that culture large numbers of spheroids in parallel are particularly effective for protein production. Three-dimensional culture allows for increased cell density, and higher protein yield per square centimeter of cell growth surface area. Any desired protein or viruses for vaccine production may be grown in the cells and isolated or purified for use as desired. In some embodiments, the protein is a native protein to the cells. In some embodiments, the protein is non-native. In some embodiments, the protein is expressed recombinantly. Preferably, the protein is overexpressed using a non-native promoter. The protein may be expressed as a fusion protein. In some embodiments, a purification or detection tag is expressed as a fusion partner to a protein of interest to facilitate its purification and/or detection. In some embodiments, fusions are expressed with a cleavable linker to allow separation of the fusion partners after purification.
- In some embodiments, the protein is a therapeutic protein. Such proteins include, but are not limited to, proteins and peptides that replace a protein that is deficient or abnormal (e.g., insulin), augment an existing pathway (e.g., inhibitors or agonists), provide a novel function or activity, interfere with a molecule or organism, or deliver other compounds or proteins (e.g., radionuclides, cytotoxic drugs, effector proteins, etc.). In some embodiments, the protein is an immunoglobulin such as an antibody (e.g., monoclonal antibody) of any type (e.g., humanized, bi-specific, multi-specific, etc.). Therapeutic protein categories include, but are not limited to, antibody-based drugs, Fc fusion proteins, anticoagulants, antigens, blood factor, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. Therapeutic proteins may be used to prevent or treat cancers, immune disorders, metabolic disorders, inherited genetic disorders, infections, and other diseases and conditions.
- In some embodiments, the protein is a diagnostic protein. Diagnostic proteins include, but are not limited to, antibodies, affinity binding partners (e.g., receptor-binding ligands), inhibitors, antagonists, and the like. In some embodiments, the diagnostic protein is expressed with or is a detectable moiety (e.g., fluorescent moiety, luminescent moiety (e.g., luciferase), colorimetric moiety, etc.).
- In some embodiments, the protein is an industrial protein. Industrial proteins include, but are not limited to, food components, industrial enzymes, agricultural proteins, analytical enzymes, etc.
- In some embodiments, the cells are used drug discovery, characterization, efficacy testing, and toxicity testing. Such testing includes, but is not limited to, pharmacological effect assessment, carcinogenicity assessment, medical imaging agent characteristic assessment, half-life assessment, radiation safety assessment, genotoxicity testing, immunotoxicity testing, reproductive and developmental testing, drug interaction assessment, dose assessment, adsorption assessment, disposition assessment, metabolism assessment, elimination studies, etc. Specific cells types may be employed for specific tests (e.g., hepatocytes for liver toxicity, renal proximal tubule epithelial cells for nephrotoxicity, vascular endothelial cells for vascular toxicity, neuronal and glial cells for neurotoxicity, cardiomyocytes for cardiotoxicity, skeletal myocytes for rhabdomyolysis, etc.). Treated cells may be assessed for any number of desired parameters including, but not limited to, membrane integrity, cellular metabolite content, mitochondrial functions, lysosomal functions, apoptosis, genetic alterations, gene expression differences, and the like.
- In some embodiments, the cell culture devices are a component of a larger system. In some embodiments, the system comprises a plurality (e.g., 2, 3, 4, 5, . . . , 10, . . . , 20, . . . , 50, . . . , 100, . . . , 1000, etc.) of such cell culture devices. In some embodiments, the system comprises an incubator for maintaining the culture devices at optimal culture conditions (e.g., temperature, atmosphere, humidity, etc.). In some embodiments, the system comprises detectors for imaging or otherwise analyzing cells. Such detectors include, but are not limited to, fluorimeters, luminometers, cameras, microscopes, plate readers (e.g., PERKIN ELMER ENVISION plate reader; PERKIN ELMER VIEWLUX plate reader), cell analyzers (e.g., GE IN Cell Analyzer 2000 and 2200; THERMO/CELLOMICS CELLNSIGHT High Content Screening Platform), and confocal imaging systems (e.g., PERKIN ELMER OPERAPHENIX high throughput content screening system; GE INCELL 6000 Cell Imaging System). In some embodiments, the system comprises perfusion systems or other components for supplying, re-supplying, and circulating culture media or other components to cultured cells. In some embodiments, the system comprises robotic components (e.g., pipettes, arms, plate movers, etc.) for automating the handing, use, and/or analysis of culture devices.
- A number of aspects of inserts, methods and assemblies have been disclosed herein. A summary of some selected aspects is presented below. In a first aspect, a cell culture insert comprises (i) a body having a first open end, a second end wherein the second end defines an opening having a diametric dimension in a range from 100 μm to 1000 μm, and one or more sidewalls extending from the first open end to the second end; wherein the one or more sidewalls are sloped; and (ii) a porous membrane disposed over the opening of the second end.
- A second aspect is a cell culture insert according the first aspect wherein at least a portion of the one or more sidewalls are non-adherent to cells.
- A third aspect is a cell culture insert according to aspect 1 or aspect 2, wherein at least a portion of the porous membrane is non-adherent to cells.
- A fourth aspect is a cell culture insert according to any one of aspects 1 to 3, wherein at least a portion of the porous membrane is adherent to cells.
- A fifth aspect is a cell culture insert according to any one of aspects 1 to 4, wherein a portion of the one or more sidewalls proximate the second end at least partially define a cell confinement volume.
- A sixth aspect is a cell culture insert according to aspect 5 wherein a depth of the confinement volume is in a range from 100 μm to 1000 μm.
- A seventh aspect is a cell culture insert according to any one of aspects 1 to 6, wherein the insert is configured such that cells cultured in the insert form a spheroid.
- An eighth aspect is a method for culturing a spheroid comprising (i) placing cell culture insert according to aspect 7 in a reservoir, the reservoir having a bottom, wherein insert is placed in the reservoir such that the second end of body of the insert is positioned above the bottom of the insert; (ii) introducing cells into the insert; (iii) introducing a cell culture medium into the insert; and (iv) culturing the cells in the cell culture medium in the insert to form the spheroid.
- A ninth aspect is a cell culture assembly, comprising (i) a reservoir defining an interior and having a bottom; and (ii) a first cell culture insert according to any one of aspects 1 to 7 configured to be positioned in the interior of the reservoir such that the second end of the body is above the bottom or the reservoir, wherein the body of the first insert defines an interior of the first insert.
- A tenth aspect is a cell culture assembly according to aspect 9, wherein the interior of the first insert, when the first insert is positioned in the interior of the reservoir, is in fluid communication with the interior of the reservoir only through the porous membrane disposed over the opening of the second end of the body of the first insert.
- An eleventh aspect is a cell culture assembly according to aspect 9 or aspect 10, further comprising a second insert having a body defining an interior, the body comprising a first open end, a second end defining an opening, and one or more sidewalls extending from the first open end to the second end, wherein the second insert is configured to be positioned in the interior of the first insert such that the second end of the body of the second insert above the second end of the body of the first insert.
- A twelfth aspect is a cell culture assembly according to aspect 11, wherein the interior of the second insert, when the second insert is positioned in the interior of the first insert, is in fluid communication with the interior of the first insert only through the porous membrane disposed over the opening of the second end of the body of the second insert.
- A thirteenth aspect is a method comprising (i) introducing target cells and a cell culture medium to an interior of a reservoir of a cell culture assembly according to aspect 12 such that the target cells grow on the bottom of the reservoir; (ii) positioning a first cell culture insert according to aspect 12 in the interior of the reservoir; (iii) introducing a plurality of a first type of cells and a cell culture medium into the interior of the first cell culture insert such that cells of the first type grow as a spheroid in proximity to the porous membrane of the first insert; (iv) positioning a second cell culture insert according to aspect 12 in the interior of the first insert; and (v) introducing a plurality of a second type of cells and a cell culture medium into the interior of the second cell culture insert such that the cells of the second type grow in proximity to the porous membrane of the second insert.
- A fourteenth aspect is a method according to aspect 13, wherein the cells of the second type cover the porous membrane of the second insert such that compounds or metabolic derivatives thereof that move from the interior of the second insert to the interior of the first insert pass through the cells of the second type.
- A fifteenth aspect is a method according to aspect 14, wherein the cells of the first type attach to the porous membrane of the first insert such that compounds or metabolic derivatives thereof that move from the interior of the first insert to the interior of the reservoir pass through the cells of the first type.
- A sixteenth aspect is a method according to aspect 15, further comprising: (i) introducing a test compound to the interior of the second insert; and (ii) identifying an effect of the test compound or a metabolic derivative thereof on the target cells.
- A seventeenth aspect is a method according to any of aspects 13 to 16, wherein the cells of the first type are hepatocytes.
- An eighteenth aspect is a method according to any of aspects 13 to 17, wherein the cells of the second type are Caco 2 cells.
- A nineteenth aspect is a cell culture insert comprising: (i) a body having a first open end, a second end wherein the second end defines an opening, and one or more sidewalls extending from the first open end to the second end; wherein the one or more sidewalls are sloped; and (ii) a porous membrane disposed over the opening of the second end, wherein the porous membrane is non-adherent to cells.
