KR20200052509A - Method of Using Microfluidic Chip for 3-Dimensional Cell Spheroid Formation - Google Patents

Abstract

The present invention relates to a co-cultivation method of a 3D cell using a 3D cell culture microfluidic chip, and a drug screening method. When using the 3D cell culture microfluidic chip, cells can be applied without additional movement while the cells are cultured three-dimensionally, thereby being useful in various fields using 3D cultured cells.

Description

Method of Using Microfluidic Chip for 3-Dimensional Cell Spheroid Formation}

The present invention relates to the formation of a 3D cell using a microfluidic chip for 3D cell culture, a co-culture method, and a drug screening method.

A large amount of cells are required for clinical applications including cell therapy or drug delivery. When culturing cells using a general culture method, a large amount of culture dish or flask should be used. However, the culture in a culture dish or flask cultures the cells in an environment of a two-dimensional hardness, so there is a risk of inducing the denaturation of the cultured cells because there is a difference from the three-dimensional ductility environment in which cells actually proliferate in vivo. have.

To reduce this risk, various three-dimensional cell culture methods have been developed, such as alginate inverse opal scaffolds and array-wells. However, such a culturing method has a limitation in that it is difficult to maintain the cells (structure) when 3D cells (structure) are formed and then moved for culture or when the culture medium is replaced.

Accordingly, while culturing cells in three dimensions, it is urgent to develop a technique for a method that can be utilized in various studies requiring three-dimensional cells without separate cell movement or that three-dimensional shapes of cells can be maintained even when the culture medium is replaced. .

Korean Patent Publication No. 10-2014-0129841

The present inventors have made extensive efforts to develop a method that can simultaneously utilize cells while forming and culturing cells in three dimensions. As a result, when using a 3D cell culture microfluidic chip for culturing cells under a specific flow condition, the present invention was completed by clarifying that the cells can be utilized simultaneously without separate movement while culturing the cells in 3D. .

Accordingly, an object of the present invention is to provide a method for spheroid formation of 3D cells using a microfluidic chip for 3D cell culture.

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

The present inventors have made extensive efforts to develop a method that can simultaneously utilize cells while forming and culturing cells in three dimensions. As a result, it was found that when a microfluidic chip for 3D cell culture in which cells are cultured under a specific flow condition is used, the cells can be simultaneously utilized without separate movement while culturing the cells in 3D.

The present invention relates to a co-culture method and a drug screening method of 3D cells using a microfluidic chip for 3D cell culture.

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

One aspect of the present invention is a three-dimensional cell co-culture (spheroid formation) method using a three-dimensional cell culture microfluidic chip comprising the step of sequentially inoculating (seeding) two or more cells in a cell culture support.

In the microfluidic chip, the cell culture solution is injected at a constant flow rate and flow rate.

In the present specification, "a microfluidic chip for three-dimensional cell culture" includes a cell culture unit consisting of one or more cell culture supports; An inlet connected to one side of the cell culture support to inject the cell culture solution; A control part connected to the inlet part to inject the cell culture solution at a constant flow rate and flow rate; Means a microfluidic chip having; an outlet portion 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 may communicate with each other to recover the cell culture solution.

As used herein, "concave" means a concave surface, and specifically, a hemisphere having a concave surface.

As used herein, "concave pattern" means a plurality of concaves arranged in a certain direction.

The concave may be arranged in multiple rows in the horizontal and vertical directions.

In a specific aspect, the concave pattern is a plurality of concaves arranged in multiple rows, and each row of the pattern may be arranged in parallel.

In this specification, "parallel" is meant to include not only complete parallelism, but also those arranged at levels that can be considered substantially parallel.

The diameter of the concave is 0.10 to 1.00 mm, 0.15 to 1.00 mm, 0.20 to 1.00 mm, 0.25 to 1.00 mm, 0.30 mm, 0.30 to 1.00 mm, 0.35 to 1.00 mm, 0.40 to 1.00 mm, 0.45 to 1.00 mm, 0.50 To be 1.00 mm, 0.55 to 1.00 mm, 0.60 to 1.00 mm, 0.65 to 1.00 mm, 0.70 to 1.00 mm, 0.75 to 1.00 mm, 0.80 to 1.00 mm, 0.85 to 1.00 mm, 0.90 to 1.00 mm or 0.95 to 1.00 mm have. When the invention is carried out within the diameter of the above-described concave, the cell growth rate may be maximized.

