How to produce exosomes

Abstract

The present invention provides a method for producing an exosome, comprising a step of applying a cell to a cell culture module to culture the cell and a step of producing an exosome by the cell, wherein the cell culture module is provided with a polymer porous film and a casing having at least two culture medium in-flow / out-flow ports and having the polymer porous film placed therein.

Description

[Technical field]

[0001] The present invention relates to a method for producing exosomes using cells. [Background technology]

[0002] Exosomes are a type of membranous vesicle structure of 50 to 300 nm that are secreted extracellularly by cells, and are formed by the collapse of the endosomal membrane inside multivesicular endosomes formed by endocytosis, and therefore contain cell membrane-derived surface proteins on the surface and cytoplasm-derived nucleic acids and proteins inside. In recent years, it has been reported that endosomes, in which the composition of proteins and nucleic acids is precisely controlled, are involved in the transmission of information between nearby or distant cells in various cell types (Non-Patent Document 1).

[0003] Exosomes are mainly used as diagnostic markers, drug delivery carriers, and biopharmaceuticals. Cancer cells and diseased cells secrete exosomes that reflect their pathological state, so disease diagnosis is possible by profiling the characteristics of exosomes (Non-Patent Document 2). Exosomes carrying drugs can also be used for drug delivery targeted at cells that receive the exosomes. Methods for carrying drugs include a method of binding or incorporating drugs into secreted exosomes, and a method of secreting drug-containing exosomes from cells that contain drugs in their cytoplasm (Non-Patent Document 3).

[0004] It has also been reported that exosomes themselves can function as physiologically active substances. It has been shown that exosomes isolated from cultured neural stem cell lines induce fibroblast migration, human umbilical vein endothelial cell branching, and neurite outgrowth in vitro (Patent Document 1). Exosomes from bone marrow dendritic cells have been reported to have immunoregulatory functions, and their therapeutic effects against infectious diseases, allergies, autoimmune diseases, etc. have been tested in mouse models (Non-Patent Documents 4 and 5).

[0005] It is believed that the physiological activity of exosomes is supported by the composition of nucleic acids and proteins controlled by the cells from which the exosomes are derived. In particular, the relationship between the miRNA contained in exosomes and physiological activity has attracted attention, and Patent Document 2 describes that exosomes isolated from cardiospheres or cardiomyocyte-derived cells (CDCs) contain miR-146a, miR-210, miR-22, and miR-24, which have a therapeutic effect on damaged or diseased cardiac tissue. In addition, Patent Document 3 describes that liposomes containing at least one or more miRNAs, miR-34b, miR-132, miR-137, miR-193a, and miR-203, act as an inhibitor of oral squamous cell carcinoma. Furthermore, Non-Patent Document 6 describes that miR-26a and miR-26b inhibit cell migration and invasion of gastric cancer cells, and Non-Patent Document 7 describes that miR-23b, miR-27b, and miR-24 significantly inhibit cell migration and invasion of prostate cancer cells.

[0006] In order to use exosomes as drug delivery carriers or biologically active substances, it is necessary to establish a method for producing sufficient amounts of exosomes of uniform quality. Exosomes are usually obtained by recovering exosomes secreted by cultured cells into a culture system using ultracentrifugation. In the prior art, such as the above-mentioned documents that present methods for using exosomes, appropriate methods are used to recover exosomes required for testing, but none of them anticipate the development of a stable and highly efficient production method that can produce pharmaceutical-level exosomes on an industrial scale.

[0007] Patent Document 4 discloses a method for producing exosomes. This method is characterized by inducing exosome secretion by contacting stem cells with a prostaglandin E receptor 4 (EP4) antagonist. As an example of a cell culture system configured for highly efficient production of exosomes with good physiological activity, Non-Patent Document 8 presents a system that combines a 3D culture system of mesenchymal stem cells with tangential flow filtration (TFF).

[0008] As described above, the composition of nucleic acids, proteins, and various signal molecules that characterize exosomes is controlled by the cells from which the exosomes are derived, so the quality of exosomes produced during the induction phase, logarithmic growth phase, stationary phase, and death phase of cell culture is thought to change. However, when considering the production of exosomes on an industrial scale, it is undesirable from the perspective of quality control if the characteristics of exosomes change with the culture time, and it is required that a production system that has been established once can produce exosomes of uniform quality over a long period of time. In conventional exosome production technologies using cultured cells, the problem of changes in exosome quality over time in cell culture systems has not been adequately addressed, and this is a technical barrier that hinders the supply of exosomes of stable quality and the establishment of an exosome production system on an industrial scale.

[0009] In the production of substances using cultured cells, not limited to exosomes, it is important to establish the most suitable cell culture method for producing the desired substance stably and efficiently. Cells generally exist in the body as a group with a three-dimensional structure. On the other hand, when culturing cells in an artificial environment, the classical plate culture method in which cells are cultured two-dimensionally in a monolayer attached to the bottom of the culture vessel, and the suspension culture method in which cells are cultured in a dispersed state in a liquid culture medium are generally used. For cells used in the plate culture method, cells with relatively high adhesiveness are suitable, but even when suitable cells are used, the properties of the cells may change significantly depending on the culture environment. There are also suitable and unsuitable cells for the suspension culture method.

[0010] As the demand for in vivo proteins for medical purposes increases, the mass production of in vivo proteins using cell culture has attracted attention. For suspension cells such as E. coli, research has been conducted on mass cultivation in large culture tanks. Mass cultivation of suspension cells using large culture tanks requires a large amount of culture medium and stirring equipment. Meanwhile, research on substance production using adherent cells is also attracting a lot of interest, along with progress in research on the cells used. In the case of adherent cells, the cells only spread two-dimensionally in the classical plate culture method, so a large cultivation area is required. In order to cultivate large amounts of cells in a more three-dimensional manner for the mass production of in vivo proteins, much research is being conducted on cell culture carriers and bioreactors.

[0011] As a representative cell culture carrier, microcarriers, which are small particles on which cells can attach and grow, have been widely studied (Patent Document 5). Various types of microcarriers have been researched and developed, and many are commercially available. They are also widely used in the production of vaccines and proteins, and are widely adopted as a methodology as a system that can withstand scale-up. However, in microcarrier culture, there is a quantitative upper limit for cell culture because the microcarriers need to be sufficiently stirred and dispersed so as not to aggregate. Furthermore, for example, when producing substances, in order to separate the carrier itself, it is necessary to separate it using a filter that separates fine particles, and this requires complicated work methodologically. Furthermore, because cells are easily detached due to shear force, the stirring conditions are extremely limited, and it is completely incompatible with the culture conditions in a strong flow channel such as those used for E. coli.

[0012] As an alternative to microcarrier culture, a method has been discovered for continuously mass culturing spheroid-shaped cells using a three-dimensional culture method using methylcellulose or gellan gum. It is certainly possible to mass-culture cells using bioreactors that use cellulose sponges. However, the system is large and closed, and there are many operational limitations, such as the inability to easily come into contact with the culture environment.

[0013] There is an increasing need to develop a novel system that is simple and suitable for automation, and that can enable cells to efficiently produce exosomes of uniform quality over a long period of time.

[0014] <Porous polyimide film> A method for culturing cells has been reported, which involves applying cells to a polyimide porous membrane and culturing the cells (Patent Document 6).

[0015] Polyimide is a general term for polymers that contain imide bonds in the repeating units. Aromatic polyimide refers to a polymer in which aromatic compounds are directly linked by imide bonds. Aromatic polyimides have a rigid and strong molecular structure because aromatics have a conjugated structure via imide bonds, and the imide bonds have strong intermolecular forces, so they have very high levels of thermal, mechanical, and chemical properties.

[0016] Polyimide porous films have been used for applications, particularly battery-related applications, such as filters, low-dielectric constant films, and electrolyte membranes for fuel cells, since before the filing of this application. Patent documents 7 to 9 describe polyimide porous films that have excellent permeability to substances such as gases, high porosity, excellent smoothness on both surfaces, relatively high strength, and a large number of macrovoids that have excellent resistance to compressive stress in the thickness direction despite their high porosity. All of these polyimide porous films are made via amic acid. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] International Publication No. 2013 / 150303 [Patent Document 2] JP 2019-38840 A [Patent Document 3] JP 2009-171876 A [Patent Document 4] JP 2018-064550 A [Patent Document 5] International Publication No. 2003 / 054174 [Patent Document 6] International Publication No. 2015 / 012415 [Patent Document 7] International Publication No. 2010 / 038873 [Patent Document 8] JP 2011-219585 A [Patent Document 9] JP 2011-219586 A [Patent Document 10] International Publication No. 2018 / 021368 [Non-patent literature]

[0018] [Non-Patent Document 1] J. of Extracellular Vesicles, 2015, 4, Article: 27066 [Non-Patent Document 2] AMA Oncology, 2016, 2, p882-889 [Non-Patent Document 3] Trends in Biotechnology, 2017, 7, p665-676 [Non-Patent Document 4] Transplantation 2003, 76: 1503-10 [Non-Patent Document 5] Am. J. Transplant. 2006, 6:1541-50 [Non-Patent Document 6] Int J Oncol. 2016 May;48(5):1837-46 [Non-Patent Document 7] Oncotarget. 2014;5:7748-7759 [Non-Patent Document 8] Molecular Therapy Vol. 26 No 12 2018 2838-2847 Summary of the Invention [Problem to be solved by the invention]

[0019] The present invention aims to provide a method for continuously producing exosomes of uniform quality at high yields over a long period of time using cells, an exosome-producing device, a kit, and uses thereof, as well as exosomes produced using the same. [Means for solving the problem]

[0020] The present inventors have found that a module constructed by housing a polymer porous membrane in a casing is suitable for mass cell culture and cell removal (Patent Document 10). As a result of intensive research aimed at solving the above-mentioned problem of providing a method capable of easily and efficiently producing exosomes in cells, a cell culture system, and exosomes produced by the cell culture system with stable quality, the inventors have found that by culturing cells using a module equipped with such a polymer porous membrane and a casing, it is possible to produce exosomes with stable production amount and quality over a long period of time, thereby arriving at the present invention.

[0021] Surprisingly, by using the method of the present invention, cultured cells can continuously produce exosomes of uniform quality over several months. This is extremely advantageous for producing exosomes to be used as biologically active substances on an industrial scale, since a production system for exosomes with desired properties can be used continuously for a long period of time once established, and exosomes of uniform quality can be prepared in large quantities.

[0022] Therefore, one of the features of the present invention is that cells are cultured using a module equipped with a porous polymer membrane and a casing.

[0023] Cells grow stably and three-dimensionally by utilizing the large-diameter interconnecting pores of the polymer porous membrane through which cells can pass, and even if the membrane contains a large number of cells compared to flat culture, the interconnecting pores ensure contact with the medium, allowing the cells to continue growing stably. Furthermore, the thin film characteristics, flexibility, shape stability, and free formability of the polymer porous membrane make it possible to coexist a large number of sheets in a small unit space or to stack multiple membranes, and the ultra-heat resistance and solvent resistance of the polymer porous membrane make it possible to sterilize the sheets easily and quickly by multiple means. Furthermore, the three-dimensional scaffold structure of the polymer porous membrane is expected to improve the amount of exosome production per cell compared to flat culture.