- A twentieth aspect is a permeable support device configured to be at least partially inserted into a reservoir of a cell culture device, the permeable support device comprising a first well having a tapered shape and bottom at least partially defined by a first permeable support.
- A twenty-first aspect is a permeable support device of aspect 20 wherein the well is configured such that cells cultured in the well form a spheroid.
- A twenty-second aspect is a permeable support device of either of aspects 20 or 21 wherein at least a portion of the first well is coated with an ultra-low binding material.
- A twenty-third aspect is a permeable support device of any of aspect 20 to 22, wherein at least a portion of the permeable support is configured to attach to cells cultured in the first well.
- A twenty-fourth aspect is a permeable support device of any of aspects 20 to 23 wherein at least a portion of the first well comprises an arcuate shape
- A twenty-fifth aspect is a permeable support device of any of aspects 20 to 24 wherein at least a portion of the first well comprises a conical shape.
- A twenty-sixth aspect is a permeable support device of any of aspects 20 to 25 wherein a portion of the well defines a confinement volume.
- A twenty-seventh aspect is a permeable support device of aspect 26 wherein a diametric dimension of the confinement volume is in a range from 200 μm to 500 μm.
- A twenty-eighth aspect The permeable support device of either of aspects 26 or 27 wherein the depth of the confinement volume is in a range from 100 μm to 500 μm.
- A twenty-ninth aspect is a permeable support device of aspect any of aspects 20 to 28 wherein the first well is configured and sized to receive a second well having a bottom, wherein the second well is located above the first well.
- A thirtieth aspect is a nested permeable support device comprising: (i) a first well having a tapered shape and a bottom portion at least partially defined by a first permeable support; and (ii) a reservoir having a bottom located below the first well.
- A thirty-first aspect is a nested permeable support device of aspect 30 wherein the well is configured such that cells cultured in the well form a spheroid.
- A thirty-second aspect is a nested permeable support device of either of aspects 30 or 31 wherein at least a portion of the first well is coated with an ultra-low binding material.
- A thirty-third aspect is a nested permeable support device of any of aspects 29 to 32, wherein at least a portion of the permeable support is configured to attach to cells cultured in the first well.
- A thirty-fourth aspect is a nested permeable support device of any of aspects 30 to 33 wherein at least a portion of the first well comprises an arcuate shape.
- A thirty-fifth aspect is a nested permeable support device of any of aspects 30 to 34 wherein at least a portion of the first well comprises a conical shape.
- A thirty-sixth aspect is a nested permeable support device of any of aspects 30 to 35 wherein a portion of the well defines a confinement volume.
- A thirty-seventh aspect is a nested permeable support device of aspect 36 wherein a diametric dimension of the confinement volume is in a range from 100 μm to 1000 μm, e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 μm, including ranges between any of the foregoing.
- A thirty eighth aspect is a nested permeable support device of either of aspects 36 or 37 wherein the depth of the confinement volume is in a range from 100 μm to 1000 μm, e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 μm, including ranges between any of the foregoing.
- A thirty-ninth aspect is a nested permeable support device of any of aspects 30 to 38 further comprising a second well having a bottom at least partially defined by a second permeable support, and wherein the first well and the first permeable support are located below the second well and the second permeable support.
- A fortieth aspect is a nested permeable support device of aspect 39 wherein the second well comprises a tapered shape.
- A forty-first aspect is a nested permeable support device of any of aspect 30 to 40 wherein the bottom of the reservoir comprises a gas permeable material.
- A forty-second aspect is a nested permeable support device comprising: (i) a first well having a bottom, wherein at least a portion of the bottom is formed by a first permeable support; (ii) a second well having a bottom, wherein the bottom of the second well comprises an arcuate shape and at least a portion of the bottom is formed by a second permeable support, and wherein the second well and the second permeable support are located below the first well and the first permeable support; and (ii) a third well having a bottom which is located below the second well and the second permeable support.
- A forty-third aspect is a nested permeable support device of aspect 42 wherein at least a portion of the second well is coated with an ultra-low binding material.
- A forty-fourth aspect is a cell culture insert comprising a body having a first open end, a second end, and one or more sidewalls extending from the first open end to the second end; and wherein the second end comprises a substrate having an array of microwells defining wells, wherein at least a portion of a substrate having an array of microwells defining a well is sloped.
- A forty-fifth aspect is a cell culture insert of aspect 44 wherein the sidewalls are sloped.
- A forty-sixth aspect is a cell culture insert of either of aspect 44 or 45 wherein the sidewalls are non-adherent to cells.
- A forty-seventh aspect is a cell culture insert of any of aspects 44 to 46 wherein at least a portion of the substrate having an array of microwells is non-adherent to cells.
- A forty-eighth aspect is a cell culture insert of any of aspects 44 to 47 wherein the wells have an inner surface defining an upper aperture and wherein the wells have a diametric dimension at the upper aperture in a range from 100 μm to 1000 μm.
- A forty-ninth aspect is a cell culture insert of any of aspects 44 to 48 wherein the wells have a depth in a range from 100 μm to 100 μm.
- A fiftieth aspect is a cell culture insert of any of aspects 44 to 49 wherein at least a portion of the substrate having an array of microwells is non-adherent to cells.
- A fifty-first aspect is a cell culture insert of any of aspects 44 to 49 wherein at least a portion of the substrate having an array of microwells is adherent to cells.
- A fifty-second aspect is a cell culture insert of any of aspects 44 to 51 wherein at least a portion of the substrate having an array of microwells is porous.
- A fifty-third aspect is a cell culture insert of any of aspects 44 to 52 wherein the substrate having an array of microwells comprises openings.
- A fifty-fourth aspect is a cell culture insert of any of aspects 44 to 53 wherein the substrate having an array of microwells is adhered to, affixed to, or juxtaposed with a porous membrane.
- A fifty-fifth aspect is a cell culture insert of any of aspects 44 to 54 wherein the second end is covered by a porous membrane.
- A fifty-sixth aspect is a cell culture insert of any of aspects 44 to 55 wherein the substrate having an array of microwells comprises a sloped surface.
- A fifty-seventh aspect is a cell culture insert of any of aspects 44 to 56 wherein the substrate having an array of microwells comprises an array of hexagonal structures.
- A fifty-eighth aspect is a cell culture assembly, comprising: (i) a reservoir defining an interior and having a bottom; and (ii) a first cell culture insert according to any one of aspects 44-57 configured to be positioned in the interior of the reservoir such that the second end of the body is above the bottom of the reservoir, wherein the body of the first insert defines an interior of the first insert.
- A fifty-ninth aspect is a cell culture assembly according to aspect 58, wherein the interior of the first insert, when the first insert is positioned in the interior of the reservoir, is in fluid communication with the interior of the reservoir only through the porous membrane disposed over the opening of the second end of the body of the first insert.
- A sixtieth aspect is a cell culture assembly according to aspect 58 or aspect 59, further comprising a second insert having a body defining an interior, the body comprising a first open end, a second end defining an opening, and one or more sidewalls extending from the first open end to the second end, wherein the second insert is configured to be positioned in the interior of the first insert such that the second end of the body of the second insert above the second end of the body of the first insert.
- A sixty first aspect is a cell culture assembly according to aspect 60, wherein the interior of the second insert, when the second insert is positioned in the interior of the first insert, is in fluid communication with the interior of the first insert only through the porous membrane disposed over the opening of the second end of the body of the second insert.
- A sixty second aspect is a cell culture insert comprising: a body sized for insert into a reservoir of a cell culture device, said body having a first open end, a second end having a porous membrane, and one or more sidewalls extending from the first open end to the second end; wherein the one or more sidewalls are sloped; and wherein said second end has an upper surface defining a plurality of microwells sized for spheroid growth.
- A sixty third aspect is the cell culture insert of aspect 62, wherein said microwells each have a diameter in a range from 100 μm to 1000 μm.
- Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that any particular order be inferred. Any recited single or multiple feature or aspect in any one claim can be combined or permuted with any other recited feature or aspect in any other claim or claims.
- It is also noted that recitations herein refer to a component being “configured” or “adapted to” function in a particular way. In this respect, such a component is “configured” or “adapted to” embody a particular property, or function in a particular manner, where such recitations are structural recitations as opposed to recitations of intended use. More specifically, the references herein to the manner in which a component is “configured” or “adapted to” denotes an existing physical condition of the component and, as such, is to be taken as a definite recitation of the structural characteristics of the component.
- While various features, elements or steps of particular embodiments may be disclosed using the transitional phrase “comprising,” it is to be understood that alternative embodiments, including those that may be described using the transitional phrases “consisting” or “consisting essentially of,” are implied. Thus, for example, implied alternative embodiments to a cell culture insert comprising body and a porous membrane include embodiments where a cell culture insert consists of a body and a porous membrane and embodiments where a cell culture insert consists essentially of a body and a porous membrane.
- It will be apparent to those skilled in the art that various modifications and variations can be made to the present inventive technology without departing from the spirit and scope of the disclosure. Since modifications, combinations, sub-combinations and variations of the disclosed embodiments incorporating the spirit and substance of the inventive technology may occur to persons skilled in the art, the inventive technology should be construed to include everything within the scope of the appended claims and their equivalents
- In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The embodiments are not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the embodiments defined by the claims.
- Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
- Notwithstanding that the numerical ranges and parameters are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
- Any direction referred to herein, such as “top,” “bottom,” “left,” “right,” “upper,” “lower,” “above,” below,” and other directions and orientations are described herein for clarity in reference to the figures and are not to be limiting of an actual device or system or use of the device or system. Many of the devices, articles or systems described herein may be used in a number of directions and orientations. Directional descriptors used herein with regard to cell culture apparatuses often refer to directions when the apparatus is oriented for purposes of culturing cells in the apparatus.
- The words “preferred” and “preferably” refer to embodiments that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments.
- As used herein, “have”, “has”, “having”, “include”, “includes”, “including”, “comprise”, “comprises”, “comprising” or the like are used in their open ended inclusive sense, and generally mean “include, but not limited to”, “includes, but not limited to”, or “including, but not limited to”.
- “Optional” or “optionally” means that the subsequently described event, circumstance, or component, can or cannot occur, and that the description includes instances where the event, circumstance, or component, occurs and instances where it does not.
- Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.
- Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, examples include from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
- For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
- A number of abbreviations are used herein. A listing of some of those abbreviations and their meaning are presented below:
- All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.
Claims (36)
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US15/492,730 US20170218321A1 (en) | 2014-10-29 | 2017-04-20 | Cell culture insert |
US17/691,511 US11976263B2 (en) | 2014-10-29 | 2022-03-10 | Cell culture insert |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201462069996P | 2014-10-29 | 2014-10-29 | |
PCT/US2015/058053 WO2016069895A1 (en) | 2014-10-29 | 2015-10-29 | Cell culture insert |
US15/492,730 US20170218321A1 (en) | 2014-10-29 | 2017-04-20 | Cell culture insert |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2015/058053 Continuation WO2016069895A1 (en) | 2014-10-29 | 2015-10-29 | Cell culture insert |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/691,511 Continuation US11976263B2 (en) | 2014-10-29 | 2022-03-10 | Cell culture insert |
Publications (1)
Publication Number | Publication Date |
---|---|
US20170218321A1 true US20170218321A1 (en) | 2017-08-03 |
Family
ID=54477377
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/492,730 Abandoned US20170218321A1 (en) | 2014-10-29 | 2017-04-20 | Cell culture insert |
US17/691,511 Active 2035-12-11 US11976263B2 (en) | 2014-10-29 | 2022-03-10 | Cell culture insert |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/691,511 Active 2035-12-11 US11976263B2 (en) | 2014-10-29 | 2022-03-10 | Cell culture insert |
Country Status (7)
Country | Link |
---|---|
US (2) | US20170218321A1 (en) |
EP (1) | EP3212759A1 (en) |
JP (1) | JP6731916B2 (en) |
KR (1) | KR102460969B1 (en) |
CN (1) | CN107109328B (en) |
SG (1) | SG11201703493SA (en) |
WO (1) | WO2016069895A1 (en) |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20170253844A1 (en) * | 2016-03-04 | 2017-09-07 | Corning Incorporated | Bowl shaped microwell |
CN107794223A (en) * | 2017-11-07 | 2018-03-13 | 广东药科大学附属第医院 | Anaerobic bacteria and the in vitro study model and method of aerobic cell interaction in cell co-culture device and analogue body |
WO2019114997A1 (en) * | 2017-12-15 | 2019-06-20 | Technische Universität Ilmenau | Cell culture carrier |
CN110903976A (en) * | 2019-12-20 | 2020-03-24 | 江苏信安佳医疗科技有限公司 | A orifice plate device for organoid spheroid is cultivateed |
WO2020091894A1 (en) * | 2018-10-28 | 2020-05-07 | Schickwann Tsai | Low-macrophage-adhesion/activation culture devices and methods thereof for continuous hematopoiesis and expansion of hematopoietic stem cells |
WO2021075810A1 (en) * | 2019-10-14 | 2021-04-22 | 연세대학교 산학협력단 | Well plate unit and multi-layer spheroid culture apparatus using same |
KR20210043968A (en) * | 2019-10-14 | 2021-04-22 | 연세대학교 산학협력단 | Well plate unit for spheroid culture and well plate unit for multilayer spheroid culture apparatus |
WO2021108346A1 (en) * | 2019-11-25 | 2021-06-03 | Wake Forest University Health Sciences | Microwell perfusion plates for organoids and related systems and methods |
CN116478818A (en) * | 2023-05-11 | 2023-07-25 | 江苏艾玮得生物科技有限公司 | Cell culture unit, device, application and culture method |
Families Citing this family (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9790465B2 (en) | 2013-04-30 | 2017-10-17 | Corning Incorporated | Spheroid cell culture well article and methods thereof |
EP3212759A1 (en) | 2014-10-29 | 2017-09-06 | Corning Incorporated | Cell culture insert |
JP6930914B2 (en) | 2014-10-29 | 2021-09-01 | コーニング インコーポレイテッド | Perfusion bioreactor platform |
US11857970B2 (en) | 2017-07-14 | 2024-01-02 | Corning Incorporated | Cell culture vessel |
CN111094536B (en) | 2017-07-14 | 2024-03-29 | 康宁股份有限公司 | Cell culture vessel for 3D culture and method for culturing 3D cells |
US11345880B2 (en) | 2017-07-14 | 2022-05-31 | Corning Incorporated | 3D cell culture vessels for manual or automatic media exchange |
EP3652291B1 (en) * | 2017-07-14 | 2021-12-29 | Corning Incorporated | Cell culture vessel |
KR102072156B1 (en) | 2017-12-08 | 2020-01-31 | 한국생산기술연구원 | Culture device of inserting type for three dimensional cell culture, kit and co-culture method of three dimensional cell using the same |
EP3728547A1 (en) * | 2017-12-20 | 2020-10-28 | Philip Morris Products S.a.s. | Improved cell culture device |
CN111868524A (en) * | 2018-03-13 | 2020-10-30 | 康宁股份有限公司 | High-density 3D hepatocyte spheroid platform for ADME research |
WO2020013847A1 (en) | 2018-07-13 | 2020-01-16 | Corning Incorporated | Microcavity dishes with sidewall including liquid medium delivery surface |
WO2020013845A1 (en) | 2018-07-13 | 2020-01-16 | Corning Incorporated | Cell culture vessels with stabilizer devices |
US11661574B2 (en) | 2018-07-13 | 2023-05-30 | Corning Incorporated | Fluidic devices including microplates with interconnected wells |
JP7271903B2 (en) | 2018-10-20 | 2023-05-12 | 東洋製罐グループホールディングス株式会社 | Sphere culture member, culture vessel, perforated member processing method, and washing vessel |
KR102441029B1 (en) * | 2020-09-21 | 2022-09-07 | 건양대학교 산학협력단 | The membrane-free micro-transwell, 3-D cell culture method and ECM analysis method using it |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030215941A1 (en) * | 2002-03-12 | 2003-11-20 | Stewart Campbell | Assay device that analyzes the absorption, metabolism, permeability and/or toxicity of a candidate compound |
US20100190197A1 (en) * | 2009-01-27 | 2010-07-29 | Martin Gregory R | Nested permeable support device and method for using the nested permeable support device |
US20130344598A1 (en) * | 2011-01-24 | 2013-12-26 | California Stem Cell, Inc. | Neural cell purification for transplantation |
US20140227784A1 (en) * | 2011-09-20 | 2014-08-14 | Kuraray Co., Ltd. | Adherent cell culture method |
Family Cites Families (272)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2947116A (en) | 1954-02-26 | 1960-08-02 | Wilton R Earle | Method of producing tissue culture flasks |
US3630849A (en) | 1969-04-24 | 1971-12-28 | David B Land | Surface micro-organism contamination assays |
