CN103103121B - Cell-culture microfluidic chip - Google Patents
Cell-culture microfluidic chip Download PDFInfo
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- CN103103121B CN103103121B CN201310017685.6A CN201310017685A CN103103121B CN 103103121 B CN103103121 B CN 103103121B CN 201310017685 A CN201310017685 A CN 201310017685A CN 103103121 B CN103103121 B CN 103103121B
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Abstract
The invention relates to a cell-culture microfluidic chip. The cell-culture microfluidic chip comprises a first main channel, first micro valves, second micro valves, third micro-valves, a second main channel and a plurality of cell culture units; the cell culture units are arrayed in parallel and arranged between the first main channel and the second main channel; each cell culture unit comprises a first culture pool, a second culture pool and a diffusion channel; the second micro valves are used for controlling the communication and non-communication of the first culture pools with the second culture pools; the first micro valves are used for controlling the communication and the non-communication of the first culture pools with the first main channel through diffusion channels; and the third micro valves are used for controlling the communication and the non-communication of the second culture pools with the second main channel through diffusion channels. By adjusting and controlling the micro valves, the spacial localization and co-culture of a variety of different cells can be realized, and the high-throughput cell culture can be realized.
Description
Technical field
The present invention relates to micro-fluidic chip technical field, particularly relate to a kind of cell culture microflow control chip.
Background technology
The running balance of cell micro-environment be ensure that cell is normally bred, the essential condition of differentiation, metabolism and functional activity, microenvironment changes, also can there is corresponding variation in cell thereupon.The pathological processes such as therefore, fetal development, tissue repair, tumour generation and the residing microenvironment of cell are closely bound up.The research of a lot of physiological and pathologicals is all cultivated and is realized based on cells in vitro, and the cultivation of traditional cells in vitro can only provide Growth of Cells environment a kind of static state, macroscopical, two dimension, under the environment that cell changes in vitro, hyperplasia has been lost original proterties gradually, does not often conform to situation in body.
Along with the emerging development of tissue culture engineering, three-dimensional cell culture technique is arisen at the historic moment, and it is the microenvironment in analogue body farthest.Traditional three-dimensional cell research method has matrix to cover cultivation, rotary flask cultivation, microcarrier cultivation, the cultivation of preset support and Rotary cell culture system etc., not only complex operation of these three-dimensional cell culture techniques, and reagent consumption is large, what is more important, with respect to the microsize of cell, this culture environment and internal milieu differ far away, are objectively difficult to truly reflect the biological property of cell under physiological status.The micro-fluidic chip that early 1990s gets up based on MEMS (micro electro mechanical system) (MEMS) Processing Technology Development, claims again chip lab, has multiple monotechnics, flexible combination, integrated feature and advantage of scale on the controlled small platform of entirety.And the fluid channel size of micro-fluidic chip design (m) (10～20 μ are m) in identical magnitude with typical cells of mamma animals size for common 10～100 μ, and under microscale, conduct heat, mass transfer is very fast, favourable Growth of Cells research environment can be provided, and micro-fluidic chip can meet the needs that high-flux cell is analyzed simultaneously.Just because of micro-fluidic chip has advantages of above-mentioned conventional cell biological study method can't be obtained, become the Important Platform of cell research, in biomedical fields wide application prospect.
Micro-fluidic chip for the dimensional culture of cell and the simulation of microenvironment makes some progress, but also has a lot of deficiencies, the problem that ubiquity flux is low.
Summary of the invention
Based on this, be necessary to provide a kind of high-throughout cell culture microflow control chip.
A kind of cell culture microflow control chip, comprise the first sprue, first micro-valve, second micro-valve, the 3rd micro-valve, the second sprue and multiple cell culture unit of arranging side by side, multiple cell culture units of arranging are side by side between the first sprue and the second sprue;
Each described cell culture unit comprises the first cultivation pool, the second cultivation pool and diffusion runner, and described second micro-valve is for controlling the first cultivation pool of described cell culture unit and the connection of the second cultivation pool or cutting out;
The first cultivation pool is connected with the first sprue by diffusion runner, and the first cultivation pool that described first micro-valve is used for controlling described cell culture unit is by spreading being communicated with or closing of runner and the first sprue;
The second cultivation pool is connected with the second sprue by diffusion runner, and the second cultivation pool that described the 3rd micro-valve is used for controlling described cell culture unit is by spreading being communicated with or closing of runner and the second sprue.
