CN103952300A - Micro-fluidic chip and cell chemotaxis movement research method - Google Patents
Micro-fluidic chip and cell chemotaxis movement research method Download PDFInfo
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- 230000034196 cell chemotaxis Effects 0.000 title claims abstract description 13
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- 239000000463 material Substances 0.000 claims description 31
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- 108010012236 Chemokines Proteins 0.000 claims description 5
- 102000019034 Chemokines Human genes 0.000 claims description 5
- 230000002427 irreversible effect Effects 0.000 claims description 5
- 239000004205 dimethyl polysiloxane Substances 0.000 claims description 3
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 3
- 229920000515 polycarbonate Polymers 0.000 claims description 3
- 239000004417 polycarbonate Substances 0.000 claims description 3
- -1 polydimethylsiloxane Polymers 0.000 claims description 3
- 210000001519 tissue Anatomy 0.000 claims description 3
- 108090000695 Cytokines Proteins 0.000 claims description 2
- 102000004127 Cytokines Human genes 0.000 claims description 2
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 2
- 102000013275 Somatomedins Human genes 0.000 claims description 2
- 238000010899 nucleation Methods 0.000 claims description 2
- 230000035605 chemotaxis Effects 0.000 abstract description 9
- 239000011664 nicotinic acid Substances 0.000 abstract description 2
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- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The aim of the invention is to provide a micro-fluidic chip and a cell chemotaxis movement research method. The micro-fluidic chip is characterized in that the micro-fluidic chip comprises a glass substrate, a first diaphragm and a second diaphragm; the first diaphragm is provided with culturing units; the second diaphragm is provided with micro-channels; a porous membrane exists between the culturing units of the first diaphragm and the micro-channels of the second diaphragm; the number of the culturing units of the first diaphragm is same to the number of the micro-channels of the second; and the micro-channels on the second diaphragm are respectively located above the culturing units of the first diaphragm. The micro-fluidic chip can be used in the research of the selective chemotaxis movement condition of cells in the micro-channels when the cells flow through the culturing units. The micro-fluidic chip likes a bionic model characterized in that a branching capilary goes through organs; and compared with the traditional cell chemotaxis research methods, the research method is a new method for researching the selective chemotaxis movement of the cells in the flowing process, and has important biomedical research values and economic values.
Description
Technical field
The present invention relates to micro-fluidic chip technology to be applied to biomedical research field.The present invention provides a kind of micro-fluidic chip and research method of studying cell chemotaxis motion especially.
Background technology
Chemotaxis is that phalangeal cell is under chemical concentrations gradient effect, towards the process of stimulator direction displacement.Chemotaxis plays a significant role in histoorgan growth, wound healing and metastases process, and for example injured tissue secretion chemokine attracts white corpuscle to arrive infection site, removes downright bad tissue, promotes wound healing; The organs such as lung, liver, bone are because a large amount of chemokine of secretion becomes modal metastases target organ etc.
At present, the conventional external model of research cell chemotaxis effect is Transwell cell, is mainly used in studying cell chemotactic process under static state.Its profile is a noggin that can be placed in orifice plate, Transwell cell is put into culture plate, in cell He in culture plate, adds nutrient solution simultaneously, by cell kind in cell, nutrient solution composition in culture plate can have influence on the cell in cell, and cell is produced to chemotaxis.But Transwell cell is a larger open space, do not possess microvascular enclosed space feature in body; And because the cell in cell is in static cultivation conditions, can not reproduce the chemotactic process of capillary blood vessel inner cell under flow state.Therefore, Transwell cell can not carry out the biological procedures of directed Chemotaxis for studying cell under flow state through wall of micrangium.
