Cell culture apparatus for culturing three-dimensional cell aggregates and cell culture method using the same

Abstract

This invention relates to a culture device and a culture method for culturing three-dimensional cell aggregates, and more specifically, to a general technology for efficiently culturing three-dimensional cell aggregates using a culture device that includes porous microwells and membranes, as well as other additional components.

Description

[Technical Field]

[0001] The present invention relates to a culture apparatus and culture method for culturing three-dimensional cell aggregates, and more specifically, to a general technique for efficiently culturing three-dimensional cell aggregates using a culture apparatus that includes porous microwells and membranes, as well as other additional components. [Background technology]

[0002] In reality, cells within the body have a three-dimensional shape, and these cells interact with their microenvironment in a three-dimensional manner. On the other hand, when culturing cells outside the body in a two-dimensional monolayer rather than a three-dimensional structure, as is done with conventional techniques, the morphological similarity to cells within the body is very poor. However, when culturing cells in three dimensions, phenomena that are more similar to those actually occurring in vivo can be observed, which is useful for drug screening and cell therapy.

[0003] Thus, in order to overcome the limitations of two-dimensional monolayer cell culture as described above, there is a growing global demand for microwell platforms that enable the culture and differentiation of three-dimensional cell aggregates (3D cell aggregations).

[0004] Conventional microwells either lacked a porous structure or used plastics, limiting the formation of three-dimensional cell aggregates. Even when three-dimensional cell aggregates were formed, the process of detaching them from the microwell platform was not easy. Furthermore, conventionally proposed culture environments relied solely on passive diffusion for the removal of waste products and the supply of nutrients generated during culture, thus limiting the growth and maturation of three-dimensional cell aggregates.

[0005] This invention was devised in view of the limitations and problems described above, and relates to a culture apparatus that can culture three-dimensional cell aggregates more efficiently than conventional methods, and a culture method using the same.

[0006] Furthermore, in addition to resolving the aforementioned technical problems of the present invention, this invention was made to provide further technical elements that cannot be easily invented by a person with ordinary skill in the art. [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention aims to provide an environment in which three-dimensional cell aggregates, or more precisely, three-dimensional cell spheroids, can be formed.

[0008] Furthermore, the present invention aims to induce the formation of three-dimensional cell aggregates at cell culture temperatures, maintaining the ease of cultivation characteristics, while simultaneously enabling the simultaneous detachment of a large number of three-dimensional cell aggregates from a microwell plate at room temperature.

[0009] Furthermore, the present invention aims to enable stable three-dimensional cell culture, efficient removal of waste products during cell culture, and smooth supply of nutrients to the lower end of porous microwells by allowing for fluid adjustment.

[0010] Furthermore, the present invention aims to minimize the loss of cells and three-dimensional cell aggregates caused by pipetting onto the cell culture surface, which is the upper surface of the microwell, for repeated replacement of cell culture medium, a method that has been widely used in the past, and to provide an environment that can continuously provide a uniform environment around cell aggregates. [Means for solving the problem]

[0011] To solve the aforementioned problems, the present invention provides a three-dimensional cell culture apparatus comprising: an upper chamber having an opening, a porous membrane, and a porous microwell for containing a cell culture medium; and a lower chamber having a space inside which the upper chamber is positioned, wherein fluid flowing into the upper part of the upper chamber flows from the upper chamber through the porous microwell into the lower chamber, and the fluid in the lower chamber is discharged, thereby providing a three-dimensional cell aggregate culture apparatus.

[0012] A method for culturing three-dimensional cell aggregates according to another embodiment of the present invention may include the steps of: seeding cells onto a porous membrane in an upper chamber using the three-dimensional cell aggregate culture apparatus; introducing cell culture medium into the upper chamber; and discharging the cell culture medium that has flowed from the upper chamber through porous microwells into the lower chamber from the lower chamber. [Effects of the Invention]

[0013] According to the present invention, an environment is provided in which three-dimensional cell condensates or cell spheroids can be efficiently formed, thereby enabling cells that have settled within a certain region to proliferate and differentiate more smoothly in three dimensions.

[0014] Furthermore, according to the present invention, it is possible to realize a microwell plate with a surface structure having a surface roughness that facilitates cell culture and harvesting through temperature changes, thereby enabling mass production and harvesting of three-dimensional cell aggregates.

[0015] Furthermore, according to the present invention, it becomes possible to realize a culture environment that does not adversely affect cell implantation, proliferation, and differentiation during cell culture, efficiently removes waste products formed during cell culture, and smoothly supplies nutrients to the lower part of the three-dimensional cells.

[0016] In addition, according to the present invention, during the process of culturing cells, not only can the possibility of disappearance of cells and cell aggregates be significantly reduced, but also a microenvironment around the persistent and uniform cell aggregates can be provided. Furthermore, by setting the height of the culture solution storage tank to correspond to the water level of the culture solution in the lower chamber, the height of the culture can be maintained constant during the culturing process.

[0017] Also, by using a porous microwell with extremely excellent hydraulic conductivity, the hydraulic pressure applied to the porous microwell and the three-dimensional cell aggregates being cultured inside can be minimized, and unnecessary stimuli other than the fluid flow can be minimized.

Brief Description of the Drawings

[0018] [Figure 1] FIG. 1 is a view showing a cell culture container according to an embodiment of the present invention. [Figure 2] FIG. 2 is a view showing a side cross-sectional view of a cell culture container according to an embodiment of the present invention. [Figure 3] FIG. 3 is a view showing a plate 30 according to an embodiment of the present invention. [Figure 4] FIG. 4 is an enlarged view showing the periphery of one opening 31 in FIG. 2. [Figure 5] FIG. 5 is a view showing the state of cell growth in a cell culture container according to another embodiment of the present invention. [Figure 6] FIG. 6 is a view showing the main body 10 and the fastening portion (40) of a cell culture container according to another embodiment of the present invention. [Figure 7] FIG. 7 is a view showing the procedure of a method for manufacturing a cell culture container according to another embodiment of the present invention. [Figure 8] FIG. 8 is a view showing an example for S10 or S100 of a method for manufacturing a cell culture container according to another embodiment of the present invention. [Figure 9] FIG. 9 is a view showing an example for S20 or S200 of a method for manufacturing a cell culture container according to another embodiment of the present invention. [Figure 10] Figure 10 is a photograph of a membrane 20 formed by a cell culture vessel manufacturing method according to one embodiment of the present invention. [Figure 11] Figure 11A shows a top view of the upper chamber of a cell culture vessel manufactured according to another embodiment of the present invention. Figure 11B is a magnified view of one of the many porous microwells in Figure 11A. [Figure 12] Figure 12 shows the change in surface roughness of the cell culture layer with respect to temperature and time. [Figure 13] Figure 13A is a magnified view of a porous microwell, and Figure 13B shows a cell culture layer containing polyisopyrucrylamide formed on the upper surface of the porous microwell. [Figure 14] Figure 14A shows an image taken during the culture process of three-dimensional cell aggregates. Figure 14B shows cell aggregates detached from the well plate. Figure 14C shows the well plate after the cell aggregates have been detached. [Figure 15] Figure 15a is an illustrative diagram of a three-dimensional cell culture apparatus according to one embodiment of the present invention, Figure 15b is an illustrative diagram of a case in which through-holes are formed in the upper chamber of the three-dimensional cell culture apparatus of the present invention in adjacent portions of porous microwells, and Figure 15c is a diagram showing an actual image of the three-dimensional cell culture apparatus. [Figure 16] Figure 16a compares the albumin expression levels measured in Example 2 and Comparative Example 2 of the present invention, and Figure 16b compares the concentrations of FITC-Dextran measured in Example 3 and Comparative Example 4 of the present invention. [Figure 17] Figure 17 shows the results of observing with the naked eye how waste products are removed over time according to Example 3 of the present invention. [Figure 18a] Figure 18a is a schematic diagram showing the fluid flow in the lower chamber of the present invention and how waste products on the upper surface of the porous microwells are removed by the porous microwells. [Figure 18b]Figure 18b shows an overview of the diffusion pathway of the culture medium into the porous microwells. [Figure 19] Figure 19 shows a cell culture apparatus according to yet another embodiment of the present invention, where Figure 19a shows the overall configuration of the cell culture apparatus, Figure 19b shows a magnified view of a combination of an upper chamber and a lower chamber, and Figure 19c shows a cross-sectional view of the cell culture apparatus including a culture medium reservoir connected to an outlet. [Figure 20] Figure 20 shows the results of a simulation of the flow of culture medium permeated through a porous membrane in the cell culture apparatus according to the present invention. [Figure 21] Figure 21 shows micrographs of porous microwells composed of a porous membrane with recesses. Figure 21a is an image magnified 4x, and Figure 21b is an image magnified 220x. Figure 21c shows another form of porous microwell with a porous membrane having recesses, where Figure 21ca) is a DSLR camera image, and Figures 21cb) and 21ccc) are micrographs magnified 4x and 20x, respectively. [Figure 22] Figure 22 shows the changes in the structure and properties of the nanofiber network of a porous membrane with respect to electrical emission time. A typical commercially available porous membrane, the Transwellinsert (Corning Corporation, USA) with an 8 μm void size, is attached for reference (far left, TW). Figure 22a is a microscope image magnified 20 times of each nanofiber network structure with respect to the commercial membrane and electrical emission time. Figures 22b, 22c, and 22d show the porosity (Figure 22b), hydraulic conductivity (Figure 22c), and induced pressure (Figure 22d) of each nanofiber network structure with respect to the commercial membrane and electrical emission time. [Figure 23]Figure 23 shows experimental results comparing the degree of cell aggregate disappearance when using the cell culture apparatus according to the present invention with the degree of cell aggregate disappearance when culturing cells using the conventional method of replacing the cell culture medium by pipetting. [Figure 24] Figure 24 shows the results of a numerical simulation comparing the nutrient concentration (glucose concentration) around cell aggregates when using the cell culture device according to the present invention with the nutrient concentration around cell aggregates when culturing cells using the conventional method of replacing the cell culture medium by pipetting. [Figure 25] Figure 25 shows how the fluid present in the cell culture device is smoothly replaced with newly injected fluid, and how the water level of the culture medium in the lower chamber is maintained at a constant level during this process. [Figure 26] Figure 26 shows a method for manufacturing porous microwells. [Figure 27] Figure 27 shows a lower chamber (a) used in the cell culture apparatus according to the present invention, and an upper chamber (b) inserted inside the lower chamber. [Figure 28] Figure 28 shows a method for manufacturing porous microwells using a compression process. [Figure 29] Figure 29 is a diagram illustrating the process of forming a fluid flow by discharging fluid through the gap region between the upper and lower chambers, where (a) is a photograph, (b) is a schematic image, and (c) is a diagram showing the fluid velocity formed in this case. [Modes for carrying out the invention]

[0019] First, with reference to Figures 1 to 10, we will describe the cell culture vessel, which is the basic component of the cell culture apparatus according to the present invention, and its manufacturing method.