In an embodiment, the height of described diffusion runner is lower than the height of described the first cultivation pool, the second cultivation pool, the first sprue and the second sprue therein.
In an embodiment, the quantity of described first micro-valve is multiple therein, corresponding one by one with multiple cell culture units, and each described first micro-valve independently controls being communicated with or cutting out of the first cultivation pool in corresponding described cell culture unit and the first sprue.
In an embodiment, the quantity of described second micro-valve is 1 therein, and described the second micro-valve interlock is controlled the connection of the first cultivation pool in described cell culture unit and the second cultivation pool or closed.
In an embodiment, the quantity of described the 3rd micro-valve is 1 therein, and the second cultivation pool in described cell culture unit being communicated with or closing by diffusion runner and the second sprue controlled in described the 3rd micro-valve interlock.
In an embodiment, the quantity of described the 3rd micro-valve is multiple therein, corresponding one by one with multiple cell culture units, and each described the 3rd micro-valve independently controls being communicated with or cutting out of the second cultivation pool in corresponding described cell culture unit and the second sprue.
In an embodiment, described the first sprue is roundabout bending structure therein, and described the second sprue is roundabout bending structure.
In an embodiment, described the first cultivation pool is connected with the first sprue by 8 diffusion runners therein; Described the second cultivation pool is connected with the second sprue by 8 diffusion runners.
In an embodiment, described the first sprue, the second sprue, the first cultivation pool and the second cultivation pool are cube structure therein, are highly 100～200 μ m.
In an embodiment, described diffusion runner is the cavity configuration with lower arcuate surface therein, is highly 45～55 μ m, and width is 225～275 μ m.
Due to first micro-valve in above-mentioned cell culture microflow control chip for the first cultivation pool of controlling each cell culture unit by being communicated with or closing of diffusion runner and the first sprue, can be the independent control of controlling or link.Suppose to have N cell culture unit, so just have N the first cultivation pool.When first micro-valve is for independent the first cultivation pool of controlling each cell culture unit during by being communicated with or cutting out of diffusion runner and the first sprue, by regulating and controlling micro-valve, N the first cultivation pool also can be realized the location of N kind difference cells; The first cultivation pool of controlling each cell culture unit for linking when first micro-valve being during by being communicated with or closing of diffusion runner and the first sprue, by regulating and controlling micro-valve, and the location that individual the first cultivation pool of N can be realized a kind of cell.The second cultivation pool is cultivated identical with the principle of the location cultivation of the first cultivation pool to cell to the location of cell.Therefore, above-mentioned cell culture microflow control chip can be realized spatial positioning and the common cultivation to multiple different cells (2～2N kind), realizes the high-throughput of cell and cultivates, and improves analysis efficiency; And the above-mentioned cell culture microflow control chip two dimension that can be used for cell cultivates and can be used for again the dimensional culture of cell, there is versatility and comparability.
Brief description of the drawings
Fig. 1 is the structural representation of the cell culture microflow control chip of an embodiment;
Fig. 2 is the structural representation of cell culture microflow control chip in other embodiments;
Fig. 3 is the aspect graph that the EpRS cell in embodiment is cultivated and grown in Matrigel dimensional culture matrix in micro-fluidic chip.
Embodiment
Below in conjunction with drawings and the specific embodiments, cell culture microflow control chip is further detailed.
As shown in Figure 1, the cell culture microflow control chip 100 of an embodiment comprises the first sprue 110, first micro-valve 120, cell culture unit 130, second micro-valve 140, the 3rd micro-valve 150 and the second sprue 160.