For solving an above-mentioned difficult problem, the present invention adopts micro-fluidic chip technology that a new research platform is provided, can simulate cell in capillary blood vessel in flow process, while especially flowing through organ different, the chemokine being produced by organ attracts to occur the process of directed chemotactic.Micro-fluidic chip technology is the new technology growing up on capillary electrophoresis basis early 1990s, in recent years to biomedical sector, permeate rapidly, shown wide application prospect, this technology has been proved to be the ideal platform that mammalian cell and microenvironment thereof are controlled, consume low, flux is high, bio-imitability is strong.Microchannel on micro-fluidic chip is micron-scale, and space relative closure is quite similar with the microvessel structure in body; And micro-fluidic chip is the ideal platform of controlling fluid, be convenient to flowing of analogue body inner blood.Therefore, the constructed micro-fluidic chip platform of the present invention is highly suitable for the cell of research under flow state through the directed Chemotaxis of capillary endothelium barrier.
Summary of the invention
The object of the present invention is to provide the research method of a kind of micro-fluidic chip and cell chemotaxis motion.
The present invention specifically provides a kind of micro-fluidic chip, it is characterized in that: this micro-fluidic chip is comprised of glass substrate, primary diaphragm and secondary diaphragm; On primary diaphragm, there is cultivation unit; On secondary diaphragm, there is microchannel; In the middle of the cultivation unit of primary diaphragm and the microchannel of secondary diaphragm, there is one deck porous-film; Cultivation unit on primary diaphragm has n, and n is greater than zero integer; On secondary diaphragm, have n bar microchannel, n value is identical with the n value of cultivating unit on primary diaphragm; Microchannel on secondary diaphragm lays respectively at the top, cultivation unit of primary diaphragm.
Micro-fluidic chip of the present invention, is characterized in that: on primary diaphragm, respectively cultivate unit separate, each cultivates unit by independently fluid inlet one, cultivation pool and liquid outlet one form; On secondary diaphragm, a side of each microchannel comes together in fluid inlet two by connecting passage, and an other side comes together in liquid outlet two by connecting passage.
Micro-fluidic chip of the present invention, is characterized in that: on primary diaphragm, respectively cultivate unit and be arranged parallel to each other, the microchannel of each on secondary diaphragm is parallel to each other.
Micro-fluidic chip of the present invention, is characterized in that: the material of described primary diaphragm and secondary diaphragm is polydimethylsiloxane; Glass substrate, primary diaphragm and secondary diaphragm are irreversible sealing-in; The degree of depth of cultivating unit is 1～30 millimeter; The degree of depth of the microchannel on secondary diaphragm is 10～1000 microns (bottom of cultivating unit on primary diaphragm can be got through, and glass substrate and primary diaphragm carry out after irreversible sealing-in, and glass substrate upper surface can be used as the bottom surface of cultivating unit); The film of described porous-film for allowing liquid and cytokine to transmit between the cultivation unit of primary diaphragm and the microchannel of secondary diaphragm, is preferably polycarbonate membrane.
The present invention also provides the method that adopts the motion of described micro-fluidic chip research cell chemotaxis, it is characterized in that: surface seeding one deck the 3rd predefined type material that is close to respectively secondary diaphragm at porous-film, in the cultivation unit of primary diaphragm, add the first predefined type material, in the microchannel of secondary diaphragm, add the second predefined type material, drive the second predefined type material to flow in microchannel, then investigate the situation of sticking of the second predefined type material.
The method of research cell chemotaxis motion of the present invention, is characterized in that: described the first predefined type material is cell, tissue, organ, chemokine or somatomedin; The second predefined type material is cell; The 3rd predefined type material is vascular endothelial cell.
The method of research cell chemotaxis motion conditions of the present invention, concrete research process is as follows:
---by fluid inlet 2, the 3rd predefined type material is added in the microchannel of secondary diaphragm, standing 12～24 hours, make the 3rd predefined type material fully be attached at porous film surface;
---by fluid inlet 1, the first predefined type material is added in the cultivation unit of primary diaphragm;
---by fluid inlet 2, the second predefined type material is added in the microchannel of secondary diaphragm;
---syringe pump is connected with fluid inlet two, to drive the second predefined type material to flow in microchannel;
---micro-fluidic chip is placed under microscope, investigates the situation of sticking of the second predefined type material.
