KR20230088311A - Well plate and method for cell culture using the same - Google Patents

Abstract

The present invention is to provide a 3D cell culture device that efficiently removes waste products generated during 3D cell culture and smoothly supplies nutrients to the bottom of 3D cells without adversely affecting the settlement, proliferation, and differentiation of cells during 3D cell culture, and a cell culture method using the same, and specifically, provides the 3D cell culture device including: an upper chamber containing a porous microwell; and a lower chamber in which the upper chamber is disposed and through which a fluid can flow to the bottom of a porous microwell by containing the fluid, and the cell culture method using the same.

Description

3D cell culture device including bottom flow and 3D cell culture method using the same {Well plate and method for cell culture using the same}

The present invention provides a cell culture device capable of stably culturing cells, efficiently removing generated waste products, and supplying nutrients to cell culture during 3-dimensional cell culture, and a 3-dimensional cell culture method using the same.

Recently, methods for forming and culturing efficient and mature three-dimensional cells are required for drug screening or disease research, and various microengineering approaches are being attempted. Among them, the microwell platform is representative.

Conventional microwell platforms rely only on passive diffusion to discharge waste products and supply nutrients during the cultivation of three-dimensional cell aggregates. However, this is insufficient to meet the increasing metabolic demands during the growth of 3D cells, and eventually limits the growth and maturation of 3D cells.

On the other hand, although a method for discharging waste materials and supplying nutrients using flow is being studied, as in Registered Patent No. 10-1403536, this method directly inhales the culture medium and discharges waste products in the area where cells are cultured. There is a risk of adversely affecting the settlement, proliferation, and differentiation of cells that have been treated, or that the cells are inhaled and discharged together with waste materials when waste materials are removed. In addition, there is a limitation in that the removal of waste materials and the supply of nutrients to the lower part of the 3-dimensional cells are not smooth.

Furthermore, a microwell platform combined with a microfluidic system has been proposed as in Patent Publication No. 10-2012-0011609, but the platform also uses a continuous flow in a cell culture chamber, and turbulence is generated inside the chamber in which cells are cultured. It can be easily generated, and furthermore, there is a problem of limiting the maturation of 3D cells by applying unnecessary physical force, and there is a limitation that waste material removal and nutrient supply to the lower part of the 3D cells are not smooth.

In order to solve the problems of the prior art as described above, the present invention is a stable three-dimensional cell culture and cell culture to efficiently discharge waste materials and smoothly supply nutrients to the lower part of the cell, flowing at the lower end of the porous microwell. Its purpose is to enable control.

In one aspect of the present invention, the present invention provides an upper chamber including an opening and a porous microwell; and a lower chamber in which the upper chamber is disposed and fluid flow is possible at a lower end of the porous microwell.

In another aspect of the present invention, the present invention is a three-dimensional cell culture method comprising the step of culturing three-dimensional cells by using the three-dimensional cell culture device of the present invention and introducing the cell culture medium and cells into porous microwells. to provide.

The 3D cell culture device of the present invention can efficiently remove waste products formed during cell culture and smoothly supply nutrients to the bottom of the 3D cells without adversely affecting the settlement, proliferation and differentiation of cells during 3D cell culture. A three-dimensional cell culture device can be provided.

Figure 1 (a) shows an exemplary view of a three-dimensional cell culture device according to an embodiment of the present invention, Figure 1 (b) is an adjacent portion of the porous microwell in the upper chamber of the three-dimensional cell culture device of the present invention It shows an example of the case where the penetrating portion is formed, and FIG. 1 (c) shows an actual image of the three-dimensional cell culture device of the present invention.
Figure 2 (a) is a comparison of the expression amount of albumin measured in Example 1 and Comparative Example 1 of the present invention, Figure 2 (b) is FITC-Dextran measured in Example 2 and Comparative Example 2 of the present invention concentration was compared.
Figure 3 shows the results of visually observing the removal of waste over time according to Example 2 of the present invention.
Figure 4 shows a schematic diagram in which waste materials on the upper surface of the porous microwell are removed by the fluid flow and the porous microwell in the lower chamber of the present invention.

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiments of the present invention may be modified in various forms, and the scope of the present invention is not limited to the embodiments described below.

The present invention provides a three-dimensional cell culture device capable of easily removing waste products generated during cell culture and smoothly supplying nutrients to the bottom of the three-dimensional cells.