The number of cell culture unit 130 is multiple, and multiple cell culture units 130 are arranged side by side, and between the first sprue 110 and the second sprue 160.The number of first micro-valve 120 is multiple, corresponding one by one with multiple cell culture units of arranging side by side 130.
Each cell culture unit 130 comprises the first cultivation pool 132, the second cultivation pool 134 and diffusion runner 136.Second micro-valve 140 controls the first cultivation pool 132 of each cell culture unit 130 and the connection of the second cultivation pool 134 or cuts out for linking.Be appreciated that, in other embodiments, multiple second micro-valves 140 also can be set, multiple second micro-valves 140 are corresponding one by one with multiple cell culture units of arranging side by side 130, and each the second micro-valve 140 is independent controls being communicated with or closing of the first cultivation pool 132 in corresponding cell culture unit 130 and the second cultivation pool 134.
The first cultivation pool 132 is connected with the first sprue 110 by diffusion runner 136.Each first micro-valve 120 is being communicated with or cutting out by diffusion runner 136 and the first sprue 110 for independent the first cultivation pool 132 of controlling corresponding cell culture unit 130.Be appreciated that in other embodiments, 1 micro-valve 120 of first micro-valve 120, the first also can be only set control for linking the first cultivation pool 132 being communicated with or closing by diffusion runner 136 and the first sprue 110 of each cell culture unit 130.
The second cultivation pool 134 is connected with the second sprue 160 by diffusion runner 136.The 3rd micro-valve 150 controls the second cultivation pool 134 being communicated with or cutting out by diffusion runner 136 and the second sprue 160 of each cell culture unit 130 for linking.Be appreciated that, in other embodiments, multiple the 3rd micro-valves 150 also can be set, multiple the 3rd micro-valves 150 are corresponding one by one with multiple cell culture units of arranging side by side 130, and the second cultivation pool 134 that each the 3rd micro-valve 150 is independent controls corresponding each cell culture units 130 is communicated with or is closed by diffusion runner 136 and the second sprue 160.
In the present embodiment, the height of diffusion runner 136 is lower than the height of the first cultivation pool 132, the second cultivation pool 134, the first sprue 110 and the second sprue 160, thereby can make the liquid rapid diffusion in the first sprue 110 and the second sprue 160 enter in the first cultivation pool 132 and the second cultivation pool 134, realize fast transport, save time.
In the present embodiment, the first sprue 110, the second sprue 160, the first cultivation pool 132 and the second cultivation pool 134 are cube structure, are highly 100～200 μ m, and its length and width as required the size and number of cultured cells design.Be appreciated that the first sprue, the second sprue, the first cultivation pool and the second cultivation pool can be other shapes, as there is cavity configuration of lower arcuate surface etc.
The first cultivation pool 132 in each cell culture unit 130 is connected with the first sprue 110 by 8 diffusion runners 136; The second cultivation pool 134 is connected with the second sprue 160 by 8 diffusion runners 136.The first sprue 110 and the second sprue 160 are roundabout warp architecture.The first sprue 110 is connected by 4 diffusion runners 136 respectively with two sidewalls of the first cultivation pool 132; The second sprue 160 with and two sidewalls of the second cultivation pool 134 be connected by 4 diffusion runners 136 respectively, thereby liquid can enter from two side the first cultivation pool 132 or the second cultivation pool 134, connect compared to one-sided wall, inoculating cell and perfusion culture effect are better, liquid communication amount is larger, is conducive to save the liquid communication time.Be appreciated that the quantity that spreads runner 136 in each cell culture unit 130 is not limited to above-mentioned 8, as can also be 4,6 etc.
Diffusion runner 136, for having the cavity configuration of lower arcuate surface, is highly 45～55 μ m, and width is 225～275 μ m.The diffusion runner 136 of cavity configuration with lower arcuate surface is better with micro-valve (first micro-valve 120, the 3rd micro-valve 150) fiting effect, and liquid is more smooth and easy in the interior circulation of diffusion runner 136.
In the present embodiment, the second all cultivation pools 134 being communicated with or closing by diffusion runner 136 and the second sprue 160 controlled in the 3rd micro-valve 150 interlock.Design can reduce the use number of micro-valve like this, can be cost-saving in meeting primary demand; In addition, be appreciated that each micro-valve needs to occupy certain space, design can also be saved space simultaneously like this.Be appreciated that in other embodiments, can be also that the 3rd micro-valve 150 is independent controls second cultivation pool 134 being communicated with or closing by diffusion runner 136 and the second sprue 160.
As shown in Figure 2, the cell culture microflow control chip 200 in other embodiments, comprises the first sprue 210, first micro-valve 220, cell culture unit 230, second micro-valve 240, the 3rd micro-valve 250 and the second sprue 260.
The number of cell culture unit 230 is multiple, and multiple cell culture units 230 are arranged side by side, and between the first sprue 210 and the second sprue 260; The number of first micro-valve 220 is multiple, and corresponding one by one with multiple cell culture units of arranging side by side 230; The number of the 3rd micro-valve 250 is multiple, and corresponding one by one with multiple cell culture units of arranging side by side 230.
Each cell culture unit 230 comprises the first cultivation pool 232, the second cultivation pool 234 and diffusion runner 236; Second micro-valve 240 controls the first cultivation pool 232 of each cell culture unit 230 and the connection of the second cultivation pool 234 or cuts out for linking.
The first cultivation pool 232 is connected with the first sprue 210 by diffusion runner 236, first micro-valve 220 for independent the first cultivation pool 232 of controlling each cell culture unit 230 by spreading being communicated with or closing of runner 236 and the first sprue 210.
The second cultivation pool 234 is connected with the second sprue 260 by diffusion runner 236, the 3rd micro-valve 250 for independent the second cultivation pool 234 of controlling each cell culture unit 230 by spreading being communicated with or closing of runner 236 and the second sprue 260.
The height of diffusion runner 236 is lower than the height of the first cultivation pool 232, the second cultivation pool 234, the first sprue 210 and the second sprue 260.
By regulating and controlling micro-valve, cell culture microflow control chip 200 as shown in Figure 2, than cell culture microflow control chip 100 as shown in Figure 1, in the same number of situation of cell culture unit, can be cultivated more kinds of different cells.
In addition, in the present embodiment, the first sprue 110 and the second sprue 150 are roundabout warp architectures all, on the one hand more diffusion admittance can be set in limited space, and more diffusion admittance is conducive to liquid to be transmitted fast.Roundabout warp architecture can alleviate the damage of liquid shear power to cell in runner in the time of inoculating cell on the one hand in addition, and cell can be evenly distributed in cultivation pool.
Cell culture microflow control chip 100 shown in Fig. 1 has multiple use.Suppose in cell culture microflow control chip 100 as shown in Figure 1 total N cell culture unit, according to serial number from small to large, be respectively cell culture unit 1, cell culture unit 2...... cell culture unit N to each cell culture unit; Suppose to have multiple different cell, be respectively cell 1, cell 2, cell 3 ... cell N.
In the time that needs are used for realizing the research of the spatial positioning of multiple different cells, altogether cultivation or the medicine chemotaxis of a kind of medicine to multiple different cells, can adopt following operating method to realize:
Close second micro-valve 140 and N first micro-valve 120, open the 3rd micro-valve 150; By the second sprue 160 to the second cultivation pool 134 transfusion cells 1 in N cell culture unit 130, be full of after the second cultivation pool 134 in N cell culture unit 130 by diffusion runner 136 until cell 1, close the 3rd micro-valve 150, pass into scavenging solution to the second sprue 160, the cell 1 residuing in the second sprue 160 is washed out, realized the spatial positioning of cell 1 in N the second cultivation pool 134.
