WO2019231265A1 - Microfluidic chip for three-dimensional cell culturing and three-dimensional cell culturing method using same - Google Patents

Abstract

The present invention relates to a microfluidic chip for three-dimensional cell culturing and a three-dimensional cell culturing method using the microfluidic chip for three-dimensional cell culturing. As the microfluidic chip for three-dimensional cell culturing comprises a cell culture support having a concave pattern, the microfluidic chip has excellent three-dimensional culturing of and maintenance effect on a cell when the cell is cultured under a specific flow condition. Accordingly, the microfluidic chip can be effectively used for three-dimensional cell culturing.

Description

Microfluidic chip for 3D cell culture and 3D cell culture method using the same

The present invention relates to a three-dimensional cell culture microfluidic chip and a three-dimensional culture method of cells using the microfluidic chip.

The present invention has been made by the task number 2015M3A9C7030091 under the support of the Ministry of Science and ICT, the research management specialized organization of the task is the Korea Research Foundation, the research project name is "bio medical technology development project", the research title is "human stem cell derived Establishment of Toxicity Evaluation Platform and Systematization of New Drug Candidates through Systematic Target Cell Production ", Organized by Konkuk University Global Industry-Academic Cooperation Group, Research Period ~ 2020.05.31. In addition, the present invention was made by the task number 71500307 under the support of the Ministry of Agriculture, Food and Rural Affairs of the Republic of Korea, the research and management agency of the project is the Ministry of Agriculture, Forestry and Food Technology Planning and Evaluation, the research project name is "Agriculture and Livestock Food Research Center Support Project", and the research project name is "Establishment of high quality black cow production system using cell utilization technology", the host institution is the University-Industry Foundation of Cheju National University, and the research period is 2015.12.02. ~ 2022.12.01. In addition, the present invention was made by the task number 2016M3A9B4919616 under the support of the Ministry of Science and Technology Information and Communication, the research management specialized organization of the project is the Korea Research Foundation, the research project name is "bio medical technology development project", the research project title is "Biofunctionality Establishment of polymer-based stem cell mass production system ", host organization is Chungnam National University Industry-Academic Cooperation Group ~ 2021.03.31.

This patent application claims priority to Korean Patent Application No. 10-2018-0063732 filed with the Korean Patent Office on June 1, 2018, the disclosure of which is hereby incorporated by reference.

Clinical applications, including cell therapy and drug delivery, require large numbers of cells. When culturing cells in a general culture method, a large amount of culture dish or flask must be used.

However, cultivation in a culture dish or flask differs from the three-dimensional soft environment in which cells proliferate in a living body by culturing cells in a two-dimensional hardness environment, and thus there is a risk of inducing denaturation of cultured cells. have.

To reduce this risk, several three-dimensional cell culture methods have been developed. For example, uniform sized three-dimensional cross-linked alginate inverse opal scaffolds can be used to create uniform sized three-dimensional cells.

However, although such scaffolds are advantageous in terms of producing cells of uniform size, there is a problem that recovery of generated cells is difficult because the scaffolds need to be collapsed in the process.

In addition, several microfluidic platforms have recently been developed to produce cells of uniform size and shape, including array-well and channel platforms, all of which are advantageous in terms of producing cells of uniform size. It is difficult to control its size extensively, and the process is complicated.

Accordingly, there is an urgent need to develop a technique relating to a method of three-dimensional culture of cells in a uniform size in a wide range.

The present inventors earnestly tried to develop a method for three-dimensional culture of cells with uniform size. As a result, the present invention was completed by clarifying that the three-dimensional culture and maintenance effect of the cells is excellent when the cells are cultured under specific flow conditions using a cell culture support including a concave pattern.

Accordingly, it is an object of the present invention to provide a microfluidic chip for three-dimensional cell culture.

Another object of the present invention is to provide a three-dimensional culture method of cells using a microfluidic chip for three-dimensional cell culture.

