CN204097450U - A kind of multidimensional many concentration susceptibility detects micro-fluidic chip - Google Patents
A kind of multidimensional many concentration susceptibility detects micro-fluidic chip Download PDFInfo
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- CN204097450U CN204097450U CN201420262631.6U CN201420262631U CN204097450U CN 204097450 U CN204097450 U CN 204097450U CN 201420262631 U CN201420262631 U CN 201420262631U CN 204097450 U CN204097450 U CN 204097450U
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Abstract
The utility model provides a kind of multidimensional many concentration susceptibility and detects micro-fluidic chip, and this micro-fluidic chip is made up of glass substrate, primary diaphragm, secondary diaphragm and porous-film; Primary diaphragm there is three-dimensional cell cultivation unit; Secondary diaphragm there are gradient concentration maker and cell two dimension culture channel; One deck porous-film is had between primary diaphragm and secondary diaphragm; The number of three-dimensional cell cultivation unit is identical with cell two dimension culture channel; Cell two dimension culture channel lays respectively at above three-dimensional cell cultivation unit.The similar many acute drug being in flow state of this micro-fluidic chip, through the bionic model of vascular endothelial cell barrier action in pipe week tissue, can investigate the effect of many acute drug to dimensional culture sick cell and the toxicity to vascular endothelial cell simultaneously.This product has the advantages such as simple to operate, low consumption, high-throughput, bio-imitability be strong, has important value and wide application prospect in biomedical research.
Description
Technical field
The utility model belongs to microfluidic chip technology is applied to field of biomedical research, relates to a kind ofly detecting the micro-fluidic chip that multiple acute drug acts on dimensional culture and two-dimentional cultured cells.
Background technology
At present, the conventional external model of detection of drugs susceptibility is 96 orifice plates, by cell cultures in orifice plate, in each hole, then adds the medicine of different sorts or different concns respectively, observes medicine to the impact of cell.But such method has a lot of shortcoming: 1) complex operation, workload is large, such as, a hole in orifice plate can only represent a kind of concentration, for three that follow biological study parallel multiple hole experiment principles, same concentration needs 3 holes, if need detection 6 kinds of drug levels, just need 18 holes, i.e. 18 sample introduction processes; 2) floorage of orifice plate is large, and reagent consumption is many; 3) medicine is in stationary state in the orifice plate, can not show drug disposition in the blood vessel with the state of blood flow; 4) medicine directly acts on cell, and medicine needs just can act on cell through after blood vessel endothelium barrier in vivo.Therefore, 96 orifice plate methods have significant limitation in the effect investigating medicine and toxicity.
Microfluidic chip technology originates in the nineties in 20th century, is a new technology of development in recent years, is also one of main technology platforms of putative systems biology research at present.It refers to as integrated in sample preparation, reaction, separation, detection etc. for the basic function of standard biologic or chemical laboratory or be substantially integrated on the chip of a piece several square centimeters (even less), network is formed by microchannel, run through a kind of technology of whole system with controlled fluid, there is the characteristics such as consumption is few, flux is high, integrated, bio-imitability is strong.Facture of microchip material has ventilative, printing opacity, the feature such as transparent, clearly can observe the upgrowth situation of cell, facilitate data gathering.
The utility model constructs a kind of multidimensional multi-medicament and detects micro-fluidic chip, this chip can generate the medicine of multiple concentration, and then the agent permeates therethrough endothelial cell barrier of different concns acts on the cell of dimensional culture, this model agent permeates therethrough endothelial cell barrier that can be used in analogue body inner blood acts on the process of pathological tissues, namely can investigate the action effect of medicine to pathological tissues, also can investigate the toxicity of drug on endothelial cells simultaneously.
Utility model content
The purpose of this utility model is to provide a kind of micro-fluidic chip simple to operation, realizes multiple types, cell that many acute drug act on two dimension and dimensional culture simultaneously, detects drug effect and drug toxicity simultaneously.
A kind of multidimensional many concentration susceptibility detects micro-fluidic chip, and this micro-fluidic chip is made up of glass substrate 1, primary diaphragm 2, secondary diaphragm 3 and porous-film 4, it is characterized in that: primary diaphragm 2 has n three-dimensional cell cultivation unit 5; Three-dimensional cell cultivation unit 5 is made up of the connected three-dimensional cell cultivation pond 7 of cell injection port 6, three and cell outlet 8; Secondary diaphragm 3 there are the gradient concentration maker 9 and n bar cell two dimension culture channel 10 that can generate n kind concentration; One end of cell two dimension culture channel 10 is connected with gradient concentration maker 9, and the other end is connected with endotheliocyte injection port 11; N bar cell two dimension culture channel 10 lays respectively at the top of n three-dimensional cell cultivation unit 5; Porous-film 4 is positioned in the middle of cell two dimension culture channel 10 and three-dimensional cell cultivation unit 5; Above n be greater than zero integer.