US4382685A (en) | 1979-07-17 | 1983-05-10 | Techne (Cambridge) Limited | Method and apparatus for stirring particles in suspension such as microcarriers for anchorage-dependent living cells in a liquid culture medium |
US4498785A (en) | 1982-06-09 | 1985-02-12 | Techne Corporation | Floating magnetic stirrer for culture medium |
DE8309876U1 (en) | 1983-04-02 | 1983-12-22 | Biotest-Serum-Institut Gmbh, 6000 Frankfurt | TWO-PHASE BLOOD CULTURE BOTTLE |
US4534656A (en) | 1983-06-07 | 1985-08-13 | Techne Corporation | Floating magnetic stirrer with driving guide rod |
GB8321239D0 (en) | 1983-08-05 | 1983-09-07 | Orbec Ltd | Innoculating means |
US4670396A (en) | 1985-05-02 | 1987-06-02 | Bioassy Systems Corporation | Vertical culture system with removable culture unit |
US4760028A (en) | 1986-07-25 | 1988-07-26 | Techne Incorporated | Bioreactor apparatus with surface aerator screen |
US5171994A (en) | 1986-08-13 | 1992-12-15 | Northrop Corporation | Infrared staring imaging array |
US5047347A (en) | 1987-08-17 | 1991-09-10 | Cline Martin J | Gas permeable culture flask and method for culturing mammalian cells |
US4839292B1 (en) | 1987-09-11 | 1994-09-13 | Joseph G Cremonese | Cell culture flask utilizing membrane barrier |
US4927764A (en) | 1987-11-17 | 1990-05-22 | Costar Corporation | Tissue culture flask |
US4980293A (en) | 1988-09-02 | 1990-12-25 | Multi-Technology Inc. | Dispensing reagents in a specimen well |
US5240854A (en) | 1989-06-05 | 1993-08-31 | Berry Eric S | Continuous high-density cell culture system |
JP2850982B2 (en) | 1989-10-25 | 1999-01-27 | ユニ・チャーム株式会社 | Disposable wearing articles |
GB9004911D0 (en) | 1990-03-05 | 1990-05-02 | Smith & Nephew | Cell culture products |
US5712137A (en) | 1990-03-05 | 1998-01-27 | Smith & Nephew Plc | Laminate of a culture substrate on a carrier for producing an apertured wound dressing |
DE4030699C1 (en) | 1990-09-28 | 1991-10-10 | Bruker Analytische Messtechnik Gmbh, 7512 Rheinstetten, De | |
US5272083A (en) * | 1990-10-10 | 1993-12-21 | Costar Corporation | Culture device and method of use having a detachable cell or tissue growth surface |
US5151366A (en) | 1991-05-24 | 1992-09-29 | Invitro Scientific Products, Inc. | Cell culture flask |
DE4132379A1 (en) | 1991-09-28 | 1993-04-08 | Kernforschungsz Karlsruhe | SUBSTRATE FOR CELL CULTURES AND CULTURE OF CELLS OR CELL AGGREGATES |
US5272084A (en) | 1991-12-18 | 1993-12-21 | Corning Incorporated | Cell culture vessels having interior ridges and method for cultivating cells in same |
US5319436A (en) | 1992-05-28 | 1994-06-07 | Packard Instrument Company, Inc. | Microplate farming wells with transparent bottom walls for assays using light measurements |
FR2697086B1 (en) | 1992-10-20 | 1994-12-09 | Thomson Csf | Method and device for inspecting transparent material. |
IT1266389B1 (en) | 1993-02-15 | 1996-12-30 | Alberto Degrassi | CONTAINER STRUCTURE, PARTICULARLY FOR CELL CULTURES |
US5374557A (en) | 1993-04-29 | 1994-12-20 | Verma; Kuldeep | Fermentation vessels and closures therefor |
DE69407923T2 (en) | 1993-05-17 | 1998-04-30 | Amersham Int Plc | DEVICE AND METHOD FOR DETECTING CELLULAR AND BIOCHEMICAL PROCESSES |
JP2716646B2 (en) | 1993-05-21 | 1998-02-18 | 住友ベークライト株式会社 | Method for forming cell aggregates |
CN2186755Y (en) | 1994-03-25 | 1995-01-04 | 中国科学院成都生物研究所 | Multipurpose culture plate |
US5487872A (en) | 1994-04-15 | 1996-01-30 | Molecular Device Corporation | Ultraviolet radiation transparent multi-assay plates |
US5707869A (en) | 1994-06-28 | 1998-01-13 | Wolf; Martin L. | Compartmentalized multiple well tissue culture plate |
US5693537A (en) | 1994-06-28 | 1997-12-02 | Wilson; John R. | Compartmentalized tissue culture flask |
US5635344A (en) | 1994-12-07 | 1997-06-03 | Cedra Corp. | Shipping medium for organ-derived cells |
US5554536A (en) | 1995-01-05 | 1996-09-10 | Millipore Investment Holdings Limited | Biological analysis device having improved contamination prevention |
GB9509487D0 (en) | 1995-05-10 | 1995-07-05 | Ici Plc | Micro relief element & preparation thereof |
US5710043A (en) | 1995-09-25 | 1998-01-20 | Becton Dickinson And Company | In vitro cell culture assembly |
US5759494A (en) | 1995-10-05 | 1998-06-02 | Corning Incorporated | Microplates which prevent optical cross-talk between wells |
US5772905A (en) | 1995-11-15 | 1998-06-30 | Regents Of The University Of Minnesota | Nanoimprint lithography |
FR2741357B1 (en) | 1995-11-22 | 1998-01-16 | Corning Inc | METHOD FOR MANUFACTURING A SUPPORT PLATE FOR A TWO-DIMENSIONAL NETWORK OF MICROWELLS, IN PARTICULAR FOR BIOLOGICAL TESTING OR CULTURE |
JPH09234811A (en) | 1995-12-27 | 1997-09-09 | Mitsubishi Gas Chem Co Inc | Film-like or sheet-like deoxidizing multilayer body and its manufacture |
JPH09173049A (en) | 1995-12-27 | 1997-07-08 | Sumitomo Bakelite Co Ltd | Culturing vessel |
US5858309A (en) | 1996-03-22 | 1999-01-12 | Corning Incorporated | Microplates with UV permeable bottom wells |
GB2314343B (en) | 1996-06-18 | 2000-08-23 | Liau Ming Yi | Method and apparatus for cultivating anchorage dependent monolayer cells |
US5783440A (en) | 1996-09-30 | 1998-07-21 | Becton Dickinson And Company | Culture vessel |
WO1998015355A2 (en) | 1996-10-10 | 1998-04-16 | Corning Incorporated | Tool and method for transfer of drops |
WO1998031466A1 (en) | 1997-01-17 | 1998-07-23 | Corning Incorporated | Multi-well plate |
JPH10210966A (en) | 1997-01-29 | 1998-08-11 | Sumitomo Bakelite Co Ltd | Culture vessel |
JPH10210866A (en) | 1997-01-30 | 1998-08-11 | Masayuki Minato | Falling prevention instrument for flowerpot not to be laid fallen even by strong wind |
US5972694A (en) | 1997-02-11 | 1999-10-26 | Mathus; Gregory | Multi-well plate |
US5859896A (en) | 1997-03-05 | 1999-01-12 | Rosen; Howard B. | Telephone line automatic prefix dialer |
DE19712484C2 (en) | 1997-03-25 | 1999-07-08 | Greiner Gmbh | Microplate with transparent bottom and process for its production |
CH692583A5 (en) | 1998-03-03 | 2002-08-15 | Weidmann H Ag | Culture vessel. |
DE19825812C1 (en) | 1998-06-09 | 2000-01-13 | Gsf Forschungszentrum Umwelt | Cell culture vessel for the cultivation of non-adherent cells |
GB9812783D0 (en) | 1998-06-12 | 1998-08-12 | Cenes Ltd | High throuoghput screen |
GB9912641D0 (en) | 1999-05-28 | 1999-07-28 | Ashby Scient Ltd | Textured and porous silicone rubber |
JP4315544B2 (en) | 1999-10-04 | 2009-08-19 | 日油株式会社 | Copolymer, production method thereof, medical material and ophthalmic material |
US6521451B2 (en) | 1999-12-09 | 2003-02-18 | California Institute Of Technology | Sealed culture chamber |
WO2003036265A2 (en) | 2001-10-26 | 2003-05-01 | Virtual Arrays, Inc. | Assay systems with adjustable fluid communication |
DE10019862A1 (en) | 2000-04-18 | 2001-11-08 | Cell Lining Ges Fuer Zellkulti | Method for automated medium exchange, useful in cell cultures, uses transparent, hollow perfusion cover for supply and removal of medium |
AU2001253655A1 (en) | 2000-04-19 | 2001-11-07 | Corning Incorporated | Multi-well plate and method of manufacture |
DE10046175A1 (en) | 2000-09-19 | 2002-03-28 | Augustinus Bader | Automatic culturing and treatment of cells, especially for diagnosis, employs cell culture plate with wells supplied with oxygen and nutrients |
US6811752B2 (en) | 2001-05-15 | 2004-11-02 | Biocrystal, Ltd. | Device having microchambers and microfluidics |
JP3809165B2 (en) | 2001-06-14 | 2006-08-16 | ミリポア・コーポレイション | Multiwell test equipment |
CA2351156A1 (en) | 2001-07-04 | 2003-01-04 | Peter W. Zandstra | A bioprocess for the generation of pluripotent cell derived cells and tissues |
ATE406436T1 (en) | 2001-07-26 | 2008-09-15 | Transparent Inc | CULTURED CELL CONSTRUCT WITH CULTURED ANIMAL CELL SPHERoids AND USE THEREOF |