Specifically, the present invention provides an upper chamber including an opening and a porous microwell; and a lower chamber in which the upper chamber is disposed and fluid flow is possible at a lower end of the porous microwell.

In the present invention, the upper chamber may mean a form in which a well-type chamber including an opening and a porous microwell is inserted through a plate. For example, as shown in FIG. 1, the upper chamber of the present invention It may include wells including openings and porous microwells, and a plate through which the wells are inserted.

Furthermore, the lower end of the porous microwell refers to a lower area of the porous microwell, and refers to an area within the lower chamber.

A penetrating part may be formed in the upper chamber to allow fluid in the lower chamber to contact the outside. In this case, the penetrating part may be formed adjacent to the porous microwell of the upper chamber, or the penetrating part may be formed at an arbitrary position in the upper chamber. can be formed As described above, when the fluid is in contact with the outside, the location of the through-hole is not particularly limited as long as it can prevent the porous membrane from being deformed by the physical force formed by the flow of the fluid.

The lower chamber may further include a fluid inlet and a fluid outlet through which fluid may flow, through which the fluid in the lower chamber may flow.

Furthermore, the fluid inlet and the fluid outlet may be further connected with a device capable of generating flow, for example, the lower chamber may be connected with a device for inducing a fluid flow so that the fluid flows in a horizontal direction of the porous microwell. there is. At this time, a pump or the like may be used as a device for inducing the flow of the fluid, but it is not limited as long as it can cause the flow of the fluid.

On the other hand, the cell culture medium may be used as the fluid, and the cell culture medium may include FBS, glucose, growth factors, etc., but is not limited as long as it is a cell culture medium used in the art.

Furthermore, the porous microwell may be formed by randomly entangling a plurality of polymer nanofibers or by molding a polymer synthetic resin. As the porous microwells are composed of a plurality of polymer nanofibers, the porous microwells may have a structure similar to that of a basement membrane in a living body, thereby providing an environment similar to a blood flow environment in a living body.

Meanwhile, the porous microwell may include a plurality of pores formed in a region between polymer nanofibers. At this time, the pores may have a size of several nm to several μm. That is, the porous microwell can act as a selective permeable membrane that selectively transmits other materials while not permeating single cells due to pores, and thus can serve as a material transfer barrier and passage. For example, the porous microwells may have a pore size of 10 nm to 8 μm in average diameter.

At this time, the polymer nanofiber or polymer synthetic resin may include at least one of a thermoplastic resin, a thermosetting resin, an elastomer, and a biopolymer. For example, the polymer nanofiber or polymer synthetic resin may include polycaprolactone It may include at least one of polyurethane, polyvinylidene fluoride (PVDF), polystyrene, collagen, gelatin, and chitosan, but is limited thereto It doesn't work.

The porous microwells may be manufactured using a mold having a recessed pattern concave downward. For example, a flat porous membrane made of polymer nanofibers is placed on the mold and formed as a separate mold for an embossing process. It can be prepared by pressing the porous membrane, but is not limited thereto.

The upper surface of the porous microwell is an area that functions as a cell culture layer, and cells are easily seated on the cell culture layer by the recessed portion, and three-dimensional cells can stably proliferate in the porous microwell .

In addition, unlike conventional cell culture vessels having a cell culture layer of a generally flat shape, the cell culture apparatus of the present invention can provide a cell culture layer of a three-dimensional three-dimensional shape, and thus a three-dimensional structure environment like in vivo. Since cells can be cultured in, it is possible to provide an environment in which three-dimensional cells can be formed.

Furthermore, the 3D cell culture device allows the fluid in the lower chamber to flow stably through a fluid flow induction device that allows the fluid flowing in the lower chamber to flow to the lower end of the porous microwell, and the pores of the porous microwell Due to the permeability of the above, the fluid having the flow is three-dimensional Waste products formed during cell culture can be discharged from the upper surface of the porous membrane to the lower chamber, and nutrients can be smoothly supplied to the lower part of the three-dimensional cells. Furthermore, in the lower chamber, three-dimensional Waste materials may be discharged to the outside of the cell culture device. For example, the fluid flow inducer may use a syringe pump, a peristaltic pump, or an agitator.

