CN103103121A - Cell-culture microfluidic chip - Google Patents
Cell-culture microfluidic chip Download PDFInfo
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- CN103103121A CN103103121A CN2013100176856A CN201310017685A CN103103121A CN 103103121 A CN103103121 A CN 103103121A CN 2013100176856 A CN2013100176856 A CN 2013100176856A CN 201310017685 A CN201310017685 A CN 201310017685A CN 103103121 A CN103103121 A CN 103103121A
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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 the 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 guarantee that cell is normally bred, the essential condition of differentiation, metabolism and functional activity, microenvironment changes, corresponding variation also can occur 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 all is based on cells in vitro and cultivates realization, and traditional cells in vitro is cultivated the Growth of Cells environment that can only provide a kind of static state, macroscopic view, two-dimentional, cell hyperplasia under the environment of external change has been lost original proterties gradually, often with body in situation do not conform to.
Along with the emerging development of tissue culture engineering, the three-dimensional cell culture technique is arisen at the historic moment, and it is the interior microenvironment of analogue body farthest.Traditional three-dimensional cell research method has matrix to cover cultivation, rotary flask cultivation, microcarrier cultivation, preset support cultivation and Rotary cell culture system etc., these three-dimensional cell culture techniques are complex operation not only, and the reagent consumption is large, what is more important, microsize with respect to cell, this culture environment and internal milieu differ far away, objectively are 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 integral body.And the fluid channel size (common 10 ~ 100 μ m) of micro-fluidic chip design is in identical magnitude with typical cells of mamma animals size (10 ~ 20 μ m), and conduct heat under microscale, mass transfer is very fast, favourable Growth of Cells research environment can be provided, and micro-fluidic chip can satisfy 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, make its Important Platform that becomes cell research, in biomedical fields wide application prospect.
The micro-fluidic chip that is used for the simulation of the dimensional culture of cell and microenvironment makes some progress, but also exist a lot of not enough, the problem that the 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 little valve, second little valve, the 3rd little valve, the second sprue and a plurality of cell culture unit of arranging side by side, a plurality of cell culture units of arranging side by side are 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 little valve is used for controlling the connection of the first cultivation pool of described cell culture unit and the second cultivation pool or cuts out;
The first cultivation pool is connected with the first sprue by the diffusion runner, described first little valve for the first cultivation pool of controlling described cell culture unit by spreading being communicated with or closing of runner and the first sprue;
The second cultivation pool is connected with the second sprue by the diffusion runner, the described the 3rd little valve for the second cultivation pool of controlling described cell culture unit by spreading being communicated with or closing of runner and the second sprue.
In 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 embodiment, the quantity of described first little valve is a plurality of, and is corresponding one by one with a plurality of cell culture units therein, and each described first little valve independently controls being communicated with or cutting out of the first cultivation pool and the first sprue in corresponding described cell culture unit.
In embodiment, the quantity of described second little valve is 1 therein, and described second little valve interlock is controlled the connection of the first cultivation pool in described cell culture unit and the second cultivation pool or closed.
In embodiment, the quantity of described the 3rd little valve is 1 therein, and the second cultivation pool being communicated with or closing by diffusion runner and the second sprue in described cell culture unit controlled in the described the 3rd little valve interlock.
In embodiment, the quantity of described the 3rd little valve is a plurality of, and is corresponding one by one with a plurality of cell culture units therein, and each the described the 3rd little valve independently controls being communicated with or cutting out of the second cultivation pool and the second sprue in corresponding described cell culture unit.
In embodiment, described the first sprue is roundabout bending structure therein, and described the second sprue is roundabout bending structure.
In 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 embodiment, 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 therein.
In embodiment, described diffusion runner is the cavity configuration with lower arcuate surface, is highly 45 ~ 55 μ m therein, and width is 225 ~ 275 μ m.