The present inventors earnestly tried to develop a method for three-dimensional culture of cells with uniform size. As a result, it was found that when the cells were cultured under specific flow conditions using a cell culture support including a concave pattern, the three-dimensional culture and maintenance effect of the cells was excellent.

The present invention relates to a three-dimensional culture method of cells using a microfluidic chip for three-dimensional cell culture and a microfluidic chip for three-dimensional cell culture.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention is a cell culture unit consisting of one or more cell culture support; An inlet connected to one side of the cell culture support for injecting the cell culture solution; A control part connected to the inlet part for injecting the cell culture fluid at a constant flow rate and flow rate; And an outlet portion connected to the other side of the cell culture support to recover the cell culture solution, wherein the cell culture support includes a concave pattern and each of the outlet portions connected to the one or more cell culture supports. Is in communication with each other relates to a three-dimensional cell culture microfluidic chip, characterized in that for recovering the cell culture.

The cell may include any cell as long as the cell requires culture in three dimensions.

The cells are, for example, embryonic stem cells (ESC), mesenchymal stem cells (MSC), endothelial progenitor cells (EPC), fibroblasts and keratin. It may be a cell (Keratinocyte), but is not limited thereto.

The flow rate (average) in the microfluidic chip is, for example, 0.001 to 0.5 mL / h, 0.010 to 0.5 mL / h, 0.010 to 0.4 mL / h, 0.010 to 0.3 mL / h, 0.010 to 0.2 mL / h, 0.015 to 0.2 mL / h, 0.015 to 0.1 mL / h, 0.02 to 0.1 mL / h, 0.02 to 0.1 mL / h, 0.02 to 0.09 mL / h, 0.03 to 0.09 mL / h, 0.03 to 0.08 mL / h, 0.04 To 0.08 mL / h, 0.04 to 0.07 mL / h, 0.05 to 0.07 mL / h, 0.05 to 0.06 mL / h, or 0.05 mL / h, but is not limited thereto.

The flow rate (average) in the microfluidic chip is, for example, 1 to 25 mm / h, 1 to 24 mm / h, 2 to 24 mm / h, 2 to 23 mm / h, 3 to 23 mm / h, 3 to 22 mm / h, 3 to 20 mm / h, 4 to 22 mm / h, 4 to 21 mm / h, 5 to 21 mm / h, 5 to 20 mm / h, 6 to 20 mm / h, 6 To 19 mm / h, 7 to 19 mm / h, 7 to 18 mm / h, 8 to 18 mm / h, 8 to 17 mm / h, 9 to 17 mm / h, 9 to 16 mm / h, 10 to 16 mm / h, 10 to 15 mm / h, 10 to 14 mm / h, 10 to 13 mm / h, 10 to 12 mm / h, 10 to 11 mm / h or 10 mm / h, but is not limited thereto. It doesn't happen.

When the invention is carried out in the above-described flow conditions (flow rate and flow rate), the physical effect applied to the cell when supplying the cell culture solution, that is, the effect due to shear stress can be minimized.

In particular, if the flow rate is exceeded, the difference in velocity occurs at least two times in the center and the edge of the cell culture scaffold (cell culture part), and the difference in the degree of the cells absorbing nutrients from the injected cell culture solution. Since it occurs, it is difficult to see that all the cells in the cell culture support have been cultured in the same environment. In other words, when the flow rate is exceeded, a problem may arise in that growth rates of cells cultured in one cell culture support are different from each other.

As used herein, "concave" refers to a concave shape of the surface, and specifically, to a hemisphere shape in which the surface is concave.

As used herein, the term "concave pattern" refers to a plurality of concaves arranged in a predetermined direction.

The concave may be arranged multiseriate in the horizontal and vertical direction. The concave pattern is arranged in multiple rows, and each row of the pattern may be specifically arranged in parallel. As used herein, "parallel" is meant to include not only complete parallelism, but also arranged at a level that can be viewed as being substantially parallel.