[0024] Although not limited thereto, the present invention preferably includes the following aspects. 1. A method for producing exosomes using cells, comprising: applying the cells to the cell culture module and culturing the cells; and Producing exosomes by cells; wherein the cell culture module comprises: A polymer porous membrane; A casing having two or more medium inlets and an inlet port, the polymer porous membrane being housed therein; The method comprising: 2. The polymer porous membrane is a three-layer structure polymer porous membrane having surface layers A and B having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B, wherein the average pore size of the pores in the surface layer A is smaller than the average pore size of the pores in the surface layer B, the macrovoid layer has partition walls bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition walls and the surface layers A and B, and the pores in the surface layers A and B communicate with the macrovoids, Here, in the casing (i) Two or more independent polymer porous membranes are aggregated, (ii) the porous polymer membrane is folded, (iii) the polymer porous membrane is wound into a roll, and / or (iv) the polymer porous membrane is knotted into a rope-like shape; The method according to item 1, which is contained in 3. The method according to any one of items 1 and 2, wherein the diameter of the medium inlet / outlet is larger than the diameter of the cells and smaller than the diameter through which the polymeric porous membrane flows. 4. The method according to any one of items 1 to 3, wherein the casing has a mesh-like structure. 5. The method according to any one of items 1 to 4, wherein the casing is made of a non-flexible material. 6. The method according to any one of items 1 to 5, wherein the polymer porous membrane has a plurality of pores having an average pore size of 0.01 to 100 μm. 7. The method according to any one of items 2 to 6, wherein the average pore size of the surface layer A is 0.01 to 50 μm. 8. The method according to any one of items 2 to 7, wherein the average pore size of the surface layer B is 20 to 100 μm. 9. The method according to any one of items 1 to 8, wherein the total thickness of the polymer porous membrane is 5 to 500 μm. 10. The method according to any one of items 1 to 9, wherein the polymer porous membrane is a polyimide porous membrane. 11. The method according to item 10, wherein the polyimide porous film is a polyimide porous film comprising a polyimide obtained from a tetracarboxylic dianhydride and a diamine. 12. The method according to any one of items 10 and 11, wherein the polyimide porous film is a colored polyimide porous film obtained by molding a polyamic acid solution composition containing a polyamic acid solution obtained from a tetracarboxylic dianhydride and a diamine and a colored precursor, and then heat-treating the resulting product at 250°C or higher. 13. The method according to any one of items 1 to 12, wherein the step of culturing the cells is carried out under static culture conditions. 14. The method according to any one of items 1 to 12, wherein the step of culturing the cells is carried out under rotary culture or agitation conditions. 15. The method according to any one of items 1 to 14, wherein the step of culturing the cells is carried out continuously. 16. The step of culturing the cells is carried out in a cell culture device installed in an incubator, wherein the cell culture device includes a culture unit that houses the cell culture module that supports the cells, the culture unit having a culture medium inlet and a culture medium outlet, a culture medium supply unit that includes a culture medium storage container, a culture medium supply line, and a liquid delivery pump that delivers the culture medium through the culture medium supply line, wherein a first end of the culture medium supply line contacts the culture medium in the culture medium storage container, and a second end of the culture medium supply line communicates with the inside of the culture unit through the culture medium supply port of the culture medium supply unit; 16. The method according to any one of items 1 to 15, comprising: 17. The method according to item 16, wherein the cell culture device does not have a medium supply line, a liquid delivery pump, an air supply port, an air exhaust port, and an oxygen-permeable membrane. 18. The method according to any one of items 1 to 17, wherein the cells are selected from the group consisting of pluripotent stem cells, tissue stem cells, somatic cells, germ cells, sarcoma cells, established cell lines and transformed cells. 19. The method according to any one of items 1 to 18, wherein the cells are selected from the group consisting of human mesenchymal stem cells, osteoblasts, chondrocytes, and cardiomyocytes. 20. The method according to any one of items 1 to 19, wherein the step of producing exosomes overlaps with at least a part of the step of culturing the cells. 21. The method according to any one of items 1 to 20, wherein the step of producing exosomes is continued for a period of 1 month, 2 months, 3 months, or 6 months or more. 22. An exosome production apparatus for use in the method according to any one of items 1 to 21, comprising the cell culture module. 23. A kit for use in the method according to any one of items 1 to 21, comprising the cell culture module. 24. Use of the cell culture module for the method according to any one of items 1 to 21. 25. Exosomes obtained by the method according to any one of items 1 to 21, comprising the cell culture module. Effect of the Invention

[0025] By using a modularized polymer porous membrane housed in a casing, suspended cells can be efficiently adsorbed, enabling stable long-term cell culture using conventional suspension culture vessels, etc. By enabling cells to be mass-cultured stably and continuously for a long period of time under appropriate conditions, a stable and efficient exosome production system can be constructed. [Brief description of the drawings]

[0026] [Figure 1] Figure 1 shows one embodiment of a cell culture module. A polymeric porous membrane is housed within a casing. [Diagram 2] Figure 2 shows one embodiment of a cell culture module. A polymeric porous membrane is housed within a casing. [Diagram 3] Figure 3 shows an embodiment of a cell culture module. (A) A polymer porous membrane is housed in a mesh casing. (B) An embodiment of a casing consisting of a mesh net and a framework is shown. [Figure 4] Figure 4 shows an embodiment of a device used in conjunction with a cell culture module in a spinner flask. (A) Rotational device. The device to which the cell culture module is applied is placed in a spinner flask, and the rotational device itself is rotated for use. (B) Fixed device. The device to which the cell culture module is applied is placed at the bottom of a spinner flask. The stirrer of the spinner flask is rotated in the space at the center of the device for use. [Diagram 5] Figure 5 shows an embodiment in which a cell culture module was applied to a flexible bag-type culture vessel for culture. The phenol red in the culture medium turned yellow (right) one hour after the start of culture (left). [Figure 6] FIG. 6 shows one embodiment of the use of a mesh module during shaking culture. [Figure 7] FIG. 7 shows a model diagram of cell culture using a polyimide porous membrane. [Figure 8] FIG. 8 is a diagram showing a dry-heat sterilization type siphon-type cell culture device (heat-resistant siphon-type reactor). [Figure 9] FIG. 9 is a diagram showing a siphon-type cell culture device. [Figure 10] 10 shows a cylindrical gas phase culture apparatus. (A) is a diagram showing the configuration of the cell culture apparatus. (B) is a diagram showing the cell culture section on which the cell culture device in (A) is placed. [Figure 11] Figure 11 shows a cylindrical gas phase culture device. The basic structure is the same as that of Figure 10, but the medium outlets of each stage are shifted by 30 degrees counterclockwise, making it easier for the medium to be discharged. [Figure 12] FIG. 12 shows a mist and shower type incubation device. [Figure 13] 13 shows an exposed-gas-phase type rotary culture apparatus. (A) shows the configuration of the exposed-gas-phase type rotary culture apparatus. (B) shows a usage mode of the exposed-gas-phase type rotary culture apparatus. [Figure 14] Figure 14 shows the cell culture section of the gas-phase exposed rotary culture device. (A) is a schematic diagram of the cell culture section, and (B) is a diagram showing how the culture section is used. (A) After 2 days of static culture, (B) After 2 days of shaking culture (without mesh). [Figure 15] 15 is a diagram showing a WAVE-type bioreactor to which the cell culture module of the present invention is applied in one embodiment, showing (A) a bag in which the cell culture module is enclosed, and (B) a process of adsorbing cells to the cell culture module using WAVE25. [Figure 16] 16 is a diagram showing a cell culture device in one embodiment of the present invention, showing (A) a cell culture module, (B) a cell culture section, and (C) a cell culture device in one embodiment. [Figure 17] FIG. 17 is a diagram showing a cell culture device in one embodiment. [Figure 18]Figure 18 shows the results of an exosome production test in cell culture using dishes. (A) shows the results of measuring the number of exosomes produced per day in the cell culture medium collected on days 6 and 13 of culture, and (B) shows the results of measuring the particle size distribution on day 13 of culture. [Figure 19] Figure 19 shows the results of an exosome production test in cell culture using a module in a 150 mL round storage bottle. (A) shows the measurement results of the number of exosomes produced per day in the cell culture medium up to day 27 of culture, and (B) shows the particle size distribution measurement results on day 27 of culture. [Figure 20] Figure 20 shows the results of an exosome production test in cell culture using a module in a 150 mL round storage bottle. (A) shows the measurement results of the number of exosomes produced per day in the cell culture medium up to day 27 of culture, and (B) shows the particle size distribution measurement results on day 27 of culture. [Figure 21] FIG. 21 shows a cell culture system (5% oxygen) using a module in an overflow reactor, where (A) shows the time course of glucose consumption and lactate production, and (B) shows the state of cell culture. [Figure 22] Figure 22 shows the results of an exosome production test in cell culture (5% oxygen) using a module in an overflow reactor. (A) shows the measurement results of the number of exosomes produced per day in the cell culture medium up to 85 days of culture, and (B) shows the particle size distribution measurement results on the 85th day of culture. [Diagram 23] FIG. 23 shows a cell culture system (normal oxygen) using a module in an overflow reactor, where (A) shows the time course of glucose consumption and lactate production, and (B) shows the state of cell culture. [Figure 24] Figure 24 shows the results of an exosome production test in cell culture (normal oxygen) using a module in an overflow reactor. (A) shows the measurement results of the number of exosomes produced per day in the cell culture medium up to day 85 of culture, and (B) shows the particle size distribution measurement results on day 85 of culture. [Diagram 25]Figure 25(A) shows the time course of glucose consumption and lactate production in a cell culture system using a module in a WAVE reactor. Figure 25(B) and (C) show the results of an exosome production test in cell culture using a module in a WAVE reactor. (B) shows the results of measuring the number of exosomes produced per day in the cell culture medium up to the 39th day of culture, and (C) shows the results of measuring the particle size distribution on the 39th day of culture. [Figure 26] FIG. 26 shows a comparison of the exosome productivity of one cell on the 13th day of culture in Comparative Example 1 and Examples 1 to 4. [Figure 27] FIG. 27 shows a comparison of the amount of exosome produced per cell between Comparative Example 1 (Dish_Day 6) and Example 1 (Bottle (5% oxygen)_Day 27). [Figure 28] FIG. 28 shows a scatterplot of the miRNA array analysis of Comparative Example 1 (Dish_Day 6) and Example 1 (Bottle (5% oxygen)_Day 27). [Figure 29] Figure 29 shows a Scatterplot of the miRNA array analysis of hsa-miR-146a, hsa-miR-210, hsa-miR-22 and hsa-miR-24. [Diagram 30] Figure 30 shows the results of miRNA array analysis of hsa-miR-34b, hsa-miR-132, hsa-miR-203, hsa-miR-137 and hsa-miR-193. [Diagram 31] Figure 31 shows the results of miRNA array analysis of hsa-miR-26a / b, hsa-miR-23b, hsa-miR-27b and hsa-miR-24-1. [Diagram 32] FIG. 32 shows a comparison of the amount of exosome produced per cell between Comparative Example 1 (Dish_Day 13) and Example 1 (Bottle (5% oxygen)_Day 27). [Diagram 33] FIG. 33 shows a scatterplot of the miRNA array analysis of Comparative Example 1 (Dish_Day 13) and Example 1 (Bottle (5% oxygen)_Day 27). [Diagram 34]Figure 34 shows the results of miRNA array analysis of hsa-miR-146a, hsa-miR-210, hsa-miR-22 and hsa-miR-24. [Diagram 35] Figure 35 shows the results of miRNA array analysis of hsa-miR-34b, hsa-miR-132, hsa-miR-203, hsa-miR-137 and hsa-miR-193. [Diagram 36] Figure 36 shows the results of miRNA array analysis of hsa-miR-26a / b, hsa-miR-23b, hsa-miR-27b and hsa-miR-24-1. [Figure 37] FIG. 37 shows a comparison of the amount of exosome produced per cell between Comparative Example 1 (Dish_Day 6) and Example 2 (Bottle (normal oxygen)_Day 27). [Figure 38] FIG. 38 shows a scatterplot of the miRNA array analysis of Comparative Example 1 (Dish_Day 6) and Example 2 (Bottle (normal oxygen)_Day 27). [Figure 39] Figure 39 shows the results of miRNA array analysis of hsa-miR-146a, hsa-miR-210, hsa-miR-22 and hsa-miR-24. [Diagram 40] Figure 40 shows the results of miRNA array analysis of hsa-miR-34b, hsa-miR-132, hsa-miR-203, hsa-miR-137 and hsa-miR-193. [Diagram 41] Figure 41 shows the results of miRNA array analysis of hsa-miR-26a / b, hsa-miR-23b, hsa-miR-27b and hsa-miR-24-1. [Diagram 42] FIG. 42 shows a comparison of the amount of exosome produced per cell between Comparative Example 1 (Dish_Day 13) and Example 2 (Bottle (normal oxygen)_Day 27). [Diagram 43] FIG. 43 shows a scatterplot of the miRNA array analysis of Comparative Example 1 (Dish_Day 13) and Example 2 (Bottle (normal oxygen)_Day 27). [Diagram 44] Table 44 shows the results of miRNA array analysis of hsa-miR-146a, hsa-miR-210, hsa-miR-22 and hsa-miR-24. [Diagram 45] Figure 45 shows the results of miRNA array analysis of hsa-miR-34b, hsa-miR-132, hsa-miR-203, hsa-miR-137 and hsa-miR-193. [Figure 46] Figure 46 shows the results of miRNA array analysis of hsa-miR-26a / b, hsa-miR-23b, hsa-miR-27b and hsa-miR-24-1. [Figure 47] FIG. 47 shows a comparison of the amount of exosome produced per cell between Comparative Example 1 (Dish_Day 6) and Example 3 (Reactor (5% oxygen)_Day 27). [Figure 48] FIG. 48 shows a scatterplot of the miRNA array analysis of Comparative Example 1 (Dish_Day 6) and Example 3 (Reactor (5% oxygen)_Day 27). [Figure 49] Figure 49 shows the results of miRNA array analysis of hsa-miR-146a, hsa-miR-210, hsa-miR-22 and hsa-miR-24. [Figure 50] Figure 50 shows the results of miRNA array analysis of hsa-miR-34b, hsa-miR-132, hsa-miR-203, hsa-miR-137 and hsa-miR-193. [Figure 51] Figure 51 shows the results of miRNA array analysis of hsa-miR-26a / b, hsa-miR-23b, hsa-miR-27b and hsa-miR-24-1. [Figure 52] FIG. 52 shows a comparison of the amount of exosome produced per cell between Comparative Example 1 (Dish_Day 13) and Example 3 (Reactor (5% oxygen)_Day 27). [Diagram 53] FIG. 53 shows a scatterplot of the miRNA array analysis of Comparative Example 1 (Dish_Day 13) and Example 3 (Reactor (5% oxygen)_Day 27). [Figure 54] Figure 54 shows the results of miRNA array analysis of hsa-miR-146a, hsa-miR-210, hsa-miR-22 and hsa-miR-24. [Figure 55] Figure 55 shows the results of miRNA array analysis of hsa-miR-34b, hsa-miR-132, hsa-miR-203, hsa-miR-137 and hsa-miR-193. [Figure 56] Figure 56 shows the results of miRNA array analysis of hsa-miR-26a / b, hsa-miR-23b, hsa-miR-27b and hsa-miR-24-1. [Figure 57] FIG. 57 shows a comparison of the amount of exosome produced per cell between Comparative Example 1 (Dish_Day 6) and Example 4 (Reactor (normal oxygen)_Day 27). [Figure 58] FIG. 58 shows a scatterplot of the miRNA array analysis of Comparative Example 1 (Dish_Day 6) and Example 4 (Reactor (normal oxygen)_Day 27). [Figure 59] Figure 59 shows the results of miRNA array analysis of hsa-miR-146a, hsa-miR-210, hsa-miR-22 and hsa-miR-24. [Figure 60] Figure 60 shows the results of miRNA array analysis of hsa-miR-34b, hsa-miR-132, hsa-miR-203, hsa-miR-137 and hsa-miR-193. [Figure 61] Figure 61 shows the results of miRNA array analysis of hsa-miR-26a / b, hsa-miR-23b, hsa-miR-27b and hsa-miR-24-1. [Figure 62] FIG. 62 shows a comparison of the amount of exosome produced per cell between Comparative Example 1 (Dish_Day 13) and Example 4 (Reactor (normal oxygen)_Day 27). [Figure 63]FIG. 63 shows a scatterplot of the miRNA array analysis of Comparative Example 1 (Dish_Day 13) and Example 4 (Reactor (normal oxygen)_Day 27). [Figure 64] Figure 64 shows the results of miRNA array analysis of hsa-miR-146a, hsa-miR-210, hsa-miR-22 and hsa-miR-24. [Figure 65] Figure 65 shows the results of miRNA array analysis of hsa-miR-34b, hsa-miR-132, hsa-miR-203, hsa-miR-137 and hsa-miR-193. [Figure 66] Figure 66 shows the results of miRNA array analysis of hsa-miR-26a / b, hsa-miR-23b, hsa-miR-27b and hsa-miR-24-1. [Figure 67] FIG. 67 is a graph showing the time course of lactate production and glucose consumption. [Figure 68] FIG. 68 is a graph showing the particle size distribution of five types of exosome samples obtained. [Figure 69] FIG. 69 shows a transmission electron microscope image of exosomes obtained by the method of the present invention in one embodiment. [Figure 70] FIG. 70 is a graph showing the amount of exosomes produced over time in various reactors. [Figure 71] FIG. 71 shows the results of miRNA array analysis (left: overall view of human miRNAs; right: selection of miR-21, 22, 23, and 24). [Figure 72] FIG. 72 is a graph showing the time course of lactate production and glucose consumption. [Figure 73] FIG. 73 shows the results of miRNA array analysis (left: overall view of human miRNAs; right: selection of miR-21, 22, 23, and 24). [Figure 74] FIG. 74 is a graph showing the time course of lactate production and glucose consumption. [Figure 75]FIG. 75 is a graph showing the time-dependent changes in cell density (left) and total cell number of human chondrocytes cultured in modules or small piece porous membranes. [Figure 76] FIG. 76 shows the results of miRNA array analysis (left: overall view of human miRNAs; right: selection of miR-21, 22, 23, and 24). [Figure 77] Figure 77 shows the usage mode of the WAVE reactor used in Example 5. Left: HDPE cap with liquid withdrawal tube used for the WAVE culture bag. Right: WAVE culture bag with HDPE cap with liquid withdrawal tube attached. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] I. Methods for Producing Exosomes The present invention relates to a method for producing exosomes using cells. The method of the present invention includes culturing cells in a module formed by housing a porous polymer membrane in a casing, and causing the cells to produce exosomes.