In particular, the size of embryoid bodies (EB) during the differentiation process of cells is an important parameter in differentiation, and the use of EB of the optimal size can improve the differentiation efficiency into desired cells. In addition, since the EB size has different differentiation efficiency according to the cell line, it is necessary to control the EB size suitable for the cell line in order to improve the differentiation efficiency. Therefore, depending on the cell line, the diameter of the concave pattern included in the cell culture support of the 3D cell culture microfluidic chip may be different.

The diameter and spacing of the concave may have a length ratio of 1.0: 1.0 to 1.5, 1.0: 1.0 to 1.4, 1.0: 1.0 to 1.3, 1.0: 1.0 to 1.2 or 1.0: 1.0 to 1.1.

When the invention is carried out within the diameter and spacing of the above-described concave, the cell yield, which is the ratio in which cells are formed in three dimensions relative to the supplied cells, can be maximized.

The number of 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 concave may vary depending on the microfluidic chip structure to be manufactured, and can be applied equally to each cell.

The cell culture support may be made of polydimethylsiloxane (PDMS), glass, and poly-based plastic materials, but is not limited thereto.

The poly-based plastic may be polystyrene, polycarbonate, polyethylene, and polypropylene, but is not limited thereto.

The method comprises an aggregation step of inoculating and culturing the first cells; And a co-culturing step of inoculating and culturing the second cells.

The aggregation step is, for example, 1 to 24 hours, 2 to 24 hours, 3 to 24 hours, 4 to 24 hours, 5 to 24 hours, 6 to 24 hours, 7 to 24 hours, 8 to 24 hours, 9 to 24 hours, 10 to 24 hours, 11 to 24 hours, 12 to 24 hours, 13 to 24 hours, 14 to 24 hours, 15 to 24 hours, 16 to 24 hours, 17 to 24 hours, 18 to 24 hours, 19 to 24 hours, 20 to 24 hours, 21 to 24 hours, 22 to 24 hours, 23 to 24 hours, 1 to 23 hours, 1 to 22 hours, 1 to 21 hours, 1 to 20 hours, 1 to 19 hours, 1 to 18 hours, 1 to 17 hours, 1 to 16 hours, 1 to 15 hours, 1 to 14 hours, 1 to 13 hours, 1 to 12 hours, 1 to 11 hours, 1 to 10 hours, 1 to 9 hours, 1 to 1 hour It may be performed for 8 hours, 1 to 7 hours, 1 to 6 hours, 1 to 5 hours, 1 to 4 hours, 1 to 3 hours, or 1 to 2 hours, but is not limited thereto. It is not.

The co-cultivation step is, for example, 1 to 48 hours, 24 to 48 hours, 25 to 48 hours, 26 to 48 hours, 27 to 48 hours, 28 to 48 hours, 29 to 48 hours, 30 to 48 hours, 31 To 48 hours, 32 to 48 hours, 33 to 48 hours, 34 to 48 hours, 35 to 48 hours, 36 to 48 hours, 37 to 48 hours, 38 to 48 hours, 39 to 48 hours, 40 to 48 hours, 41 To 48 hours, 42 to 48 hours, 43 to 48 hours, 44 to 48 hours, 45 to 48 hours, 46 to 48 hours, 47 to 48 hours, 1 to 47 hours, 1 to 46 hours, 1 to 45 hours, 1 To 44 hours, 1 to 43 hours, 1 to 42 hours, 1 to 41 hours, 1 to 40 hours, 1 to 39 hours, 1 to 38 hours, 1 to 37 hours, 1 to 36 hours, 1 to 35 hours, 1 To 34 hours, 1 to 33 hours, 1 to 32 hours, 1 to 31 hours, 1 to 30 hours, 1 to 29 hours, 1 to 28 hours, 1 to 27 hours, 1 to 26 hours Liver, but it can be carried out for 1 to 25 hours, or 1 to 24 hours, and the like.