EP1279728A1 (en) | 2001-07-27 | 2003-01-29 | Schuler, Gerold | Generation of fully mature and stable dentritic cells from leukapheresis products for clinical applications |
US6767607B2 (en) | 2001-08-09 | 2004-07-27 | Corning Incorporated | Multiwell plate having transparent well bottoms |
FR2830107B1 (en) | 2001-09-24 | 2004-09-24 | Gemplus Card Int | ELECTRONIC KEY FOR CONNECTION TO A PORT OF A TELECOMMUNICATION DEVICE AND METHOD FOR MANUFACTURING THE KEY |
JP2003135056A (en) | 2001-10-30 | 2003-05-13 | Mitsuo Ochi | Method for producing tissue equivalent for transplantation and instrument for producing the same |
JP2003180335A (en) | 2001-12-21 | 2003-07-02 | Sumitomo Bakelite Co Ltd | Storable culture vessel |
US7262601B2 (en) | 2002-02-12 | 2007-08-28 | Bhp Billiton Innovation Pty Ltd | Aircraft equipped for airborne vector magnetic exploration surveys |
US20050147959A1 (en) | 2002-03-25 | 2005-07-07 | Frondoza Carmelita G. | Tissue analogs for in vitro testing and method of use therefor |
US20030183958A1 (en) | 2002-03-28 | 2003-10-02 | Becton, Dickinson And Company | Multi-well plate fabrication |
ATE360057T1 (en) | 2002-09-20 | 2007-05-15 | Becton Dickinson Co | ROLLER BOTTLE |
JP3764959B2 (en) | 2002-10-10 | 2006-04-12 | 独立行政法人理化学研究所 | Cell culture container and cell culture method |
EP1416303B8 (en) | 2002-10-30 | 2010-10-13 | Hitachi, Ltd. | Method for manufacturing functional substrates comprising columnar micro-pillars |
US20040091397A1 (en) | 2002-11-07 | 2004-05-13 | Corning Incorporated | Multiwell insert device that enables label free detection of cells and other objects |
FI115060B (en) | 2003-04-22 | 2005-02-28 | Chip Man Technologies Oy | Analysis and breeding equipment |
KR20060004958A (en) | 2003-04-25 | 2006-01-16 | 제이에스알 가부시끼가이샤 | Biochip and biochip kit, and method of producing the same and method of using the same |
US7425440B2 (en) | 2003-06-10 | 2008-09-16 | The Automation Partnership (Cambridge) Limited | Culture flask |
US20050032208A1 (en) | 2003-06-18 | 2005-02-10 | Oh Steve Kah Weng | Materials and methods to produce stem cells |
US8597597B2 (en) | 2003-06-26 | 2013-12-03 | Seng Enterprises Ltd. | Picoliter well holding device and method of making the same |
AU2004256209B2 (en) | 2003-07-08 | 2010-04-08 | Axiogenesis Ag | Novel method for the preparation of embryoid bodies (EBs) and uses thereof |
US20050112030A1 (en) | 2003-08-21 | 2005-05-26 | Gaus Stephanie E. | Meshwell plates |
US20050074873A1 (en) | 2003-09-09 | 2005-04-07 | Shanler Michael S. | Tissue culture vessel |
CA2542116C (en) | 2003-10-08 | 2015-01-27 | Wilson Wolf Manufacturing Corporation | Cell culture methods and devices utilizing gas permeable materials |
JP4749155B2 (en) | 2003-10-16 | 2011-08-17 | 株式会社 ジャパン・ティッシュ・エンジニアリング | Cultured cell sheet packaging |
US8658349B2 (en) | 2006-07-13 | 2014-02-25 | Seahorse Bioscience | Cell analysis apparatus and method |
US7186548B2 (en) | 2003-11-10 | 2007-03-06 | Advanced Pharmaceutical Sciences, Inc. | Cell culture tool and method |
EP1702052A4 (en) | 2003-12-19 | 2009-02-18 | Univ Waterloo | Cultured cell and method and apparatus for cell culture |
PL1720972T3 (en) | 2004-03-05 | 2014-06-30 | Dpx Holdings Bv | Process for cell culturing by continuous perfusion and alternating tangential flow |
US20080268515A1 (en) | 2004-03-30 | 2008-10-30 | Roy Cullimore | Method and apparatus for production and refinement of microbial consortia for the generation of selective therapeutic chemical agents |
US8318479B2 (en) | 2004-05-19 | 2012-11-27 | Massachusetts Institute Of Technology | Perfused three-dimensional cell/tissue disease models |
WO2006019836A1 (en) | 2004-07-22 | 2006-02-23 | Corning Incorporated | Culture flask |
US7767446B2 (en) | 2004-09-16 | 2010-08-03 | Becton, Dickinson And Company | Perfusion bioreactors for culturing cells |
EP1810004A1 (en) | 2004-10-18 | 2007-07-25 | Molecular Cytomics Ltd. | Current damper for the study of cells |
JP3981929B2 (en) | 2004-10-29 | 2007-09-26 | 財団法人北九州産業学術推進機構 | Cell tissue microchip |
EP2166344A1 (en) | 2004-11-24 | 2010-03-24 | Asahi Glass Company, Limited | Defect inspection method and apparatus for transparent plate materials |
JP4672376B2 (en) | 2005-01-11 | 2011-04-20 | 株式会社クラレ | Cell culture method with controlled extension direction |
GB2427688A (en) | 2005-06-28 | 2007-01-03 | Inogen S A | Microwell plate |
EP1910300A2 (en) | 2005-07-15 | 2008-04-16 | Laboratorios del Dr. Esteve S.A. | Prodrugs of pyrazoline compounds, their preparation and use as medicaments |
US7745209B2 (en) | 2005-07-26 | 2010-06-29 | Corning Incorporated | Multilayered cell culture apparatus |
DE202006020796U1 (en) | 2005-08-01 | 2010-06-24 | Life Technologies Corp., Carlsbad | container |
EP1976972B1 (en) | 2006-01-24 | 2014-03-12 | Brown University | Cell aggregation and encapsulation device and method |
JP5397934B2 (en) | 2006-02-21 | 2014-01-22 | Scivax株式会社 | Spheroids and method for producing the same |
WO2007105418A1 (en) | 2006-02-24 | 2007-09-20 | Kuraray Co., Ltd. | Cell culture container and method of producing the same |
ITMI20061063A1 (en) | 2006-05-31 | 2007-12-01 | Mindseeds Lab S R L | METRODO AND PE SYSTEM RLA SELECTION AND MODIFICATION OF SINGLE CELLS AND THEIR SMALL AGGREGATES |
DE102006030068A1 (en) | 2006-06-28 | 2008-01-03 | M2P-Labs Gmbh | Apparatus and method for the supply and removal of fluids in shaken microreactors arrays |
US7745210B2 (en) | 2006-06-30 | 2010-06-29 | Corning Incorporated | Fluid flow diverter for cell culture vessel |
JP2009542230A (en) | 2006-07-07 | 2009-12-03 | ユニバーシティ オブ マイアミ | Oxygen-enhanced cell culture platform |
US9175254B2 (en) | 2006-07-07 | 2015-11-03 | University Of Miami | Enhanced oxygen cell culture platforms |
US8053230B2 (en) | 2006-09-07 | 2011-11-08 | Nalge Nunc International Corporation | Culture dish with lid |
US8486692B2 (en) | 2006-11-14 | 2013-07-16 | Acme Biosystems Llc | Cell culture apparatus and associated methods |
US20080118974A1 (en) | 2006-11-20 | 2008-05-22 | Gregory Roger Martin | Large scale cell culture vessel |
DE202006017853U1 (en) | 2006-11-23 | 2007-01-18 | Forschungszentrum Karlsruhe Gmbh | Synthetic thermoplastic insert for a mocrotiter plate for the cultivation of different cells, comprises carrier structure, conically tapered recesses arranged in the carrier structure, and perforation and foil joint between the recesses |
US20080176318A1 (en) | 2006-12-07 | 2008-07-24 | Wilson John R | Highly efficient devices and methods for culturing cells |
US7897379B2 (en) | 2007-02-26 | 2011-03-01 | Corning Incorporated | Device and method for reducing bubble formation in cell culture |
US20110086375A1 (en) | 2007-03-02 | 2011-04-14 | Mark Ungrin | Devices and methods for production of cell aggregates |
WO2008118500A1 (en) | 2007-03-27 | 2008-10-02 | Wafergen, Inc. | Nutrient perfusion plate with heater & gas exchange for high content screening |
KR100836827B1 (en) | 2007-04-09 | 2008-06-10 | 전남대학교산학협력단 | Cell culture dish for the embryoid body formation from embryonic stem cells |
WO2008140295A1 (en) | 2007-05-16 | 2008-11-20 | Erasmus University Medical Center Rotterdam | Cell culture substrate, culture flasks and methods for cell cultivation employing said substrate |
DE102007027273A1 (en) | 2007-05-24 | 2008-11-27 | Heipha Gmbh | Container for receiving nutrient media |
FR2916451A1 (en) | 2007-05-25 | 2008-11-28 | Mhs Ind Soc Par Actions Simpli | SYSTEM FOR CULTIVATION OF BIOLOGICAL CELLS |
US9309491B2 (en) | 2007-05-29 | 2016-04-12 | Corning Incorporated | Cell culture apparatus for co-culture of cells |