[0020] Figure 1 shows a cell culture vessel according to one embodiment of the present invention, and Figure 2 shows a one-sided cross-sectional view. Figure 2 is a one-sided cross-sectional view along the line A-A' in Figure 1. Figure 3 shows a plate 30 according to one embodiment of the present invention.

[0021] As shown in Figures 1 and 2, the cell culture vessel may include a main body 10 and a membrane 20, and may further include a plate 30. For the sake of clarity, the plate 30 will be described first, followed by the main body 10 and membrane 20 in subsequent descriptions.

[0022] The plate 30 has one or more openings 31 which are open in shape, and the main body 10 can be inserted into these openings 31. The openings 31 may be open in shape with the bottom closed and the top open, as shown in Figure 3a, or they may be through-shaped openings that penetrate the plate 30, as shown in Figure 3b. If the openings 31 are through-shaped, the cell culture container may have a housing space with the top open, and the housing space may further include a cover container on which the plate 30 is mounted.

[0023] If the plate 30 is provided with multiple openings 31, these openings 31 can be arranged spaced apart from each other. This allows for the isolation of the influence between samples in each opening 31 during cell culture, and as a result, multiple independent experimental data can be obtained using a single plate 30.

[0024] As shown in Figures 1 and 2, the main body 10 includes a spacer SP, an inlet 11 formed at the other end, and a through-hole that penetrates both the spacer and the inlet 11. Here, the spacer refers to the part of the main body 10 that is on the side or side wall, and is indicated by the symbol SP in the drawings. As can be seen from the drawings, the outer surface of the spacer faces the inner surface of the opening 31, and it can be confirmed that a predetermined gap or space exists between the spacer and the inner surface of the opening.

[0025] The spacer SP of the main body 10 can be inserted into the opening 31 of the plate 30. In this case, the main body 10 may include one insertion part or may include multiple insertion parts. That is, if it includes one insertion part, the main body 10 can have that insertion part inserted into one opening 31. If it includes multiple insertion parts, the main body 10 can have each insertion part inserted into a number of openings 31. In this case, if it includes multiple insertion parts, the main body 10 has a structure in which each insertion part is connected to each other, and each insertion part can be inserted into a number of openings 31.

[0026] Figures 1 to 6 illustrate a main body 10 that includes one insertion portion, but the present invention is not limited to this, and the contents of the present invention can of course be applied when the main body 10 includes multiple spacers and through portions.

[0027] When the spacer of the main body 10 is inserted into the opening 31, the spacer of the main body 10 is located at the bottom, and the inlet portion 11 of the main body 10 is located at the top. The spacer of the main body 10 may be a vertical type with a uniform width from one end to the other, a funnel type with a gradually widening width from one end to the other, or a combination of the vertical and funnel types. The through portion of the main body 10 can be formed in various shapes, such as a circular or polygonal cross-section, and its size can also be varied.

[0028] Figure 4 is a magnified view of the area around one opening 31. In particular, when the spacer of the main body 10 is attached to the opening 31 of the plate 30, it is preferable that the inlet portion 11 of the main body 10 has a wider cross-section than the spacer of the main body 10 and the inlet of the opening 31, as shown in Figure 4, so that one end of the spacer of the main body 10 is positioned a certain distance away from the bottom surface of the cell culture vessel. This allows a fluid passage containing nutrients for supplying cells to be formed in the space between the spacer of the main body 10 and the bottom surface of the cell culture vessel. At this time, the bottom surface of the cell culture vessel can be the bottom surface of the opening in the case of an open-type plate, or the bottom surface of the cover container in the case of a through-type plate.

[0029] The membrane 20 is a layer that provides a cell culture surface on which cells are cultured, and is used in the through-hole on the spacer side of the main body 10. For example, the membrane 20 can be formed by an electrochemical method to cover one end of the spacer of the main body 10. In this case, the membrane 20 can be made up of multiple polymer nanofibers randomly intertwined or made from a molded polymer synthetic resin. For example, each polymer nanofiber can have a diameter of 1 nm or more and less than 1000 nm. By being made up of multiple polymer nanofibers, the membrane 20 can provide a blood flow environment in the body by having a structure similar to the basement membrane in the body.

[0030] For example, polymer nanofibers or polymer synthetic resins may contain at least one of the following: thermoplastic resins, thermosetting resin elastic polymers, and biopolymers. For example, polymer nanofibers or polymer synthetic resins may contain at least one of the following: polycaprolactone, polyurethane, polyvinylidene fluoride (PVDF), polystyrene, collagen, gelatin, and chitosan.

[0031] The membrane 20 may include porous microwells 21, connecting portions 22, and fixed portions 23. In this case, the microwells 21, connecting portions 22, and fixed portions 23 have a structure in which multiple polymer nanofibers are intertwined and connected to each other.

[0032] The microwells 21 are regions that function as cell culture surfaces and are formed as downward-facing depressions. This depression shape makes it easier for cells to settle into the microwells 21, allowing them to grow stably within the microwells 21 regardless of fluid movement. At this time, at least one of the microwells 21 is located within the region formed by the penetration of the main body 10. That is, when viewed from above or below, the microwells 21 are smaller in size than the penetration of the main body 10 and are included within the region formed by the penetration.

[0033] By forming the microwells 21, which are the cell culture surfaces, in a concave shape, the membrane 20 can concentrate cells more firmly on the cell culture surface and stably attach them, thereby increasing the surface area of ​​the cell culture surface and improving cell adhesion efficiency. Furthermore, unlike conventional cell culture vessels that usually have a flat cell culture surface, the body 10 of the cell culture vessel according to the present invention has a three-dimensional cell culture surface, which allows cells to be cultured in a three-dimensional structural environment similar to that in vivo, and enables the formation of three-dimensional cell spheroids. When multiple microwells 21 are located within the area formed by the penetration portion of the body 10, the spacers of the body 10 have multiple cell culture surfaces, which can further improve cell adhesion efficiency.

[0034] The connecting portion 22 is a region formed around any microwell 21 and connecting the microwells 21, and can have a flat shape. The connecting portion 22 may also be thicker than the microwell 21. This is because, due to the embossing process described later, the microwell 21 corresponds to a region that extends into a recessed shape at the bottom within the membrane 20 and becomes thinner than before, while the connecting portion 22 corresponds to a region that does not stretch and maintains its original thickness.

[0035] The fixed portion 23 is a region fixed to the edge of one end of the spacer of the main body 10. Furthermore, the fixed portion 23 is thinner and less dense than the connecting portion 22. This is because the membrane 20 can be formed by an electrochemical emission method using an electrolyte solution. In other words, the microwells 21 and the connecting portion 22 are regions that are generated at the location where the electrolyte solution is present during electrochemical emission, so more polymer nanofibers are formed there than in the fixed portion 23, and therefore they can be formed with a higher density and greater thickness compared to the fixed portion 23.

[0036] Figure 5 shows how cells proliferate in the cell culture vessel according to the present invention. Figures 5a, 5b, and 5c sequentially show how cells proliferate over time under fluid concentration conditions.

[0037] On the other hand, the membrane 20 contains numerous voids (holes) formed in the regions between polymer nanofibers. In this case, the voids can have dimensions ranging from a few micrometers to tens of micrometers. The membrane 20 can act as a selective permeable membrane, allowing other substances to pass through while preventing single cells from passing through, thereby fulfilling the role of a mass transport barrier and passage.

[0038] The first porosity formed by the first voids in the microwells 21 and the second porosity formed by the second voids in the connecting portion 22 can be different. In this case, the porosity is the ratio of the void area present in a unit area. In particular, the first porosity is larger than the second porosity. This is because, as the region corresponding to the microwells 21 in the membrane 20 extends into a downward concave shape by the embossing process, a phenomenon occurs in which many parts that were filled with entangled polymer nanofibers in that region become open, or the area of ​​already open parts expands. However, although these two types of porosity are described in order to explain the difference between the first and second porosity, the present invention is not limited to having only these two types of porosity. That is, the present invention may have a variety of porosity other than the first and second porosity.

[0039] As shown in Figure 5, this difference in porosity causes a fluid concentration phenomenon in the peripheral region of the microwell 21, which in turn allows for more active cell proliferation in the microwell 21. This is because, according to Darcy's equation, regions with higher porosity have higher permeability.

[0040] For cell culture, the openings 31 of the plate 30 are filled with a fluid (for example, a mixture of cell culture medium, distilled water, PBS solution, etc.), but this fluid must be replaced periodically using a spuit or the like. At this time, the fluid replacement can be carried out, for example, by suctioning the fluid from the outside of the main body 10 while simultaneously supplying new fluid to the inside of the main body 10. This is because if the fluid is suctioned from the inside of the main body 10, there is a risk that it may adversely affect cells that are settling in the microwells 21 and growing and differentiating, or that the cells themselves may be discharged.

[0041] In the fluid exchange process described above, the fluid filling the containment space that houses the microwell 21 passes from above to below the microwell 21 and the connecting portion 22. At this time, since the microwell 21 has a larger porosity than the connecting portion 22, it allows more fluid to pass through than the connecting portion 22, as shown in Figure 5. As a result, a phenomenon occurs around the microwell 21 in which the fluid moves more concentratedly than around the connecting portion 22, i.e., a fluid concentration phenomenon.

[0042] When such fluid concentration occurs, oxygen and nutrients contained in the fluid can be supplied more smoothly to cells that are proliferating and differentiating within the microwells 21, thereby further promoting cell proliferation and differentiation. Furthermore, this fluid concentration phenomenon also causes cells to accumulate inside the microwells 21. In other words, by having multiple regions with different porosities, and by forming the microwells 21, which are the cell culture surfaces within these regions, to have a higher porosity than the connecting portion 22, the membrane 20 can increase the efficiency of cell proliferation and differentiation in the microwells 21.