Multidimensional many concentration susceptibility described in the utility model detects micro-fluidic chip, it is characterized in that: described gradient concentration maker 9 is made up of 2 medicine injection port 9a, 2 sample intake passage 9b and medicament mixed passage 9c; Wherein medicament mixed passage 9c is made up of multilayer medicine mixed cell, every layer of medicament mixed unit comprises an interconnection and many curved shape medicament mixed passages that are connected vertical with this interconnection, and every layer of curved shape medicament mixed passage is connected with the interconnection of lower one deck simultaneously; The first layer medicament mixed unit has three curved shape medicament mixed passages, and most end one deck has n bar curved shape medicament mixed passage, and every bar curved shape medicament mixed passage is all connected with a cell two dimension culture channel 10; One end of every bar sample intake passage 9b is provided with medicine injection port 9a, and the other end is connected with the Article 1 interconnection of medicament mixed unit.
Multidimensional many concentration susceptibility described in the utility model detects micro-fluidic chip, it is characterized in that: on primary diaphragm 2, each three-dimensional cell cultivation unit 5 is separate, and arranged in parallel; Each cell two dimension culture channel 10 on secondary diaphragm 3 is separate, and arranged in parallel.
Multidimensional many concentration susceptibility described in the utility model detects micro-fluidic chip, it is characterized in that: described porous-film 4 is the film allowing liquid and cytokine to transmit between the three-dimensional cell cultivation unit 5 and the cell two dimension culture channel 10 of secondary diaphragm 3 of primary diaphragm 2, is preferably polycarbonate membrane.
Multidimensional many concentration susceptibility described in the utility model detects micro-fluidic chip, it is characterized in that: the material of primary diaphragm 2 and secondary diaphragm 3 is polydimethylsiloxane; Glass substrate 1, primary diaphragm 2 and secondary diaphragm 3 is irreversible sealing-in.
Adopt micro-fluidic chip described in the utility model to study multidimensional many concentration susceptibility detection method to be: in the three-dimensional cell cultivation unit 5 of primary diaphragm 2, add the first predefined type material respectively, the second predefined type material is added in the passage of secondary diaphragm 3, porous-film 4 surface seeding one deck the 3rd predefined type material in secondary diaphragm cell two dimension culture channel, drive the second predefined type material to flow in passage, then investigate the operative condition of the second predefined type material to the first predefined type material and the 3rd predefined type material.
Described first predefined type material is cell, tissue, organ; Second predefined type material is substratum, medicine, chemokine or somatomedin; 3rd predefined type material is vascular endothelial cell.
Concrete research process is as follows:
---by cell injection port 6, first predefined type material and extracellular matrix surrogate are added in primary diaphragm three-dimensional cell cultivation unit 5;
---by medicine injection port 9a, the second predefined type material is added in the passage of secondary diaphragm;
---by endotheliocyte injection port 11, the 3rd predefined type material is added in the cell two dimension culture channel 10 of secondary diaphragm, leave standstill 12 ~ 24 hours, make the 3rd predefined type material fully be attached at porous-film 4 surface;
---syringe pump is connected with medicine injection port 9a, drives the second predefined type material to flow in passage.
Micro-fluidic chip described in the utility model is similar to acute drug more than through the bionic model of blood vessel endothelium barrier action in pipe Zhou Sanwei pathological tissues, and investigates the effect of medicine to pathological tissues and the toxicity to vascular endothelial cell simultaneously.The utility model solve conventional model flux low, consume the problems such as large, bio-imitability is poor, there is the advantages such as simple to operate, low consumption, high-throughput, bio-imitability are strong, there is important value and wide application prospect in biomedical research.