The average flow rate in the microfluidic chip is 0.001 to 0.100 mL / h, 0.002 to 0.100 mL / h, 0.003 to 0.100 mL / h, 0.004 to 0.100 mL / h, 0.005 to 0.100 mL / h, 0.006 to 0.100 mL / h, 0.007 to 0.100 mL / h, 0.008 to 0.100 mL / h, 0.009 to 0.100 mL / h, 0.010 to 0.100 mL / h, 0.020 to 0.100 mL / h, 0.030 to 0.100 mL / h, 0.040 to 0.100 mL / h, 0.050 To 0.100 mL / h, 0.060 to 0.100 mL / h, 0.070 to 0.100 mL / h, 0.080 to 0.100 mL / h, 0.090 to 0.100 mL / h, 0.001 to 0.090 mL / h, 0.001 to 0.080 mL / h, 0.001 to 0.070 mL / h, 0.001 to 0.060 mL / h, 0.001 to 0.050 mL / h, 0.001 to 0.040 mL / h, 0.001 to 0.030 mL / h, 0.001 to 0.020 mL / h, 0.001 to 0.010 mL / h, 0.001 to 0.009 mL / h, 0.001 to 0.008 mL / h, 0.001 to 0.007 mL / h, 0.001 to 0.006 mL / h, 0.001 to 0.005 mL / h, 0.001 to 0.004 mL / h, 0.001 to 0.003 mL / h, 0.001 to 0.002 mL / h, but is not limited thereto.

The average flow rate in the microfluidic chip is 0.2 to 20.0 mm / h, 0.3 to 20.0 mm / h, 0.4 to 20.0 mm / h, 0.5 to 20.0 mm / h, 0.6 to 20.0 mm / h, 0.7 to 20.0 mm / h, 0.8 to 20.0 mm / h, 0.9 to 20.0 mm / h, 1.0 to 20.0 mm / h, 2.0 to 20.0 mm / h, 3.0 to 20.0 mm / h, 4.0 to 20.0 mm / h, 5.0 to 20.0 mm / h, 6.0 To 20.0 mm / h, 7.0 to 20.0 mm / h, 8.0 to 20.0 mm / h, 9.0 to 20.0 mm / h, 10.0 to 20.0 mm / h, 11.0 to 20.0 mm / h, 12.0 to 20.0 mm / h, 13.0 to 20.0 mm / h, 14.0 to 20.0 mm / h, 15.0 to 20.0 mm / h, 16.0 to 20.0 mm / h, 17.0 to 20.0 mm / h, 18.0 to 20.0 mm / h, 19.0 to 20.0 mm / h, 0.2 to 19.0 mm / h, 0.2 to 18.0 mm / h, 0.2 to 17.0 mm / h, 0.2 to 16.0 mm / h, 0.2 to 15.0 mm / h, 0.2 to 14.0 mm / h, 0.2 to 13.0 mm / h, 0.2 to 12.0 mm / h, 0.2 to 11.0 mm / h, 0.2 to 10.0 mm / h, 0.2 to 9.0 mm / h, 0.2 to 8.0 mm / h, 0.2 to 7.0 mm / h, 0.2 to 6.0 mm / h, 0.2 to 5.0 mm / h, 0.2 to 4.0 mm / h, 0.2 To 3.0 mm / h, 0.2 to 2.0 mm / h, 0.2 to 1.0 mm / h, 0.2 to 0.9 mm / h, 0.2 to 0.8 mm / h, 0.2 to 0.7 mm / h, 0.2 to 0.6 mm / h, 0.2 to 0.5 mm / h, 0.2 to 0.4 mm / h, or 0.2 to 0.3 mm / h, but is not limited thereto.

When the invention is carried out within the above-described flow rate and flow rate, the physical effect applied to the cells when supplying the cell culture solution may be minimized.

The cell may include any cell as long as it can be cultured in three dimensions in the art, for example, embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), intermediate Mesenchymal stem cells (MSC) and fibroblasts (Fibroblast), but are not limited thereto.