[0043] If the plate 30 is provided with multiple openings 31, the cell culture vessel according to the present invention can derive multiple independent experimental data obtained under a three-dimensional structural environment similar to that in vivo, using a single plate 30.

[0044] Figure 6 shows the main body 10 and fastening part 40 of a cell culture vessel according to another embodiment of the present invention.

[0045] On the other hand, a cell culture vessel according to another embodiment of the present invention may further include a fastening portion 40, which has one end and the other end that penetrates and fastens to one end of the spacer of the main body 10, as shown in Figure 6.

[0046] In this case, the fastening portion 40 may include one through portion or may include multiple through portions. That is, when it includes one through portion, the fastening portion 40 is fastened to each spacer of the main body 10 and formed into a ring shape. When it includes multiple through portions, the fastening portion 40 is fastened so that each through portion corresponds to each spacer of the main body 10. Figure 6 shows the case where the fastening portion 40 includes one through portion, but the present invention is not limited to this, and the contents of the present invention are of course applicable when the fastening portion 40 includes multiple through portions.

[0047] If a fastening portion 40 is also included, either the membrane 20 is not provided at one end of the spacer of the main body 10, but at one end of the fastening portion 40, or the membrane 20 is provided at both one end of the spacer of the main body 10 and one end of the fastening portion 40. When the fastening portion 40 is fastened to the spacer of the main body 10, the through portion of the fastening portion 40 and the through portion of the main body 10 are connected to each other.

[0048] The fastening portion 40 can be provided on one end of the spacer of the main body 10 in a form that can be attached and detached. For example, threads may be formed inside or outside the fastening portion 40, and threads corresponding to the threads of the fastening portion 40 may be formed on the outside or inside of one end of the main body. Alternatively, the fastening portion 40 may be fitted to the inner circumferential surface of the through portion at one end of the spacer of the main body 10, as shown in Figure 6b, or to the outer circumference of one end of the spacer of the main body 10, as shown in Figure 6c.

[0049] The membrane 20 provided at one end of the fastening portion 40 is the same as the membrane 20 provided at one end of the spacer of the main body 10, except that the spacer of the main body 10 is replaced by the fastening portion 40. In other words, the membrane 20 provided at one end of the fastening portion 40 is only different in that its position has changed from one end of the spacer of the main body 10 to one end of the fastening portion 40. Therefore, a detailed explanation of the membrane 20 provided at one end of the fastening portion 40 will be omitted below and replaced with the explanation of the membrane 20 provided at one end of the spacer of the main body 10 described above.

[0050] The following describes a method for manufacturing a cell culture vessel according to one embodiment of the present invention. This method for manufacturing a cell culture vessel includes a method for manufacturing a microwell 21 or a membrane 20.

[0051] Figure 7 shows the procedure for manufacturing a cell culture vessel according to the present invention.

[0052] A method for manufacturing a cell culture vessel according to one embodiment of the present invention includes preparation steps S10 and S100 and formation steps S20 and S200, as shown in Figure 7. At this time, S10 and S20 are the methods for manufacturing the main body 10 and membrane 20 described above, and S100 and S200 are the methods for manufacturing the main body 10, membrane 20 and fastening part 40 described above.

[0053] S10 is the step of preparing the main body 10 and the membrane 20. S100 is the step of preparing the main body 10, the membrane 20 and the fastening part 40. At this time, the main body 10, the membrane 20 and the fastening part 40 are the same as those described above based on Figures 1 to 6, so their explanation will be omitted below. However, the membrane 20 can be formed by an electrochemical emission method using an electrolyte solution, and the electrochemical emission method using an electrolyte solution will be explained next.

[0054] Figure 8 shows an example of a method for producing a cell culture vessel according to another embodiment of the present invention, corresponding to S10 or S100.

[0055] The electro-radiation method using an electrolyte solution involves forming a membrane 20 so as to cover one end of the spacer or fastening portion 40 of the main body 10, and can be performed inside a chamber. The chamber is a space in which the work is performed and can prevent leakage of the polymer solution to the outside when the membrane 20 is formed. Hereafter, the electro-radiation method using an electrolyte solution to form a membrane 20 on one end of the spacer of the main body 10 will be called the "first electro-radiation method," and the electro-radiation method using an electrolyte solution to form a membrane 20 on one end of the fastening portion 40 will be called the "second electro-radiation method." First, the first electro-radiation method will be explained.

[0056] The first electrical discharge method may sequentially include an electrolyte filling step, a voltage application step, and a membrane formation step.

[0057] The electrolyte filling stage is the stage in which the electrolyte solution 50 is filled into the spacer of the main body 10, which is formed so that one end and the other end are through each other, as shown in Figure 8a. At this time, one end of the spacer of the main body 10 is positioned upward and the other end is blocked. Thereafter, the electrolyte solution 50 is filled into one end of the spacer of the main body 10. At this time, a lid is provided to close the inlet 11 of the main body 10, and the lid may be equipped with electrodes for applying a voltage to the electrolyte solution 50. That is, the electrodes are formed to penetrate the lid and can be connected to the electrolyte solution 50 filling the spacer of the main body 10.

[0058] Alternatively, during the electrolyte filling stage, as shown in Figure 8b, the spacer of the main body 10 may be placed in the electrolyte container 60 filled with the electrolyte solution 50, with one end of the spacer of the main body 10 facing upward, thereby filling the through-hole of the main body 10 with the electrolyte solution 50. When the spacer of the main body 10 is placed in the containment space of the electrolyte container 60, pressure is generated as the spacer of the main body 10 touches the surface of the electrolyte solution 50 and presses against the surface of the electrolyte solution 50, and this pressure causes the electrolyte solution 50 to fill the spacer of the main body 10. At this time, in order to more easily generate pressure on the surface of the electrolyte solution 50, the containment space of the electrolyte container 60 may be formed to match the shape of the spacer of the main body 10.

[0059] Since the electrolyte solution 50 is conductive, when a voltage is applied during the voltage application stage, it becomes negatively charged, attracting positively charged particles with electrical force. As a result, the positively charged particles can accumulate at the top of the electrolyte. The electrolyte solution 50 can be divided into strong electrolytes and weak electrolytes depending on the degree of dissociation. The degree of dissociation varies depending on the solvent.

[0060] For example, as the electrolyte solution 50, a solution of potassium chloride and distilled water mixed in a 3% mol ratio can be used. Furthermore, any substance and concentration that dissolves in water or an organic solvent (ethanol, methanol) and exhibits an electrical conductivity higher than 1 mS / cm can be used as the electrolyte solution 50. Additionally, any substance and concentration that dissolves in water and has a relative dielectric constant higher than 80 F / m can be used as the electrolyte solution 50.

[0061] The voltage application stage, as shown in Figure 8c, is the stage in which a voltage is applied between the electrolyte solution 50 and the metal needle 71 of the electric radiator 70. At this time, the voltage is supplied via a power supply, but changes in the strength of the applied voltage may cause changes in the structure of the membrane 20 formed in the membrane formation stage.

[0062] An electric field is formed between the electrolyte solution 50 and the metal needle 71 of the electric radiator 70. If the strength of the electric field formed is too low, the polymer aqueous solution will not be continuously discharged, making it difficult to produce polymer nanofibers of uniform thickness. Furthermore, the produced polymer nanofibers may not be smoothly focused onto the electrolyte solution 50. Conversely, if the strength of the electric field is too high, the polymer fibers may not adhere accurately to the upper surface of the electrolyte solution 50, making it difficult for them to maintain a normal shape. In light of these considerations, the voltage applied to the electrolyte solution 50 and the metal needle 71 of the electric radiator 70 can be between 5kV and 30kV.

[0063] A negative voltage can be applied to the electrolyte solution 50, and a positive voltage can be applied to the metal needle 71. As a result, the electrolyte solution 50 becomes negatively charged, and the polymer solution emitted during the membrane formation stage becomes positively charged.

[0064] The membrane formation step, as shown in Figure 8c, involves applying a voltage and radiating a polymer solution onto the spacer of the main body 10 via the electric radiator 70 to form the membrane 20. At this time, the membrane 20 is formed in a network-like structure in which multiple polymer nanofibers are randomly intertwined due to the high degree of freedom of the electrolyte solution 50.

[0065] On the other hand, the electric radiator 70 is a device that supplies the polymer solution. That is, the electric radiator 70 stores the polymer solution to a viscosity suitable for electric discharge, and then discharges the polymer solution through the metal needle 71. At this time, the discharged polymer solution can harden simultaneously with scattering to form polymer nanofibers.

[0066] The metal needle 71 is configured to dispense a polymer solution. Being made of metal, the metal needle 71 can be easily connected to a power supply, and the charge charging efficiency of the polymer solution dispensed when voltage is applied from the power supply can be improved. In particular, the metal needle 71 is located at the top of the main body 10, spaced apart from the spacer, and can discharge the polymer solution with its discharge end facing the spacer of the main body 10.

[0067] For example, the electric discharger 70 can consist of a syringe, a syringe pump, and a metal needle 71. That is, the polymer solution is placed in the syringe and the polymer solution can be discharged into the air by the metal needle 71 using the power of the syringe pump. In this case, a 23 gauge needle can be used for the metal needle 71, but its size may vary depending on the polymer solution. In particular, the polymer solution can be discharged at a discharge rate of 0.01 ml / h to 3 ml / h so as to maintain the surface shape of the electrolyte solution 50 while placing polymer fibers on the surface of the electrolyte solution 50.

[0068] When a polymer solution is discharged within the aforementioned voltage application range (5kV to 30kV) and discharge speed range (0.01ml / h to 3ml / h), polymer nanofibers with a diameter of 10nm to 900nm can be formed.

[0069] As a polymer solution, a 5% to 25% solution can be used, which is prepared by mixing chloroform and methanol in a 1:1 mass ratio and then adding polycarprolactone. Alternatively, a 25% to 30% solution can be used, which is prepared by mixing acetone and dimethylformamide in a 3:7 volume ratio and then adding polyvinylidene fluoride (PVDF). In addition, polymer solutions can be produced using polystyrene, polycarbonate, a collagen / polycarbonate blending solution, gelatin, and other materials.