[0028] 1.Cells The type of cells that can be used in the method of the present invention is not particularly limited as long as they produce exosomes. It is known that exosomes are produced in various cells, and the profile of the exosomes they produce also varies depending on the cells of origin and the conditions during production. Therefore, in the present invention, cells that can produce exosomes with the desired quality and yield can be appropriately selected based on each embodiment.

[0029] In one embodiment, in the present invention, pluripotent stem cells are used as cultured cells. Pluripotent stem cells are intended to be a general term for stem cells that have the ability to differentiate into cells of any tissue (differentiation pluripotency). Pluripotent stem cells include, but are not limited to, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ stem cells (EG cells), germ stem cells (GS cells), and the like. ES cells or iPS cells are preferred. iPS cells are particularly preferred for reasons such as the absence of ethical issues. Any known pluripotent stem cells can be used, but for example, the pluripotent stem cells described in International Publication WO2009 / 123349 (PCT / JP2009 / 057041) can be used.

[0030] In one embodiment, in the present invention, tissue stem cells are used as cultured cells. The term "tissue stem cells" refers to stem cells whose cell lineage is limited to a specific tissue, but which have the ability to differentiate into various cell types (multipotency). For example, hematopoietic stem cells in bone marrow become blood cells, and neural stem cells differentiate into nerve cells. There are various other types, such as hepatic stem cells that create the liver and skin stem cells that become skin tissue. Preferably, the tissue stem cells are selected from mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, neural stem cells, skin stem cells, and hematopoietic stem cells.

[0031] In one embodiment, somatic cells are used as cultured cells in the present invention. Somatic cells refer to cells other than germ cells that constitute a multicellular organism. In sexual reproduction, somatic cells are not passed on to the next generation. Preferably, the somatic cells are selected from hepatocytes, pancreatic cells, muscle cells, bone cells, osteoblasts, osteoclasts, chondrocytes, adipocytes, skin cells, fibroblasts, pancreatic cells, kidney cells, lung cells, or blood cells such as lymphocytes, erythrocytes, leukocytes, monocytes, macrophages, or megakaryocytes.

[0032] In one embodiment, in the present invention, reproductive cells are used as cultured cells. Reproductive cells refer to cells that play a role in transmitting genetic information to the next generation in reproduction. For example, gametes for sexual reproduction, i.e., eggs, egg cells, sperm, sperm cells, and spores for asexual reproduction are included.

[0033] The cells may be selected from the group consisting of sarcoma cells, cell lines, and transformed cells. "Sarcoma" refers to cancer occurring in connective tissue cells derived from non-epithelial cells such as bone, cartilage, fat, muscle, and blood, and includes soft tissue sarcoma, malignant bone tumor, and the like. Sarcoma cells are cells derived from sarcoma. "Cell lines" refer to cultured cells that have been maintained outside the body for a long period of time, have acquired certain stable properties, and can be subcultured semi-permanently. There are cell lines derived from various tissues of various species, including humans, such as PC12 cells (derived from rat adrenal medulla), CHO cells (derived from Chinese hamster ovary), HEK293 cells (derived from human fetal kidney), HL-60 cells (derived from human white blood cells), HeLa cells (derived from human cervical cancer), Vero cells (derived from African green monkey kidney epithelial cells), MDCK cells (derived from dog kidney tubular epithelial cells), and HepG2 cells (derived from human hepatoma). The term "transformed cell" refers to a cell whose genetic properties have been altered by introducing nucleic acid (such as DNA) from outside the cell.

[0034] Exosomes are known to be actively produced in undifferentiated cells such as stem cells and cancer cells, and in advantageous cases, stem cells are used as cultured cells in the methods of the present invention.

[0035] 2. Exosomes In the present invention, exosomes refer to membranous vesicle structures of 50 to 300 nm secreted extracellularly by cells. Since the type of cells from which exosomes are derived and the conditions under which they are produced affect the quality of exosomes, the conditions under which exosomes can be produced with the desired quality and yield can be appropriately selected in the method of the present invention. When advantageous, the obtained exosomes are evaluated for quality suitable for their intended use, such as use as a drug delivery carrier or a physiologically active substance.

[0036] 3. Cell Culture Module The cell culture module used in the method of the present invention comprises: A polymer porous membrane; A casing having two or more medium inlets and an inlet port, the polymer porous membrane being housed therein; wherein the polymer porous membrane is a three-layer structure polymer porous membrane having surface layers A and B having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B, wherein the average pore size of the pores present in the surface layer A is smaller than the average pore size of the pores present in the surface layer B, the macrovoid layer has partition walls bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition walls and the surface layers A and B, and the pores in the surface layers A and B communicate with the macrovoids, and wherein (i) Two or more independent polymer porous membranes are aggregated, (ii) the porous polymer membrane is folded, (iii) the polymer porous membrane is wound into a roll, and / or (iv) the polymer porous membrane is knotted into a rope-like shape; The cell culture module is hereinafter also referred to as the "cell culture module of the present invention." In this specification, the term "cell culture module" can be simply referred to as "module," and these terms have the same meaning even when interchanged.

[0037] In this specification, the term "cell culture module" refers to a cell culture substrate applicable to cell culture vessels, cell culture devices, and cell culture systems, particularly cell culture vessels, cell culture devices, and cell culture systems used for suspension culture. Some embodiments of the cell culture module are shown in Figs. 1 to 3 and Fig. 16(A). The cell culture module of the present invention can be used in the embodiments shown in Figs. 4 to 6 and 8 to 17. It can also be used in the embodiments shown in the examples described later.

[0038] In the cell culture module of the present invention, the polymer porous membrane is housed in a casing, which prevents the membrane-like polymer porous membrane from continuously deforming within the casing, thereby preventing stress from being applied to the cells grown within the polymer porous membrane and suppressing apoptosis and the like, making it possible to stably culture a large number of cells.

[0039] The casing of the cell culture module of the present invention has two or more medium inlets, so that the cell culture medium is supplied to the inside of the casing and discharged to the outside. The diameter of the medium inlet of the casing is preferably larger than the diameter of the cells so that the cells can flow into the inside of the casing. In addition, the diameter of the medium inlet is preferably smaller than the diameter at which the polymer porous membrane flows out from the medium inlet. The diameter smaller than the diameter at which the polymer porous membrane flows out can be appropriately selected depending on the shape and size of the polymer porous membrane contained in the casing. For example, when the polymer porous membrane is string-shaped, there is no particular limitation as long as it is smaller than the width of the short side of the polymer porous membrane and has an appropriate diameter at which the polymer porous membrane does not flow out. The number of the medium inlet and outlet is preferably as many as possible so that the cell culture medium can be easily supplied and / or discharged to the inside and outside of the casing. The number is preferably 5 or more, preferably 10 or more, preferably 20 or more, preferably 50 or more, and preferably 100 or more. The medium inlet and outlet of the casing may have a mesh-like structure in part or all of the casing. In addition, the casing itself may be mesh-like. In the present invention, a mesh-shaped structure refers to, for example, a vertical, horizontal, and / or diagonal lattice structure in which each opening forms a medium inlet / outlet port large enough for fluid to pass through, but is not limited to this.

[0040] The casing of the cell culture module of the present invention is preferably strong enough not to be deformed by the movement of the culture medium under normal culture conditions, such as stirring culture and shaking culture conditions, and is preferably made of a non-flexible material. In addition, the casing is preferably made of a material that does not affect the growth of cells in cell culture. Examples of such materials include, but are not limited to, polymers such as polyethylene, polypropylene, nylon, polyester, polystyrene, polycarbonate, polymethyl methacrylate, and polyethylene terephthalate, and metals such as stainless steel and titanium. If the casing has a certain degree of strength, the shape of the polymer porous membrane inside the casing is prevented from being continuously deformed, and the effects of the present invention are more effectively exhibited. In this specification, "the casing does not deform" means that it does not deform under the load received under a normal culture environment, and does not mean that it absolutely does not deform.

[0041] The cell culture module of the present invention includes a casing. (i) Two or more independent polymer porous membranes are aggregated, (ii) the porous polymer membrane is folded, (iii) the polymer porous membrane is wound into a roll, and / or (iv) the polymer porous membrane is knotted into a rope-like shape; It is contained.

[0042] In this specification, "two or more independent polymer porous membranes are aggregated and housed in a casing" refers to a state in which two or more independent polymer porous membranes are aggregated and housed in a certain space surrounded by a casing. In the present invention, the two or more independent polymer porous membranes may be fixed by any method at least one place of the polymer porous membrane and at least one place in the casing, so that the polymer porous membrane is fixed in a state in which it does not move in the casing. In addition, the two or more independent polymer porous membranes may be small pieces. The shape of the small pieces may be any shape, such as a circle, an ellipse, a square, a triangle, a polygon, a string, etc., but is preferably approximately square. In the present invention, the size of the small pieces may be any size, but when it is approximately square, the length may be any length, but for example, it is preferably 80 mm or less, preferably 50 mm or less, more preferably 30 mm or less, even more preferably 20 mm or less, and may be 10 mm or less. In addition, when the small piece of the polymer porous membrane is substantially square, the length of one side may be formed along the inner wall of the casing or shorter than the length of one side of the inner wall (for example, about 0.1 mm to 1 mm shorter) so that the polymer porous membrane does not move in the casing. This prevents stress from being applied to the cells grown in the polymer porous membrane, suppresses apoptosis, etc., and enables stable and large-scale cell culture. In the case of a string-shaped polymer porous membrane, as described below, (ii) the polymer porous membrane may be folded, (iii) the polymer porous membrane may be wound into a roll, and / or (iv) the polymer porous membrane may be tied into a rope and housed in the casing. In addition, in order to house two or more independent polymer porous membranes in a concentrated manner in the casing, any number of polymer porous membranes may be stacked. In this case, an insole may be provided between the polymer porous membranes. By providing an insole, the culture medium can be efficiently supplied between the stacked polymer porous membranes.The insole is not particularly limited as long as it has the function of forming an arbitrary space between the laminated polymer porous membranes and efficiently supplying the culture medium, and for example, a planar structure having a mesh structure can be used. The material of the insole can be, for example, polystyrene, polycarbonate, polymethyl methacrylate, polyethylene terephthalate, stainless steel mesh, but is not limited thereto. When the insole has a mesh structure, it is sufficient that the mesh has a mesh size large enough to supply the culture medium between the laminated polymer porous membranes, and can be appropriately selected.