The cell culture medium may include any culture medium as long as it is a culture medium that can be used for culturing and / or differentiating cells (stem cells) in the art, and the type and / or concentration of components or factors included in the culture medium is determined by a person skilled in the art. It can be easily adjusted.

The microfluidic chip for three-dimensional cell culture used in the three-dimensional cell co-cultivation method of the present invention is stable even without separate cell movement for two or more cells by using the cell culture solution because it is injected and released at a constant flow rate and flow rate. As a result, there is an advantage in that aggregation and co-culturing of multi-layered 3D cells can be performed at once.

Another aspect of the present invention is a drug screening method using a microfluidic chip for 3D cell culture, comprising inoculating cells in a cell culture support and processing drug candidates.

In the microfluidic chip, the cell culture solution is injected at a constant flow rate and flow rate.

The drug screening method may include comparing cells of the cell culture support treated with the drug candidate and cells of a cell culture support not treated with the drug candidate.

The drug screening method may be a comparison of cell viability, oxygen consumption rate (OCR), and / or cell function.

The cell culture support treated with the drug candidate and the cell culture support not treated with the drug candidate may be included in one three-dimensional cell culture microfluidic chip.

The average flow rate in the microfluidic chip is 0.001 to 0.100 mL / h, 0.002 to 0.100 mL / h, 0.003 to 0.100 mL / h, 0.004 to 0.100 mL / h, 0.005 to 0.100 mL / h, 0.006 to 0.100 mL / h, 0.007 to 0.100 mL / h, 0.008 to 0.100 mL / h, 0.009 to 0.100 mL / h, 0.010 to 0.100 mL / h, 0.020 to 0.100 mL / h, 0.030 to 0.100 mL / h, 0.040 to 0.100 mL / h, 0.050 To 0.100 mL / h, 0.060 to 0.100 mL / h, 0.070 to 0.100 mL / h, 0.080 to 0.100 mL / h, 0.090 to 0.100 mL / h, 0.001 to 0.090 mL / h, 0.001 to 0.080 mL / h, 0.001 to 0.070 mL / h, 0.001 to 0.060 mL / h, 0.001 to 0.050 mL / h, 0.001 to 0.040 mL / h, 0.001 to 0.030 mL / h, 0.001 to 0.020 mL / h, 0.001 to 0.010 mL / h, 0.001 to 0.009 mL / h, 0.001 to 0.008 mL / h, 0.001 to 0.007 mL / h, 0.001 to 0.006 mL / h, 0.001 to 0.005 mL / h, 0.001 to 0.004 mL / h, 0.001 to 0.003 mL / h, 0.001 to 0.002 mL / h, but is not limited thereto.

The average flow rate in the microfluidic chip is 0.2 to 20.0 mm / h, 0.3 to 20.0 mm / h, 0.4 to 20.0 mm / h, 0.5 to 20.0 mm / h, 0.6 to 20.0 mm / h, 0.7 to 20.0 mm / h, 0.8 to 20.0 mm / h, 0.9 to 20.0 mm / h, 1.0 to 20.0 mm / h, 2.0 to 20.0 mm / h, 3.0 to 20.0 mm / h, 4.0 to 20.0 mm / h, 5.0 to 20.0 mm / h, 6.0 To 20.0 mm / h, 7.0 to 20.0 mm / h, 8.0 to 20.0 mm / h, 9.0 to 20.0 mm / h, 10.0 to 20.0 mm / h, 11.0 to 20.0 mm / h, 12.0 to 20.0 mm / h, 13.0 to 20.0 mm / h, 14.0 to 20.0 mm / h, 15.0 to 20.0 mm / h, 16.0 to 20.0 mm / h, 17.0 to 20.0 mm / h, 18.0 to 20.0 mm / h, 19.0 to 20.0 mm / h, 0.2 to 19.0 mm / h, 0.2 to 18.0 mm / h, 0.2 to 17.0 mm / h, 0.2 to 16.0 mm / h, 0.2 to 15.0 mm / h, 0.2 to 14.0 mm / h, 0.2 to 13.0 mm / h, 0.2 to 12.0 mm / h, 0.2 to 11.0 mm / h, 0.2 to 10.0 mm / h, 0.2 to 9.0 mm / h, 0.2 to 8.0 mm / h, 0.2 to 7.0 mm / h, 0.2 to 6.0 mm / h, 0.2 to 5.0 mm / h, 0.2 to 4.0 mm / h, 0.2 To 3.0 mm / h, 0.2 to 2.0 mm / h, 0.2 to 1.0 mm / h, 0.2 to 0.9 mm / h, 0.2 to 0.8 mm / h, 0.2 to 0.7 mm / h, 0.2 to 0.6 mm / h, 0.2 to 0.5 mm / h, 0.2 to 0.4 mm / h, or 0.2 to 0.3 mm / h, but is not limited thereto.