Accompanying drawing explanation
Microfluidic chip structure schematic diagram described in Fig. 1 embodiment 1, comprising glass substrate 1, primary diaphragm 2, secondary diaphragm 3, porous-film 4;
Fig. 2 primary diaphragm 2 structural representation;
Fig. 3 secondary diaphragm 3 structural representation;
Fig. 4 shows micro-fluidic chip integrated operation process;
Fig. 5 display is inoculated in the vascular endothelial cell on porous-film 4 surface;
Fig. 6 shows secondary diaphragm 3 gradient concentration and generates situation; After the medicine injection port 9a both sides of secondary diaphragm 3 add phosphate buffered saline buffer (PBS) and fluorescent dyes rhodamine-123 with syringe pump driving respectively simultaneously, fluorescent dyes rhodamine-123 forms the fluorescence intensity situation (being followed successively by cell two dimension culture channel 10a, 10b, 10c, 10d, 10e, 10f by left-to-right in figure) of the concentration that six kinds strengthen gradually by gradient concentration maker 9;
Fig. 7 shows the medicament sensitivity test of tumor cell line in micro-fluidic chip (ACC-M); The tumor cell line (ACC-M) of three dimensional matrix glue parcel is inoculated in the three-dimensional cell cultivation unit 5 of primary diaphragm 2, inoculation vascular endothelial cell strain HUVEC in cell two dimension culture channel 10, starting point concentration is that the taxol (PTX) of 0.5 μ g/ml forms six kinds of different drug levels through gradient concentration maker 9, and then the situation (being followed successively by three-dimensional cell cultivation unit 5a, 5b, 5c, 5d, 5e, 5f by left-to-right in figure) of effect tumor cell line (ACC-M).
Embodiment
Below in conjunction with accompanying drawing, embodiment of the present utility model is described in further detail.
Embodiment 1
The microfluidic chip structure of this laboratory designed, designed and preparation as Figure 1-3, this micro-fluidic chip is made up of glass substrate 1, primary diaphragm 2, secondary diaphragm 3 and porous-film 4, wherein the material of primary diaphragm 2 and secondary diaphragm 3 is polydimethylsiloxane, porous-film 4 is polycarbonate membrane, is positioned in the middle of primary diaphragm 2 and secondary diaphragm 3.
Primary diaphragm 2 has the three-dimensional cell cultivation unit 5 that six separate, each three-dimensional cell cultivation unit 5 is made up of the connected three-dimensional cell cultivation pond 7 of cell injection port 6, three and cell outlet 8; Secondary diaphragm 3 there are the gradient concentration maker 9 and six cell two dimension culture channel 10 that can generate 6 kinds of concentration; One end of cell two dimension culture channel 10 is connected with gradient concentration maker 9, and the other end is connected with endotheliocyte injection port 11; Wherein gradient concentration maker 9 is made up of two medicine injection port 9a, two sample intake passage 9b and medicament mixed passage 9c; Article six, cell two dimension culture channel 10 lays respectively at the top of six three-dimensional cell cultivation unit 5.Wherein medicament mixed passage 9c is made up of four layers of medicament mixed unit, every layer of medicament mixed unit comprises an interconnection and many curved shape medicament mixed passages that are connected vertical with this interconnection, and every layer of curved shape medicament mixed passage is connected with the interconnection of lower one deck simultaneously; The first layer has three curved shape medicament mixed passages, and the 4th layer has six articles of curved shape medicament mixed passages; One end of sample intake passage 9b is provided with medicine injection port 9a, and the other end is connected with the Article 1 interconnection of medicament mixed unit.
The preparation process of described chip is: first, by irreversible sealing technology by primary diaphragm 2 and glass substrate 1 sealing-in; Then, above the three-dimensional cell cultivation unit 5 porous-film 4 being covered in primary diaphragm 2; Finally, the three-dimensional cell cultivation unit 5 that the cell of secondary diaphragm 3 two dimension culture channel 10 is aimed on primary diaphragm 2 is carried out irreversible sealing-in.
As Fig. 4, add tumour cell by cell injection port 6, add cell culture medium by medicine injection port 9a, 37 DEG C, 5%CO
2cultivate in incubator, become spheroid to cell; By endotheliocyte injection port 11, vascular endothelial cell strain HUVEC is inoculated in the surface of porous-film 4.Then chip is placed in 37 DEG C, 5%CO
2cultivate 24 hours in incubator, HUVEC is fully attached (Fig. 5).
Embodiment 2
Chip described in embodiment 1 is adopted to study: by both sides medicine injection port 9a, syringe pump is utilized to drive phosphate buffered saline buffer (PBS) and fluorescent dyes rhodamine-123 by gradient concentration maker 9 respectively, after for some time, the situation that fluorescent dyes rhodamine-123 forms strengthen gradually six kinds of different concns fluorescence intensities of monolithic stability in six cell two dimension culture channel 10 is shown in Fig. 6.