US7800749B2 (en) | 2007-05-31 | 2010-09-21 | Corning Incorporated | Inspection technique for transparent substrates |
KR20160005142A (en) | 2007-06-29 | 2016-01-13 | 셀룰러 다이내믹스 인터내셔널, 인코포레이티드 | Automated method and apparatus for embryonic stem cell culture |
JP5233187B2 (en) | 2007-07-11 | 2013-07-10 | パナソニック株式会社 | Cell electrophysiological sensor |
JP2009050194A (en) | 2007-08-27 | 2009-03-12 | Sumitomo Bakelite Co Ltd | Vessel for cell aggregate-forming culture |
JP3139350U (en) | 2007-11-27 | 2008-02-14 | 株式会社クラレ | Cell culture vessel |
EP2918670A1 (en) | 2008-01-25 | 2015-09-16 | Corning Incorporated | Limited access multi-layer cell culture system |
ATE481472T1 (en) | 2008-02-01 | 2010-10-15 | Eppendorf Ag | CULTURE PLATE WITH FLAP FOR SIDE VENTILATION |
US20100074515A1 (en) | 2008-02-05 | 2010-03-25 | Kla-Tencor Corporation | Defect Detection and Response |
US20100112014A1 (en) | 2008-04-11 | 2010-05-06 | Gilbert Ryan J | Novel hydrogel compositions and methods of using |
CN102046773A (en) | 2008-05-30 | 2011-05-04 | 康宁股份有限公司 | Cell culture apparatus having different micro-well topography |
US8216828B2 (en) | 2008-05-30 | 2012-07-10 | Corning Incorporated | Assembly of cell culture vessels |
WO2009148507A1 (en) | 2008-05-30 | 2009-12-10 | Corning Incorporated | Cell culture apparatus having variable topography |
US8178345B2 (en) | 2008-05-30 | 2012-05-15 | Corning Incorporated | Multilayer cell culture vessels |
AU2009268666B2 (en) | 2008-07-08 | 2014-11-13 | Wilson Wolf Manufacturing, LLC | Improved gas permeable cell culture device and method of use |
US9783769B2 (en) | 2008-07-16 | 2017-10-10 | Emd Millipore Corporation | Layered flask cell culture system |
EP2342317B1 (en) | 2008-09-22 | 2012-12-19 | University Of Zurich Prorektorat Forschung | Hanging drop plate |
JP5578779B2 (en) | 2008-10-08 | 2014-08-27 | 国立大学法人東北大学 | Spheroid culture method and spheroid culture vessel |
EP2344622B1 (en) | 2008-10-08 | 2017-12-20 | Agency for Science, Technology And Research | Apparatus for culturing anchorage dependent cells |
JP5288171B2 (en) | 2008-10-31 | 2013-09-11 | 旭硝子株式会社 | Cultivation container |
US20110229961A1 (en) | 2008-11-05 | 2011-09-22 | Nanopoint, Inc. | Active microfluidic system for in vitro culture |
US8956867B2 (en) | 2008-11-07 | 2015-02-17 | Wisconsin Alumni Research Foundation | Method for culturing stem cells |
ES2343721B1 (en) | 2008-12-19 | 2011-06-06 | Histocell, S.L. | CELL TRANSPORTATION SYSTEM. |
JP2010158214A (en) * | 2009-01-09 | 2010-07-22 | Olympus Corp | Culture vessel |
DE102009005526A1 (en) | 2009-01-20 | 2010-07-22 | Universität Duisburg-Essen | Bioreactor for biomass production by living species, comprises flat beds, which are provided for medium containing living species and have transparent and/or gas-permeable bottom, lighting device between each of the beds, and light guide |
WO2010085751A2 (en) | 2009-01-26 | 2010-07-29 | The Regents Of The University Of California | Apparatus and method for culturing stem cells |
DE102009013673A1 (en) | 2009-03-09 | 2010-09-16 | Eppendorf Ag | Cell culture dish |
US20120129208A1 (en) | 2009-03-18 | 2012-05-24 | Michelle Khine | Honeycomb shrink wells for stem cell culture |
SG174435A1 (en) | 2009-03-26 | 2011-10-28 | Agency Science Tech & Res | Apparatus for cell or tissue culture |
EP2422003A4 (en) | 2009-04-24 | 2012-10-31 | Univ Ohio State | Interactive microenvironment system |
US9169460B2 (en) | 2009-05-19 | 2015-10-27 | Lifeglobal Group Llc | Flooding dish and method for changing media in the dish in the preparation of mammalian specimen culture and for cryo-preservation, freezing, vitrification and the thawing and warming of such specimens |
US7929129B2 (en) | 2009-05-22 | 2011-04-19 | Corning Incorporated | Inspection systems for glass sheets |
US8778669B2 (en) | 2009-07-22 | 2014-07-15 | Corning Incorporated | Multilayer tissue culture vessel |
US8143053B2 (en) | 2009-10-26 | 2012-03-27 | Biomerieux, Inc. | Lockable cell growth chamber with antilocking feature |
WO2011060244A1 (en) | 2009-11-12 | 2011-05-19 | The Texas A & M University System | Spheroidal aggregates of mesenchymal stem cells |
EP2499500B1 (en) | 2009-11-13 | 2020-12-30 | Ventana Medical Systems, Inc. | Thin film processing apparatuses for adjustable volume accommodation |
WO2011083768A1 (en) | 2010-01-08 | 2011-07-14 | 住友ベークライト株式会社 | Culture vessel for formation of aggregated cell mass |
US9493733B2 (en) | 2010-01-18 | 2016-11-15 | Becton, Dickinson And Company | Container assembly |
DE202011110503U1 (en) | 2010-01-28 | 2014-12-04 | The Regents Of The University Of Michigan | Hanging drop devices and systems |
CN201626959U (en) | 2010-02-01 | 2010-11-10 | 中国农业科学院北京畜牧兽医研究所 | Micro perfusion device for cell culture |
JP2011172533A (en) * | 2010-02-25 | 2011-09-08 | Fusao Komada | Method for three-dimensional high-density cell culture using microspace structure |
US20130122539A1 (en) | 2010-05-04 | 2013-05-16 | Mo-Huang Li | Microsieve for cells and particles filtration |
JP5703302B2 (en) | 2010-09-08 | 2015-04-15 | 株式会社島津製作所 | Cell culture container and cell culture method using the container |
CN103119151B (en) | 2010-09-14 | 2015-02-04 | Agc科技玻璃株式会社 | Culture substrate |
US9260684B1 (en) | 2010-11-11 | 2016-02-16 | Stemcell Technologies Inc. | Cell culture device |
CN103250047A (en) | 2010-12-09 | 2013-08-14 | 旭硝子株式会社 | Method and system for measuring defect in glass ribbon |
TWI438273B (en) | 2011-03-08 | 2014-05-21 | Univ Chang Gung | High-throughput perfusative microfluidic cell culture wafers for miniaturized three-dimensional cell culture |
GB201105226D0 (en) | 2011-03-29 | 2011-05-11 | Univ Leiden | Methods |
KR101802908B1 (en) | 2011-03-30 | 2017-11-29 | 도레이 카부시키가이샤 | Membrane-separation-type culture device, membrane-separation-type culture kit, stem cell separation method using same, and separation membrane |
JP2012249547A (en) | 2011-05-31 | 2012-12-20 | Oji Holdings Corp | Cell culture substrate and method for manufacturing the same |
WO2012170232A1 (en) | 2011-06-09 | 2012-12-13 | Bellbrook Labs, Llc | Device for washing suspended cells or particles |
JP5853512B2 (en) | 2011-09-08 | 2016-02-09 | 大日本印刷株式会社 | Cell culture container and manufacturing method thereof |
KR101306289B1 (en) | 2011-09-15 | 2013-09-09 | (주) 인텍플러스 | Method of inspecting plat panel |
US9200246B2 (en) | 2011-12-01 | 2015-12-01 | Acea Biosciences, Inc. | Co-culture device assembly |
EP2806261B1 (en) | 2012-01-19 | 2019-06-05 | Yamaha Hatsudoki Kabushiki Kaisha | Wellplate and suction device provided with said wellplate |
US20150004686A1 (en) | 2012-02-02 | 2015-01-01 | Corning Incorporated | Automatic continuous perfusion cell culture microplate consumables |
EP2623204A1 (en) | 2012-02-03 | 2013-08-07 | F. Hoffmann-La Roche AG | Sample handling system |
JP5882072B2 (en) | 2012-02-06 | 2016-03-09 | 株式会社日立ハイテクノロジーズ | Defect observation method and apparatus |
CN202450098U (en) | 2012-03-09 | 2012-09-26 | 山东省农业科学院畜牧兽医研究所 | Culture bottle for primary explant culture cells |
EP2653531A1 (en) | 2012-04-18 | 2013-10-23 | Oxyphen AG | Culture assembly |
US20140004086A1 (en) | 2012-06-29 | 2014-01-02 | Tissue Genesis Inc. | Formation of cell aggregates |
JP6111673B2 (en) | 2012-07-25 | 2017-04-12 | 住友電気工業株式会社 | Silicon carbide semiconductor device |
USD685497S1 (en) | 2012-08-31 | 2013-07-02 | Corning Incorporated | Cell culture flask |
US9573128B1 (en) | 2012-09-06 | 2017-02-21 | SciKon Innovation, Inc. | Fluidics device allowing fluid flow between a plurality of wells |
KR20150047598A (en) | 2012-09-14 | 2015-05-04 | 스미또모 베이크라이트 가부시키가이샤 | Microwell plate |
CN202849407U (en) | 2012-09-26 | 2013-04-03 | 无锡耐思生物科技有限公司 | Cell culture flask structure |