The diameter of the concave is, for example, 0.1 to 1.0 mm, 0.15 to 1.0 mm, 0.2 to 1.0 mm, 0.25 to 1.0 mm, 0.3 to 1.0 mm, 0.35 to 1.0 mm, 0.4 to 1.0 mm, 0.45 to 1.0 mm , 0.5 to 1.0 mm, 0.55 to 1.0 mm, 0.6 to 1.0 mm, 0.65 to 1.0 mm, 0.7 to 1.0 mm, 0.75 to 1.0 mm, 0.8 to 1.0 mm, 0.85 to 1.0 mm, 0.9 to 1.0 mm, 0.95 to 1.0 mm Or 0.3 mm, but is not limited thereto.

When the invention is carried out within the diameter of the above-mentioned concave, the cell growth rate can be maximized.

Specifically, when the diameter of the concave exceeds the above range, three-dimensional cultured cells have a problem of cell necrosis (necrocytosis), and when the diameter of the concave is less than the above range, there is a problem that the three-dimensional culture of the cells itself is impossible.

In particular, the size of embryonic bodies (EB) during the differentiation of cells is an important parameter for differentiation, the use of the optimal size of EB can improve the efficiency of differentiation into cells. In addition, since different cell lines have different optimal EB sizes, it is necessary to control the EB size on a cell-by-cell basis to improve differentiation efficiency. Therefore, the diameter of the concave pattern included in the cell culture support for each cell may be different. When the invention is carried out within the diameter of the above-mentioned concave, the cell growth rate can be maximized.

The diameter and spacing of the concave may be a length ratio of 1: 1 to 1.5.

When the invention is carried out within the diameter and spacing of the above-mentioned concave, the cell yield, which is the rate at which cells are formed in three dimensions with respect to the supplied cells, can be maximized.

The number of the concave may be 1 to 34 in one direction, for example, 1 to 34 in the horizontal direction and 1 to 10 in the vertical direction, but is not limited thereto.

The horizontal-vertical ratio according to the number of the concave may vary depending on the microfluidic chip structure to be manufactured, and may be equally applied to each cell.

The cell culture support is polydimethylsiloxane (PDMS), glass, glass, and polystyrene-based plastic materials such as polystyrene, polycarbonate, polyethylene, and polypropylene. It may be, but is not limited thereto.

Another aspect of the invention relates to a three-dimensional cell culture method comprising the following steps.

Preparing a microfluidic chip for cell culture comprising a cell culture support; And

Injecting the cell culture solution to the microfluidic chip.

The injection step may be to inject the cell culture at a constant flow rate and flow rate.

The cell may include any cell as long as the cell requires culture in three dimensions.

The cells are, for example, embryonic stem cells (ESC), mesenchymal stem cells (MSC), endothelial progenitor cells (EPC), fibroblasts and keratin. It may be a cell (Keratinocyte), but is not limited thereto.

The overlapping contents of the microfluidic chip for cell culture are omitted in consideration of the complexity of the present specification.

The microfluidic chip according to the present invention is characterized by being capable of culturing and maintaining cells in three dimensions by injecting a cell culture solution into a cell culture support at a constant flow rate and flow rate.

In other words, the microfluidic chip according to the present invention can supply a culture medium at a low speed, thereby minimizing physical effects on the cells and continuously supplying a fresh medium to extend the culture period of the cells and increase the growth rate of the cells. It features.

Hereinafter, with reference to the accompanying drawings will be described in detail for the three-dimensional cell culture microfluidic chip according to an embodiment of the present invention.

1 is a plan view schematically illustrating a microfluidic chip for three-dimensional cell culture according to an embodiment of the present invention. Specifically, FIG. 1A is a plan view illustrating a microfluidic chip including one cell culture support, and FIG. 1B is a plan view illustrating a microfluidic chip including three cell culture supports.