[0070] During the membrane formation stage, the electrical attractive force generated between the perforation on the spacer side of the main body 10, which is filled with the electrolyte solution 50, and the polymer solution is constant and can be greater than the electrical attractive force generated between the edge of the spacer of the main body 10 and the polymer solution. As a result, the region of the membrane 20 that accumulates in the perforation on the spacer side of the main body 10, which is filled with the electrolyte solution 50 (hereinafter referred to as the "perforation region") is constant and has a relatively high density and thickness.

[0071] In contrast, the magnitude of the electrical attraction between the edge of the spacer of the main body 10 and the polymer solution is smaller than the magnitude of the electrical attraction between the penetration portion of the spacer of the main body 10 and the polymer solution, and it gradually decreases as you move away from the penetration portion of the spacer of the main body 10. As a result, the fixed portion 23, which is the membrane 20 accumulated at the edge of the spacer of the main body 10, has a relatively small density and thickness, although it is not constant.

[0072] The membrane formation step may further include adjusting the radiation time of the electric emitter 70 to adjust one or more of the thickness, porosity, and transparency of the membrane 20 formed at one end of the spacer of the main body 10. That is, the longer the radiation time of the electric emitter 70, the greater the amount of polymer nanofibers that are accumulated. As a result, the membrane 20 formed at one end of the spacer of the main body 10 becomes thicker, and its porosity and transparency become smaller.

[0073] Furthermore, the membrane formation step may further include a step of adjusting the diameter of the polymer nanofibers of the membrane 20 formed by adjusting the concentration of the polymer solution. That is, as the concentration of the polymer solution increases, its viscosity increases, so the diameter of the polymer nanofibers of the membrane 20 formed at one end of the spacer of the main body 10 increases.

[0074] Next, we will explain the second method of electrical emission.

[0075] The second electrical discharge method, like the first electrical discharge method, includes an electrolyte filling step, a voltage application step, and a membrane formation step, and may further include a fastening step. In this case, the electrolyte filling step, the voltage application step, and the membrane formation step are the same as in the first electrical discharge method described above, except that the spacer of the main body 10 is replaced with the fastening part 40. Therefore, a detailed explanation of the electrolyte filling step, voltage application step, and membrane formation step of the second electrical discharge method is omitted below and replaced by the explanation of the electrolyte filling step, voltage application step, and membrane formation step of the first electrical discharge method described above.

[0076] In the second electrolysis method, a membrane 20 can be formed on one end of the fastening portion 40 through the electrolyte filling step, the voltage application step, and the membrane formation step. Subsequently, the fastening portion fastening step is the step of fastening the fastening portion 40, on which the membrane 20 is formed, to one end of a spacer of the main body 10 that is formed to penetrate from one end to the other. For example, the fastening portion fastening step can be performed by a transfer device that transfers the spacer of the main body 10 or the fastening portion 40 and fastens the spacer of the main body 10 to the fastening portion 40.

[0077] However, the first and second electrical emission methods may further include the step of cutting the membrane 20 after it has been formed to match the shape of the spacer or fastening portion 40 of the main body 10.

[0078] Figure 9 shows an example of a method for producing a cell culture vessel according to another embodiment of the present invention, corresponding to S20 or S200.

[0079] S20 is the step in which an embossing process is performed on the membrane 20 formed on the spacer of the main body 10. S200 is the step in which an embossing process is performed on the membrane 20 formed on the fastening portion 40.

[0080] In other words, in S20 or S200, as shown in Figure 9, an embossing process is performed on the membrane 20 prepared in S10 or S100 using a mold M on which the pattern of microwells 21 has been formed. At this time, the embossing process is a process in which the membrane 20 is pressed with the mold M and a pattern corresponding to the pattern of the mold M is formed on the membrane 20. That is, as a result of the embossing process, microwells 21 and connecting portions 22 can be formed on the membrane 20.

[0081] The embossing process may be a hot embossing process in which the membrane 20 is pressurized with a heated mold M, but is not limited to this. When using a hot embossing process, the result of S20 can be obtained more quickly.

[0082] The mold M may include a first mold M1 whose lower part protrudes due to the pattern of microwells 21, and a second mold M2 whose upper part is recessed due to the pattern of microwells 21. That is, a membrane 20 can be placed between the first mold M1 and the second mold M2, and the first mold M1 and the second mold M2 can be pressed together and then separated to form microwells 21 and a connecting portion 22 on the membrane 20. When joined, the protruding portion of the first mold M1 contacts one surface of the membrane 20, and the recessed portion of the second mold M2 contacts the other surface of the membrane 20.

[0083] In the hot embossing process, either the first mold M1 or the second mold M2 may be heated before bonding, or both the first mold M1 and the second mold M2 may be heated. However, since the protruding shape of the first mold M1 has a greater influence on the formation of the microwells 21, it is preferable to heat and use only the first mold M1. Furthermore, it is preferable that the temperature of the heated first mold M1 or second mold M2 is lower than the melting point of the polymer nanofibers forming the membrane 20.

[0084] Figure 10 shows a photograph of a membrane 20 formed by a cell culture vessel manufacturing method according to one embodiment of the present invention. In this case, Figure 10c shows a plan view of the membrane 20, and Figure 10d shows a plan view of the microwells 21 and connecting portion 22, which is an enlarged view of Figure 10c. Figure 10a shows an enlarged photograph of the microwells 21, and Figure 10b shows the first void formed in the microwells 21 of Figure 10a. Figure 10e shows an enlarged photograph of the connecting portion 22, and Figure 10f shows the second void formed in the connecting portion 22 of Figure 10e.

[0085] As shown in Figures 10a and 10e, the membrane 20 formed by the cell culture vessel manufacturing method according to one embodiment of the present invention can be confirmed to be formed in a network-like structure in which multiple polymer nanofibers are intertwined. Furthermore, referring to Figures 10b and 10f, it can be confirmed that in the membrane 20 formed by the cell culture vessel manufacturing method according to one embodiment of the present invention, the number of first voids formed in the microwells 21 is greater than the number of second voids formed in the connecting portion 22. At this time, it can be confirmed that the porosity of the first voids is increased by approximately 10 times or more compared to the porosity of the second voids.

[0086] A cell culture vessel and its manufacturing method have been described with reference to Figures 1 to 10. In the examples with reference to Figures 1 to 10, the conditions under which the porosity of a part of the membrane, such as microwells or connecting parts, must be met were mentioned. However, the cell culture vessel according to the present invention only needs to have voids large enough for fluids or substances to pass through, and the porosity can be any value without any constraints. In other words, in all the examples mentioned in this detailed description, the presence of voids is naturally acknowledged, but it should be understood that differences in porosity are not an essential limitation for realizing the cell culture vessel.

[0087] Other embodiments of the microwells and plates will be described below with reference to Figures 11 to 14.

[0088] First, in the explanation of Figure 1, the porous microwells 21 and plate 30 were described, but in another embodiment, a coating composition having a surface structure that changes with temperature can be coated onto the porous microwells 21 to realize a cell culture vessel containing a cell culture layer whose surface structure changes with temperature.

[0089] A cell culture layer having a surface structure that changes with temperature can be formed on the upper surface of the porous microwell 21, having a surface structure that is suitable for cell culture with high adhesion to cells at temperatures above 32°C and below 40°C, and a surface structure that is suitable for cell detachment at temperatures above 0°C and below 32°C, making it suitable for cell retrieval.

[0090] The cell culture layer may contain polyisopropylacrylamide (Poly(N-isopropylacrylamide), PNIPAAm) and may have a temperature-dependent surface roughness change characteristic such that the surface roughness is 4 to 37 nm, preferably 20 to 32 nm, at temperatures above 32°C and below 40°C, and 4 μm or more at temperatures above 0°C and below 32°C. In this case, the surface roughness refers to the measurement taken using a non-contact atomic force microscope (Park Systems, South Korea) that utilizes a PPP-NCHR cantilever beam with a frequency of 300 kHz or a BL-AC40TS cantilever beam with a frequency of 25 kHz.

[0091] The cell culture layer may contain 1 to 5% by weight of a crosslinking agent based on the total weight of the cell culture layer, preferably more than 1% by weight but less than 3% by weight. If the crosslinking agent content is less than 1% by weight, a problem may arise in which cells do not adhere well to the cell culture layer, and if it exceeds 5% by weight, a problem may arise in which cell spheroids do not adhere well at temperatures below the LCST (lower critical solution temperature).

[0092] Furthermore, while the porous microwells 21 are permeable to fluids, the areas excluding them are not permeable to fluids. In this case, the pores of the porous microwells 21 can have a pore size with an average diameter of 100 nm to 20 μm, and preferably a pore size of 100 nm to 5 μm. With such a pore size, the porous microwells can act as a selective permeable membrane that selectively allows other substances to pass through while preventing single cells from passing through, thereby fulfilling the role of a mass transport barrier and passage.

[0093] Furthermore, as described above, due to the difference in permeability between the porous microwell 21 and its surrounding area, when fluid flows through the porous microwell 21 and the cell culture layer, a fluid concentration phenomenon can occur in the porous microwell. When such a fluid concentration phenomenon occurs, oxygen and nutrients contained in the fluid can be smoothly supplied to cells that are proliferating and differentiating in the porous microwell, thereby further promoting cell proliferation and differentiation. In addition, this fluid concentration phenomenon can cause cells to accumulate in the porous microwell, facilitating the formation of cell aggregates or spheroids.

[0094] Furthermore, the cell culture vessel of the present invention may include an upper chamber 100 and a lower chamber 200, as shown in Figure 1. The lower chamber 200 may be a chamber in which the upper chamber 100 is arranged or fitted and through which cell culture medium or the like flows. Moreover, the present invention can use a lower chamber in which multiple upper chambers are arranged so that many cells can be cultured simultaneously, and the number of upper chambers can be used without limitation as long as it is a number that is common in the industry.

[0095] The following describes a method for manufacturing a three-dimensional cell culture vessel. Specifically, the method comprises an N-isopropylacrylamide monomer, a crosslinking agent, and the remainder being water, wherein the crosslinking agent comprises a step of providing a coating aqueous solution in an amount of 1 to less than 5 parts by weight based on 100 parts by weight of a mixture of the N-isopropylacrylamide monomer and water; a step of forming a coating layer on the upper surface of the porous microwells of a well plate chamber containing porous microwells at the bottom using the coating aqueous solution; and a step of irradiating the coating layer with UV light to form a cell culture layer.