US9701938B2 (en) | 2012-10-12 | 2017-07-11 | Lena Biosciences, Inc. | Intra-culture perfusion methods and applications thereof |
KR20140048733A (en) * | 2012-10-16 | 2014-04-24 | 삼성전자주식회사 | Multiwell plate and method for analyzing target material using the same |
US9176115B2 (en) | 2012-10-26 | 2015-11-03 | The University Of Akron | Engineering individually addressable cellular spheroids using aqueous two-phase systems |
GB2507744A (en) | 2012-11-07 | 2014-05-14 | Universitaetsklinikum Freiburg | Matrix for generating and cultivating uniform cell aggregations |
US9494577B2 (en) | 2012-11-13 | 2016-11-15 | Seahorse Biosciences | Apparatus and methods for three-dimensional tissue measurements based on controlled media flow |
US9389187B2 (en) | 2012-11-29 | 2016-07-12 | Corning Incorporated | Glass-sheet optical inspection systems and methods with illumination and exposure control |
JP2014132869A (en) * | 2013-01-11 | 2014-07-24 | Sumitomo Bakelite Co Ltd | Cell culture vessel |
US20140240489A1 (en) | 2013-02-26 | 2014-08-28 | Corning Incorporated | Optical inspection systems and methods for detecting surface discontinuity defects |
US10072241B2 (en) * | 2013-03-13 | 2018-09-11 | Innovative Surface Technologies, Inc. | Conical devices for three-dimensional aggregate(s) of eukaryotic cells |
KR20140113139A (en) * | 2013-03-15 | 2014-09-24 | 고려대학교 산학협력단 | Cell spheroid culture plate |
EP2781591B1 (en) | 2013-03-19 | 2017-05-03 | Unisense Fertilitech A/S | A tray, a system and a method for monitoring and culturing of a cell culture |
JPWO2014156455A1 (en) | 2013-03-28 | 2017-02-16 | 富士フイルム株式会社 | Cell culture tool |
USD748812S1 (en) | 2013-04-12 | 2016-02-02 | Corning Incorporated | Arched cell culture flask |
KR102234887B1 (en) | 2013-04-19 | 2021-04-01 | 주식회사 에이엔케이 | Substrate For Cell Culture |
US9790465B2 (en) | 2013-04-30 | 2017-10-17 | Corning Incorporated | Spheroid cell culture well article and methods thereof |
WO2014197550A1 (en) | 2013-06-05 | 2014-12-11 | David Kranbuehl | Multi point method and apparatus for monitoring the aging and changes in corresponding tensile performance properties of a polymer |
AU2014276229B2 (en) | 2013-06-07 | 2019-11-21 | Corning Incorporated | Culture vessel and culture method |
JP6307802B2 (en) | 2013-07-05 | 2018-04-11 | 株式会社Ihi | Culture vessel |
JP2015029431A (en) | 2013-07-31 | 2015-02-16 | 大日本印刷株式会社 | Cell culture vessel |
CN203513696U (en) | 2013-08-27 | 2014-04-02 | 浙江硕华医用塑料有限公司 | Cell culture flask |
DE102013109450B4 (en) | 2013-08-30 | 2015-04-02 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | exposure apparatus |
ES2795974T3 (en) | 2013-09-05 | 2020-11-25 | Univ Bern | Device for in vitro modeling of organ tissue in vivo |
JP2015050964A (en) | 2013-09-06 | 2015-03-19 | 住友ベークライト株式会社 | Multistage culture flask and production method of cell culture flask |
JP6185816B2 (en) | 2013-10-11 | 2017-08-23 | Agcテクノグラス株式会社 | Cell culture vessel |
KR102343744B1 (en) | 2013-10-30 | 2021-12-24 | 밀리차 라디식 | Devices and methods for three-dimensional tissue culturing |
JPWO2015087369A1 (en) | 2013-12-12 | 2017-03-16 | ヤマハ発動機株式会社 | Well plate, object sorting apparatus equipped with the well plate |
EP3114207A4 (en) | 2014-03-03 | 2017-10-25 | Kiyatec Inc. | 3d tissue culture devices and systems |
WO2015132729A1 (en) | 2014-03-04 | 2015-09-11 | Pluristem Ltd. | Systems and methods for growing and harvesting cells |
US9933373B2 (en) | 2014-04-29 | 2018-04-03 | Glasstech, Inc. | Glass sheet acquisition and positioning mechanism for an inline system for measuring the optical characteristics of a glass sheet |
JP6386258B2 (en) | 2014-06-16 | 2018-09-05 | 日東電工株式会社 | Cell culture sheet and method for producing the same |
US10067065B1 (en) | 2014-06-23 | 2018-09-04 | Amazon Technologies, Inc. | Electronic device inspecting system and method |
DE102014214077A1 (en) | 2014-07-18 | 2016-01-21 | Hamilton Bonaduz Ag | Laboratory containers, in particular cell culture containers, with a gas compensation line extending into the container volume |
WO2016020992A1 (en) | 2014-08-05 | 2016-02-11 | ヤマハ発動機株式会社 | Culture apparatus, culture method using same, and method for selecting aggregated cell mass |
EP2985343A1 (en) | 2014-08-11 | 2016-02-17 | Karlsruher Institut für Technologie | In vitro-co-culturesystem |
WO2016064757A1 (en) | 2014-10-19 | 2016-04-28 | Nortis, Inc. | Modular microfluidic system for perfused cell culture |
JP6930914B2 (en) | 2014-10-29 | 2021-09-01 | コーニング インコーポレイテッド | Perfusion bioreactor platform |
EP3212759A1 (en) | 2014-10-29 | 2017-09-06 | Corning Incorporated | Cell culture insert |
CN107109340B (en) | 2014-10-29 | 2021-10-22 | 康宁股份有限公司 | Spheroid capture insert |
EP3212761A1 (en) | 2014-10-29 | 2017-09-06 | Corning Incorporated | Microwell design and fabrication for generation of cell culture aggregates |
US20170342363A1 (en) | 2014-10-29 | 2017-11-30 | Corning Incorporated | Devices and methods for generation and culture of 3d cell aggregates |
CN113265332A (en) | 2014-10-29 | 2021-08-17 | 康宁股份有限公司 | Method and apparatus for generating and culturing 3D cell aggregates |
JP2016093149A (en) | 2014-11-14 | 2016-05-26 | 真志 池内 | Cell culture apparatus, and cell culture method |
WO2016085787A1 (en) | 2014-11-25 | 2016-06-02 | Corning Incorporated | Cell culture media extending materials and methods |
DE102014017728A1 (en) | 2014-12-01 | 2016-06-02 | Merck Patent Gmbh | Culture dish for micro-organisms |
US20170326545A1 (en) | 2014-12-10 | 2017-11-16 | Corning Incorporated | Reinforced microplate |
JP2016136920A (en) | 2015-01-29 | 2016-08-04 | 大日本印刷株式会社 | Cell culture container |
JP2016136921A (en) | 2015-01-29 | 2016-08-04 | 大日本印刷株式会社 | Cell culture container |
CN204752742U (en) | 2015-03-12 | 2015-11-11 | 广州医科大学 | Cell culture's cell culture ware can be used to to grow slowly |
WO2016157322A1 (en) | 2015-03-27 | 2016-10-06 | 株式会社日立製作所 | Closed-system culture vessel, transport method, and automated culturing device |
CN204702760U (en) | 2015-04-15 | 2015-10-14 | 上海诺狄生物科技有限公司 | A kind of culture dish |
CN204714819U (en) | 2015-04-21 | 2015-10-21 | 北京青藤谷禧干细胞科技研究院有限公司 | A kind of Tissue Culture Flask of belt supporting frame |
CN204608026U (en) | 2015-04-22 | 2015-09-02 | 广州洁特生物过滤股份有限公司 | A kind of cell with super hydrophilic growth surface embeds ware |
CN204803327U (en) | 2015-06-30 | 2015-11-25 | 广州洁特生物过滤股份有限公司 | Ventilative cell culture dish |
KR101709203B1 (en) | 2015-07-14 | 2017-02-22 | 재단법인 포항산업과학연구원 | Solid electrolyte, method for manufacturing the same, and all solid state rechargeable lithium battery including the same |
WO2017025584A1 (en) | 2015-08-12 | 2017-02-16 | Cytovac A/S | A cell cultivation method, a cell cultivation vessel, and uses thereof |
US10227556B2 (en) | 2015-09-04 | 2019-03-12 | Wayne State University | Cell culture devices for biomimetic and pathomimetic cell cultures |
US20170067019A1 (en) | 2015-09-07 | 2017-03-09 | Bioreactor Sciences Llc | Method of Continuous Mass Production of Progenitor Stem-like Cells Using a Bioreactor System |
CN205990396U (en) | 2015-09-09 | 2017-03-01 | 广州市迪景微生物科技有限公司 | A kind of lid culture dish difficult for drop-off |
CN108026498A (en) | 2015-09-17 | 2018-05-11 | Agc科技玻璃株式会社 | Cell culture container |
ES2615512B1 (en) | 2015-11-06 | 2018-03-15 | Universidad De Zaragoza | DEVICE AND MICROFLUIDIC SYSTEM FOR THE STUDY OF CELL CULTURES |
CN205170866U (en) | 2015-12-08 | 2016-04-20 | 湖南人文科技学院 | No clear tape embryo culture ware |
NL2016281B1 (en) | 2016-02-18 | 2017-08-24 | Perkinelmer Health Sciences B V | Means and methods for spheroid cell culturing and analysis. |
US10774296B2 (en) | 2016-04-05 | 2020-09-15 | Corning Incorporated | Lidded cell culture devices with improved handling performance and methods for using same |