Referring to the drawings, the three-dimensional cell culture microfluidic chip 1 according to an embodiment of the present invention, the cell culture unit 10, the inlet 20, the control unit 30 and the outlet 40 It may be configured to include.

Cell culture unit 10 may be configured to consist of one or more cell culture support (11). When composed of one or more cell culture supports, each cell culture support may be configured to be connected in parallel.

The cell culture support may comprise a concave pattern.

The concave pattern may include a plurality of concave arranged in a predetermined direction.

The concave may be arranged multiseriate in the horizontal and vertical direction.

The diameter of the concave is, for example, 0.1 to 1.0 mm, 0.15 to 1.0 mm, 0.2 to 1.0 mm, 0.25 to 1.0 mm, 0.3 to 1.0 mm, 0.35 to 1.0 mm, 0.4 to 1.0 mm, 0.45 to 1.0 mm , 0.5 to 1.0 mm, 0.55 to 1.0 mm, 0.6 to 1.0 mm, 0.65 to 1.0 mm, 0.7 to 1.0 mm, 0.75 to 1.0 mm, 0.8 to 1.0 mm, 0.85 to 1.0 mm, 0.9 to 1.0 mm, 0.95 to 1.0 mm Or 0.3 mm, but is not limited thereto.

When the invention is carried out within the diameter of the above-mentioned concave, the cell growth rate can be maximized.

The diameter and spacing of the concave may be a length ratio of 1: 1 to 1.5.

When the invention is carried out within the diameter and spacing of the above-mentioned concave, the cell yield, which is the rate at which cells are formed in three dimensions with respect to the supplied cells, can be maximized.

The number of the concave may be 1 to 34 in one direction, for example, 1 to 34 in the horizontal direction and 1 to 10 in the vertical direction, but is not limited thereto.

The horizontal-vertical ratio according to the number of the concave may vary depending on the microfluidic chip structure to be manufactured, and may be equally applied to each cell.

The cell culture support is polydimethylsiloxane (PDMS), glass, glass, and polystyrene-based plastic materials such as polystyrene, polycarbonate, polyethylene, and polypropylene. It may be, but is not limited thereto.

The inlet 20 is an injection port for injecting the cell culture solution, and may be configured to be connected to one side of the cell culture support.

The tube connecting the inlet and the cell culture support may be made of a pipe (tube) structure, the outer diameter (outer diameter) of 1 to 1.5 mm, the inner diameter (inner diameter) may be 0.1 to 0.5 mm. When the invention is carried out within the outer / inner diameter range of the tube described above, there is an advantage that the cell culture solution can be injected under the flow conditions (flow rate and flow rate).

The material of the tube may include any material used to manufacture a tube in the art.

The control unit 30 is configured to inject the cell culture fluid at a constant flow rate and flow rate, and may be configured to be connected to the inlet.

The flow rate (average) in the microfluidic chip is, for example, 0.001 to 0.5 mL / h, 0.010 to 0.5 mL / h, 0.010 to 0.4 mL / h, 0.010 to 0.3 mL / h, 0.010 to 0.2 mL / h, 0.015 to 0.2 mL / h, 0.015 to 0.1 mL / h, 0.02 to 0.1 mL / h, 0.02 to 0.1 mL / h, 0.02 to 0.09 mL / h, 0.03 to 0.09 mL / h, 0.03 to 0.08 mL / h, 0.04 To 0.08 mL / h, 0.04 to 0.07 mL / h, 0.05 to 0.07 mL / h, 0.05 to 0.06 mL / h, or 0.05 mL / h, but is not limited thereto.