Close second micro-valve 140, the 3rd micro-valve 150 and the first micro-valve 120 of the N-1 except the first cultivation pool 132 of controlling in cell culture unit 1, open first micro-valve 120 of controlling the first cultivation pool 132 in cell culture unit 1; By the first sprue 110 to the first cultivation pool 132 transfusion cells 2 in cell culture unit 1, be full of after the first cultivation pool 132 in cell culture unit 1 by diffusion runner 136 until cell 2, first micro-valve 120 of the first cultivation pool 132 in closing control cell culture unit 1, pass into scavenging solution to the first sprue 110, the cell 2 residuing in the first sprue 110 is washed out, realize the spatial positioning in first cultivation pool 132 of cell 2 in cell culture unit 1.
Close second micro-valve 140, the 3rd micro-valve 150 and the first micro-valve 120 of the N-1 except the first cultivation pool 132 of controlling in cell culture unit 2, open first micro-valve 120 of controlling the first cultivation pool 132 in cell culture unit 2; By the first sprue 110 to the first cultivation pool 132 transfusion cells 3 in cell culture unit 2, be full of after the first cultivation pool 132 in cell culture unit 2 by diffusion runner 136 until cell 3, first micro-valve 120 of the first cultivation pool 132 in closing control cell culture unit 2, pass into scavenging solution to the first sprue 110, the cell 3 residuing in the first sprue 110 is washed out, realize the spatial positioning in first cultivation pool 132 of cell 3 in cell culture unit 2.
Remaining the like, above-mentioned cell culture microflow control chip 100 can be realized the spatial positioning of the different cells of N+1 kind; Transmit identical substratum by the first sprue 110 and the second sprue 160, can realize the common cultivation of the different cells of N+1 kind; Transmit same medicine by the first sprue 110 and the second sprue 160, can realize the research of the medicine chemotaxis of a kind of medicine to the different cells of N+1 kind.
In the time that needs are realized high-flux medicaments sifting, can adopt following operating method to realize:
By regulating and controlling micro-valve, in the first cell culture insert 132 in N cell culture unit and the second cell culture insert 134, location is cultivated same cell altogether.
By regulating and controlling micro-valve, can in N the first cell culture insert 132, input the substratum containing the same medicine of different concns through the first sprue 110, between the first cell culture insert 132, form drug level gradient; By the second sprue 160 to input in N the second cell culture insert 136 containing the substratum of medicine, experimental group in contrast, invasion and attack and the chemotaxis of the different concns that can study same medicine to same cell, realizes high-flux medicaments sifting.
Can also be by the micro-valve of regulation and control, in N the first cell culture insert 132, input the substratum containing the different pharmaceutical of same concentrations by the first sprue 110, in N the second cell culture insert 134, input the substratum containing medicine by the second sprue 160, experimental group in contrast, can realize the research of the impact of different pharmaceutical on same cell, realize high-flux medicaments sifting.
In the time need to realizing the two dimension of cell or dimensional culture, can adopt following operating method to realize:
To need behind cultured cells location, if need to carry out two dimension cultivation, directly allow cell located growth in the first cultivation pool and the second cultivation pool; If need to carry out dimensional culture, first the three dimensional matrixes such as Matrigel are passed in the first cultivation pool and the second cultivation pool, the effectively chemical component of analog cell microenvironment, and then introduce cell, it is adhered in the three dimensional matrixes such as Matrigel and differential growth.Cultured cells can be for various biological analysis research.
Dimensional culture is cultivated and is more approached internal milieu than two dimension, and it is more accurate that the cellular form obtaining is cultivated than two dimension.
Cell culture microflow control chip 200 shown in Fig. 2, except having the purposes of cell culture microflow control chip 100 as shown in Figure 1, also has some other purposes.
Because the second all cell culture insert in cell culture microflow control chip 100 is controlled by a second micro-valve interlock, therefore, the second all cell culture insert in cell culture microflow control chip 100 can only be realized a kind of location of cell and cultivate, and tests for control group.And cell culture microflow control chip 200 arranges independently second micro-valve to each the second cell culture insert, can realize the location of various kinds of cell is cultivated.Therefore, the in the situation that of cell culture unit invariable number, cell culture microflow control chip 200 can be located and cultivate more kinds of different cells than cell culture microflow control chip 100, can realize the location of different cells in 2N and cultivate, and be more suitable for the carrying out of control group experiment.