CN205669029U (en) | 2016-05-09 | 2016-11-02 | 浙江硕华生命科学研究股份有限公司 | A kind of cell culture ware |
CN205839030U (en) | 2016-07-27 | 2016-12-28 | 浙江译美生物科技有限公司 | Tissue Culture Dish |
EP3296018A1 (en) | 2016-09-19 | 2018-03-21 | Ecole Polytechnique Fédérale de Lausanne (EPFL) | Organoid arrays |
US10854930B2 (en) | 2016-10-07 | 2020-12-01 | The Regents Of The University Of Michigan | Stabilization coatings for solid state batteries |
KR20180068115A (en) | 2016-12-13 | 2018-06-21 | 삼성전자주식회사 | Composite electrolyte structure and lithium metal battery comprising the same |
JP2018108032A (en) | 2016-12-28 | 2018-07-12 | Agcテクノグラス株式会社 | Culture vessel |
CN108727025A (en) | 2017-04-17 | 2018-11-02 | 中国科学院上海硅酸盐研究所 | Lithium garnet composite ceramics, Its Preparation Method And Use |
JP2020517575A (en) | 2017-04-27 | 2020-06-18 | コーニング インコーポレイテッド | Curved glass forming process and system via differential heating of glass sheets |
KR20200014890A (en) | 2017-06-06 | 2020-02-11 | 더 리젠츠 오브 더 유니버시티 오브 미시건 | Method for Suppressing Metal Diffusion in Solid Electrolyte |
KR20200027462A (en) | 2017-07-07 | 2020-03-12 | 코닝 인코포레이티드 | Vehicle interior system with curved cover glass and display or touch panel and method of forming the same |
EP3652291B1 (en) | 2017-07-14 | 2021-12-29 | Corning Incorporated | Cell culture vessel |
JP7250755B2 (en) | 2017-07-14 | 2023-04-03 | コーニング インコーポレイテッド | cell culture vessel |
US11345880B2 (en) | 2017-07-14 | 2022-05-31 | Corning Incorporated | 3D cell culture vessels for manual or automatic media exchange |
CN111094534A (en) | 2017-07-14 | 2020-05-01 | 康宁股份有限公司 | Processing features for microcavity cell culture vessels |
CN107460125A (en) | 2017-10-12 | 2017-12-12 | 李慧杰 | Suspending cell culture bottle |
CN111868524A (en) | 2018-03-13 | 2020-10-30 | 康宁股份有限公司 | High-density 3D hepatocyte spheroid platform for ADME research |
WO2020013847A1 (en) | 2018-07-13 | 2020-01-16 | Corning Incorporated | Microcavity dishes with sidewall including liquid medium delivery surface |
US10871400B2 (en) | 2018-08-27 | 2020-12-22 | Corning Incorporated | Retardation profile for stress characterization of tubing |
JP2022534507A (en) | 2019-05-30 | 2022-08-01 | コーニング インコーポレイテッド | Cell storage and transport media and systems, and methods for transport of cell aggregates |
-
2015
- 2015-10-29 EP EP15791189.2A patent/EP3212759A1/en active Pending
- 2015-10-29 SG SG11201703493SA patent/SG11201703493SA/en unknown
- 2015-10-29 WO PCT/US2015/058053 patent/WO2016069895A1/en active Application Filing
- 2015-10-29 CN CN201580071727.1A patent/CN107109328B/en active Active
- 2015-10-29 JP JP2017523446A patent/JP6731916B2/en active Active
- 2015-10-29 KR KR1020177014157A patent/KR102460969B1/en active IP Right Grant
-
2017
- 2017-04-20 US US15/492,730 patent/US20170218321A1/en not_active Abandoned
-
2022
- 2022-03-10 US US17/691,511 patent/US11976263B2/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030215941A1 (en) * | 2002-03-12 | 2003-11-20 | Stewart Campbell | Assay device that analyzes the absorption, metabolism, permeability and/or toxicity of a candidate compound |
US20100190197A1 (en) * | 2009-01-27 | 2010-07-29 | Martin Gregory R | Nested permeable support device and method for using the nested permeable support device |
US20130344598A1 (en) * | 2011-01-24 | 2013-12-26 | California Stem Cell, Inc. | Neural cell purification for transplantation |
US20140227784A1 (en) * | 2011-09-20 | 2014-08-14 | Kuraray Co., Ltd. | Adherent cell culture method |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20170253844A1 (en) * | 2016-03-04 | 2017-09-07 | Corning Incorporated | Bowl shaped microwell |
CN107794223A (en) * | 2017-11-07 | 2018-03-13 | 广东药科大学附属第医院 | Anaerobic bacteria and the in vitro study model and method of aerobic cell interaction in cell co-culture device and analogue body |
WO2019114997A1 (en) * | 2017-12-15 | 2019-06-20 | Technische Universität Ilmenau | Cell culture carrier |
WO2020091894A1 (en) * | 2018-10-28 | 2020-05-07 | Schickwann Tsai | Low-macrophage-adhesion/activation culture devices and methods thereof for continuous hematopoiesis and expansion of hematopoietic stem cells |
CN113015784A (en) * | 2018-10-28 | 2021-06-22 | 蔡士宽 | Low macrophage adhesion and activation culture device and method for culturing bone marrow by using same |
WO2021075810A1 (en) * | 2019-10-14 | 2021-04-22 | 연세대학교 산학협력단 | Well plate unit and multi-layer spheroid culture apparatus using same |
KR20210043968A (en) * | 2019-10-14 | 2021-04-22 | 연세대학교 산학협력단 | Well plate unit for spheroid culture and well plate unit for multilayer spheroid culture apparatus |
KR102309955B1 (en) | 2019-10-14 | 2021-10-07 | 연세대학교 산학협력단 | Well plate unit for spheroid culture and well plate unit for multilayer spheroid culture apparatus |
WO2021108346A1 (en) * | 2019-11-25 | 2021-06-03 | Wake Forest University Health Sciences | Microwell perfusion plates for organoids and related systems and methods |
CN110903976A (en) * | 2019-12-20 | 2020-03-24 | 江苏信安佳医疗科技有限公司 | A orifice plate device for organoid spheroid is cultivateed |
CN116478818A (en) * | 2023-05-11 | 2023-07-25 | 江苏艾玮得生物科技有限公司 | Cell culture unit, device, application and culture method |
Also Published As
Publication number | Publication date |
---|---|
EP3212759A1 (en) | 2017-09-06 |
WO2016069895A9 (en) | 2017-04-20 |
KR20170073686A (en) | 2017-06-28 |
WO2016069895A1 (en) | 2016-05-06 |
JP6731916B2 (en) | 2020-07-29 |
CN107109328A (en) | 2017-08-29 |
JP2017536817A (en) | 2017-12-14 |
US20220195366A1 (en) | 2022-06-23 |
KR102460969B1 (en) | 2022-10-31 |
US11976263B2 (en) | 2024-05-07 |
CN107109328B (en) | 2021-02-05 |
SG11201703493SA (en) | 2017-05-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11976263B2 (en) | Cell culture insert | |
JP7071553B2 (en) | Equipment and methods for the generation and culture of 3D cell aggregates | |
KR102470509B1 (en) | Spheroid trap insert | |
JP5847733B2 (en) | Hanging drop array plate | |
Liang et al. | In situ sensors for blood-brain barrier (BBB) on a chip | |
US20190322969A1 (en) | Devices and methods for generation and culture of 3d cell aggregates | |
US9243221B2 (en) | Compositions and methods of functionally enhanced in vitro cell culture system | |
JP2021518110A (en) | High Density 3D Hepatocyte Spheroid Platform for Drug ADME Research | |
Hamon et al. | New tools and new biology: recent miniaturized systems for molecular and cellular biology | |
Busche et al. | HepaChip-MP–a twenty-four chamber microplate for a continuously perfused liver coculture model | |
Mai et al. | MatriGrid® Based Biological Morphologies: Tools for 3D Cell Culturing | |
Mueller et al. | 3D hepatic in vitro models as tools for toxicity studies | |
Kaisar et al. | In vitro BBB models: Working with static platforms and microfluidic systems | |
US20220290080A1 (en) | Cell culture assemblies and methods of using the same | |
Keshavarz et al. | Cell culture techniques in microfluidic chips | |
Yi et al. | Cell Culture and Observation on Microfluidics |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: CORNING INCORPORATED, NEW YORK Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:MARTIN, GREGORY ROGER;TANNER, ALLISON JEAN;REEL/FRAME:042088/0324 Effective date: 20170329 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
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: NON FINAL ACTION MAILED Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION 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: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION 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: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |