CN106811409A - Multiple organ tumor-targeting drug test platform and its application based on micro-fluidic chip - Google Patents
Multiple organ tumor-targeting drug test platform and its application based on micro-fluidic chip Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
Abstract
The present invention provides a kind of multiple organ tumor-targeting drug test platform and its application based on micro-fluidic chip, the micro-fluidic chip is mainly made up of upper strata chip, lower layer chip and porous membrane, a layer chip is connected on porous membrane, connect lower layer chip under porous membrane, upper strata chip structure is big cell culture chamber, lower layer chip structure is independent multichannel, and number of channels is 1-9 bars.Multiple organ tumor-targeting drug test platform based on micro-fluidic chip, builds as follows：(1) chip manufacturing and modification, inoculation and the culture of (2) upper strata chip inner cell, the inoculation and culture of (3) lower layer chip inner cell.The hepatic metabolism process in upper strata chip inoculation liver cell analogue body, lower layer chip is inoculated with different tumour cells and normal tissue cell, medicine can simultaneously be observed after hepatic metabolism to the effect of different cells, medicine can also be observed after hepatic metabolism to the effect of multigroup cell, the screening and test of tumor-targeting drug are realized, the patent medicine rate of screening medicine is greatly improved.
Description
Technical field
The invention belongs to micro fluidic chip technical field, and in particular to a kind of multiple organ based on micro-fluidic chip
Tumor-targeting drug test platform and its application.
Background technology
Chemotherapy is one of Main Means of Multimodality Therapy of Malignant Tumors.The conventional method of drug test
Generally relying on bio-toxicity is carried out, and does not consider metabolic process and biological selectivity in medicine body, is so tested
The active medicine for obtaining Chang Yinwei toxicity and metabolic problems and stranded, patent medicine rate pole after research and development enter clinic
It is low.Preferable targeted drug test platform should be able to be to killing tumour cell without damaging normal tissue cells
Medicine carries out test screen, can be while effect of the testing drug to different tumours and normal structure, also may be used
To simulate the real process of oral drugs increased activity or decrease after hepatic metabolism.Build such a platform energy
Reach for medicament research and development and instruct clinical application important in inhibiting.
Microfluid based Lab on a chip is referred to biological and chemical etc. also known as chip lab or micro-fluidic chip
Involved sample preparation, reaction, separation, detection, cell culture, sorting, cracking etc. are basic in field
Operating unit is integrated or is integrated into substantially on one piece of chip of several square centimeters (even more small), is formed by microchannel
Network, whole system is run through with controlled fluid, is used to replace the various functions of conventional chemical or biology laboratory
A kind of technology.Microfluidic chip technology as a science and technology for developing rapidly, in biology
Medical domain presents its unique advantage, more because its with cell size matching, environment with physiological environment it is close,
More accurate manipulation can be provided on time and Spatial Dimension, it is easy to realized by flexible design various thin
The features such as born of the same parents' functional study and turn into the Important Platform of bionic of new generation and cell research.At present, new
During medicine is evaluated, the simulation of organizational project is carried out using micro-fluidic chip, especially build multiple organ cancer target
Drug test study of platform is analyzed also in blank stage.If successfully building, can add in medicament research and development
The screening of fast effectively new drug has great application prospect.
The content of the invention
It is an object of the invention to provide a kind of multiple organ tumor-targeting drug test platform based on micro-fluidic chip
And its application, and the pharmacodynamic study of tumor-targeting drug is applied to, particular for being truly metabolized in analogue body
Under the conditions of drug test, can simultaneously observe medicine after hepatic metabolism to the effect of different cells.
The present invention provides a kind of micro-fluidic chip, is mainly made up of upper strata chip, lower layer chip and porous membrane,
Connected on porous membrane and connect under a layer chip, porous membrane lower layer chip, the upper strata chip structure is cell training
Room is supported, lower layer chip structure is independent multichannel, and number of channels is 1-9 bars
The chip material is the PDMS polymer that light-permeable is breathed freely, and porous membrane material is polycarbonate membrane,
Aperture is 0.01um-4um..
The multiple organ tumor-targeting drug test platform based on micro-fluidic chip that the present invention is provided, according to following
The foundation of method：
(1) chip manufacturing and modification
Upper strata chip and lower layer chip are designed and produced, porous membrane is clipped in the middle carries out sealing-in, is placed in culture dish
In, ultraviolet sterilization overnight after, the collagen working solution that will be prepared with pipettor adds upper strata chip cell culture
Room and each passage of lower layer chip, the culture dish normal temperature of fixed chip is stood overnight, and is removed within 12-36 hours
Collagen working solution, cell culture fluid irrigation channel 2-4 times, modifies bottom surface, promotes cell preferably
It is adherent；
(2) inoculation of upper strata chip inner cell and culture
After liver cell digestion, adjust to suitable cell density, be added in the chip cell culture chamber of upper strata,
Under the modification of collagen, cell is rapid adherent and uniformly spreads in cell culture chamber bottom surface, when in optics
Basis of microscopic observation to cell be uniformly distributed in cell culture chamber when, immediately by chip move into carbon dioxide training
Continuation is cultivated in supporting case；
(3) inoculation of lower layer chip inner cell and culture
When liver cell is paved with cell culture chamber 60% by propagation, different tumour cells is thin with normal structure
Born of the same parents distinguish inoculated and cultured in each passage of lower layer chip, after 12 hours, the training containing liquid are added to upper strata chip
Base is supported to continue to cultivate 48 hours.
A kind of application of the multiple organ tumor-targeting drug test platform based on micro-fluidic chip, for tumor target
Investigated to the chip inner cell activity of medicine sorting platform, its procedure is as follows：
(1) detection of cytoactive, CCK-8 reagents are carried out using CCK-8 kits：Cell culture medium=1:9
It is mixed into detection working solution, adds lower layer chip passage, put in incubator after 3 hours, is transferred to 96 holes
In plate, ELIASA determines absorbance at 450nm.
(2) porous membrane that lower layer chip has upper strata chip with envelope is separated, the cell of lower layer chip can be carried out
Microexamination, routine immunization fluorescence colour is characterized.
A kind of application of the multiple organ tumor-targeting drug test platform based on micro-fluidic chip, for tumor target
Method to drug metabolism processes research is applied in the chip of medicine sorting platform, the method process is as follows：
Culture supernatants and lower floor's nutrient solution are collected, mass spectral analysis can be respectively carried out, can detect that medicine is former
The content of type and metabolin.
The present invention relates to the medicine for being applied to build by microfluidic chip technology in analogue body under true metabolic conditions
The technical field of test platform, can simultaneously observe medicine after hepatic metabolism to the effect of different cells, and should
For the test screen of tumor-targeting drug, the patent medicine rate of screening medicine can be greatly improved.
Brief description of the drawings
Fig. 1 is micro-fluidic chip schematic diagram of the present invention, and a is schematic diagram in kind；B is micro-fluidic chip section
Schematic diagram, wherein 1 upper strata chip, 2 lower layer chips, 3 porous membranes, 4 cell culture chambers, 5 for 1# it is logical
Road, 6 be 2# passages, 7 be 3# passages.
Fig. 2 is cell fluorescence figure, and wherein a is that micro-fluidic chip upper strata chip culturing room of the present invention inner cell is glimmering
Light figure；Upper strata chip cell；B is micro-fluidic chip lower layer chip passage inner cell fluorogram of the present invention；C sheets
Invent is micro-fluidic chip upper strata chip culturing room inner cell fluorogram and lower layer chip passage inner cell fluorogram
Merge figure.
Fig. 3：A capecitabines act on the micro- light field figure and dead/vital staining fluorogram of different cells；B cards
Train his cell viability influence of the shore on different cells.
Fig. 4：A be various dose capecitabine act on breast cancer cell micro- light field figure and dead/vital staining it is glimmering
Light figure；B is inhibiting rate of the various dose capecitabine to breast cancer cell.