[0043] In this specification, the term "folded porous polymer membrane" refers to a porous polymer membrane that is folded in the casing and is immobilized in the casing due to friction between each surface of the porous polymer membrane and / or the surface inside the casing. In this specification, "folded" may refer to a state in which the porous polymer membrane has creases or does not have creases.

[0044] In this specification, the term "polymer porous membrane wound into a roll" refers to a polymer porous membrane that is wound into a roll and does not move in the casing due to friction between each surface of the polymer porous membrane and / or the surface inside the casing. In addition, in the present invention, the term "polymer porous membrane woven into a rope" refers to a polymer porous membrane in which, for example, a plurality of strip-shaped polymer porous membranes are woven into a rope by any method and do not move relative to each other due to friction between the polymer porous membranes. (i) A polymer porous membrane in which two or more independent polymer porous membranes are aggregated, (ii) a folded polymer porous membrane, (iii) a polymer porous membrane wound into a roll, and (iv) a polymer porous membrane tied into a rope may be combined and housed in a casing.

[0045] In this specification, "a state in which the polymer porous membrane does not move within the casing" refers to a state in which the polymer porous membrane is housed within the casing so that it does not continuously change shape when the cell culture module is cultured in a cell culture medium. In other words, the polymer porous membrane itself is restrained by the fluid so that it does not continuously undulate. Since the polymer porous membrane is kept in a state in which it does not move within the casing, stress is prevented from being applied to the cells growing within the polymer porous membrane, and the cells can be stably cultured without the cells being killed by apoptosis or the like.

[0046] The cell culture module of the present invention can be applied to commercially available culture devices and systems for culturing cells. For example, it can be applied to a culture device in which the culture vessel is made of a flexible bag, and can be used in a state where the cells are suspended in the culture vessel. The cell culture module of the present invention can be applied to an agitation culture vessel such as a spinner flask, and can be cultured. In addition, the cell culture module can be applied to an open vessel or a closed vessel as the culture vessel. For example, a petri dish, flask, plastic bag, test tube, or large tank for cell culture can be appropriately used. For example, the cell culture dish made by BD Falcon and the Nunc Cell Factory made by Thermo Scientific are included.

[0047] 4. Application of cell culture module to cell culture device In this specification, the term "cell culture device" is generally a term that is treated as synonymous with a cell culture system, a bioreactor, or a reactor, and has the same meaning even when used interchangeably. The cell culture module of the present invention can be applied to the cell culture devices exemplified below. It can also be applied to commercially available devices other than the devices exemplified below.

[0048] (1) Siphon-type culture device The cell culture module of the present invention is applicable to a siphon-type culture device shown in Figures 8 and 9. The siphon-type culture device includes a polymer porous membrane, a cell culture section in which the polymer porous membrane is housed, a liquid reservoir section in which the cell culture section is housed, a culture medium supplying means disposed in the upper part of the liquid reservoir section, an inverted U-shaped tube communicated with the bottom of the liquid reservoir section, a culture medium collecting means installed at the lower part of the other end of the inverted U-shaped tube, and a culture medium discharging means disposed in the culture medium collecting means, wherein the polymer porous membrane is a three-layer structure polymer porous membrane having surface layers A and B having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B. wherein the average pore size of the pores present in the surface layer A is smaller than the average pore size of the pores present in the surface layer B, the macrovoid layer has a partition wall bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition wall and the surface layers A and B, the pores in the surface layers A and B communicate with the macrovoids, and when the liquid level of the culture medium supplied from the culture medium supply means to the liquid reservoir reaches the top of the inverted U-shaped tube, the culture medium is intermittently discharged to the culture medium recovery means according to the siphon principle. In the cell culture device, the polymer porous membrane can be used in place of a cell culture module.

[0049] (2) Cylindrical gas phase culture device The cell culture module of the present invention is applicable to a cylindrical gas phase culture device shown in Figures 10, 11 and 17. In one embodiment, the cylindrical gas phase culture device is a cell culture device comprising a polymer porous membrane, a cell culture section in which the polymer porous membrane is housed, a medium supplying means arranged in the upper part of the cell culture section, and a medium recovery means arranged in the lower part of the cell culture section, wherein the polymer porous membrane is a three-layered polymer porous membrane having surface layers A and B having a plurality of holes, and a macrovoid layer sandwiched between the surface layers A and B, wherein the average pore size of the holes present in the surface layer A is smaller than the average pore size of the holes present in the surface layer B, and the macrovoid layer has a partition wall bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition wall and the surface layers A and B, wherein the cell culture section is a bottom portion having one or more medium outlets, and a side portion arranged approximately perpendicular to the bottom. In one embodiment, the cylindrical gas phase culture device is a cell culture device comprising a polymer porous membrane, a cell culture section containing the polymer porous membrane, a medium supplying means arranged in the upper part of the cell culture section, and a medium collecting means arranged in the lower part of the cell culture section, wherein the polymer porous membrane has a three-layer structure having surface layers A and B having a plurality of holes, and a macrovoid layer sandwiched between the surface layers A and B, wherein the average pore size of the holes in the surface layer A is smaller than the average pore size of the holes in the surface layer B, the macrovoid layer has a partition wall bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition wall and the surface layers A and B, the pores in the surface layers A and B communicate with the macrovoids, and the medium collecting means is a part of an outer cylinder containing the cell culture section. In this device, the polymer porous membrane can be used in place of a cell culture module.

[0050] (3) Mist and shower type culture equipment The cell culture module of the present invention is applicable to a mist and shower type culture device shown in Fig. 12. The mist and shower type culture device includes a polymer porous membrane, a polymer porous membrane mounting part on which the polymer porous membrane is mounted, a housing in which the polymer porous membrane mounting part is housed, a liquid medium supply part disposed inside the housing, a medium supply line communicating with the liquid medium supply part, a medium storage part communicating with the medium supply line, and a pump provided in a part of the medium supply line, wherein the polymer porous membrane is sandwiched between surface layers A and B having a plurality of pores, and The cell culture device is a polymer porous membrane having a three-layer structure having a macrovoid layer surrounded by a partition wall and a surface layer B, wherein the average pore size of the pores in the surface layer A is smaller than the average pore size of the pores in the surface layer B, the macrovoid layer has a partition wall bonded to the surface layers A and B and a plurality of macrovoids surrounded by the partition wall and the surface layers A and B, the pores in the surface layers A and B communicate with the macrovoids, and the polymer porous membrane mounting part has a plurality of slit-shaped or mesh-shaped medium outlets. In the device, the polymer porous membrane can be used in place of a cell culture module.

[0051] (4) Gas-phase exposed type rotary culture device The cell culture module of the present invention can be applied to the rotary culture apparatus shown in FIGS. The gas-phase exposed rotary culture device is a cell culture device comprising a polymer porous membrane, a cell culture section having the polymer porous membrane, a shaft penetrating the cell culture section, a rotary motor for rotating the shaft, and a medium tank for immersing at least a part of the cell culture section, wherein the polymer porous membrane is a three-layer structure having surface layers A and B having a plurality of holes, and a macrovoid layer sandwiched between the surface layers A and B, wherein the average pore size of the holes in the surface layer A is smaller than the average pore size of the holes in the surface layer B, the macrovoid layer has a partition wall bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition wall and the surface layers A and B, the pores in the surface layers A and B communicate with the macrovoids, the cell culture section rotates around the shaft, and the cells supported on the polymer porous membrane are cultured alternately in the gas phase and the liquid phase. In the device, the polymer porous membrane can be used in place of a cell culture module.

[0052] 5.Polymer Porous Membranes The average pore size of the pores present in the surface layer A (hereinafter also referred to as "side A" or "mesh side") in the polymer porous membrane used in the present invention is not particularly limited, and is, for example, 0.01 μm or more and less than 200 μm, 0.01 to 150 μm, 0.01 to 100 μm, 0.01 to 50 μm, 0.01 μm to 40 μm, 0.01 μm to 30 μm, 0.01 μm to 20 μm, or 0.01 μm to 15 μm, and preferably 0.01 μm to 15 μm.

[0053] The average pore size of the pores present in surface layer B (hereinafter also referred to as "side B" or "large hole side") in the polymer porous membrane used in the present invention is not particularly limited as long as it is larger than the average pore size of the pores present in surface layer A, and is, for example, more than 5 μm and not more than 200 μm, 20 μm to 100 μm, 30 μm to 100 μm, 40 μm to 100 μm, 50 μm to 100 μm, or 60 μm to 100 μm, and preferably 20 μm to 100 μm.

[0054] The average pore size of the polymer porous membrane surface can be determined by measuring the pore area of ​​200 or more openings in a scanning electron microscope photograph of the porous membrane surface, and calculating the average diameter from the average pore area according to the following formula (1), assuming that the pore shape is a perfect circle.

number

[0055] The thickness of the surface layers A and B is not particularly limited, but is, for example, 0.01 to 50 μm, and preferably 0.01 to 20 μm.

[0056] The average pore size of the macrovoids in the macrovoid layer in the polymer porous membrane in the membrane plane direction is not particularly limited, but is, for example, 10 to 500 μm, preferably 10 to 100 μm, and more preferably 10 to 80 μm. The thickness of the partition walls in the macrovoid layer is not particularly limited, but is, for example, 0.01 to 50 μm, and preferably 0.01 to 20 μm. In one embodiment, at least one partition wall in the macrovoid layer has one or more holes with an average pore size of 0.01 to 100 μm, preferably 0.01 to 50 μm, which communicates with adjacent macrovoids. In another embodiment, the partition walls in the macrovoid layer have no pores.

[0057] The total thickness of the polymer porous membrane surface used in the present invention is not particularly limited, but may be 5 μm or more, 10 μm or more, 20 μm or more, or 25 μm or more, and may be 500 μm or less, 300 μm or less, 100 μm or less, 75 μm or less, or 50 μm or less, preferably 5 to 500 μm, and more preferably 25 to 75 μm.

[0058] The thickness of the porous polymer membrane used in the present invention can be measured using a contact type thickness meter.

[0059] The porosity of the porous polymer membrane used in the present invention is not particularly limited, but is, for example, not less than 40% and less than 95%.

[0060] The porosity of the porous polymer membrane used in the present invention can be calculated from the basis weight by measuring the thickness and mass of a porous film cut to a predetermined size according to the following formula (2).

number

[0061] The polymer porous membrane used in the present invention is preferably a three-layer structure polymer porous membrane having surface layers A and B each having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B, wherein the average pore size of the pores in the surface layer A is 0.01 μm to 15 μm, and the average pore size of the pores in the surface layer B is 20 μm to 100 μm, the macrovoid layer has partition walls bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition walls and the surface layers A and B, the partition walls of the macrovoid layer and the surface layers A and B have thicknesses of 0.01 to 20 μm, the pores in the surface layers A and B are connected to the macrovoids, the total thickness is 5 to 500 μm, and the porosity is 40% or more and less than 95%. In one embodiment, at least one partition wall in the macrovoid layer has one or more pores that connect adjacent macrovoids and have an average pore size of 0.01 to 100 μm, preferably 0.01 to 50 μm. In another embodiment, the partition wall does not have such pores.

[0062] The polymer porous membrane used in the present invention is preferably sterilized. The sterilization method is not particularly limited, but may be any sterilization method such as dry heat sterilization, steam sterilization, sterilization with a disinfectant such as ethanol, or electromagnetic sterilization using ultraviolet rays or gamma rays.

[0063] The polymer porous membrane used in the present invention is not particularly limited as long as it has the above-mentioned structural characteristics, but is preferably a polyimide porous membrane or a polyethersulfone (PES) porous membrane.

[0064] 5-1.Porous polyimide film Polyimide is a general term for polymers containing imide bonds in the repeating units, and usually refers to aromatic polyimides in which aromatic compounds are directly linked by imide bonds. Aromatic polyimides have a rigid and strong molecular structure because aromatic compounds have a conjugated structure via imide bonds, and the imide bonds have strong intermolecular forces, so they have very high levels of thermal, mechanical, and chemical properties.

[0065] The polyimide porous film that can be used in the present invention is preferably a polyimide porous film containing (as a main component) a polyimide obtained from a tetracarboxylic dianhydride and a diamine, and more preferably a polyimide porous film consisting of a polyimide obtained from a tetracarboxylic dianhydride and a diamine. "Containing as a main component" means that the polyimide porous film does not essentially contain any component other than the polyimide obtained from the tetracarboxylic dianhydride and the diamine as a constituent component, or may contain such a component, but it is an additional component that does not affect the properties of the polyimide obtained from the tetracarboxylic dianhydride and the diamine.

[0066] In one embodiment, the polyimide porous film that can be used in the present invention also includes a colored polyimide porous film obtained by molding a polyamic acid solution composition containing a polyamic acid solution obtained from a tetracarboxylic acid component and a diamine component and a colored precursor, and then heat-treating the molded product at 250° C. or higher.