[0096] In this embodiment, the rate of cell desorption due to temperature changes in the cell culture support can be adjusted by adjusting the content of the crosslinking agent. As mentioned above, the crosslinking agent can be 1 part by weight or more and less than 5 parts by weight, preferably more than 1 part by weight and less than 3 parts by weight, based on 100 parts by weight of the mixture of N-isopropylacrylamide monomer and water. If the content of the crosslinking agent is less than 1 part by weight based on 100 parts by weight of the mixture of N-isopropylacrylamide monomer and water, the problem of cells not easily adhering to the cell culture support may occur. If it is 5 parts by weight or more, the problem of cells not easily desorbing at temperatures below LCST may occur.

[0097] On the other hand, the crosslinking agent plays a role in polymerizing the N-isopropylacrylamide monomer with polyisopyrucrylamide, and any crosslinking agent that can be used in the usual methods used to produce polyisopyrucrylamide, namely homopolymerization, copolymerization and terpolymerization, and cross-linked polymerization, can be used without limitation. Preferably, N,N'-methylenebisacrylamide (MBAAm) and tetramethylethylenediamine (TEMED) or a mixture thereof can be used as the crosslinking agent.

[0098] The coating aqueous solution may further contain a photoinitiator so that monomers can crosslink in the coating aqueous solution by UV light. In this case, the amount of the photoinitiator may be 0.01 to 0.1 parts by weight, preferably 0.01 to 0.05 parts by weight, based on 100 parts by weight of the mixture of N-isopropylacrylamide monomer and water. If the amount of the photoinitiator is less than 0.01 parts by weight, the problem of UV-induced crosslinking not occurring may occur, and if it exceeds 0.05 parts by weight, the problem of cells dying due to toxicity during subsequent cell culture may occur due to the toxicity of the crosslinking agent itself.

[0099] On the other hand, the photoinitiator can be used without limitation as long as it can initiate crosslinking through UV light, for example, 2-hydroxy-1-1[4-(hydroxyethoxy]phenyl]-2-methyl-1-propanone can be used.

[0100] The step of forming the coating layer is not particularly limited, as long as the coating layer formed by the coating aqueous solution is such that fluid can permeate into the porous microwells. For example, the step of forming the coating layer can be performed using a spin coating method or a bar coating method. Furthermore, since the cell culture layer formed by the coating layer is formed in a hydrogel-like form, the movement of the solution is smooth, and fluid can flow through the porous microwells and the hydrogel.

[0101] The step of irradiating the coating layer with UV light to form a cell culture layer is a step of polymerization of monomers and is not particularly limited as long as the UV light is irradiated to the extent that a polymer of N-isopropylacrylamide (polyisopropylacrylamide) can be formed. For example, it can be carried out by irradiating with UV light at 1800 w for 10 minutes.

[0102] On the other hand, for the attachment and detachment of the cultured cells, the method for culturing three-dimensional cell aggregates may include the step of attaching the three-dimensional cell aggregates to a cell culture layer and culturing them at a temperature of over 32°C to 40°C, and then detaching the three-dimensional cell aggregates from the cell culture layer at a temperature of 0°C to less than 32°C. The cells may be, but are not limited to, myoblasts, embryonic fibroblasts, human umbilical vein endothelial cells, or human epidermal cells.

[0103] <Example 1>

[0104] In Example 1, an upper chamber equipped with porous microwells at the bottom was fabricated. Specifically, porous microwells were fabricated by perforating holes of a certain size into polymethyl methacrylate (PMMA) and bonding polymer nanofibers to the perforated PMMA together with an adhesive.

[0105] Next, using the phase separation phenomenon that occurs in a solution containing a high concentration of N-isopropylacrylamide monomer, aqueous solutions of N-isopropylacrylamide with a high content and aqueous solutions of N-isopropylacrylamide with a low content were produced. A cell culture vessel was then manufactured in which a cell culture layer was formed on the upper surface of the porous microwells of an upper chamber equipped with the produced porous microwells, using the aforementioned coating aqueous solution, so that cells adhere to the surface of the porous microwells at temperatures above 32°C and below 40°C, and desorb at temperatures above 0°C and below 32°C.

[0106] Specifically, N-isopropylacrylamide monomer and distilled water were mixed in a 1:1 mass ratio and stirred for 5 minutes to ensure that the N-isopropylacrylamide monomer could dissolve sufficiently in the distilled water. Over time, the low-concentration and high-concentration N-isopropylacrylamide aqueous solutions became stably separated. Finally, when the N-isopropylacrylamide aqueous solution was stably separated, each solution was transferred to a vial using pipetting to obtain 5 ml of N-isopropylacrylamide aqueous solution with a high content, where the mass ratio of N-isopropylacrylamide to water was 87:13.

[0107] Next, 0.05 g of N,N'-methylenebisacrylamide (MBAAm), a crosslinking agent for reacting with ultraviolet light during UV irradiation treatment (100 parts by weight of a mixture of N-isopropylacrylamide monomer and water: 1 part by weight), and 0.005 g of 2-hydroxy-1-1[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone, a photoinitiator (100 parts by weight of a mixture of N-isopropylacrylamide monomer and water: 0.01 parts by weight), were added to the respective aqueous solutions of N-isopropylacrylamide.

[0108] A cell culture vessel was manufactured in which a composition prepared by adding a crosslinking agent and a photoinitiator to an aqueous solution of N-isopropylacrylamide was thinly coated onto the polymer nanofibers using a bar coat, and then irradiated with a UV light source for 10 minutes to form a cell culture layer on the upper surface of the porous microwells, in which cells adhere at temperatures above 32°C and below 40°C, and desorb at temperatures above 0°C and below 32°C.

[0109] Figures 11A and 11B show a cell culture vessel in which a cell culture layer is formed on the upper surface of the manufactured porous microwells, where cells adhere at temperatures above 32°C and below 40°C, and desorb at temperatures above 0°C and below 32°C.

[0110] <Experimental Example 1>

[0111] To measure the change in surface roughness, the changes in surface roughness of the cell culture layer in Example 1 with respect to temperature and time were measured using an atomic force microscope (ParkSystems, South Korea), and the results are shown in Figure 12.

[0112] As shown in Figure 12, at 37°C, a temperature exceeding the LSCT, there was almost no change in the surface roughness of the cell culture layer in Example 1, whereas at 20°C, a temperature below the LSCT, a rapid change in the surface roughness of the cell culture layer in Example 1 was observed.

[0113] <Experimental Example 2>

[0114] In the cell culture vessels manufactured in Example 1, surface shapes were compared using a scanning electron microscope to confirm the morphological differences in the cell culture layers where cells adhere at temperatures above 32°C and below 40°C, and where cells detach at temperatures above 0°C and below 32°C.

[0115] Figure 13A shows the polymer nanofibers before the composition containing N-isopropylacrylamide is coated onto them, and Figure 13B shows the cell culture layer after the polymer nanofibers have been coated with the composition containing N-isopropylacrylamide. The cell culture layer is in a form in which polymer nanofibers and hydrogel are bonded together.

[0116] <Experimental Example 3>

[0117] Human hepatoma cell line (HepG2) was seeded in the cell culture vessel of Example 1 at 36°C, and three-dimensional cell aggregates were cultured after 3 days. To harvest the cultured three-dimensional human hepatoma cell line aggregates, the upper chamber containing the three-dimensional human hepatoma cell line aggregates was moved to an environment of 20°C.

[0118] Figure 14A shows an image of a cell culture vessel in which cells are being cultured, and Figure 14B shows an image of cultured cells. Figure 14C shows an image of a cell culture vessel from which cultured cells have been detached. As shown in Figure 5C, it was confirmed that cells cultured in the cell culture vessel of the present invention are easily detached.

[0119] In the following, with reference to Figures 15 to 18, we will describe a cell culture apparatus according to one embodiment of the present invention, more precisely, a cell culture apparatus that enhances the cell culture effect by utilizing bottom flow, and a cell culture method using the same.

[0120] This embodiment relates to a three-dimensional cell culture apparatus that can easily remove waste products generated during cell culture and smoothly supply nutrients to the lower part of the three-dimensional cells. Specifically, the present invention relates to a three-dimensional cell culture apparatus comprising an upper chamber 100 having an opening and porous microwells, and a lower chamber 200 in which the upper chamber is located and fluid flow is possible at the lower end of the porous microwells.

[0121] Referring to Figure 15, the upper chamber 100 can mean a configuration in which a well-shaped chamber, including the opening 11 and the porous microwell 21, is fitted through the plate. The lower end of the porous microwell 21 refers to the lower region of the porous microwell, which is the region within the lower chamber 200.

[0122] The upper chamber 100 may have a perforation so that the fluid in the lower chamber 200 can come into contact with the outside. In this case, the perforation may be formed in a portion adjacent to the porous microwell 21 of the upper chamber 100, or it may be formed at any position in the upper chamber 100. When the fluid comes into contact with the outside as described above, the position of the perforation is not particularly limited as long as it can prevent the shape of the porous membrane from being deformed by the physical force formed by the fluid flow.

[0123] The lower chamber 200 may further comprise a fluid inlet 310 and a fluid outlet 330 through which fluid can flow, thereby enabling fluid to flow within the lower chamber 200. Furthermore, the fluid inlet 310 and the fluid outlet 330 may be further connected to a device capable of inducing fluid flow. For example, the lower chamber 200 may be connected to a device that guides fluid flow so that the fluid flows horizontally through the porous microwells 21. In this case, a pump or the like can be used as the device that guides fluid flow, but it is not particularly limited as long as it is capable of inducing fluid flow.

[0124] Thus, the three-dimensional cell culture apparatus ensures stable fluid flow in the lower chamber 200 by using a fluid flow guidance device that directs the fluid flowing through the lower chamber 200 to the lower end of the porous microwells 21. Due to the permeability of the voids in the porous microwells 21, the fluid with the aforementioned flow can discharge waste products formed during three-dimensional cell culture from the upper surface of the porous membrane into the lower chamber 200, thereby smoothly supplying nutrients to the lower part of the three-dimensional cells. Consequently, waste products can be discharged from the lower chamber 200 to the outside of the three-dimensional cell culture apparatus. For example, the fluid flow guidance device can be a syringe pump, a peristaltic pump, or a stirrer.