Embodiment 3
Chip described in embodiment 1 is adopted to study: the tumor cell line (ACC-M) being added three dimensional matrix glue parcel by cell injection port 6 in 6 three-dimensional cell cultivation unit 5 of primary diaphragm 2,37 DEG C, 5%CO
2cultivate three days in incubator.By endotheliocyte injection port 11, vascular endothelial cell strain HUVEC is inoculated in the surface of porous-film 4, chip is placed in 37 DEG C, 5%CO
2cultivate 24 hours in incubator, HUVEC is fully attached.Syringe pump is utilized to drive cell culture medium and taxol (PTX, concentration is 0.5 μ g/ml) medicine by gradient concentration maker 9 respectively by both sides medicine injection port 9a, continous perfusion 24 hours.Remove secondary diaphragm 3, with Hoechst33342 and PI fluorescent dye, take pictures under fluorescent microscope, calculate ACC-M cells survival situation (Fig. 7).
Above-described embodiment, only for technical conceive of the present utility model and feature are described, its object is to person skilled in the art can be understood content of the present utility model and implement according to this, can not limit protection domain of the present utility model with this.All equivalences done according to the utility model spirit change, retrofit or modify, and all should be encompassed within protection domain of the present utility model.
Claims (6)
1. multidimensional many concentration susceptibility detects micro-fluidic chip, this micro-fluidic chip is made up of glass substrate (1), primary diaphragm (2), secondary diaphragm (3) and porous-film (4), it is characterized in that: primary diaphragm (2) has n three-dimensional cell cultivation unit (5); Three-dimensional cell cultivation unit (5) is made up of cell injection port (6), 3 connected three-dimensional cell cultivation ponds (7) and cell outlet (8); Secondary diaphragm (3) there are the gradient concentration maker (9) and n bar cell two dimension culture channel (10) that can generate n kind concentration; One end of cell two dimension culture channel (10) is connected with gradient concentration maker (9), and the other end is connected with endotheliocyte injection port (11); N bar cell two dimension culture channel (10) lays respectively at the top of n three-dimensional cell cultivation unit (5); Porous-film (4) is positioned in the middle of cell two dimension culture channel (10) and three-dimensional cell cultivation unit (5); Above n be greater than zero integer.
2. detect micro-fluidic chip according to multidimensional many concentration susceptibility according to claim 1, it is characterized in that: described gradient concentration maker (9) is made up of 2 medicine injection ports (9a), 2 sample intake passages (9b) and medicament mixed passage (9c); Wherein medicament mixed passage (9c) is made up of multilayer medicine mixed cell, every layer of medicament mixed unit comprises an interconnection and many curved shape medicament mixed passages that are connected vertical with this interconnection, and every layer of curved shape medicament mixed passage is connected with the interconnection of lower one deck simultaneously; The first layer medicament mixed unit has three curved shape medicament mixed passages, and most end one deck has n bar curved shape medicament mixed passage, and every bar curved shape medicament mixed passage is all connected with cell two dimension culture channel (10); One end of every bar sample intake passage (9b) is provided with medicine injection port (9a), and the other end is connected with the Article 1 interconnection of medicament mixed unit.
3. detect micro-fluidic chip according to the multidimensional many concentration susceptibility described in claim 1 or 2, it is characterized in that: the upper each three-dimensional cell cultivation unit (5) of primary diaphragm (2) is separate, and arranged in parallel; Cell two dimension culture channel (10) on secondary diaphragm (3) is separate, and arranged in parallel.
4. detect micro-fluidic chip according to the multidimensional many concentration susceptibility described in claim 1 or 2, it is characterized in that: described porous-film (4) is the film allowing liquid and cytokine to transmit between the three-dimensional cell cultivation unit (5) and cell two dimension culture channel (10) of secondary diaphragm (3) of primary diaphragm (2).
5. detect micro-fluidic chip according to multidimensional many concentration susceptibility according to claim 4, it is characterized in that: described porous-film is polycarbonate membrane.
6. detect micro-fluidic chip according to the multidimensional many concentration susceptibility described in claim 1 or 2, it is characterized in that: the material of primary diaphragm (2) and secondary diaphragm (3) is polydimethylsiloxane; Glass substrate (1), primary diaphragm (2) and secondary diaphragm (3) are irreversible sealing-in.
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