[0067] Polyamic acid is obtained by polymerizing a tetracarboxylic acid component and a diamine component. Polyamic acid is a polyimide precursor that can be converted into a polyimide by thermal or chemical imidization to close the ring.

[0068] The polyamic acid may be partially imidized as long as the imidization does not affect the present invention. That is, the polyamic acid may be partially thermally or chemically imidized.

[0069] When the polyamic acid is thermally imidized, an imidization catalyst, an organic phosphorus-containing compound, fine particles such as inorganic fine particles and organic fine particles, etc., can be added to the polyamic acid solution as necessary. When the polyamic acid is chemically imidized, a chemical imidization agent, a dehydrating agent, fine particles such as inorganic fine particles and organic fine particles, etc. can be added to the polyamic acid solution as necessary. It is preferable to carry out the process under conditions in which the color precursor does not precipitate even if the above-mentioned components are mixed into the polyamic acid solution.

[0070] In this specification, the term "colored precursor" refers to a precursor that is partially or completely carbonized by heat treatment at 250° C. or higher to produce a colored product.

[0071] The colored precursor that can be used in the production of the polyimide porous film is preferably one that is uniformly dissolved or dispersed in a polyamic acid solution or a polyimide solution, and is thermally decomposed by heat treatment at 250° C. or higher, preferably 260° C. or higher, more preferably 280° C. or higher, and more preferably 300° C. or higher, preferably in the presence of oxygen such as air, at 250° C. or higher, preferably 260° C. or higher, more preferably 280° C. or higher, and more preferably 300° C. or higher, and carbonized to produce a colored product, more preferably one that produces a black colored product, and more preferably a carbon-based colored precursor.

[0072] When the colored precursor is heated, it becomes what appears to be a carbonized product at first glance, but in terms of structure it contains elements other than carbon, and includes layered structures, aromatic bridged structures, and disordered structures including tetrahedral carbon.

[0073] The carbon-based coloring precursor is not particularly limited, and examples thereof include tar or pitch such as petroleum tar, petroleum pitch, coal tar, and coal pitch, coke, a polymer obtained from a monomer containing acrylonitrile, a ferrocene compound (ferrocene and ferrocene derivatives), etc. Among these, a polymer and / or a ferrocene compound obtained from a monomer containing acrylonitrile is preferred, and as a polymer obtained from a monomer containing acrylonitrile, polyacrylonitrile is preferred.

[0074] In another embodiment, the polyimide porous film that can be used in the present invention also includes a polyimide porous film obtained by molding a polyamic acid solution obtained from a tetracarboxylic acid component and a diamine component, without using the above-mentioned colored precursor, and then heat-treating the molded product.

[0075] The polyimide porous film produced without using a colored precursor may be produced, for example, by casting a polyamic acid solution consisting of 3-60% by mass of a polyamic acid having an intrinsic viscosity of 1.0-3.0 and 40-97% by mass of an organic polar solvent into a film, immersing or contacting the polyamic acid solution with a water-based coagulation solvent to produce a polyamic acid porous film, and then heat-treating the polyamic acid porous film to imidize it. In this method, the water-based coagulation solvent may be water or a mixture of 5% by mass or more and less than 100% by mass of water and more than 0% by mass and 95% by mass or less of an organic polar solvent. After the imidization, at least one side of the obtained porous polyimide film may be subjected to a plasma treatment.

[0076] The tetracarboxylic dianhydride that can be used in the production of the polyimide porous film may be any tetracarboxylic dianhydride, and may be appropriately selected depending on the desired properties, etc. Specific examples of tetracarboxylic dianhydrides include pyromellitic dianhydride, biphenyl tetracarboxylic dianhydrides such as 3,3',4,4'-biphenyl tetracarboxylic dianhydride (s-BPDA) and 2,3,3',4'-biphenyl tetracarboxylic dianhydride (a-BPDA), oxydiphthalic dianhydride, diphenylsulfone-3,4,3',4'-tetracarboxylic dianhydride, bis(3,4-dicarboxyphenyl)sulfide dianhydride, 2,2-bis(3,4-dicarboxyphenyl)-1,1,1,3,3,3-hexafluoropropane dianhydride, 2,3,3',4'-benzophenone tetracarboxylic dianhydride, 3,3',4,4'-benzophenone tetracarboxylic dianhydride, bis(3,4-dicarboxyphenyl)methane dianhydride, and 2,2-bis(3,4-dicarboxyphenyl)propane dianhydride, p-phenylene bis(trimellitic acid monoester acid anhydride), p-biphenylene bis(trimellitic acid monoester acid anhydride), m-terphenyl-3,4,3',4'-tetracarboxylic acid dianhydride, p-terphenyl-3,4,3',4'-tetracarboxylic acid dianhydride, 1,3-bis(3,4-dicarboxyphenoxy)benzene dianhydride, 1,4-bis(3,4-dicarboxyphenoxy)benzene dianhydride, 1,4-bis(3,4-dicarboxyphenoxy)biphenyl dianhydride, 2,2-bis[(3,4-dicarboxyphenoxy)phenyl]propane dianhydride, 2,3,6,7-naphthalene tetracarboxylic acid dianhydride, 1,4,5,8-naphthalene tetracarboxylic acid dianhydride, 4,4'-(2,2-hexafluoroisopropylidene)diphthalic dianhydride, and the like. It is also preferable to use aromatic tetracarboxylic acids such as 2,3,3',4'-diphenylsulfonetetracarboxylic acid, etc. These may be used alone or in combination of two or more kinds.

[0077] Among these, at least one aromatic tetracarboxylic dianhydride selected from the group consisting of biphenyltetracarboxylic dianhydride and pyromellitic dianhydride is particularly preferred. As the biphenyltetracarboxylic dianhydride, 3,3',4,4'-biphenyltetracarboxylic dianhydride can be suitably used.

[0078] The diamine that can be used in the production of the polyimide porous film may be any diamine. Specific examples of the diamine include the following: 1) Benzenediamines with one benzene nucleus, such as 1,4-diaminobenzene (paraphenylenediamine), 1,3-diaminobenzene, 2,4-diaminotoluene, and 2,6-diaminotoluene; 2) Diaminodiphenyl ethers such as 4,4'-diaminodiphenyl ether and 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenylmethane, 3,3'-dimethyl-4,4'-diaminobiphenyl, 2,2'-dimethyl-4,4'-diaminobiphenyl, 2,2'-bis(trifluoromethyl)-4,4'-diaminobiphenyl, 3,3'-dimethyl-4,4'-diaminodiphenylmethane, 3,3'-dicarboxy-4,4'-diaminodiphenylmethane, 3 ,3',5,5'-Tetramethyl-4,4'-diaminodiphenylmethane, Bis(4-aminophenyl)sulfide, 4,4'-diaminobenzanilide, 3,3'-Dichlorobenzidine, 3,3'-Dimethylbenzidine, 2,2'-Dimethylbenzidine, 3,3'-Dimethoxybenzidine, 2,2'-Dimethoxybenzidine, 3,3'-Diaminodiphenyl ether, 3,4'-Diaminodiphenyl ether, 4,4'-Diaminodiphenyl ether, 3,3'-Diaminodiphenyl Sulfide, 3,4'-diaminodiphenyl sulfide, 4,4'-diaminodiphenyl sulfide, 3,3'-diaminodiphenyl sulfone, 3,4'-diaminodiphenyl sulfone, 4,4'-diaminodiphenyl sulfone, 3,3'-diaminobenzophenone, 3,3'-diamino-4,4'-dichlorobenzophenone, 3,3'-diamino-4,4'-dimethoxybenzophenone, 3,3'-diaminodiphenylmethane, 3,4'-diaminodiphenylmethane, 4,4'-di Diamines with two benzene nuclei, such as aminodiphenylmethane, 2,2-bis(3-aminophenyl)propane, 2,2-bis(4-aminophenyl)propane, 2,2-bis(3-aminophenyl)-1,1,1,3,3,3-hexafluoropropane, 2,2-bis(4-aminophenyl)-1,1,1,3,3,3-hexafluoropropane, 3,3'-diaminodiphenyl sulfoxide, 3,4'-diaminodiphenyl sulfoxide, and 4,4'-diaminodiphenyl sulfoxide; 3) 1,3-bis(3-aminophenyl)benzene, 1,3-bis(4-aminophenyl)benzene, 1,4-bis(3-aminophenyl)benzene, 1,4-bis(4-aminophenyl)benzene, 1,3-bis(4-aminophenoxy)benzene, 1,4-bis(3-aminophenoxy)benzene, 1,4-bis(4-aminophenoxy)benzene, 1,3-bis(3-aminophenoxy)-4-trifluoromethylbenzene, 3,3'-diamino-4-(4-phenyl)phenoxybenzophenone, 3,3'-diamino-4,4'-di(4-phenylphenoxy)benzophenone, 1,3 -Diamines with three benzene nuclei, such as bis(3-aminophenylsulfide)benzene, 1,3-bis(4-aminophenylsulfide)benzene, 1,4-bis(4-aminophenylsulfide)benzene, 1,3-bis(3-aminophenylsulfone)benzene, 1,3-bis(4-aminophenylsulfone)benzene, 1,4-bis(4-aminophenylsulfone)benzene, 1,3-bis[2-(4-aminophenyl)isopropyl]benzene, 1,4-bis[2-(3-aminophenyl)isopropyl]benzene, and 1,4-bis[2-(4-aminophenyl)isopropyl]benzene; 4) 3,3'-bis(3-aminophenoxy)biphenyl, 3,3'-bis(4-aminophenoxy)biphenyl, 4,4'-bis(3-aminophenoxy)biphenyl, 4,4'-bis(4-aminophenoxy)biphenyl, bis[3-(3-aminophenoxy)phenyl]ether, bis[3-(4-aminophenoxy)phenyl]ether, bis[4-(3-aminophenoxy)phenyl]ether, bis[4-(4-aminophenoxy)phenyl]ether, bis[3-(3-aminophenoxy)phenyl]ketone, bis[3-(4-aminophenoxy)phenyl]ketone, bis[4-(3-aminophenoxy)phenyl]ketone, bis[4-(4-aminophenoxy)phenyl]ketone, bis[3-(3-aminophenoxy)phenyl]sulfide, bis[3-(4-aminophenoxy)phenyl]sulfide, bis[4-(3-aminophenoxy)phenyl]sulfide, bis[4-(4-aminophenoxy)phenyl]sulfide, bis[3-(3-aminophenoxy)phenyl]sulfone, bis[3-(4-aminophenoxy)phenyl]sulfone, bis[4-(3-aminophenoxy)phenyl]sulfone bis[4-(4-aminophenoxy)phenyl]sulfone, bis[3-(3-aminophenoxy)phenyl]methane, bis[3-(4-aminophenoxy)phenyl]methane, bis[4-(3-aminophenoxy)phenyl]methane, bis[4-(4-aminophenoxy)phenyl]methane, 2,2-bis[3-(3-aminophenoxy)phenyl]propane, 2,2-bis[3-(4-aminophenoxy)phenyl]propane, 2,2-bis[4-(3-aminophenoxy)phenyl]propane, 2,2- Diamines with four benzene nuclei such as bis[4-(4-aminophenoxy)phenyl]propane, 2,2-bis[3-(3-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, 2,2-bis[3-(4-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, 2,2-bis[4-(3-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, and 2,2-bis[4-(4-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane.

[0079] These may be used alone or in combination of two or more. The diamine to be used may be appropriately selected depending on the desired properties.

[0080] Among these, aromatic diamine compounds are preferred, and 3,3'-diaminodiphenyl ether, 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenyl ether, paraphenylenediamine, 1,3-bis(3-aminophenyl)benzene, 1,3-bis(4-aminophenyl)benzene, 1,4-bis(3-aminophenyl)benzene, 1,4-bis(4-aminophenyl)benzene, 1,3-bis(4-aminophenoxy)benzene, and 1,4-bis(3-aminophenoxy)benzene can be suitably used. In particular, at least one diamine selected from the group consisting of benzenediamine, diaminodiphenyl ether, and bis(aminophenoxy)phenyl is preferred.

[0081] From the viewpoints of heat resistance and dimensional stability at high temperatures, the polyimide porous film that can be used in the present invention is preferably formed from a polyimide obtained by combining a tetracarboxylic dianhydride and a diamine, which has a glass transition temperature of 240° C. or higher or has no clear transition point at 300° C. or higher.

[0082] The polyimide porous film that can be used in the present invention is preferably a polyimide porous film made of the following aromatic polyimides, from the viewpoints of heat resistance and dimensional stability at high temperatures. (i) an aromatic polyimide comprising at least one tetracarboxylic acid unit selected from the group consisting of biphenyltetracarboxylic acid units and pyromellitic acid units, and an aromatic diamine unit; (ii) an aromatic polyimide comprising a tetracarboxylic acid unit and at least one aromatic diamine unit selected from the group consisting of a benzenediamine unit, a diaminodiphenyl ether unit and a bis(aminophenoxy)phenyl unit; and / or (iii) An aromatic polyimide comprising at least one tetracarboxylic acid unit selected from the group consisting of biphenyltetracarboxylic acid units and pyromellitic acid units, and at least one aromatic diamine unit selected from the group consisting of benzenediamine units, diaminodiphenyl ether units, and bis(aminophenoxy)phenyl units.