[0125] Furthermore, in order to disperse the pressure applied to the porous microwells during three-dimensional cell culture and to efficiently remove and supply the waste products and nutrients that are formed, the upper chamber 100 can be formed with a through portion 400 so that the surface of the fluid in the lower chamber 200 is in contact with the outside.

[0126] The aforementioned through-hole 400 provides a structure that allows the fluid surface of the lower chamber 200 to come into contact with the outside. This structure causes pressure to be applied to the upper chamber 100 during fluid flow, deforming the porous microwells 21, thereby solving the problem of adverse effects on cell implantation, proliferation, and differentiation during three-dimensional cell culture.

[0127] On the other hand, the present invention provides a three-dimensional cell culture method that includes the step of introducing a cell culture medium and cells into porous microwells using the three-dimensional cell culture apparatus of the present invention and culturing the cells, wherein the cells may be myoblasts, embryonic fibroblasts, human umbilical vein endothelial cells, human hepatocellular carcinoma cells (HepG2 cells), or human epidermal cells, but are not limited to these.

[0128] The present invention will be described in more detail below with reference to specific examples. The following examples are merely illustrative to aid in understanding the present invention, and the scope of the present invention is not limited thereto.

[0129] <Example 2>

[0130] In a three-dimensional cell culture apparatus comprising an upper chamber 100 and a lower chamber 200, each having porous microwells as shown in Figure 15a, HepG2 cells and DMEM (Dulbeco's Modified Eagle's Media, FBS 10%) culture medium were injected into the porous microwells 21. This mixture was then stabilized at 37°C for 48 hours to form three-dimensional spheroids. Subsequently, the cells were cultured in the lower chamber for 9 days under flow conditions.

[0131] <Comparative Example 2>

[0132] Except for not providing lower-end flow as in Example 2, three-dimensional HepG2 spheroids were cultured in the same manner as in Example 2.

[0133] In Example 2 and Comparative Example 2, the albumin expression levels of HepG2 spheroids were measured on days 6 and 9 during the culture period. The measured albumin expression levels are shown in Figure 16a. As shown in Figure 16a, the albumin expression levels of HepG2 spheroids cultured in Comparative Example 2 were 1182.64 mg / ml on day 6 and 2966.505 mg / ml on day 9, while the albumin expression levels of HepG2 spheroids cultured in Example 2 were 4813.99 mg / ml on day 6 and 6644.55 mg / ml on day 9. In other words, it was found that HepG2 spheroids cultured in a 3D cell culture device with bottom-end flow had higher albumin expression levels, and from this, it was confirmed that bottom-end flow improved the functionality of 3D HepG2 spheroids.

[0134] <Example 3>

[0135] In the three-dimensional cell culture apparatus of the present invention using bottom-end flow, to confirm that waste products can be efficiently removed, 200 μg / ml of FITC-Dextran 20kDa was placed in the porous microwells of a three-dimensional cell culture apparatus as shown in Figure 15a, and bottom-end flow was performed for 3 hours. After that, the concentration of FITC-Dextran remaining in the porous microwells was measured.

[0136] <Comparative Example 3>

[0137] Except for not providing bottom-end flow as in Example 3, the concentration of FITC-Dextran remaining in the porous microwells was measured 3 hours after adding FITC-Dextran, similar to Example 3.

[0138] Figure 17 shows the results of visual observation of the extent of waste accumulation in Example 3, and Figure 16b shows the concentrations of FITC-Dextran measured in Example 3 and Comparative Example 3. As shown in Figure 16b, in Example 4, where there was bottom flow, the concentration of FITC-Dextran remaining in the porous microwells was 55.6199 ± 3.3429 mg / ml, while in Comparative Example 3, the concentration of FITC-Dextran remaining in the porous microwells was confirmed to be 131.435 ± 7.80245 mg / ml.

[0139] In other words, when there is bottom-end flow, the concentration of FITC-dextran remaining in the porous microwells is significantly lower than when there is no bottom-end flow. This confirms that even with the same amount of culture medium, cellular waste remaining in the porous microwells can be removed more efficiently when bottom-end flow is present. This was confirmed, as shown in Figure 18a, that in the lower chamber, waste on the upper surface of the porous microwells is removed by the fluid flow induction device and the porous microwells. Figure 18a shows how waste present in the porous microwells is removed by passing through the voids when there is bottom-end flow of the culture medium; in other words, waste is removed from inside the microwells by passing through the voids along with the fluid flow. On the other hand, Figure 18b shows the phenomenon in which the culture medium naturally diffuses into the microwells when bottom-end flow is present in the lower chamber. This figure explains that when there is bottom-end flow of the culture medium, not only can waste be removed through the voids of the membrane, but diffusion of the culture medium can also occur simultaneously. Referring to Figures 18a and 18b, it goes without saying that the direction in which waste products pass through the voids and the direction in which the culture medium passes through the voids are opposite to each other.

[0140] The following describes a cell culture apparatus and cell culture method according to another embodiment of the present invention with reference to Figures 19 to 29.

[0141] According to this embodiment, when using the existing cell culture medium replacement method, in which used cell culture medium is removed by pipetting at the top of the microwells and new cell culture medium is injected during the cell culture process, the problem of loss of cells and cell aggregates due to the flow of the culture medium by pipetting can be solved. Furthermore, the continuous flow around the cell aggregates induced by permeating the porous microwells makes it possible to realize a uniform cellular microenvironment such as nutrients. Therefore, according to the present invention, it is possible to minimize the loss of cells during the culture process, induce a continuously uniform microenvironment around the cell aggregates, and culture three-dimensional cell aggregates that exhibit phenomena more similar to those in vivo compared to two-dimensional culture.

[0142] Referring to Figure 19, the cell culture apparatus according to this embodiment includes an upper chamber 110 having an opening, a porous membrane, and porous microwells 21 having a cell culture space for containing culture medium. It also includes a lower chamber 210 having a space inside which the upper chamber 110 is placed. Fluid flowing into the upper part of the upper chamber 110 flows from the upper chamber 110 through the porous microwells 21 into the lower chamber 210, and the fluid in the lower chamber 210 is discharged to facilitate fluid flow.

[0143] Furthermore, this detailed description will refer to a three-dimensional cell aggregate culture method using the cell culture apparatus shown in Figure 19, which includes the steps of: seeding cells onto a porous membrane in the upper chamber 110; introducing cell culture medium into the upper chamber 110 through an inlet 311 or opening; and discharging the cell culture medium that has flowed from the upper chamber 110 through the porous microwells into the lower chamber 210 from the lower chamber 210. The three-dimensional cell aggregate culture method may also further include the step of introducing the cell culture medium into the lower chamber 210 as the cell culture medium is made to flow by the cell culture medium being discharged from the lower chamber 210.

[0144] In a three-dimensional cell culture apparatus according to one embodiment of the present invention, fluid flowing into the upper part of the upper chamber 110 flows from the upper chamber 110 through the porous microwells 21 into the lower chamber 210, and consequently, the fluid in the lower chamber 210 is discharged, causing the fluid to flow. At this time, the fluid in the lower chamber 210 may be discharged through a fluid outlet provided in the lower chamber 210, or through the gap region between the upper chamber 110 and the lower chamber 210, for example, through the upper part of the gap region.

[0145] The fluid outlet can be formed at any position in the lower chamber 210, for example, at the lower end of the lower chamber 210 as shown in Figure 1c, but is not limited thereto. On the other hand, if the lower chamber 210 is not provided with such a fluid outlet, a fluid flow can be formed by discharging the fluid through the gap region between the upper chamber 110 and the lower chamber 210, as shown in Figures 29a to 29c. In this case, because a gap region exists between the upper chamber 110 and the lower chamber 210, the size of each chamber can be adjusted so that a gap exists between the walls of each side of the upper chamber 110 and the lower chamber 210, and the fluid can be discharged from the top of the interface that contacts the air in the gap region between them.

[0146] In other words, the three-dimensional cell culture apparatus according to the present invention can be equipped with a discharge structure to discharge fluid from the lower chamber 210, and in this case, the discharge structure can consist of a structure that causes fluid to flow through a fluid outlet 331 into the lower chamber 210, or a structure that causes fluid to flow by discharging it through the gap region between the upper chamber 110 and the lower chamber 210, in other words, through the gap that exists between the upper chamber 110 and the lower chamber 210.

[0147] On the other hand, while other discharge structures not specifically mentioned in this detailed description may exist, there are no limitations on the specific detailed conditions of the discharge structure, as long as the fluid flow containing the cell culture medium can maintain a downward flow from the upper chamber 110 to the lower chamber 210.

[0148] In this embodiment, the porous membrane consists of a nanofiber network and may be a porous membrane with a porosity of 20% to 60%, for example, a porous membrane with a porosity of 30% to 50%. If the porosity is less than 20%, the water permeability coefficient becomes low, which increases the water pressure applied to the porous membrane, leading to the problem of a decrease in the viability of cell aggregates being cultured on the porous membrane.

[0149] Furthermore, the porous membrane can have an average void particle size of 10 nm to 10 μm. That is, since the porous membrane includes an average void size within the range described above, it can act as a selective permeable membrane that prevents single cells from passing through while selectively allowing other substances such as nutrients and growth factors in the cell culture medium to pass through. As a result, it is possible to induce aggregation of single cells on the porous membrane to form a three-dimensional aggregate, and after the aggregate is formed, it can act as a mass transfer barrier and passage.

[0150] The porous membrane can maintain the characteristics of a high water permeability coefficient within the range of porosity and average void particle size described in the present invention. Furthermore, the porous membrane can have a density of 1 to 20 μms. -1 It can have a hydraulic conductivity of 1 μm². -1 If the pressure is less than 1000 Pa, it can lead to high water pressure on the porous membrane, and if water pressure of 1000 Pa or more is applied to the porous membrane and cell aggregates during culture, it can have adverse effects such as reducing cell viability.

[0151] The porous membrane is a nanofiber network for cell culture, and its manufacturing method is not particularly limited, but for example, it may be made of polymer nanofibers formed by electrical radiation.