The flow rate (average) in the microfluidic chip is, for example, 1 to 25 mm / h, 1 to 24 mm / h, 2 to 24 mm / h, 2 to 23 mm / h, 3 to 23 mm / h, 3 to 22 mm / h, 3 to 20 mm / h, 4 to 22 mm / h, 4 to 21 mm / h, 5 to 21 mm / h, 5 to 20 mm / h, 6 to 20 mm / h, 6 To 19 mm / h, 7 to 19 mm / h, 7 to 18 mm / h, 8 to 18 mm / h, 8 to 17 mm / h, 9 to 17 mm / h, 9 to 16 mm / h, 10 to 16 mm / h, 10 to 15 mm / h, 10 to 14 mm / h, 10 to 13 mm / h, 10 to 12 mm / h, 10 to 11 mm / h or 10 mm / h, but is not limited thereto. It doesn't happen.

When the invention is carried out in the above-described flow conditions (flow rate and flow rate), the physical effect applied to the cell when supplying the cell culture solution, that is, the effect due to shear stress can be minimized.

The outlet portion 40 is an outlet for recovering the cell culture solution, and may be configured to be connected to the other side of the cell culture support.

The tube connecting the outlet and the cell culture support may be made of a pipe (tube) structure, the outer diameter (outer diameter) of 1 to 1.5 mm, the inner diameter (inner diameter) may be 0.1 to 0.5 mm. When the invention is carried out within the outer / inner diameter range of the tube described above, there is an advantage that the cell culture solution can be injected under the flow conditions (flow rate and flow rate).

The material of the tube may include any material used to manufacture a tube in the art.

In particular, in the configuration in which the cell culture portion is composed of one or more cell culture supports, each of the outlet portions connected to the cell culture support may be in communication with each other to recover the cell culture solution.

The present invention relates to a three-dimensional culture method of cells using a three-dimensional cell culture microfluidic chip and a three-dimensional cell culture microfluidic chip, wherein the three-dimensional cell culture microfluidic chip is a cell containing a concave pattern By including a culture support, when culturing cells under specific flow conditions, the three-dimensional culture and maintenance effect of the cells are excellent, and thus can be usefully used as three-dimensional cell culture applications.

1A is a plan view schematically illustrating a three-dimensional cell culture microfluidic chip according to an embodiment of the present invention, and is a plan view schematically illustrating a microfluidic chip including one cell culture support.

Figure 1b is a plan view of the three-dimensional cell culture microfluidic chip according to an embodiment of the present invention, a plan view of a microfluidic chip containing three cell culture support.

FIG. 2 is a diagram (unit: mm, R: radius) of an entire modeling shape for deriving an optimal flow condition according to an exemplary embodiment of the present invention.

3 is a view showing the final modeling shape of the concave pattern for deriving the optimum flow conditions according to an experimental example of the present invention.

Figure 4a is a diagram confirming the velocity distribution when the flow rate is 0.1 mL / h, in order to derive the optimum flow conditions according to an experimental example of the present invention.

Figure 4b is a diagram confirming the velocity distribution when the flow rate is 0.05 mL / h, in order to derive the optimum flow conditions according to an experimental example of the present invention.

Figure 4c is a diagram confirming the velocity distribution when the flow rate is 0.015 mL / h, in order to derive the optimum flow conditions according to an experimental example of the present invention.

Figure 5a is a diagram confirming the local velocity distribution at the inlet and outlet when the flow rate is 0.1 mL / h, in order to derive the optimum flow conditions according to an experimental example of the present invention.

Figure 5b is a diagram confirming the local velocity distribution at the inlet and outlet when the flow rate is 0.05 mL / h, in order to derive the optimum flow conditions according to an experimental example of the present invention.

Figure 5c is a diagram confirming the local velocity distribution at the inlet and outlet when the flow rate is 0.015 mL / h, in order to derive the optimum flow conditions according to an experimental example of the present invention.

Figure 6a shows the local velocity distribution at the inlet side when the flow rate is 0.1 mL / h, 0.05 mL / h and 0.015 mL / h in order to derive the optimum flow conditions according to an experimental example of the present invention It is also.

Figure 6b is a side view of the local velocity distribution at the outlet when the flow rate is 0.1 mL / h, 0.05 mL / h and 0.015 mL / h in order to derive the optimum flow conditions according to an experimental example of the present invention It is also.