Be below specific embodiment part:
EpRs cell cultured continuously 5 days in Matrigel three dimensional matrix, realizes the dimensional culture of EpRs cell.Matrigel is rich in various kinds of cell epimatrix composition, as: layer is adhered albumen, collagen iv, TGF-β etc., the effectively chemical component of analog cell microenvironment, inducing cell adhering to and breaking up in three dimensional matrix, by the cultivation of continuous 5 days, EpRs cell was assembled the cell ball that formation is made up of multiple cells in Matrigel, as shown in Figure 3, wherein, upper left side the first width figure is the virgin state figure of EpRs cell; Upper right side the second width figure is the cultured continuously state graph after 3 days in Matrigel three dimensional matrix of EpRs cell; Lower left the 3rd width figure is the cultured continuously state graph after 4 days in Matrigel three dimensional matrix of EpRs cell; Lower right the 4th width figure is the cultured continuously state graph after 5 days in Matrigel three dimensional matrix of EpRs cell.
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (2)
1. a cell culture microflow control chip, it is characterized in that, comprise the first sprue, first micro-valve, second micro-valve, the 3rd micro-valve, the second sprue and multiple cell culture unit of arranging side by side, multiple cell culture units of arranging are side by side between the first sprue and the second sprue;
Each described cell culture unit comprises the first cultivation pool, the second cultivation pool and diffusion runner, and described second micro-valve is for controlling the first cultivation pool of described cell culture unit and the connection of the second cultivation pool or cutting out;
The first cultivation pool is connected with the first sprue by diffusion runner, and the first cultivation pool that described first micro-valve is used for controlling described cell culture unit is by spreading being communicated with or closing of runner and the first sprue;
The second cultivation pool is connected with the second sprue by diffusion runner, and the second cultivation pool that described the 3rd micro-valve is used for controlling described cell culture unit is by spreading being communicated with or closing of runner and the second sprue;
The quantity of described first micro-valve is multiple, corresponding one by one with multiple cell culture units, and each described first micro-valve independently controls being communicated with or cutting out of the first cultivation pool in corresponding described cell culture unit and the first sprue;
The quantity of described second micro-valve is 1, and described the second micro-valve interlock is controlled the connection of the first cultivation pool in described cell culture unit and the second cultivation pool or closed;
The quantity of described the 3rd micro-valve is 1, and the second cultivation pool in described cell culture unit being communicated with or closing by diffusion runner and the second sprue controlled in described the 3rd micro-valve interlock;
Described the first sprue is roundabout bending structure, and described the second sprue is roundabout bending structure;
Described the first sprue, the second sprue, the first cultivation pool and the second cultivation pool are cube structure, are highly 100～200 μ m;
Described diffusion runner is the cavity configuration with lower arcuate surface, is highly 45～55 μ m, and width is 225～275 μ m;
Wherein, described the first sprue is connected by diffusion runner respectively with two sidewalls of described the first cultivation pool, and described the second sprue is connected by diffusion runner respectively with two sidewalls of described the second cultivation pool.
2. cell culture microflow control chip according to claim 1, is characterized in that, described the first cultivation pool is connected with the first sprue by 8 diffusion runners; Described the second cultivation pool is connected with the second sprue by 8 diffusion runners.
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| CN103627635B (en) * | 2013-11-18 | 2015-05-20 | 辽宁中医药大学 | Multifunctional micro-fluidic chip for cell migration and invasion assay |
| CN105062890A (en) * | 2015-09-11 | 2015-11-18 | 温州梅塔光学科技有限公司 | Single cell experiment device |
| CN105838606B (en) * | 2016-06-08 | 2018-10-02 | 牛海涛 | A kind of high-flux cell Combined culture chip |
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| CN101275114A (en) * | 2008-04-22 | 2008-10-01 | 北京大学 | Microflow cell culture array and application thereof |
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