The first cultivation pool of be used for controlling each cell culture unit due to first little valve in above-mentioned cell culture microflow control chip is communicated with or closes by diffusion runner and the first sprue, can be independent control or interlock control.Suppose to have N cell culture unit, N the first cultivation pool so just arranged.When first little valve be used for independent the first cultivation pool of controlling each cell culture unit by diffusion runner and the first sprue be communicated with or when closing, by regulating and controlling little valve, N the first cultivation pool also can be realized the location of N kind difference cells; When first little valve be used for the first cultivation pool that interlock controls each cell culture unit by diffusion runner and the first sprue be communicated with or when closing, by regulating and controlling little valve, the location that individual the first cultivation pool of N can be realized a kind of cell.The second cultivation pool is cultivated identical to the principle of the location cultivation of cell with the first cultivation pool 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 that namely the high-throughput of cell is cultivated, and improves analysis efficiency; And above-mentioned cell culture microflow control chip can be cultivated again and can for the dimensional culture of cell, have versatility and comparability for the two dimension of cell.
Description of 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 in micro-fluidic chip and grown in Matrigel dimensional culture matrix.
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 little valve 120, cell culture unit 130, second little valve 140, the 3rd little valve 150 and the second sprue 160.
The number of cell culture unit 130 is a plurality of, and a plurality of cell culture units 130 are arranged side by side, and between the first sprue 110 and the second sprue 160.The number of first little valve 120 is a plurality of, and is corresponding one by one with a plurality of 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.The second little valve 140 is used for interlock and controls the connection of the first cultivation pool 132 of each cell culture unit 130 and the second cultivation pool 134 or cut out.Be appreciated that, in other embodiments, a plurality of second little valve 140 also can be set, a plurality of second little valve 140 is corresponding one by one with a plurality of cell culture units of arranging side by side 130, and the first cultivation pool 132 in the cell culture unit 130 of each the second independent control correspondence of little valve 140 is communicated with or closes with the second cultivation pool 134.
The first cultivation pool 132 is connected with the first sprue 110 by diffusion runner 136.Each first little valve 120 is used for independent the first cultivation pool 132 of controlling corresponding cell culture unit 130 being communicated with or cutting out by diffusion runner 136 and the first sprue 110.Be appreciated that in other embodiments, 1 the first little valve of little valve 120, the first 120 also can only be set be used for the first cultivation pool 132 that interlock controls each cell culture unit 130 being communicated with or closing by diffusion runner 136 and the first sprue 110.
The second cultivation pool 134 is connected with the second sprue 160 by diffusion runner 136.The 3rd little valve 150 is used for the second cultivation pool 134 that interlock controls each cell culture unit 130 being communicated with or cutting out by diffusion runner 136 and the second sprue 160.Be appreciated that, in other embodiments, the a plurality of the 3rd little valve 150 also can be set, the a plurality of the 3rd little valve 150 is corresponding one by one with a plurality of cell culture units of arranging side by side 130, and the second cultivation pool 134 that each the 3rd little valve 150 is independent controls each corresponding cell culture unit 130 is communicated with or closes 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 the liquid rapid diffusion in the first sprue 110 and the second sprue 160 is entered 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 have 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 be connected cultivation pool 134 two sidewalls respectively by 4 the diffusion runners 136 be connected, thereby liquid can enter from the 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, the liquid communication amount is larger, is conducive to save the liquid communication time.The quantity that is appreciated that in each cell culture unit 130 diffusion runner 136 is not limited to above-mentioned 8, as being 4,6 etc.
The cavity configuration of diffusion runner 136 for having 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 little valve (first little valve 120, the 3rd little 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 little valve 150 interlock.Design can reduce the use number of little valve like this, can save cost when satisfying primary demand; In addition, be appreciated that each little valve needs to occupy certain space, design simultaneously can also conserve space like this.Be appreciated that in other embodiments, can be also that the 3rd little 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 little valve 220, cell culture unit 230, second little valve 240, the 3rd little valve 250 and the second sprue 260.
The number of cell culture unit 230 is a plurality of, and a plurality of cell culture units 230 are arranged side by side, and between the first sprue 210 and the second sprue 260; The number of first little valve 220 is a plurality of, and corresponding one by one with a plurality of cell culture units of arranging side by side 230; The number of the 3rd little valve 250 is a plurality of, and corresponding one by one with a plurality of 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; The second little valve 240 is used for interlock and controls the connection of the first cultivation pool 232 of each cell culture unit 230 and the second cultivation pool 234 or cut out.