[0152] The method for producing the nanofiber network may include, for example, a step of electrically emitting a solution prepared by dissolving polycaprolactone in a chloroform / methanol 3 / 1 vol / vol mixture to a concentration of 4-10 wt%. The electrical emission is performed at a voltage of 10-30 kV for 0.1-2.0 mlhr. -1 It is preferable that the polymer solution is discharged at the specified rate. If the voltage is below the range, there may be problems in producing uniform nanofibers, and if it exceeds the range, there may be problems in producing stable nanofibers, such as uneven stacking of nanofibers.

[0153] The porous microwell may have a porous membrane in whole or in part in the recess formed by a downward indentation, and preferably, the porous microwell, i.e., when the opening of the upper chamber is positioned upward, the surface forming the bottom is formed of a porous membrane.

[0154] On the other hand, the porous membrane forming the lower surface of the upper chamber 110 of the present invention can be formed to have convex portions and / or recesses. For example, recesses can be formed using the porous membrane as shown in Figure 28, or recesses can be formed by further adding side walls or convex portions that can define a section corresponding to the recess on the porous membrane as shown in Figure 26. In this case, the material of the side walls or convex portions may be porous or not, and is not particularly limited. Preferably, the lower surface of the recess is a porous membrane, and the side walls or convex portions may be porous or not. For example, Figures 21a and 21b are examples showing a form in which the side walls of the recess are not made of a porous material by laminating a further layer in which through holes are formed, and Figure 21c shows a case in which recesses are formed in the porous membrane itself, and both the recessed and non-recessed portions are made of a material that is porous overall.

[0155] Porous microwells can be manufactured, for example, by combining a porous membrane fabricated by electrical radiation with an array of through-holes, as shown in Figure 26, but are not limited to this. On the other hand, as shown in Figure 21c, the formation of recesses and protrusions in porous materials can be carried out, for example, by using a compression process utilizing a mold, as shown in Figure 28.

[0156] The upper surface of the porous membrane of the porous microwell is a region that functions as a cell culture layer. As described above, when the porous membrane has convex and concave portions, cells can more easily settle into the formed concave portions, inducing aggregation of single cells within the porous microwell, which then allows for the stable formation of three-dimensional cell aggregates for cultivation. More preferably, the porous membrane of the present invention has convex and concave portions, and all surfaces consist of a porous membrane.

[0157] On the other hand, the lower chamber 210 has a space inside which the upper chamber 110 is positioned, and if the lower chamber 210 is equipped with a fluid outlet, the fluid in the lower chamber 210 containing the culture medium can flow while the fluid in the lower chamber 210 is positioned inside the upper chamber 110, with the fluid being discharged through the fluid outlet 331 from which the fluid can be discharged. For this purpose, devices capable of causing flow can be further connected to the upper and / or lower chambers 210, for example, a device for injecting cell culture medium into the upper chamber 110 at a constant flow rate may be added, and the lower chamber 210 can be connected to a device that controls the flow of fluid, which causes the fluid flowing into the upper chamber 110 to flow through the porous membrane of the upper chamber 110 into the lower chamber 210 and be discharged at a constant flow rate, and this can be, for example, a culture medium storage tank. At this time, there are no particular restrictions on the device that guides the fluid flow, as long as it can cause the fluid to flow. For example, the fluid flow guiding device can be a syringe pump, a peristaltic pump, or an agitator.

[0158] The three-dimensional cell culture apparatus according to the present invention may further include a culture medium storage tank 65 arranged on the same plane as the lower chamber 210 and in fluid communication with the lower chamber 210, and the height of the culture medium in the culture medium storage tank 65 can be set to correspond to the water level of the culture medium in the lower chamber 210.

[0159] The water level of the culture medium in the lower chamber 210 can be 1 to 20 mm above the porous membrane at the lower end of the upper chamber 110, for example, 1 to 19 mm, preferably 1 to 18 mm, for example, 1 to 8 mm. If the water level of the culture medium is below the desired range, it may not be possible to smoothly supply nutrients to the cell aggregates during culture, and if it exceeds the desired range, it may lead to flooding and overuse of the culture medium.

[0160] In addition, the distance between the porous membrane at the lower end of the upper chamber 110 and the bottom of the lower chamber 210 can be 0.1 to 8 mm, for example, 0.2 to 7 mm, preferably 1 to 5 mm. If the distance is less than the desired range, the flow of the cell culture medium may be restricted and there may be a risk that it cannot be discharged through the discharge port. Also, if it exceeds the desired range, it can lead to overuse of the culture medium.

[0161] At this time, the fluid, that is, the cell culture medium, can flow while maintaining a constant speed. For this purpose, the inflow and discharge of the fluid can be adjusted at a constant speed. At this time, for example, the fluid can flow at a speed of 0.0001 to 1 ml / hr -1 , for example, 0.001 to 1 ml / hr -1 , preferably at a speed of 0.01 to 1 ml / hr -1 and can flow at a speed of. If the flow rate of the fluid is less than 0.0001 ml / hr -1 , there can be a problem that the amount of cell culture medium required by the cell aggregates is not supplied, and if it exceeds 1 ml / h r-1 , excessive shear stress due to flow may act on the cell aggregates and may have an adverse effect on the cell aggregates. Shear stress that does not have an adverse effect on cells, for example, a shear stress of 0.001 to 10 dyne / cm -2 can have a positive effect on cell differentiation and proliferation.

[0162] On the other hand, the cell culture medium is preferably applied to the porous membrane at a water pressure of less than 1000 Pa, and the lower the pressure, the more preferable, and for example, it can be 1 Pa to 100 Pa. Incidentally, if it exceeds 1000 Pa, there is a problem that the cell survival rate decreases.

[0163] The three-dimensional cell aggregate culture apparatus and the tertiary cell aggregate culture method using the apparatus of the present invention can use cell lines, such as myoblasts, embryonic fibroblasts, umbilical vein endothelial cells, hepatocellular carcinoma cells (HepG2 cells), epidermal cells, or a mixture of at least one of these. In addition to cell lines, primary cells from the liver, pancreas, spleen, small intestine, etc., can also be used. Finally, while stem cells, such as induced pluripotent stem cells, mesenchymal stem cells, or a mixture of at least one of these, can be cultured, the invention is not limited to these, and spheroids or organoids, which are three-dimensional cell aggregates, can also be cultured.

[0164] The method for culturing three-dimensional cell aggregates according to this embodiment may include the steps of seeding cells onto a porous membrane in the upper chamber 110 using the three-dimensional cell culture apparatus of the present invention, and introducing cell culture medium into the upper part of the upper chamber 110 through an inlet 311 or opening.

[0165] The cells can be seeded and cultured on the porous membrane, and the seeding can be performed by seeding the cells to be cultured on the porous membrane prior to the step of introducing fluid into the upper chamber 110. This makes the cells applicable to the three-dimensional cell culture apparatus, and the method of seeding the cells can be carried out by methods used in the industry, for example, by using micropipette.

[0166] Cells seeded using the aforementioned method form three-dimensional cell aggregates within a few hours to a few days. After cell aggregate formation, the cell culture medium can be flowed using the developed device.

[0167] Next, the present invention will be described in more detail through specific examples. The following examples are merely illustrative to aid in understanding the present invention, and the scope of the present invention is not limited thereto. [Examples]

[0168] 1. Manufacturing of porous membranes

[0169] Polycaprolactone (PCL; Mn = 80,000 gmol) -1 Chloroform and methanol were purchased from Sigma-Aldrich (USA). The PCL solution for electrolysis was prepared by dissolving PCL in a 7.5 wt% chloroform / methanol mixture of 3 / 1 vol / vol. The prepared PCL solution was placed in a 5 ml precision syringe (Gastight syringe, Hamilton) and administered via a commercial electrolysis device (ES-robot, NanoNC, South Korea) using a 23-gauge metal needle positioned 10 cm away from a 5 cm diameter annular electrode. -1 The material was discharged at a flow rate of [value missing]. Electrical emission was performed by applying a high voltage of 15kV between the metal needle and the annular electrode using the aforementioned commercial electric radiator. The electrically emitted PCL nanofibers (As-electrospun PCL nanofibers) were deposited between the annular electrode and the PCL nanofiber to create a gas and mass-permeable nanofiber membrane. Electrical emission was carried out at a relative humidity of 50-60% and a temperature of 20-25°C. Micrographs of the nanofiber network during the emission time are shown in Figure 22, confirming that the diameter of the nanofibers was approximately 800-1000 nm. As the emission time increased, the average void size and porosity decreased, while the average thickness increased.

[0170] Figure 22 shows the changes in the structure of the nanofiber network due to radiation time during the manufacturing of the aforementioned nanofiber network (Figure 22a), the change in porosity (Figure 22b), the hydraulic conductivity of the porous membrane (Figure 22c), and the induced pressure (Figure 22d), respectively.

[0171] In this case, the method for calculating the porosity involved converting the magnified microscope image into a binary image, and then using ImageJ software (NIH, USA) to calculate the area fraction of voids generated by the nanofiber network relative to the nanofiber network area.

[0172] 2. Manufacturing of an upper chamber with porous microwells

[0173] To manufacture the upper chamber equipped with porous microwells that contain the porous membrane and cell culture space for the culture medium obtained in step 1 above, a series of steps were carried out, which are shown in Figure 26. Specifically, the porous membrane region at the lower end of the upper chamber was formed by applying adhesive to a 500 μm thick polymethyl methacrylate (PMMA) plate (Acryl Choika, South Korea), then using a laser cutter (ML-7050A, Machineshop, South Korea) to perforate an array of through-holes in the PMMA plate coated with adhesive, and finally, by using the applied adhesive to bond the through-holes to the porous membrane, a recess with a porous membrane at the bottom was formed as shown in Figure 21.

[0174] Figure 28 shows a series of steps for fabricating the porous membrane region at the lower end of the upper chamber in another configuration. More specifically, the lower end of the upper chamber, which has a porous membrane with convex and concave sections formed on all surfaces, was completed by a compression process using a convex mold and a concave mold on the flat porous membrane obtained in step 1 above. At this time, the concave mold was fabricated by perforating a 10 mm thick polymethyl methacrylate (PMMA) plate (Acryl Choika, South Korea) using machining equipment (EGX-350, Roland, USA). To fabricate the convex mold composed of polydimethylsiloxane (PDMS), a mixture of PDMS and a hardener (Sylgard 184, Dow Corning, USA) in a weight ratio of 10:1 was poured into the concave mold and cured at 55°C for 12 hours.