When the invention is carried out within the above-described flow rate and flow rate, the physical effect applied to the cells when supplying the cell culture solution may be minimized.

The cell may include any cell as long as it can be cultured in three dimensions in the art, for example, hepatocyte, cardiomyocyte, endothelial cell, and neural cell. It may be, but is not limited to this.

The cell culture medium may include any culture medium as long as it is a culture medium that can be used for culturing and / or differentiating cells (stem cells) in the art, and the type and / or concentration of components or factors included in the culture medium is determined by a person skilled in the art. It can be easily adjusted.

The overlap of the three-dimensional cell culture microfluidic chip is omitted in consideration of the complexity of the present specification.

Since the microfluidic chip for 3D cell culture used in the drug screening method of the present invention is injected and released at a constant flow rate and flow rate, aggregation and drug of 3D cells can be performed without using a separate cell. It has the advantage of being able to perform cell response assays at once.

In addition, since the microfluidic chip for 3D cell culture used in the drug screening method of the present invention has several cell culture supports (channels), it is used to analyze cell responses by drugs in multiple concentration conditions at once. There is an advantage to do.

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

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

Referring to the above 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.

The cell culture unit 10 may be configured to be made of one or more cell culture supports 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 include a concave pattern.

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

The concave may be arranged in multiple rows in the horizontal and vertical directions.

The diameter of the concave is 0.10 to 1.00 mm, 0.15 to 1.00 mm, 0.20 to 1.00 mm, 0.25 to 1.00 mm, 0.30 mm, 0.30 to 1.00 mm, 0.35 to 1.00 mm, 0.40 to 1.00 mm, 0.45 to 1.00 mm, 0.50 To be 1.00 mm, 0.55 to 1.00 mm, 0.60 to 1.00 mm, 0.65 to 1.00 mm, 0.70 to 1.00 mm, 0.75 to 1.00 mm, 0.80 to 1.00 mm, 0.85 to 1.00 mm, 0.90 to 1.00 mm or 0.95 to 1.00 mm However, it is not limited thereto. When the invention is carried out within the diameter of the above-described concave, the cell growth rate may 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-described concave, the cell yield, which is the ratio in which cells are formed in three dimensions relative to the supplied cells, can be maximized.

The number of cone caves 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 concave may vary depending on the microfluidic chip structure to be manufactured, and can be applied equally to each cell.

The cell culture support may be made of polydimethylsiloxane (PDMS), glass, and poly-based plastic materials, but is not limited thereto.

The poly-based plastic may be polystyrene, polycarbonate, polyethylene, and polypropylene, but is not limited thereto.

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

A culture medium storage container capable of storing the culture medium may be additionally disposed at the inlet.

The tube connecting the inlet and the cell culture support may be made of a pipe (tube) structure, and the outer diameter (outer diameter) may be 1.0 to 1.5 mm, and the inner diameter (inner diameter) may be 0.1 to 0.5 mm.

The material of the tube may include any material used to manufacture the 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 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 portion and the cell culture support may be made of a pipe (tube) structure, an outer diameter (outer diameter) of 1.0 to 1.5 mm, and an inner diameter (inner diameter) of 0.1 to 0.5 mm.

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

In particular, in a configuration in which the cell culture part is composed of one or more cell culture supports, each of the outlet parts connected to the cell culture support can communicate with each other to recover the cell culture solution.