The first cultivation pool 232 is connected with the first sprue 210 by diffusion runner 236, and the first cultivation pool 232 that first little valve 220 is used for independent each cell culture unit 230 of control is 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, and the second cultivation pool 234 that the 3rd little valve 250 is used for independent each cell culture unit 230 of control is 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 little valve, cell culture microflow control chip 200 as shown in Figure 2 in the same number of situation of cell culture unit, can be cultivated more kinds of different cells than as shown in Figure 1 cell culture microflow control chip 100.
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 when 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 Figure 1 has multiple use.Suppose in cell culture microflow control chip 100 as shown in Figure 1 total N cell culture unit, according to from small to large serial number, 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.
When needs are used for realizing the spatial positioning of multiple different cells, cultivate or a kind of medicine during to the research of the medicine chemotaxis of multiple different cells altogether, can adopt following operating method to realize:
Close second little valve 140 and N first little valve 120, open the 3rd little valve 150; The second cultivation pool 134 transfusion cells 1 by the second sprue 160 in N the cell culture unit 130, be full of the second cultivation pool 134 in N cell culture unit 130 by diffusion runner 136 until cell 1 after, close the 3rd little valve 150, pass into scavenging solution to the second sprue 160, the cell 1 that residues in the second sprue 160 is washed out, namely realized the spatial positioning of cell 1 in N the second cultivation pool 134.
Close N-1 first little valve 120 second little valve 140, the 3rd little valve 150 and the first cultivation pool 132 in controlling cell culture unit 1, open first little valve 120 of the first cultivation pool 132 in control cell culture unit 1; The first cultivation pool 132 transfusion cells 2 by the first sprue 110 in the cell culture unit 1, be full of the first cultivation pool 132 in cell culture unit 1 by diffusion runner 136 until cell 2 after, first little 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 that residues in the first sprue 110 is washed out, namely realize the spatial positioning in first cultivation pool 132 of cell 2 in cell culture unit 1.
Close N-1 first little valve 120 second little valve 140, the 3rd little valve 150 and the first cultivation pool 132 in controlling cell culture unit 2, open first little valve 120 of the first cultivation pool 132 in control cell culture unit 2; The first cultivation pool 132 transfusion cells 3 by the first sprue 110 in the cell culture unit 2, be full of the first cultivation pool 132 in cell culture unit 2 by diffusion runner 136 until cell 3 after, first little 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 that residues in the first sprue 110 is washed out, namely 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; By the first sprue 110 and the second sprue 160 identical substratum of transmission, can realize the common cultivation of the different cells of N+1 kind; By the first sprue 110 and the second sprue 160 transmission same medicines, can realize that a kind of medicine is to the research of the medicine chemotaxis of the different cells of N+1 kind.
When needs are realized high-flux medicaments sifting, can adopt following operating method to realize:
By regulating and controlling little valve, in the first cell culture insert 132 in N cell culture unit and the second cell culture insert 134, the location is cultivated the same cell altogether.
By regulating and controlling little valve, can contain the substratum of the same medicine of different concns to input in N the first cell culture insert 132 through the first sprue 110, form the drug level gradient between the first cell culture insert 132; Input the substratum that does not contain medicine by the second sprue 160 in N the second cell culture insert 136, experimental group, can study the different concns of same medicine to invasion and attack and the chemotaxis of same cell in contrast, realizes high-flux medicaments sifting.
Can also be by the little valve of regulation and control, the substratum that contains the different pharmaceutical of same concentrations by the input in N the first cell culture insert 132 of the first sprue 110, input the substratum that does not contain medicine by the second sprue 160 in N the second cell culture insert 134, experimental group in contrast, can realize that different pharmaceutical on the research of the impact of same cell, realizes high-flux medicaments sifting.
When needs are realized the two dimension of cell or dimensional culture, can adopt following operating method to realize:
After needing 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 introducing cell adheres to it and differential growth in the three dimensional matrixes such as Matrigel.Cultured cells can be used for various biological analysis researchs.
Dimensional culture is cultivated more near internal milieu than two dimension, and the cellular form that obtains is than two-dimentional cultivate more accurate.