Micro-fluidic chip of the present invention can be for research the Chemotaxis optionally in a plurality of cultivation unit processes of the stream of cells in microchannel.With respect to traditional Transwell cell, the present invention is similar to a branched capillary blood vessel by the bionic model of a plurality of organs, and a kind of novel method of studying cell selectivity Chemotaxis in flow process is provided, in biomedical research, there is important value and wide application prospect.
Accompanying drawing explanation
The schematic diagram of Fig. 1 micro-fluidic chip of the present invention, comprising glass substrate 1, primary diaphragm 2, secondary diaphragm 3, porous-film 4;
Fig. 2 primary diaphragm structural representation, wherein has four relatively independent cultivation unit (5a, 5b, 5c, 5d) on primary diaphragm, each cultivates unit fluid inlet 1, cultivation pool 7, liquid outlet 1;
Fig. 3 secondary diaphragm structural representation, wherein on secondary diaphragm, there are four parallel microchannels (9a, 9b, 9c, 9d), microchannel comes together in fluid inlet 2 11 at one deck by dendritic connecting passage one 10a of a component, and microchannel comes together in liquid outlet 2 12 at other one deck by connecting passage two 10b;
The micro-fluidic chip photo that Fig. 4 has assembled, this chip is connected with syringe pump 13 by fluid inlet 2 11;
Fig. 5 shows the vascular endothelial cell that is inoculated in porous-film 4 contiguous secondary diaphragm 3 surfaces;
Fig. 6 is presented in the cultivation pool of primary diaphragm 2 and adds FITC-Dextran(molecular weight to be about 12KD) after, FITC-Dextran enters the situation (be followed successively by from left to right in figure at 0 minute, 10 minutes, 30 minutes, 120 minutes FITC-Dextran and enter the situation in secondary diaphragm microchannel) in secondary diaphragm microchannel by porous-film and vascular endothelial cell;
Fig. 7 is presented in the cultivation pool of primary diaphragm and adds after different concns Chemokine CXCL12, and tumor cell line (ACC-M) attaches to the situation (in figure concentration be followed successively by 0ng/ml, 25ng/ml, 50ng/ml, 100ng/ml) from top to bottom of vascular endothelial cell.
Fig. 8 is presented in the different cultivation pools of primary diaphragm and adds and extract after the different cells of mouse, and tumor cell line (ACC-M) attaches to the situation (being followed successively by from top to bottom the myocyte, pneumonocyte, liver cell, the medullary cell that extract from mouse in figure) of vascular endothelial cell.
Embodiment
Embodiment 1
Micro-fluidic chip used is this laboratory designed, designed and preparation.
As Figure 1-3, micro-fluidic chip of the present invention is comprised of glass substrate 1, primary diaphragm 2, secondary diaphragm 3 and porous-film 4, and wherein the material of primary diaphragm 2 and secondary diaphragm 3 is polydimethylsiloxane, and porous-film 4 is polycarbonate membrane.On primary diaphragm 2, have four unit that are parallel to each other and independently cultivate (5a, 5b, 5c, 5d), each cultivates unit (5a, 5b, 5c, 5d) respectively independently fluid inlet 1 and liquid outlet 1, between fluid inlet 1 and liquid outlet 1, is provided with cultivation pool 7; On secondary diaphragm 3, there are four microchannels that are parallel to each other (9a, 9b, 9c, 9d), one side of microchannel comes together in fluid inlet 2 11 at one deck by dendritic connecting passage one 10a of a component, opposite side comes together in liquid outlet 2 12 at other one deck by connecting passage two 10b, and microchannel (9a, 9b, 9c, 9d) lays respectively at the top, cultivation unit (5a, 5b, 5c, 5d) of primary diaphragm 2.
The preparation process of described chip is: first, by irreversible sealing technology by primary diaphragm 2 and glass substrate 1 sealing-in; Second step, is covered in porous-film 4 above the cultivation unit of primary diaphragm 2; The 3rd step, the cultivation unit that the microchannel of secondary diaphragm 3 is aimed on primary diaphragm 2 carries out irreversible sealing-in.