The present invention relates to a co-cultivation method and a drug screening method of a 3D cell using a microfluidic chip for 3D cell culture, and when the microfluidic chip for 3D cell culture is used, the cells are separately cultured in 3D. Since it can be utilized simultaneously without the movement of, it can be usefully used in various fields using 3D cultured cells.

1A is a top plan view of a 3D cell culture microfluidic chip containing one cell culture support according to an embodiment of the present invention.
Figure 1b is a plan view of a microfluidic chip for three-dimensional cell culture containing three cell culture support according to an embodiment of the present invention.
2 is a diagram (model: mm, R: radius) for the modeling shape for the production of a cell culture support according to an embodiment of the present invention.
3 is a diagram showing the results of co-culturing adipose-derived mesenchymal stem cells and fibroblasts using a microfluidic chip according to an embodiment of the present invention.
Figure 4a is a diagram showing a drug (azatyoprine) screening results (cell death effect) for hepatocytes using a microfluidic chip according to an embodiment of the present invention.
Figure 4a is a diagram showing a drug (azatyoprine) screening results (oxygen consumption reduction effect) for hepatocytes using a microfluidic chip according to an embodiment of the invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention more specifically, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Manufacturing example. Preparation of concave pattern

The concave pattern included in the cell culture support was prepared by a known method (Moon et al., Optimizing human embryonic stem cells differentiation efficiency by screening size-tunable homogenous embryoid bodies, Biomaterials, 2014; 35: 5987-97. ).

Example. Preparation of microfluidic chip comprising cell culture support

Based on the modeling shape standard shown in FIG. 2, a cell culture support containing a concave pattern was prepared, and 3 to 5 cell culture supports were connected to prepare a microfluidic chip.

Ready. Cell culture

Human Adipose-derived Mesenchymal stem cells (human AD-MSC, LONZA, Swiss) are RPMI 1640 (Gibco) supplemented with 10% Fetal bovine serum (FBS, Gibco, USA) ) Cultured in the medium.

Human fibroblasts (ATCC, USA) were maintained in Dulbecco's Modified Eagle's medium (DMEM, Sigma-Aldrich, USA) with 10% fetal bovine serum (Gibco) added.

Each medium includes 1% non-essential amino acid (NEAA, Gibco), 1% penicillin-streptomycin (PS, Gibco) and 100 mM β-mercaptoethanol (β-mercaptoethanol, Gibco). ) Was added.

The adipose-derived mesenchymal stem cells and fibroblasts were used after 5-6 passages using a 0.25% trypsin solution, and maintained at 5 ° C ₂ , 37 ° C in a fluid chamber.

Human hepatocytes were purchased from ScienCell (ScienCell Research Laboratories, USA) and cultured in hepatocyte medium containing 5% FBS and hepatocyte growth supplement, respectively.

Experimental Example 1. Spheroid formation using microfluidic chips

In a microfluidic chip comprising three cell culture supports, adipose-derived mesenchymal stem cells and fibroblasts were co-cultured under the following three conditions. Each cell was stained with different fluorescent labels (Molecular Probes) DiI and DiO.

-Condition 1 (first cell culture support): After seeding and agglutinating the fibroblasts, on the second day, the fat-derived mesenchymal stem cells are inoculated and co-culturing.

-Condition 2 (second cell culture support): After inoculating and agglutinating adipose-derived mesenchymal stem cells, fibroblasts were inoculated on day 2 and co-cultured.

-Condition 3 (Third cell culture support): Inoculated with adipose-derived mesenchymal stem cells and fibroblasts and co-cultured after aggregation.

More specifically, adipose-derived mesenchymal stem cells and fibroblasts were treated with 0.25% trypsin, incubated at 37 ° C for 2 minutes, and centrifuged for 5 minutes at 1000 rpm, and then reproduced in the medium to a final concentration of 5x10 ⁵ cells / mL. It was clouded. In the case of conditions 1 and 2, the fat-derived mesenchymal stem cells or the same amount of fibroblasts were inoculated into the cell culture support 200 mL each, and cultured at 37 ° C. for one day, followed by different (opposite) cells to each cell. The culture support was further inoculated and co-cultured. In the case of condition 3, the fat-derived mesenchymal stem cells and the same amount of fibroblasts were mixed and cultured by inoculating 200 mL of the cell culture support.