[0083] The polyimide porous film used in the present invention is preferably a three-layered polyimide porous film having surface layers A and B each having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B, wherein the average pore size of the pores in the surface layer A is 0.01 μm to 15 μm, and the average pore size of the pores in the surface layer B is 20 μm to 100 μm, the macrovoid layer has partition walls bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition walls and the surface layers A and B, the partition walls of the macrovoid layer and the surface layers A and B have thicknesses of 0.01 to 20 μm, the pores in the surface layers A and B are connected to the macrovoids, the polyimide porous film has a total thickness of 5 to 500 μm, and a porosity of 40% or more and less than 95%. At least one partition wall in the macrovoid layer has one or more pores that communicate with adjacent macrovoids and have an average pore size of 0.01 to 100 μm, preferably 0.01 to 50 μm.

[0084] For example, the polyimide porous films described in WO 2010 / 038873, JP 2011-219585 A, and JP 2011-219586 A can also be used in the present invention.

[0085] 5-2.Polyethersulfone (PES) porous membrane The PES porous membrane that can be used in the present invention comprises polyethersulfone, and typically consists essentially of polyethersulfone.Polyethersulfone can be synthesized by a method known to those skilled in the art, for example, by a method of polycondensation reaction of dihydric phenol, alkali metal compound and dihalogenodiphenyl compound in organic polar solvent, or a method of synthesizing an alkali metal di-salt of dihydric phenol in advance and polycondensation reaction of dihalogenodiphenyl compound in organic polar solvent, etc.

[0086] Examples of the alkali metal compound include alkali metal carbonates, alkali metal hydroxides, alkali metal hydrides, alkali metal alkoxides, etc. In particular, sodium carbonate and potassium carbonate are preferred.

[0087] Examples of the dihydric phenol compound include hydroquinone, catechol, resorcin, 4,4'-biphenol, bis(hydroxyphenyl)alkanes (e.g., 2,2-bis(hydroxyphenyl)propane and 2,2-bis(hydroxyphenyl)methane), dihydroxydiphenylsulfones, dihydroxydiphenylethers, and compounds in which at least one hydrogen atom on the benzene ring of these compounds is substituted with a lower alkyl group such as a methyl group, an ethyl group, or a propyl group, or a lower alkoxy group such as a methoxy group or an ethoxy group. Two or more of the above compounds can be mixed and used as the dihydric phenol compound.

[0088] The polyethersulfone may be a commercially available product, and examples of the commercially available product include Sumikaexcel 7600P and Sumikaexcel 5900P (both manufactured by Sumitomo Chemical Co., Ltd.).

[0089] From the viewpoint of favorable formation of macrovoids in the porous polyethersulfone membrane, the inherent viscosity of the polyethersulfone is preferably 0.5 or more, more preferably 0.55 or more, and from the viewpoint of ease of production of the porous polyethersulfone membrane, it is preferably 1.0 or less, more preferably 0.9 or less, even more preferably 0.8 or less, and particularly preferably 0.75 or less.

[0090] In addition, from the viewpoints of heat resistance and dimensional stability at high temperatures, the PES porous membrane or the polyethersulfone used as the raw material thereof preferably has a glass transition temperature of 200°C or higher, or no clear glass transition temperature is observed.

[0091] The method for producing the PES porous membrane that can be used in the present invention is not particularly limited, but may be, for example, A step of casting a polyethersulfone solution containing 0.3% by mass to 60% by mass of polyethersulfone having an inherent viscosity of 0.5 to 1.0 and 40% by mass to 99.7% by mass of an organic polar solvent into a film, and immersing or contacting the film with a coagulation solvent containing a poor solvent or a non-solvent for polyethersulfone as an essential component to produce a coagulated film having pores; A process of heat-treating the coagulated membrane having pores obtained in the process to coarsen the pores and obtain a PES porous membrane. and the heat treatment includes heating the coagulated film having the pores to a temperature equal to or higher than the glass transition temperature of the polyethersulfone, or to 240° C. or higher.

[0092] The PES porous membrane that can be used in the present invention is preferably a PES porous membrane having a surface layer A, a surface layer B, and a macrovoid layer sandwiched between the surface layer A and the surface layer B, the macrovoid layer has partition walls bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition walls and the surface layers A and B, the average pore size in the membrane plane direction being 10 μm to 500 μm, The partition walls of the macrovoid layer have a thickness of 0.1 μm to 50 μm, The surface layers A and B each have a thickness of 0.1 μm to 50 μm, One of the surface layers A and B has a plurality of pores having an average pore size of more than 5 μm and not more than 200 μm, and the other has a plurality of pores having an average pore size of 0.01 μm or more and less than 200 μm, One of the surface layers A and B has a surface opening ratio of 15% or more, and the other surface layer has a surface opening ratio of 10% or more; the pores of the surface layer A and the surface layer B communicate with the macrovoids, The PES porous membrane has a total thickness of 5 μm to 500 μm and a porosity of 50% to 95%. It is a PES porous membrane.

[0093] 6. Application of Cells to the Cell Culture Module The specific steps of applying the cells to the cell culture module are not particularly limited. The steps described herein or any method suitable for applying the cells to a membrane-like carrier can be adopted. In the method of the present invention, the application of the cells to the cell culture module includes, but is not limited to, the following aspects. (1) applying a first medium containing suspended cells to a cell culture module; (2) maintaining the cell culture module at a temperature suitable for cell culture and adsorbing the cells onto a polymer porous membrane in the cell culture module; and (3) culturing the cell culture module to which the cells have been adsorbed in a second medium in a culture vessel; Includes.

[0094] A model diagram of cell culture using a cell culture module is shown in FIG. 7. FIG. 7 is a diagram for aiding understanding, and each element is not drawn to scale. In the cell culture method of the present invention, cells are applied to the cell culture module and cultured, so that a large amount of cells grow in the multifaceted interconnected porous parts and surfaces of the polymer porous membrane, making it possible to culture a large amount of cells easily. In addition, in the cell culture method of the present invention, it is possible to culture a large amount of cells while significantly reducing the amount of medium used for cell culture compared to conventional methods. For example, a large amount of cells can be cultured for a long period of time even if a part or the entire polymer porous membrane is not in contact with the liquid phase of the cell culture medium. In addition, it is also possible to significantly reduce the total volume of the cell culture medium contained in the cell culture vessel relative to the total volume of the polymer porous membrane including the cell survival area.

[0095] In this specification, the volume occupied by the polymer porous membrane without cells, including the volume of its internal gap, is referred to as the "apparent polymer porous membrane volume" (see FIG. 7). Then, when cells are applied to the polymer porous membrane and the cells are supported on the surface and inside of the polymer porous membrane, the volume occupied by the polymer porous membrane, the cells, and the medium infiltrated inside the polymer porous membrane as a whole in the space is referred to as the "polymer porous membrane volume including the cell survival area" (see FIG. 1). In the case of a polymer porous membrane with a thickness of 25 μm, the polymer porous membrane volume including the cell survival area is a value up to about 50% larger than the apparent polymer porous membrane volume. In the method of the present invention, a plurality of polymer porous membranes can be accommodated in one cell culture vessel and cultured. In this case, the sum of the polymer porous membrane volumes including the cell survival area for each of the plurality of polymer porous membranes supporting cells may be simply described as the "sum of the polymer porous membrane volumes including the cell survival area".

[0096] By using the cell culture module, cells can be cultured well for a long period of time even under conditions where the total volume of the cell culture medium contained in the cell culture vessel is 10,000 times or less than the total volume of the polymer porous membrane including the cell survival area. Also, cells can be cultured well for a long period of time even under conditions where the total volume of the cell culture medium contained in the cell culture vessel is 1,000 times or less than the total volume of the polymer porous membrane including the cell survival area. Furthermore, cells can be cultured well for a long period of time even under conditions where the total volume of the cell culture medium contained in the cell culture vessel is 100 times or less than the total volume of the polymer porous membrane including the cell survival area. And, cells can be cultured well for a long period of time even under conditions where the total volume of the cell culture medium contained in the cell culture vessel is 10 times or less than the total volume of the polymer porous membrane including the cell survival area.

[0097] That is, according to the present invention, the space (container) for cell culture can be made as small as possible compared to conventional cell culture devices for two-dimensional culture. In addition, when the number of cells to be cultured is to be increased, the volume for cell culture can be flexibly increased by a simple operation such as increasing the number of polymer porous membranes to be laminated. With a cell culture device equipped with the polymer porous membrane used in the present invention, it is possible to separate the space (container) for cell culture and the space (container) for storing the cell culture medium, and it is possible to prepare the required amount of cell culture medium according to the number of cells to be cultured. The space (container) for storing the cell culture medium may be enlarged or small depending on the purpose, or may be a replaceable container, and is not particularly limited.

[0098] In the method of the present invention, for example, the number of cells contained in the cell culture vessel after the culture using the polymer porous membrane is 1.0 × 10 per milliliter of medium, assuming that all the cells are uniformly dispersed in the cell culture medium contained in the cell culture vessel. 5 pcs or more, 1.0×10 6 pcs or more, 2.0×10 6 pcs or more, 5.0×10 6 pcs or more, 1.0×10 7 pcs or more, 2.0×10 7 pcs or more, 5.0×10 7 pcs or more, 1.0×10 8 pcs or more, 2.0×10 8 pcs or more, 5.0×10 8 pcs or more, 1.0×10 9 pcs or more, 2.0×10 9 pcs or more, or 5.0×10 9 The cells can be cultured until they have more than one cell.

[0099] In this specification, the term "medium" refers to a cell culture medium for culturing cells, particularly animal cells. The term "medium" is used synonymously with the term "cell culture fluid." Therefore, the medium used in the present invention refers to a liquid medium. The type of medium may be any commonly used medium, and is appropriately determined depending on the type of cells to be cultured.

[0100] In culturing the cells of the present invention, the first medium used in step (1) is not particularly limited as long as it is a medium capable of culturing cells. For example, when culturing CHO cells, BalanCD (trademark) CHO GROWTH A manufactured by Irvine Scientific can be used.

[0101] In the cell culture of the present invention, the temperature at which cells can be cultured in step (2) may be any temperature at which cells can be adsorbed to the polymer porous membrane, and is 10° C. to 45° C., preferably 15° C. to 42° C., more preferably 20° C. to 40° C., and even more preferably 25° C. to 39° C. In addition, in the cell culture method of the present invention, the time for adsorbing cells in step (2) is, for example, 5 minutes to 24 hours, preferably 10 minutes to 12 hours, and more preferably 15 minutes to 500 minutes.

[0102] In the cell culture of the present invention, in the step (2), the cells may be adsorbed to the polymer porous membrane of the cell culture module while shaking and / or stirring, or may be left stationary to allow the cells to be adsorbed to the polymer porous membrane of the cell culture module. The shaking method is not particularly limited, but for example, a culture vessel containing the cell culture module of the present invention and the cells may be placed on a commercially available shaking device and shaken. The shaking may be performed continuously or intermittently, for example, shaking and leaving stationary may be repeated alternately, and the shaking method may be appropriately adjusted. The stirring method is not particularly limited, but for example, the cell culture module of the present invention and the cells may be placed in a commercially available spinner flask and stirred by rotating a stirrer. The stirring may be performed continuously or intermittently, for example, stirring and leaving stationary may be repeated alternately, and the stirring method may be appropriately adjusted.

[0103] In the cell culture of the present invention, the second medium used in the step (3) is selected from the medium used for culturing adherent cells, and may be, but is not limited to, D-MEM, E-MEM, IMDM, Ham's F-12, etc. The second medium is preferably a medium that does not contain a component that inhibits the adhesion of cells to a substrate, such as a surfactant. The second medium used is appropriately selected depending on the type of cells. In the step (3), culturing in the second medium promotes the adhesion of the cells adsorbed to the polymer porous membrane in the step (2) to the inside of the polymer porous membrane. This allows the cells to be stably cultured without falling off the polymer porous membrane. In addition, the cell culture method of the present invention uses a cell culture module equipped with the above-mentioned polymer porous membrane. In the cell culture module used in the cell culture method of the present invention, the polymer porous membrane is housed in a casing, thereby preventing the membrane-like polymer porous membrane from continuously deforming in the casing. This prevents stress from being applied to the cells grown in the polymer porous membrane, and apoptosis, etc. are suppressed, making it possible to stably culture a large number of cells.

[0104] In the cell culture of the present invention, the step (3) can be performed using commercially available culture devices and systems for culturing cells. For example, the culture vessel may be a flexible bag-type culture vessel, and the culture can be performed in an agitation-type culture vessel, such as a spinner flask. In addition, the culture vessel can be an open vessel or a closed vessel. For example, a petri dish, flask, plastic bag, test tube, or large tank for cell culture can be appropriately used. For example, a cell culture dish manufactured by BD Falcon and a Nunc Cell Factory manufactured by Thermo Scientific are included. In addition, by using a cell culture module in the cell culture method of the present invention, it is possible to culture cells that were not originally capable of suspension culture in a suspension culture-like state using a device for suspension culture. For example, a spinner flask or a rotary culture manufactured by Corning can be used as a device for suspension culture. In addition, the step (3) can be performed using a cell culture device described in this specification.

[0105] In the cell culture of the present invention, the step (3) can also be carried out using a continuous circulation type device that continuously adds and recovers medium to and from a culture vessel including the cell culture module.