As Fig. 4, by fluid inlet 1, in cultivating unit, add cell culture fluid, by fluid inlet 2 11, in microchannel, add cell culture fluid.By fluid inlet 2 11, vascular endothelial cell strain HUVEC is inoculated in to the surface of porous-film 4.Then chip is placed in the CO2 incubator of 37 ℃ and cultivates 24 hours, make HUVEC fully attach (Fig. 5), stand-by.
Embodiment 2
Described in employing embodiment 1, chip is studied: by fluid inlet 1, in cultivation pool 7, add FITC-Dextran(molecular weight to be about 12KD), along with the prolongation of time, FITC-Dextran diffuses into by porous-film 4 and vascular endothelial cell in the microchannel of secondary diaphragm 3 (Fig. 6).
Embodiment 3
Described in employing embodiment 1, chip is studied: the interior Chemokine CXCL12 that adds respectively concentration to be divided into 0ng/ml, 25ng/ml, 50ng/ml, 100ng/ml of four cultivation pools 7 by fluid inlet 1 at primary diaphragm 2, hatch 2 hours.The tumor cell line (ACC-M) that adds red fluorescence probe mark by fluid inlet 2 11 in microchannel.By the syringe pump 13 being connected with fluid inlet 2 11, drive the tumor cell line (ACC-M) of red fluorescence probe mark, it is flowed in the microchannel of secondary diaphragm 3, after 30 minutes, stop, after rinsing gently with phosphate buffered saline buffer, under fluorescent microscope, take pictures, record ACC-M cell adhesion in the situation (Fig. 7) of vascular endothelial cell.
Embodiment 4
Described in employing embodiment 1, chip is studied: interior myocyte, pneumonocyte, liver cell, the medullary cell extracting from mouse that add respectively of four cultivation pools 7 by fluid inlet 1 at primary diaphragm 2, hatch 2 hours.The tumor cell line (ACC-M) that adds red fluorescence probe mark by fluid inlet 2 11 in microchannel.By syringe pump 13, drive the tumor cell line (ACC-M) of red fluorescence probe mark, it is flowed in the microchannel of secondary diaphragm 3, after 30 minutes, stop, after rinsing gently with phosphate buffered saline buffer, under fluorescent microscope, take pictures, record ACC-M cell adhesion in the situation (Fig. 8) of vascular endothelial cell.
Above-described embodiment is only explanation technical conceive of the present invention and feature, and its object is to allow person skilled in the art can understand content of the present invention and implement according to this, can not limit the scope of the invention with this.All equivalences that spirit is done according to the present invention change or modify, within all should being encompassed in protection scope of the present invention.
Claims (10)
1. a micro-fluidic chip, is characterized in that: this micro-fluidic chip is comprised of glass substrate, primary diaphragm and secondary diaphragm; On primary diaphragm, there is cultivation unit; On secondary diaphragm, there is microchannel; In the middle of the cultivation unit of primary diaphragm and the microchannel of secondary diaphragm, there is one deck porous-film; Cultivation unit on primary diaphragm has n, and n is greater than zero integer; On secondary diaphragm, have n bar microchannel, n value is identical with the n value of cultivating unit on primary diaphragm; Microchannel on secondary diaphragm lays respectively at the top, cultivation unit of primary diaphragm.
2. according to micro-fluidic chip described in claim 1, it is characterized in that: on primary diaphragm, respectively cultivate unit separate, each cultivates unit by independently fluid inlet one, cultivation pool and liquid outlet one form.
3. according to micro-fluidic chip described in claim 1 or 2, it is characterized in that: on secondary diaphragm, a side of each microchannel comes together in fluid inlet two by connecting passage, an other side comes together in liquid outlet two by connecting passage.
4. according to the arbitrary described micro-fluidic chip of claim 1～3, it is characterized in that: on primary diaphragm, respectively cultivate unit and be arranged parallel to each other, the microchannel of each on secondary diaphragm is parallel to each other.