[0175] The remaining side portions within the upper chamber, which have an opening shape, were manufactured using an injection molding machine (SE50D, Sumitomo Corporation, Japan). The side portions thus obtained and the lower end of the upper chamber obtained above were joined using adhesive to complete the upper chamber equipped with porous microwells.

[0176] 3. Manufacturing of a 3D cell aggregate culture system

[0177] To fabricate the lower chamber and its lid, which are made of polydimethylsiloxane (PDMS), a mixture of PDMS and a hardener (Sylgard 184, Dow Corning, USA) in a 10:1 weight ratio was poured into a mold and cured at 55°C for 12 hours. The molds for the lower chamber and lid were fabricated using a 20mm thick PMMA plate with machining equipment (EGX-350, Roland, USA). The lower chamber was fabricated to a size of 30mm x 30mm x 30mm, with a 2mm deep groove designed on the top surface (Figure 27a), and the distance from the bottom surface of the lower chamber to the porous membrane of the porous microwell fabricated in step 2 was set to 8mm (Figure 27b). A 1mm diameter hole was punched in the center of the bottom surface of the lower chamber and the lid using a Biopsy punch (Miltex, USA) to form an outlet.

[0178] A tube (manufactured by Biokonvision, South Korea) is connected through a perforation in the opening of the cap, and a syringe pump (KDS200, manufactured by KD Scientific, USA) dispenses 0.062 mlh through the tube. -1 The cell culture medium was injected at the specified flow rate. A perforation in the bottom of the lower chamber was also connected to a tube to transfer the cell culture medium to the culture medium storage tank. Furthermore, a Z-stage (manufactured by Sciencetown, South Korea) was installed at the bottom of the culture medium storage tank, and the height of the storage tank was set to correspond to the water level of the culture medium in the lower chamber. This was designed to maintain a constant water level of the culture medium in the lower chamber despite the continuous inflow of culture medium from the syringe pump during the culture process.

[0179] 4. Cell culture using aggregate culture device

[0180] <Example 4>

[0181] A three-dimensional cell culture apparatus was provided as shown in Figure 19, which included an upper chamber and a lower chamber equipped with porous microwells manufactured in step 1, as well as a culture medium storage tank capable of storing the discharged culture medium. At this time, the height of the culture medium storage tank was set so that the water level in the lower chamber was 12 mm from the bottom surface of the lower chamber. The cell culture apparatus thus obtained will be referred to interchangeably as "the cell culture apparatus of the present invention" and "the cell culture apparatus of Example 4".

[0182] In the aforementioned three-dimensional cell culture apparatus, hepatocytes (HepG2) and DMEM (Dulbeco's Modified Eagle's Media, FBS 10%) culture medium were injected together into porous microwells. This mixture was then subjected to a static environment at 37°C for 48 hours without applying any flow, allowing three-dimensional HepG2 aggregates, i.e., HepG2 spheroids, to form. Subsequently, a constant flow rate of 0.062 mlhr was maintained using a syringe pump. -1 The cell culture medium was continuously injected into the upper chamber. Subsequently, to discharge the injected fluid, it was discharged into the culture medium reservoir through the lower chamber, which has a downward-facing fluid outlet 331, and the HepG2 spheroids were cultured for 8 days.

[0183] <Comparative Example 4>

[0184] HepG2 spheroids were cultured in the same manner as in Example 4, except that an impermeable microwell consisting of an impermeable PMMA plate bottom surface (not a porous membrane) was used, and the cell culture medium was replaced by pipetting without using devices to apply fluid flow to the cell culture medium, such as syringe pumps or culture medium reservoirs. Experimental Example

[0185] (1) Confirmation of fluid exchange within the 3D cell culture device over time.

[0186] In the 3D cell culture apparatus of Example 4, a new fluid of 0.062 mlhr was added to the upper chamber. -1Experiments were conducted to confirm the fluid flow changes (confirmation of fluid exchange) within the 3D cell culture device when the fluid was injected at the specified flow rate.

[0187] As shown in Figure 25, we were able to confirm that the blue solution already present inside the cell culture aggregate was gradually replaced by a newly injected clear solution (in the black and white diagram, you can see that the darker solution gradually changes to a lighter color as time progresses from left to right in Figure 25). We were also able to confirm that the water level in the lower chamber remained constant during this process.

[0188] ( 2) Interpretation of the cell culture medium flow in Example 4

[0189] To apply the computerized fluid interpretation method for measuring the flow of cell culture medium in the 3D cell culture apparatus of the present invention, the flow velocity (Surface velocity: 0 ms) in the 3D cell culture apparatus of Example 4 was measured using COMSOL Multiphysics software (Version 5.0, USA). -1 ~8.11e -6 ms -1 The following elements were interpreted and calculated: the flow direction (black cone shape), and the streamlines (white lines).

[0190] As a result, as shown in Figure 20, we were able to confirm that the cell culture medium flows smoothly from the upper chamber to the lower chamber in the three-dimensional cell culture apparatus of the present invention.

[0191] (3) Comparison of the degree to which cells disappear during cell culture

[0192] In Example 4 and Comparative Example 4, the degree of hepatocyte spheroid disappearance was compared at 2-day intervals, and the results are shown in Figure 23.

[0193] As shown in Figure 23, in Example 4 there was no loss of stem cell spheroids at all, but in Comparative Example 4, spheroids were continuously lost, and it was confirmed that about 15% of HepG2 cell spheroids were lost by day 8.

[0194] (4) Comparison of nutrient concentrations around three-dimensional cell aggregates over time

[0195] In Example 4 and Comparative Example 4, the concentration of nutrients (glucose) surrounding HepG2 spheroids over time was measured, and COMSOL Multiphysics software (Version 5.0, USA) was used for comparative analysis. The values ​​used in this process are shown in Table 1 below.

[0196] [Table 1]

[0197] As shown in Figure 24, in the cell culture apparatus of Example 4, it was numerically predicted that a uniform nutrient concentration would be induced around the HepG2 cell aggregates after 36 hours. However, in Comparative Example 4, the nutrient concentration changed drastically before and after the pipetting interval, and it was numerically predicted that a non-uniform nutrient concentration would be induced around the cell aggregates.

[0198] (5) Measurement of the permeability coefficient and water pressure of the porous membrane in the three-dimensional cell culture apparatus of Example 4

[0199] New fluid flows into the upper chamber at a rate of 0.062 mlhr. -1 Experiments were conducted to confirm the applied pressure on the porous membrane in the 3D cell culture apparatus when injected at a certain flow rate. To confirm this, the hydraulic conductivity was measured beforehand outside the invented apparatus using the falling-head method as described below.

[0200] Specifically, initial pressure head (h i Water containing ) permeates through a porous membrane to reach the final pressure head (h f The time taken to reach ) was checked and the permeability coefficient was measured using an equation based on Darcy's law.

[0201]

number

[0202] Here, K is the water permeability coefficient of the porous membrane, L is the thickness of the porous membrane, and t is h i de h f This is the time it takes to reach that point, and in this experiment, h was 100 mm and 10 mm respectively. i and h f The following was used. Based on the calculated water permeability coefficient, the permeability of the porous membrane was calculated using Equation 2 below.

[0203]

number

[0204] Here, k is the permeability of the porous membrane, μ is the viscosity of the cell culture medium, ρ is the density of the cell culture medium, and g is the acceleration due to gravity.

[0205] 0.062 mlhr -1 Based on the measured permeability coefficient and calculated permeability, the water pressure applied to the porous membrane was calculated using the following equation (Kozeny-Carman equation) when the cell culture medium permeated the porous membrane at a given flow rate.

[0206]

number

[0207] Here, the fluid velocity k currently permeating the porous membrane is the permeability of the porous membrane, μ is the viscosity of the cell culture medium, L is the thickness of the porous membrane, and p is the water pressure applied to the porous membrane.

[0208] As shown in Figure 22, we confirmed that the water pressure applied to the porous membrane, i.e., the hydraulic repulsion force, is lower the more sparse the nanofiber network constituting the porous membrane is, that is, the shorter the time it takes for electrical radiation to occur, i.e., the higher the porosity and hydraulic conductivity. When this was compared and analyzed with a commercially available porous membrane that is not the porous membrane of the present invention, we were able to confirm that a much lower hydraulic repulsion force is applied to the porous membrane of the present invention, and that a porous membrane composed of a nanofiber network is even more suitable for the apparatus of the present invention.

Claims

1. A three-dimensional cell culture device, An upper chamber comprising a porous microwell having an opening, a porous membrane, and a cell culture space for containing culture medium, The upper chamber comprises a lower chamber having a space inside which the upper chamber is arranged, An outlet provided at the lower end of the lower chamber, A culture medium storage tank is arranged on the same plane as the lower chamber and is in fluid communication with the outlet, Includes, The fluid that flows into the upper part of the upper chamber flows from the upper chamber through the porous microwells into the lower chamber, and the fluid in the lower chamber is discharged through the outlet. The water level of the culture medium in the culture medium storage tank is set to correspond to the water level of the culture medium in the lower chamber. A culture apparatus for three-dimensional cell aggregates characterized by the following features.

2. The step of seeding cells onto a porous membrane in the upper chamber using the three-dimensional cell culture apparatus described in Claim 1, The step of introducing the cell culture medium into the upper chamber through the opening, The steps include: discharging the cell culture medium that has flowed from the upper chamber through porous microwells into the lower chamber from the lower chamber; including A method for culturing three-dimensional cell aggregates.

3. The aforementioned cells are Stem cells including induced pluripotent stem cells and mesenchymal stem cells, Cell lines including myoblasts, embryo fibroblasts, umbilical vein endothelial cells, hepatocellular carcinoma cells (HepG2-2 cells), and epidermal cells, It is a primary cell obtained from the liver, pancreas, or small intestine, and at least one selected from the group consisting of these. A method for culturing a three-dimensional cell aggregate according to claim 2.

4. The step further includes introducing the cell culture medium into the lower chamber. A method for culturing a three-dimensional cell aggregate according to claim 2.

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