[0106] In the cell culture of the present invention, the step (3) may be a system in which the cell culture medium is continuously or intermittently supplied into the cell culture vessel from a cell culture medium supplying means installed outside the culture vessel including the cell culture module, and the cell culture medium may be circulated between the cell culture medium supplying means and the cell culture vessel.

[0107] 7. Cell culture, exosome production system and culture conditions In the method of the present invention, the cell culture / exosome production system and culture conditions can be appropriately determined depending on the type of cells, etc. Culture methods suitable for each cell are known, and a person skilled in the art can culture the cells applied to the cell culture module using any known method. The cell culture medium can also be appropriately prepared depending on the type of cells.

[0108] In the method of the present invention, the shape and size of the system used for culture are not particularly limited, and cell culture dishes, flasks, plastic bags, test tubes, and large tanks can be used as appropriate. Examples include cell culture dishes manufactured by BD Falcon and Nunc Cell Factory manufactured by Thermo Scientific. In addition, by using a polyimide porous film in the present invention, it has become possible to culture cells that were not originally capable of suspension culture in a suspension culture-like state using a suspension culture device. As a suspension culture device, for example, a spinner flask or a rotary culture device manufactured by Corning can be used.

[0109] In the present invention, the cells may be cultured under static culture conditions. By intermittently exchanging the medium, the produced exosomes can be isolated. In addition, in the present invention, the cells may be cultured under rotary culture or agitation conditions. The same applies to agitation culture in a rotary culture / spinner flask. In addition, each of these methods can be equipped with a continuous or intermittent medium exchange system to aim for long-term culture.

[0110] In the present invention, cells may be continuously cultured. For example, the culture in the method of the present invention can be carried out in a continuous circulation or open type apparatus in which a medium is continuously added to and collected from a cell culture module, and the polymer porous membrane is exposed to air.

[0111] In the present invention, the cells may be cultured in a system in which the cell culture medium is continuously or intermittently supplied into the cell culture vessel from a cell culture medium supplying means installed outside the cell culture vessel, in which the cell culture medium circulates between the cell culture medium supplying means and the cell culture vessel.

[0112] The present invention includes an embodiment including installing a cell culture device in an incubator and culturing cells. When the cell culture device is installed in a system in which a cell culture medium is continuously or intermittently supplied into the cell culture container from a cell culture medium supplying means installed outside the cell culture container, the system may be a cell culture device including a culture unit which is a cell culture container and a culture medium supplying unit which is a cell culture medium supplying means, wherein the culture unit is a culture unit that houses one or more cell culture modules for supporting cells and is provided with a culture medium inlet and a culture medium outlet, and the culture medium supplying unit is a cell culture device that includes a culture medium storage container, a culture medium supplying line, and a liquid delivery pump that delivers the culture medium through the culture medium supplying line, wherein a first end of the culture medium supplying line contacts the culture medium in the culture medium storage container and a second end of the culture medium supplying line communicates with the inside of the culture unit through the culture medium supplying port of the culture unit.

[0113] In the above cell culture device, the culture unit may be a culture unit that does not include a culture medium supply line, a liquid feed pump, an air supply port, and an air exhaust port, or may be a culture unit that includes a culture medium supply line, a liquid feed pump, an air supply port, and an air exhaust port. The culture unit may not include an air supply port and an air exhaust port. Furthermore, in the above cell culture device, the culture unit may further include a culture medium discharge line, where a first end of the culture medium discharge line is connected to a culture medium storage container and a second end of the culture medium discharge line is connected to the inside of the culture unit via the culture medium exhaust port of the culture unit, and the culture medium may be circulated between the culture medium supply unit and the culture unit.

[0114] 8. Cellular production of exosomes In the present invention, exosomes are produced from cells by culturing the cells as described above. The produced exosomes can be collected by known methods. Since exosomes are secreted from cells, the substances can be collected from the cell culture medium.

[0115] In the method of the present invention, the cell culture process and the exosome production process may or may not be separate. For example, when the cells are applied to the cell culture module and cell culture is started, the cells produce exosomes, so when the production system is designed to continuously collect exosomes from the cells from the start to the end of the culture, the cell culture process and the exosome production process are carried out simultaneously. Alternatively, when the medium composition or the settings of the culture device are different between the cell culture and the exosome production, the culture conditions are switched to those designed for exosome production after the cell culture process is completed, so that the two processes are separate.

[0116] In the method of the present invention, the cultured cells can continuously produce exosomes of uniform quality over a long period of time. Preferably, in the method of the present invention, the step of producing exosomes is continued for one month, two months, three months, six months or more. Preferably, in the method of the present invention, the molecular profile and physiological activity of exosomes produced by the cells are evaluated during the exosome production period.

[0117] II. Exosome production device The present invention also relates to an apparatus for producing exosomes by cell culture for use in the method of the present invention, comprising a cell culture module. In the exosome production apparatus of the present invention, the cell culture module may be used in a fixed state or suspended in a cell culture medium, or may be placed in the medium or exposed from the medium.

[0118] The exosome production device in the cell culture of the present invention may take any form as long as it includes a cell culture module, and a known cell culture device can be used. The shape and size of the culture device are not particularly limited, and anything from a petri dish or test tube to a large tank can be used as appropriate. For example, cell culture dishes manufactured by BD Falcon and Nunc Cell Factory manufactured by Thermo Scientific are included. In addition, by using a polymer porous membrane in the present invention, it has become possible to culture cells that were not originally capable of suspension culture in a suspension culture-like state using a suspension culture device. For example, a spinner flask or rotary culture device manufactured by Corning can be used as a suspension culture device.

[0119] The exosome production device of the present invention using cultured cells can also be implemented as a continuous circulation or open type device in which culture medium is continuously added and collected on a cell culture module, and in a form in which a polymer porous membrane sheet is exposed to the air.

[0120] In the method of the present invention, a means for recovering exosomes produced by cells at a higher concentration may be further provided. For example, by directly connecting a semipermeable membrane or the like to the circulating feed medium, it is possible to construct an efficient long-term culture method and a method for removing unnecessary substances, by adding carbohydrates and amino acids while removing unnecessary substances such as lactic acid.

[0121] III. Kit The present invention further relates to a kit for use in the method of the present invention, comprising a cell culture module. In addition to the cell culture module, the kit of the present invention may appropriately comprise components necessary for cell culture, exosome production, and exosome recovery. For example, the kit may include cells to be applied to the cell culture module, a cell culture medium, a continuous medium supply device, a continuous medium circulation device, a cell culture device, an evaluation means for confirming exosome production, an exosome recovery means (e.g., ultracentrifugation, ultrafiltration, immunoprecipitation, chromatography, etc.), and an instruction manual for the kit.

[0122] IV.Use The present invention further comprises the use of a cell culture module for the above-mentioned method of the present invention.

[0123] V. Exosomes The present invention further includes exosomes obtained by the methods of the present invention.

[0124] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Those skilled in the art can easily modify and alter the present invention based on the description in this specification, and such modifications and alterations are within the technical scope of the present invention. EXAMPLES

[0125] The polymer porous membrane used in the following examples is a polyimide porous membrane, which was prepared by molding a polyamic acid solution composition containing a polyamic acid solution obtained from 3,3',4,4'-biphenyltetracarboxylic dianhydride (s-BPDA) as a tetracarboxylic acid component and 4,4'-diaminodiphenyl ether (ODA) as a diamine component, and polyacrylamide as a coloring precursor, and then heat-treating the polyimide porous membrane at 250°C or higher. The obtained polyimide porous membrane was a three-layered polyimide porous membrane having a surface layer A and a surface layer B having a plurality of pores, and a macrovoid layer sandwiched between the surface layer A and the surface layer B, and the average pore size of the pores present in the surface layer A was 19 μm, the average pore size of the pores present in the surface layer B was 42 μm, the membrane thickness was 25 μm, and the porosity was 74%.

[0126] The modularized polymer porous membrane used in the following examples (referred to as "module" in the following examples) was a polyethylene casing having 16 medium inlets of 2 mm x 2 mm on one side, containing the following in this order: 6 square polyimide porous membranes of 1.0 x 1.0 cm; 1.0 x 1.0 cm insole (polypropylene / polyethylene, manufactured by NBC Meshtec, product number: ESP10TC); 6 square polyimide porous membranes of 1.0 x 1.0 cm; 1.0 x 1.0 cm insole (polypropylene / polyethylene, manufactured by NBC Meshtec, product number: ESP10TC); 6 square polyimide porous membranes of 1.0 x 1.0 cm. Unless otherwise specified, the module refers to the above structure.

[0127] In the embodiment specified as "Type 2 module" in the description, a polyethylene casing having 16 medium inlet / outlet ports of 2 mm x 2 mm on one side was provided with the following: 4 sheets of 1.0 x 1.0 cm square polyimide porous membranes; 1.0 x 1.0 cm insole (polypropylene / polyethylene, manufactured by NBC Meshtec, product number: ESP10TC); 4 sheets of 1.0 x 1.0 cm square polyimide porous membranes; 1.0 x 1.0 cm insole (polypropylene / polyethylene, manufactured by NBC Meshtec, product number: ESP10TC); 4 sheets of 1.0 x 1.0 cm square polyimide porous membranes; 1.0 x 1.0 cm insole (polypropylene / polyethylene, manufactured by NBC Meshtec, product number: ESP10TC); 4 sheets of 1.0 x 1.0 cm square polyimide porous membranes; 1.0 x 1.0 cm insole (polypropylene / polyethylene, manufactured by NBC Meshtec, product number: ESP10TC ); Four 1.0 x 1.0 cm square polyimide porous membranes were stacked in this order and used.

[0128] Comparative Example 1: Cell culture method using dishes Second-passage mesenchymal stem cells (Y25 male_451491) cultured on IWAKI collagen I-coated dishes were detached using TrypLE (trademark) Select manufactured by Thermo Fisher Scientific and suspended in medium (Kohjin Bio Co., Ltd. xeno-free medium; KBM ADSC-4). The cell suspension (Total; 3.0 × 10 5 cells) in IWAKI Collagen I coated dish 60cm 2 The cells were seeded on the cells, and allowed to stand in a CO2 incubator set at 5% CO2 and 37°C for approximately 16 hours to allow the cells to adhere, after which the medium was replaced with 12 mL of the above medium (KBM ADSC-4).The medium was then replaced on the 6th day after the start of the culture, and the culture was continued for 13 days.

[0129] The number of exosomes produced per day in the cell culture medium collected on days 6 and 13 of culture was measured using the procedure described below in "Measurement of exosome amount using a zeta potential / particle size distribution analyzer." The results are shown in Figure 18A, and the particle size distribution measurement results on day 13 of culture are shown in Figure 18B.

[0130] Example 1: Cell culture method using a module in a Corning 150 mL round storage bottle (Bottle (5% oxygen)) After pouring 50 mL of medium (Kohjin Bio's xeno-free medium; KBM ADSC-4) into a 150 mL round Corning storage bottle containing five modules, the bottle was placed in a Kuhner incubator shaker (Lab-Therm LT-XC) set to 5% CO2, 5% O2, and 37°C, and the bottle was stirred at a rotation speed of 100 rpm for approximately 1 hour to wet the modules.

[0131] The second-passage mesenchymal stem cells (Y25 male_451491) cultured on IWAKI collagen I-coated dishes were detached using TrypLE (trademark) Select manufactured by Thermo Fisher Scientific and suspended in the above-mentioned medium (KBM ADSC-4). The area of ​​the culture substrate polyimide porous membrane inside the module was 1 cm 22.0 x 10 per 4 cells (Total; 1.80 × 10 6 A cell suspension of KBM ADSC-4 cells was poured into a storage bottle, and the cells were allowed to adhere to the module for approximately 16 hours while stirring at 100 rpm, after which the medium was replaced with 50 mL of the above-mentioned medium (KBM ADSC-4). The medium was replaced 6 days after the start of culture, and thereafter, it was replaced every 3 or 4 days.

[0132] The number of exosomes produced per day in the cell culture medium from the start of culture to the 27th day of culture was measured using the procedure described below in "Measurement of exosome amount using a zeta potential / particle size distribution analyzer," and the results are shown in Figure 19A. The results of particle size distribution measurement on the 27th day of culture are shown in Figure 19B. It was confirmed that exosomes were produced stably even in long-term continuous culture lasting approximately one month.

[0133] Example 2: Cell culture method using a module in a Corning 150 mL round storage bottle (Bottle (normal oxygen)) Cell culture was performed in the same manner as in Example 1 (Bottle (5% oxygen)), except that a Corning 150 mL round storage bottle was placed in a CO2 incubator (aniCell) equipped with a humidified shaker manufactured by N-BIOTEK, set at 5% CO2 and 37°C.

[0134] The number of exosomes produced per day in the cell culture medium from the start of culture to the 27th day of culture was measured using the procedure described below in "Exosome quantity measurement using a zeta potential / particle size distribution measurement device," and the results are shown in Figure 20A. The results of particle size distribution measurement on the 27th day of culture are shown in Figure 20B. It was confirmed that exosomes were produced stably even during long-term continuous culture spanning approximately one month.

[0135] Example 3: Cell culture method using a module in an overflow reactor (Reactor (5% oxygen)) 40 mL of medium (Kohjin Bio's xeno-free medium; KBM ADSC-4) was poured into an overflow reactor culture vessel containing 40 modules, and the vessel was placed in a Kuhner incubator shaker (Lab-Therm LT-XC) set to 5% CO2, 5% O2, and 37°C. The vessel was then stirred at a rotation speed of 80 rpm for approximately 1 hour to moisten the modules.