Figure 7a is a diagram confirming the shear stress (Shear stress) at the inlet in order to derive the optimum flow conditions according to an experimental example of the present invention.

Figure 7b is a diagram confirming the shear stress (Shear stress) at the outlet in order to derive the optimum flow conditions according to an experimental example of the present invention.

8 is a view evaluating the effectiveness of the three-dimensional cell culture microfluidic chip according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Preparation example. Fabrication of Concave Patterns

Concave patterns included in the cell culture support were prepared by known methods (Moon et al., Optimizing human embryonic stem cells differentiation efficiency by screening size-tunable homogenous embryoid bodies, Biomaterials, 2014; 35: 5987-97. ).

Experimental Example 1. Analysis of Optimal Flow Conditions through Computer Simulation

In order to explore the optimal flow conditions for culturing three-dimensional cells, modeling shapes of cell culture scaffolds containing concave patterns were prepared (see FIG. 1). At this time, the cells are cultured in a three-dimensional spheroid (spheroid) form in the concave pattern of the cell culture support, it was used in the modeling shape by proceeding to simplify the form (see Fig. 2).

Using this modeling shape, pressure distribution, velocity distribution and shear stress were evaluated under various flow conditions as follows. The results (velocity distribution, local velocity distribution, shear stress, etc.) are shown in Figures 4-7.

Flow analysis program used: Commercial Computational Fluid Dynamics (CFD) program

Flow rate: 0.015 mL / h to 0.1 mL / h

Speed: 3 mm / h to 20 mm / h

Shaped concave (cell) diameter: 0.3 mm

Distance between the shaped concave (cell) and the concave (cell): 0.4 mm

Result measuring position: 0.05 mm from bottom

Number of cells shaped in the channel: 34 * 10

Optimum flow conditions refer to conditions that exhibit the maximum flow rate with minimal effect on the cell.

As can be seen from Figures 4a to 4c, the velocity distribution was the fastest distribution at the flow rate 0.1 mL / h, there was a difference in the flow rate distribution of the upper and bottom layers, a medium distribution at the flow rate 0.05 mL / h, and a flow rate of 0.015 The slowest flow at mL / h shows that the flow rate distributions of the top and bottom layers are almost identical.

As can be seen in Figures 5a to 5c, it can be seen how the flow flows by showing the local velocity distribution in the form of a vector in the middle of the cell is cultured. The fastest distribution at the flow rate of 0.1 mL / h showed a difference in the flow rate distribution between the upper and the bottom layers, with a medium distribution at the flow rate of 0.05 mL / h and the slowest flow at the flow rate of 0.015 mL / h. It can be seen that the flow velocity distribution of the bed is almost the same.

As can be seen in Figures 6a and 6b, the local velocity distribution on the side of the inlet and outlet is the fastest distribution at the flow rate 0.1 mL / h, there was a difference in the flow rate distribution of the upper layer and the bottom layer, at a flow rate of 0.05 mL / h The median distribution and the slowest flow at a flow rate of 0.015 mL / h indicated that the flow rates of the top and bottom layers were nearly identical. At this time, it was confirmed that the even flow distribution was formed even at the inlet and the outlet without the cell culture section.

As can be seen in Figures 7a and 7b, the shear stress of the inlet and outlet is almost insignificant difference between the cell portion on the side of the flow directly and the opposite cell portion, the effect on the cell at this flow or flow rate is very small And it was found.

As a result of the synthesis, it was confirmed that the optimum flow condition was a rate of 22 mm / h to 3 mm / h at a flow rate of 0.1 to 0.015 mL / h.

Example. Preparation of microfluidic chip comprising cell culture support

Based on the modeling shape specification of Experimental Example 1, a cell culture support was prepared, and the five cell culture supports were connected to prepare a microfluidic chip.

Experimental Example 2. Evaluation of the effectiveness of the microfluidic chip

The formation of three-dimensional cells and the survival of cells when the formed cells were maintained for 5 days were confirmed.