5. according to the arbitrary described micro-fluidic chip of claim 1～3, it is characterized in that: the material of described primary diaphragm and secondary diaphragm is polydimethylsiloxane; Glass substrate, primary diaphragm and secondary diaphragm are irreversible sealing-in, and the degree of depth of cultivating unit on primary diaphragm is 1～30 millimeter; The degree of depth of the microchannel on secondary diaphragm is 10～1000 microns.
6. according to the arbitrary described micro-fluidic chip of claim 1～3, it is characterized in that: the film of described porous-film for allowing liquid and cytokine to transmit between the cultivation unit of primary diaphragm and the microchannel of secondary diaphragm.
7. according to micro-fluidic chip described in claim 6, it is characterized in that: described porous-film is polycarbonate membrane.
8. a method that adopts micro-fluidic chip research cell chemotaxis motion described in claim 1, it is characterized in that: surface seeding one deck the 3rd predefined type material that is close to respectively secondary diaphragm at porous-film, in the cultivation unit of primary diaphragm, add the first predefined type material, in the microchannel of secondary diaphragm, add the second predefined type material, drive the second predefined type material to flow in microchannel, then investigate the situation of sticking of the second predefined type material.
9. according to the method for studying cell chemotaxis motion described in claim 8, it is characterized in that: described the first predefined type material is cell, tissue, organ, chemokine or somatomedin; The second predefined type material is cell; The 3rd predefined type material is vascular endothelial cell.
10. according to the method for studying cell chemotaxis motion described in claim 8 or 9, it is characterized in that, concrete research process is as follows:
---by fluid inlet 2, the 3rd predefined type material is added in the microchannel of secondary diaphragm, standing 12～24 hours, make the 3rd predefined type material fully be attached at porous film surface;
---by fluid inlet 1, the first predefined type material is added in the cultivation unit of primary diaphragm;
---by fluid inlet 2, the second predefined type material is added in the microchannel of secondary diaphragm;
---syringe pump is connected with fluid inlet two, to drive the second predefined type material to flow in microchannel;
---micro-fluidic chip is placed under microscope, investigates the situation of sticking of the second predefined type material.
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Cited By (12)
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| CN104342360A (en) * | 2014-10-14 | 2015-02-11 | 大连理工大学 | Micro-fluidic chip for realizing chemotaxis of white blood cell |
| CN104513787A (en) * | 2015-01-07 | 2015-04-15 | 东北大学 | Integrated micro-fluidic chip and system for capture, culture and administration of single cells |
| CN104524652A (en) * | 2015-01-29 | 2015-04-22 | 中国科学技术大学 | Artificial liver in-vitro working platform |
| CN104774747A (en) * | 2015-04-14 | 2015-07-15 | 浙江大学 | Microfluidic liquid drop chip device for cell migration analysis experiment and method |
| CN104830683A (en) * | 2015-04-30 | 2015-08-12 | 大连医科大学附属第二医院 | Bionic micro-fluidic chip for simulating in vivo tumor cells and metastasis microenvironment |
| CN105176816A (en) * | 2015-10-30 | 2015-12-23 | 东南大学 | Micro-vessel liver chip based on cell clusters and making method and using method thereof |
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| CN107955782A (en) * | 2016-10-14 | 2018-04-24 | 中国科学院大连化学物理研究所 | Liver-blood-brain barrier system of analogue body intracellular metabolite process based on micro-fluidic chip |
| CN109504604A (en) * | 2018-10-24 | 2019-03-22 | 大连医科大学 | A kind of micro-fluidic chip and the Chemotaxis research method of delicate tissues slice induction |
| CN110117524A (en) * | 2019-04-17 | 2019-08-13 | 华南理工大学 | A kind of micro flow control chip device of hydrodynamic shear induction tumour cell cross-film migration |
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| CN112501238A (en) * | 2020-11-30 | 2021-03-16 | 中国科学院深圳先进技术研究院 | Method for tracking cell movement based on three-dimensional environment and microfluidic chip |
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