Specific embodiment
The following examples will be further described to the present invention, but not thereby limiting the invention.
Embodiment 1
The foundation of multiple organ tumor-targeting drug test platform chip
Chip is designed and produced, upper strata chip 1 is a cell culture chamber 4, and lower layer chip 2 is three parallel only
Vertical passage, structure is as shown in Figure 1 b.Porous membrane 3 is placed in UV activation 1 hour, silanization on slide
Treatment 30 minutes, oxygen plasma sealing-in is together carried out with upper strata chip 1, puts 80 degree of baking ovens, 30 minutes.
The use of monomer and ratio of initiator is 20:1 PDMS polymer, gets rid of 10um-50um thickness on slide, under
After layer chip 2 upper surface dips thin PDMS, bonding of being alignd with the porous membrane 3 for being sealed with upper strata chip,
80 degree, 30 minutes completion of cures.Result is as shown in Figure 1a.
Embodiment 2
The sign of multiple organ tumor-targeting drug test platform
The micro-fluidic chip made of laboratory designed, designed, structure is as shown in Figure 1 b.Hepg2 cells are used
After green cell film dyestuff treatment 30min, upper strata chip cell culture chamber 4 is accessed.MDA-MB-231 is thin
After born of the same parents, A549 cells, GES-1 cells process 30min using red cell film dyestuff, lower floor is respectively connected to
Chip three passage 1# passages 5,2# passages 6,3# passages 7.It is thin upper strata chip to be shot using fluorescence microscope
The cell of born of the same parents culturing room 4, as a result as shown in Figure 2 a, lower layer chip passage 1# is shot using fluorescence microscope
Passage 5,2# passages 6, the cell of 3# passages 7, as a result as shown in Figure 2 b.By in the chip culturing room of upper strata
Cell fluorescence figure merges with lower layer chip passage inner cell fluorogram, as a result as shown in Figure 2 c.
Embodiment 3
Effect of the capecitabine to different cells is tested using multiple organ tumor-targeting drug test platform
The micro-fluidic chip made using laboratory designed, designed, structure is as shown in Figure 1 b.Compound concentration is 100
The I type Collagen type-I working solution of μ g/mL, via in cell entry pond injection chip, normal temperature stands overnight,
Collagen working solution, cell culture fluid irrigation channel 3 times are removed after 24 hours.On upper strata, chip be inoculated with/is not inoculated with
During Hepg2 cells, MDA-MB-231, U87, GES-1 cell are inoculated with respectively in 3 units of lower layer chip,
Upper strata culturing room is added containing 80 μM of H-DMEM culture mediums of capecitabine.After 48 hours, by dead/living dye
Color reagent box and CCK8 kits detect toxic action of the medicine to different cells.Result such as Fig. 3 a and figure
Shown in 3b, the capecitabine of 80uM concentration is when without hepatic metabolism, and the treatment of 48 hours is equal to each cytotoxicity
It is smaller；And through hepatic metabolism after, the treatment normal tissue cell GES-1 lethal effects of 48 hours are weaker, and
There is obvious lethal effect to MB231 and U87, and medicine is stronger to the lethal effect of MB231.
Embodiment 4
Effect of the various concentrations capecitabine to breast cancer is tested using multiple organ tumor-targeting drug test platform
The micro-fluidic chip made using laboratory designed, designed, structure is as shown in Figure 1 b.After chip modification,
Diabetic angiopathy model is set up using cell inoculation same as Example 1 and training method.On upper strata
Chip be inoculated with/is not inoculated with Hepg2 cells, and lower layer chip unit is inoculated with MDA-MB-231 cells, containing not
Upper strata culturing room is added with the H-DMEM culture mediums of concentration capecitabine.48 as a child, by dead/living dye
Color reagent box and CCK8 kits detect toxic action of the medicine to different cells.Result such as Fig. 4 a and figure
Shown in 4b, MB231 cell viabilities are reduced with the increase of capecitabine concentration.