First, DiO (Lipophilic Tracers; DiO, THERMO FISHER, USA) was added to human Adipose-derived Mesenchymal stem cells (human AD-MSC, LONZA, Swiss) at a concentration of 1 μg / ml. Incubated for 4 hours.

After washing with phosphate buffer saline (PBS, WELGENE, Republic of Korea) and removing bubbles with 70% ethanol (Ethanol, MERCK, USA), washed three times with PBS and then three-dimensional cells The medium required for formation was replaced. The medium is Dulbecco's Modified Eagle's medium (DMEM, SIGMA-ALDRICH; USA), 15% Fetal bovine serum (FBS, GIBCO, USA), 1% Non-essential amino acid acid; NEAA, GIBCO, USA), 1% Penicillin-streptomycin (PS, GIBCO, USA) and 100 mM β-mercaptoethanol (β-mercaptoethanol, GIBCO, USA) The same medium was used for all experiments.

1 x 10 ⁵ cells were injected into the inlet to allow the medium to emerge toward the outlet. After cell seeding, only the inlet was introduced into the inlet to wash out the cells remaining outside the concave toward the outlet. Medium was replaced by adding 200 μL to 300 μL of medium at the inlet at 24 hour intervals.

As can be seen in Figure 9, it was confirmed that the three-dimensional cells were formed on the first day after the cell inoculation, the formed embryoid body was not found a problem such as cell death even if further cultured for 5 days.

[Description of the code]

1: Microfluidic chip for three-dimensional cell culture

10: cell culture unit

11: cell culture support

20: entrance

30: control unit

40: exit section

The present invention relates to a three-dimensional cell culture microfluidic chip and a three-dimensional culture method of cells using the microfluidic chip.

Claims (13)

1. A cell culture unit comprising one or more cell culture supports;

   An inlet connected to one side of the cell culture support for injecting the cell culture solution;

   A control part connected to the inlet part for injecting the cell culture fluid at a constant flow rate and flow rate; And

   And an outlet connected to the other side of the cell culture support to recover the cell culture solution.

   The cell culture support comprises a concave pattern,

   Each of the outlet portions connected to the one or more cell culture supports is in communication with each other microfluidic chip for three-dimensional cell culture, characterized in that for recovering the cell culture.
2. According to claim 1, wherein the flow rate is 0.015 to 0.1 mL / h, three-dimensional cell culture microfluidic chip.
3. The microfluidic chip of claim 1, wherein the flow rate is 3 to 22 mm / h.
4. The microfluidic chip of claim 1, wherein the concave-shaped pattern includes a plurality of concave arranged in a predetermined direction.
5. 5. The microfluidic chip of claim 4, wherein the concave is arranged in a multiseriate in a horizontal and longitudinal direction.
6. The microfluidic chip of claim 4, wherein the concave has a diameter of 0.3 to 1.0 mm.
7. 5. The microfluidic chip of claim 4, wherein the diameter and interval of the concave are 1: 1 to 1.5 in length ratio.
8. The microfluidic chip of any one of claims 4 to 7, wherein the concave is 1 to 34 in one direction and 1 to 10 in the other direction.
9. The method of claim 1, wherein the cell culture support is made of polydimethylsiloxane (PDMS), glass, glass, polystyrene, polycarbonate, polyethylene, and polypropylene. It is any one or more materials selected from, 3D cell culture microfluidic chip.
10. Cell three-dimensional culture method comprising the following steps:

    Preparing a microfluidic chip for cell culture comprising a cell culture support; And

    Injecting the cell culture solution to the microfluidic chip.
11. The method of claim 10, wherein the injecting step is injecting the cell culture at a constant flow rate and flow rate, cell three-dimensional culture method.
12. The method of claim 1, wherein the flow rate is 0.015 to 0.1 mL / h.
13. The method of claim 1, wherein the flow rate is 3 to 22 mm / h.

Images