Cell culture microflow control chip 200 shown in Figure 2 also has some other purposes except the purposes with cell culture microflow control chip 100 as shown in Figure 1.
Because the second all cell culture insert in cell culture microflow control chip 100 is controlled by second a little valve interlock, therefore, the second all cell culture insert in cell culture microflow control chip 100 can only be realized a kind of location cultivation of cell, and is used for the control group experiment.And cell culture microflow control chip 200 arranges independently second little valve for each second cell culture insert, can realize the location of various kinds of cell is cultivated.Therefore, in the situation that cell culture unit invariable number, cell culture microflow control chip 200 can be located than cell culture microflow control chip 100 and be cultivated more kinds of different cells, can realize that the location of different cells in 2N is cultivated, and be more suitable for the carrying out of control group experiment.
Be below the specific embodiment part:
EpRs cell cultured continuously 5 days in the Matrigel three dimensional matrix realizes the dimensional culture of EpRs cell.Matrigel is rich in various kinds of cell epimatrix composition, as: glutinous albumen, collagen iv, the TGF-β etc. of connecting of layer, the effective chemical component of analog cell microenvironment, inducing cell adhering to and breaking up in three dimensional matrix, by the cultivation of continuous 5 days, the EpRs cell was assembled the cell ball that formation is comprised of a plurality of 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 the Matrigel three dimensional matrix of EpRs cell; Lower left the 3rd width figure is the cultured continuously state graph after 4 days in the Matrigel three dimensional matrix of EpRs cell; Lower right the 4th width figure is the cultured continuously state graph after 5 days in the 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.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 (10)
1. cell culture microflow control chip, it is characterized in that, comprise the first sprue, first little valve, second little valve, the 3rd little valve, the second sprue and a plurality of cell culture unit of arranging side by side, a plurality of cell culture units of arranging side by side are 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 little valve is used for controlling the connection of the first cultivation pool of described cell culture unit and the second cultivation pool or cuts out;
The first cultivation pool is connected with the first sprue by the diffusion runner, described first little valve for the first cultivation pool of controlling described cell culture unit by spreading being communicated with or closing of runner and the first sprue;
The second cultivation pool is connected with the second sprue by the diffusion runner, the described the 3rd little valve for the second cultivation pool of controlling described cell culture unit by spreading being communicated with or closing of runner and the second sprue.
2. cell culture microflow control chip according to claim 1, is characterized in that, 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.
3. cell culture microflow control chip according to claim 1, it is characterized in that, the quantity of described first little valve is a plurality of, corresponding one by one with a plurality of cell culture units, each described first little valve independently controls being communicated with or cutting out of the first cultivation pool and the first sprue in corresponding described cell culture unit.
4. cell culture microflow control chip according to claim 1, is characterized in that, the quantity of described second little valve is 1, and the described second little valve interlock is controlled the connection of the first cultivation pool in described cell culture unit and the second cultivation pool or closed.
5. cell culture microflow control chip according to claim 1, it is characterized in that, the quantity of described the 3rd little valve is 1, and the second cultivation pool being communicated with or closing by diffusion runner and the second sprue in described cell culture unit controlled in the described the 3rd little valve interlock.
6. cell culture microflow control chip according to claim 1, it is characterized in that, the quantity of described the 3rd little valve is a plurality of, corresponding one by one with a plurality of cell culture units, each the described the 3rd little valve independently controls being communicated with or cutting out of the second cultivation pool and the second sprue in corresponding described cell culture unit.
7. cell culture microflow control chip according to claim 1, is characterized in that, described the first sprue is roundabout bending structure, and described the second sprue is roundabout bending structure.
8. 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.
9. cell culture microflow control chip according to claim 1, is characterized in that, described the first sprue, the second sprue, the first cultivation pool and the second cultivation pool are cube structure, is highly 100 ~ 200 μ m.
10. cell culture microflow control chip according to claim 1, is characterized in that, described diffusion runner is the cavity configuration with lower arcuate surface, is highly 45 ~ 55 μ m, and width is 225 ~ 275 μ m.
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| CN105838606A (en) * | 2016-06-08 | 2016-08-10 | 牛海涛 | High-flux cell co-culture chip |
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