At this time, the cell culture solution was injected through the inlet of the microfluidic chip at a rate of 0.8 μL / min, and 500-1,000 μL of new culture solution was added to the culture medium storage container arranged at the inlet at a period of 12-24 hours.

As can be seen in Figure 3, in the sequential co-culture group (condition 1 and condition 2), it was confirmed that the cells inoculated on the second day were cultured while surrounding the surface of the aggregated cell culture body (EB) on the first day. Did. On the other hand, in the simple co-culture group (condition 3), it was confirmed that aggregates are formed in one compact form by fusion without distinction of each cell.

Experimental Example 2. Drug screening using a microfluidic chip

In a microfluidic chip comprising four cell culture supports, hepatocytes are seeded to form embryoid bodies (EBs) while simultaneously in multiple concentrations (1 μM, 10 μM and 100 μM) of azathioprine. Drug screening was performed.

More specifically, hepatocytes were treated with 0.25% trypsin, incubated at 37 ° C. for 2 minutes, centrifuged at 1000 rpm for 5 minutes, and then resuspended in the medium to a final concentration of ⁵ × 10 ⁵ cells / mL. The hepatocytes were inoculated into 4 cell culture supports by 200 μL and cultured at 37 ° C. for one day to form embryoid bodies. After 24 hours, the drug response was confirmed for 3 days by treating azathioprine with multiple concentrations in fresh 200 μL medium.

At this time, the cell culture solution was injected through the inlet of the microfluidic chip at a rate of 0.8 μL / min, and 500-1,000 μL of new culture solution was added to the culture medium storage container arranged at the inlet at a period of 12-24 hours.

As can be seen in Figure 4a, it was confirmed that apoptosis due to the drug reaction occurs within 3 days only in hepatocytes treated with azathioprine at a concentration of 100 μM.

In addition, as can be seen in Figure 4b, the oxygen consumption of hepatocytes treated with azatyoprine at concentrations of 1 μM and 10 μM was somewhat higher or similar to that of the control group, but the oxygen of hepatocytes treated with azatyoprine at a concentration of 100 μM. It was confirmed that the consumption amount appeared lower than the control group.

1: Microfluidic chip for 3D cell culture
10: cell culture section
11: Cell culture support
20: entrance
30: control unit
40: outlet

Claims (12)

A three-dimensional cell co-culture (spheroid formation) method using a three-dimensional cell culture microfluidic chip comprising the step of sequentially inoculating (seeding) two or more cells to the cell culture support,
The microfluidic chip is a cell culture medium is injected at a constant flow rate and flow rate, a three-dimensional cell co-culture method. The method of claim 1, wherein the method
Aggregation step of inoculating and culturing the first cells; And
And a co-culturing step of inoculating and culturing the second cells. The method of claim 2, wherein the aggregation step is performed for 1 to 24 hours. The method of claim 2, wherein the co-culturing step is performed for 1 to 48 hours. The method of claim 1, wherein the flow rate is 0.001 to 0.100 mL / h. The method of claim 1, wherein the flow rate is 0.2 to 20.0 mm / h. The method of claim 1, wherein the cells are embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), mesenchymal stem cells (MSC) and fibroblasts (Fibroblast). It is selected from the group consisting of, 3D cell co-culture method. A method for drug screening using a microfluidic chip for 3D cell culture, comprising inoculating cells into a cell culture support and processing drug candidates,
The microfluidic chip is a method for drug screening, wherein the cell culture solution is injected at a constant flow rate and flow rate. The method of claim 8, wherein the method comprises comparing the cells of the cell culture support treated with the drug candidate and the cells of the cell culture support without treating the drug candidate with each other. 9. The method of claim 8, wherein the flow rate is 0.001 to 0.100 mL / h. The method of claim 8, wherein the flow rate is 0.2 to 20.0 mm / h. The method of claim 8, wherein the cells are selected from the group consisting of hepatocytes, cardiomyocytes, endothelial cells, and neural cells.

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