Claims (5)
1. a kind of micro-fluidic chip, it is characterised in that：The chip is main by upper strata chip, lower layer chip and many
Hole filter membrane composition, to connect on porous membrane and connect lower layer chip, upper strata chip structure under a layer chip, porous membrane
It is big cell culture chamber, lower layer chip structure is independent multichannel, and number of channels is 1-9 bars.
2. according to a kind of micro-fluidic chip described in claim 1, it is characterised in that：The chip material is
The ventilative PDMS polymer of light-permeable, porous membrane material is polycarbonate membrane, and aperture is 0.01um-4um.
3. a kind of multiple organ tumor-targeting drug test platform based on micro-fluidic chip described in claim 1,
It is characterized in that the platform builds as follows：
(1) chip manufacturing and modification
Upper strata chip and lower layer chip are designed and produced, porous membrane is clipped in the middle carries out sealing-in, will with pipettor
The collagen working solution for preparing adds upper strata chip cell culture chamber and each passage of lower layer chip, and bottom surface is carried out
Modification, promotes cell preferably adherent；
(2) inoculation of upper strata chip inner cell and culture
After liver cell digestion, adjust to suitable cell density, be added in the chip of upper strata, in repairing for collagen
Under decorations effect, cell is rapid adherent and uniformly spreads in cell culture chamber bottom surface, when seeing under an optical microscope
When observing cell and being uniformly distributed in cell culture chamber, immediately will chip move into CO2gas incubator in continue
Culture；
(3) inoculation of lower layer chip inner cell and culture
When liver cell is paved with cell culture chamber 60% by propagation, different tumour cells is thin with normal structure
Born of the same parents' inoculated and cultured after 12 hours, adds the culture medium containing liquid to continue to cultivate in lower layer chip to upper strata chip
48 hours.
4. according to the application of multiple organ tumor-targeting drug test platform described in claim 3, it is characterised in that
The platform can be used for chip inner cell activity and investigate, and procedure is as follows：
(1) detection of cytoactive, CCK-8 reagents are carried out using CCK-8 kits：Cell culture medium=1:9
It is mixed into detection working solution, adds lower layer chip passage, put in incubator after 3 hours, is transferred to 96 holes
In plate, ELIASA determines absorbance at 450nm；
(2) porous membrane that lower layer chip has upper strata chip with envelope is separated, the cell of lower layer chip can be carried out
Microexamination, routine immunization fluorescence colour is characterized.
5. according to the application of multiple organ tumor-targeting drug test platform described in claim 3, it is characterised in that
Method the method process that the platform can be used for drug metabolism processes research is as follows：Collect culture supernatants and under
Layer nutrient solution, can respectively carry out mass spectral analysis, can detect the content of medicine prototype and metabolin.
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Cited By (7)
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| CN109055204A (en) * | 2018-10-19 | 2018-12-21 | 杭州捷诺飞生物科技股份有限公司 | Drug screening organ chip |
| CN110373377A (en) * | 2019-07-11 | 2019-10-25 | 杭州原生生物科技有限公司 | A kind of construction method and application thereof of in vitro vascularized tissue |
| CN110878285A (en) * | 2019-11-29 | 2020-03-13 | 许传亮 | Chip organ model for screening bladder tumor chemotherapy drugs and manufacturing method thereof |
| CN110940808A (en) * | 2019-11-18 | 2020-03-31 | 汕头大学医学院 | Establishment method of CXCR4 targeted drug therapy triple negative breast cancer platform |
| CN111921572A (en) * | 2020-07-15 | 2020-11-13 | 广东药科大学 | Microfluidic chip and multi-target active ingredient screening method |
| CN113362690A (en) * | 2021-05-31 | 2021-09-07 | 中国科学技术大学 | Liver lobule chip |
| WO2022104626A1 (en) * | 2020-11-19 | 2022-05-27 | 深圳先进技术研究院 | Micro-fluidic technology-based multifunctional organ chip, preparation method therefor and use thereof |
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| CN103952300A (en) * | 2014-03-31 | 2014-07-30 | 大连医科大学 | Micro-fluidic chip and cell chemotaxis movement research method |
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