In addition, the three-dimensional cell culture device In order to disperse the pressure applied to the porous microwells during cell culture and to efficiently remove and supply formed waste products and nutrients, the upper chamber may have a through portion formed so that the surface of the fluid in the lower chamber can contact the outside. there is.

The penetrating portion provides a structure through which the surface of the fluid in the lower chamber can be brought into contact with the outside, and the porous microwell is deformed by applying pressure to the upper chamber when the fluid flows through the structure, thereby deforming the 3D cell culture. It is possible to solve problems that adversely affect the settlement, proliferation, and differentiation of cells upon application.

On the other hand, the present invention is a three-dimensional cell culture comprising the step of culturing cells by introducing a cell culture medium and cells into a porous microwell using the three-dimensional cell culture device of the present invention. Provided is a cell culture method, wherein the cells are myoblasts, fetal fibroblasts (embryo fibroblasts), human umbilical vein endothelial cells, human liver cancer cells (HepG2 cells) or human epidermal cells (human epidermal cells), but is not limited thereto.

Furthermore, the 3D cell culture device of the present invention can culture 3D spheroids or 3D organoids.

Hereinafter, the present invention will be described in more detail through specific examples. The following examples are merely examples to aid understanding of the present invention, and the scope of the present invention is not limited thereto.

Example

Example 1

In a three-dimensional cell culture apparatus including an upper chamber and a lower chamber including porous microwells of the form shown in FIG. 1 (a), HepG2 cells and DMEM (Dulbeco's Modified Eagle's Media, FBS 10%) culture medium and After injection together, it was stabilized at 37 ° C. for 48 hours to form a three-dimensional spheroid. After that, flow was applied to the lower chamber and cultured for 9 days.

Comparative Example 1

Three-dimensional HepG2 spheroids were cultured in the same manner as in Example 1, except that the bottom flow was not applied in Example 1.

Albumin expression levels of Hep G2 spheroids were measured on days 6 and 9 during the culture period in Example 1 and Comparative Example 1. The measured albumin expression level is shown in FIG. 2 (a), and as shown in FIG. 2 (a), the albumin expression level of Hep G2 spheroids cultured in Comparative Example 1 was 1182.64mg/ml on the 6th day and 2966.505 on the 9th day. On the other hand, it was confirmed that the albumin expression level of Hep G2 spheroids cultured in Example 1 was 4813.99mg/ml on the 6th day and 6644.55mg/ml on the 9th day. That is, it was found that the albumin expression level of Hep G2 spheroids cultured in a three-dimensional cell culture apparatus with a bottom flow was high, and through this, it was confirmed that the functionality of the three-dimensional Hep G2 spheroids was improved due to the bottom flow.

Example 2

In order to confirm that waste materials can be efficiently removed from the three-dimensional cell culture device of the present invention by the bottom flow, FITC-Dextran 20 kDa was added to the porous microwell of the three-dimensional cell culture device of the form shown in FIG. 1 (a). 200ug/ml was added, the bottom flow was performed for 3 hours, and the concentration of FITC Dextran remaining in the porous microwell was measured.

Comparative Example 2

FITC-Dextran was added in the same manner as in Example 2, except that the bottom flow was not applied in Example 2, and the concentration of FITC-Dextran remaining in the porous microwell was measured after 3 hours.

The result of visually examining the degree of waste accumulation in Example 2 is shown in FIG. 3, and the concentration of FITC-Dextran measured in Example 2 and Comparative Example 2 is shown in FIG. 2(b). As shown in Figure 2 (b), in the case of Example 2 with bottom flow, the concentration of FITC-Dextran remaining in the porous microwell was 55.6199 ± 3.3429 mg / ml, whereas in the case of Comparative Example 2, the concentration of FITC-Dextran remained in the porous microwell It was confirmed that the concentration of FITC-Dextran present was 131.435±7.80245mg/ml.

That is, when there is bottom flow, the concentration of FITC-dextran remaining in the porous microwell is significantly lower than that when there is no bottom flow. was found to be able to effectively remove As shown in FIG. 4 , it was confirmed that the waste materials on the upper surface of the porous microwells were removed by the fluid flow inducing device and the porous microwells in the lower chamber.

Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations are possible without departing from the technical spirit of the present invention described in the claims. It will be obvious to those skilled in the art.

Claims (1)

3D cell spheroid culture device.

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