[0136] The second-passage mesenchymal stem cells (Y25 male_451491) cultured on IWAKI collagen I-coated dishes were detached using TrypL (trademark) Select manufactured by Thermo Fisher Scientific and suspended in the above-mentioned medium (KBM ADSC-4). The area of ​​the culture substrate polyimide porous membrane inside the module was 1 cm 2 2.0 x 10 per 4 cells (Total; 1.44 × 10 7 After pouring the cell suspension of 1000 cells into the overflow reactor culture vessel, medium was added so that the liquid volume in the overflow reactor culture vessel became 80 mL, and the cells were allowed to adsorb for approximately 26 hours under stirring conditions at a rotation speed of 80 rpm.

[0137] After cell adsorption, glucose solution (45 w / v% D(+)-glucose solution manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the medium (KBM ADSC-4), and the total glucose concentration was adjusted to 2000 mg / L (glucose-adjusted KBM ADSC-4) was pumped into the overflow reactor culture vessel at a rate of 40 mL / day using a tube pump. The medium overflowing from the medium collection port installed on the wall of the culture vessel was collected in a collection bottle. The collected medium was observed for cell growth behavior by changes in each metabolic parameter of the collected medium using CedexBio manufactured by Roche, and the liquid supply amount and rotation speed were appropriately changed according to the consumption status of glucose, etc. of the cells, and long-term cell culture was performed. The time-dependent changes in glucose consumption and lactate production are shown in Figure 21A, and the state of cell culture using the overflow reactor is shown in Figure 21B. Stable cell proliferation and growth were observed over a long period of time.

[0138] The number of exosomes produced per day in the collected cell culture medium was measured using the procedure described below in "Exosome quantity measurement using a zeta potential / particle size distribution analyzer," and the results are shown in Figure 22A. The results of particle size distribution measurement on the 85th day of culture are shown in Figure 22B. It was confirmed that exosomes were produced stably even in long-term continuous culture over approximately 3 months.

[0139] Example 4: Cell culture method using a module in an overflow reactor (Reactor (normal oxygen)) Cell culture was carried out in the same manner as in Example 3 (Reactor (5% oxygen)), except that the overflow reactor was placed in a CO2 incubator (aniCell) equipped with a humidified shaker manufactured by N-BIOTEK, set at 5% CO2 and 37°C. The time course of glucose consumption and lactate production is shown in Figure 23A, and the state of cell culture using the overflow reactor is shown in Figure 23B. Stable cell proliferation and growth over a long period of time was observed.

[0140] The number of exosomes produced per day in the collected cell culture medium was measured using the procedure described below in "Exosome quantity measurement using a zeta potential / particle size distribution analyzer," and the results are shown in Figure 24A. The results of particle size distribution measurement on the 85th day of culture are shown in Figure 24B. It was confirmed that exosomes were produced stably even in long-term continuous culture over approximately 3 months.

[0141] Example 5: Cell culture using modules in a WAVE reactor To perform continuous medium extraction, a liquid extraction tube was attached to the cap of a GE WAVE culture bag (CELLBAG DISPOSABLE BIOREACTOR Part# CB0002L11-33), which was then sterilized and replaced with a gamma-ray sterilized Nalgene HDPE cap (product number: 342151-0384) and connected to a Harvest tube pump (Figure 77). In the following examples, when performing continuous culture in a WAVE reactor, a similar extraction line was installed and culture was performed.

[0142] 200 modules that had been previously wetted for more than 24 hours in Ham's F-12 medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., containing glutamine and phenol red) were transferred in a sterile manner into the bag, 200 mL of medium (Kohjin Bio's xeno-free medium; KBM ADSC-4) was poured in, and the modules were then wetted for approximately 1 hour under conditions of 5% CO2, 37°C temperature, 15 rpm vibration speed, and 9° vibration angle.

[0143] The third-passage mesenchymal stem cells (Y25 male_18TL262066) cultured in a dish were detached using Gibco Trypsin-EDTA (0.05%) and phenol red, and suspended in culture medium (Fujifilm Wako Pure Chemical Industries, Ltd.; DMEM). The area of ​​the culture substrate polyimide porous membrane inside the module was 1 cm 2 2.0 x 10 per 4 cells(Total:7.20×10 7 Immediately after seeding the cell suspension of 100 cells, medium (Kohjin Bio's xeno-free medium; KBM ADSC-4) was added so that the liquid volume in the GE WAVE culture bag became 300 mL, and the cells were allowed to adhere for approximately 27 hours under the same vibration conditions as when the module was wetted.

[0144] After that, using the perfusion function (bag weight management function) of the reactor, the cell culture medium was extracted from the GE WAVE culture bag via the Harvest tube pump at a rate of 350 mL / day, and the same volume of the extracted medium (KBM ADSC-4) was pumped into the GE WAVE culture bag via the Feed tube pump. The collected medium was used to observe the cell growth behavior by changes in each metabolic parameter using Roche CedexBio, and stable cell proliferation and growth over a long period of time was observed. The time course of glucose consumption and lactate production is shown in Figure 25A.

[0145] The number of exosomes produced per day in the collected cell culture medium was measured using the procedure described below in "Exosome quantity measurement using a zeta potential / particle size distribution analyzer," and the results are shown in Figure 25B. The particle size distribution measurement results on the 39th day of culture are shown in Figure 25C. It was confirmed that exosomes were produced stably even in long-term continuous culture lasting approximately one month.

[0146] A comparison of the number of exosomes produced per cell per day in various culture methods shown in Comparative Examples and Examples on the 13th day of cell culture is shown in Figure 26. It was confirmed that module culture had a much higher exosome production capacity per cell than dish culture.

[0147] Exosome quantity measurement using a zeta potential / particle size distribution analyzer The cell culture fluids collected under the culture conditions of Comparative Example 1 and Examples 1 to 5 were centrifuged at 10,000 g for 30 minutes at 4° C. using a high-speed refrigerated centrifuge (Kubota Shoji Co., Ltd., Model 6000) to remove debris (Preparation Solution 1). "Preparation Solution 1" was diluted to a predetermined concentration using PBS(-) (FUJIFILM Wako Pure Chemical Co., Ltd., distributor code: 166-23555) from which fine particles had been removed by suction filtration using a 0.025 μm filter (Merck Co., Ltd., product name: MF-Millipore Membrane Filter, model number: VSWP04700) (Preparation Solution 2). Next, "Preparation Solution 2" was diluted to a predetermined concentration using a 0.2 μm syringe filter (Sartorius Co., Ltd., product name: Minisart, model number: 16534K) to remove particles of 200 nm or more (Preparation Solution 3). Using a zeta potential / particle size distribution measuring device (Microtech Nichion Co., Ltd., ZEECOM ZC-3000), the particle size distribution of the particles contained in "Preparation Solution 3" and the number of particles in the exosome fraction were measured by Brownian motion trajectory analysis.

[0148] When collecting the medium for measuring exosomes, the cell number was measured using Cell Counting Kit-8 (Dojindo Laboratories), and the amount of exosomes produced per cell per day was calculated using the following "Formula 1," and the exosome production ability of cells under each cell culture condition was compared. Formula 1: Number of particles x sample dilution ratio x percentage of particles below 150 nm x volume of recovered medium / total number of cells / number of days medium was stored The change in exosome production amount per day over time under each cell culture condition was measured using the following "Equation 2." Formula 2: Number of particles x sample dilution rate x percentage of particles below 150 nm / number of days the medium was stored

[0149] miRNA Array Analysis Using GeneChip™ miRNA 4.0 Array The cell culture fluids collected under the culture conditions of Comparative Example 1 and Examples 1 to 5 were centrifuged at 10,000 g for 30 minutes at 4° C. using a high-speed refrigerated centrifuge (Kubota Shoji Co., Ltd., Model 6000) to remove debris. Exosomes were extracted from the resulting supernatant by polymer precipitation using Total Exosome Isolation Regent (from cell culture media) (Thermo Fisher Scientific, Cat. #4478359). Total RNA was then extracted and purified from the exosome extract using Total Exosome RNA and Protein Isolation Kit (Thermo Fisher Scientific, Cat. #4478545), and the concentration of the total RNA solution was measured using Nano Drop 2000 (Thermo Fisher Scientific). Using the obtained total RNA (390 ng), human miRNA array analysis was performed using GeneChip™ miRNA 4.0 Array (Thermo Fisher Scientific, Cat. #902445).

[0150] Comparison of exosome production amount between Comparative Example 1 (Dish_Day 6) and Example 1 (Bottle (5% oxygen)_Day 27) and miRNA array analysis A comparison of the amount of exosome production per cell between Comparative Example 1 (Dish_Day 6) and Example 1 (Bottle (5% oxygen)_Day 27) is shown in Figure 27. It was confirmed that the exosome production capacity was 2.8 times higher in Bottle (5% oxygen) culture using a polyimide porous membrane module than in Dish_Day 6, even in a long-term continuous stable culture state.

[0151] FIG. 28 shows a scatterplot of the miRNA array analysis of Comparative Example 1 (Dish_Day 6) and Example 1 (Bottle (5% oxygen)_Day 27), and Table 1 shows the miRNAs for which a difference in expression ratio of 10-fold or more was observed. [Table 1]

[0152] 29 shows a scatterplot of the miRNA array analysis of hsa-miR-146a, hsa-miR-210, hsa-miR-22, and hsa-miR-24, which are miRNAs effective in treating damaged or diseased cardiac tissue as described in Patent Document 2. Even in a long-term continuous stable culture state, Bottle (5% oxygen) culture using a polyimide porous membrane module showed a 4.0-fold higher production of hsa-miR-210-3p and a 17.5-fold higher production of hsa-miR-24a-3p compared to Dish_Day6, and the expression levels of other miRNAs were comparable.

[0153] The results of miRNA array analysis of hsa-miR-34b, hsa-miR-132, hsa-miR-203, hsa-miR-137, and hsa-miR-193, which are miRNAs effective in suppressing oral squamous cell carcinoma as described in Patent Document 3, are shown in Figure 30. Even in a long-term continuous stable culture state, Bottle (5% oxygen) culture using a polyimide porous membrane module showed a 5.8-fold higher production amount of hsa-miR-193a-5p compared to Dish_Day6, and the expression amounts of other miRNAs were comparable.

[0154] The results of miRNA array analysis of hsa-miR-26a / b, hsa-miR-23b, hsa-miR-27b, and hsa-miR-24-1, which are miRNAs effective in suppressing gastric cell carcinoma or prostate cancer cells as described in Non-Patent Documents 6 and 7, are shown in Figure 31. Even in a long-term continuous stable culture state, Bottle (5% oxygen) culture using a polyimide porous membrane module showed a 36.6-fold higher production of hsa-miR-26a-5p and a 15.4-fold higher production of hsa-miR-23b-3p compared to Dish_Day6, and the expression levels of other miRNAs were comparable.

[0155] Comparison of exosome production amount between Comparative Example 1 (Dish_Day 13) and Example 1 (Bottle (5% oxygen)_Day 27) and miRNA array analysis A comparison of the amount of exosome production per cell between Comparative Example 1 (Dish_Day 13) and Example 1 (Bottle (5% oxygen)_Day 27) is shown in Figure 32. It was confirmed that the exosome production ability was 2.9 times higher in Bottle (5% oxygen) culture using a polyimide porous membrane module than in Dish_Day 13, even in a long-term continuous stable culture state.

[0156] FIG. 33 shows a scatterplot of the miRNA array analysis of Comparative Example 1 (Dish_Day 13) and Example 1 (Bottle (5% oxygen)_Day 27), and Table 2 shows the miRNAs for which a difference in expression ratio of 10-fold or more was observed. [Table 2]

[0157] The results of miRNA array analysis of hsa-miR-146a, hsa-miR-210, hsa-miR-22, and hsa-miR-24, which are miRNAs effective for treating damaged or diseased cardiac tissue as described in Patent Document 2, are shown in Figure 34. Even in Bottle (5% oxygen) culture using a polyimide porous membrane module in a long-term continuous stable culture state, hsa-miR-24-3p showed a 5.3-fold higher production amount compared to Dish_Day13 just before reaching confluence, and the expression amounts of other miRNAs were comparable.

[0158] The results of miRNA array analysis of hsa-miR-34b, hsa-miR-132, hsa-miR-203, hsa-miR-137, and hsa-miR-193, which are miRNAs effective in suppressing oral squamous cell carcinoma as described in Patent Document 3, are shown in Figure 35. In comparison with Dish_Day13 just before reaching confluence, Bottle (5% oxygen) culture using a polyimide porous membrane module in a long-term continuous stable culture state also showed results equivalent to those of Dish_Day13.

[0159] The results of miRNA array analysis of hsa-miR-26a / b, hsa-miR-23b, hsa-miR-27b, and hsa-miR-24-1, which are miRNAs effective in suppressing gastric cell carcinoma or prostate cancer cells as described in Non-Patent Documents 6 and 7, are shown in Figure 36. Even in a long-term continuous stable culture state, Bottle (5% oxygen) culture using a polyimide porous membrane module showed a high production amount of hsa-miR-26a-5p of 29.1 times and hsa-miR-23b-3p of 6.1 times higher than Dish_Day13 just before reaching confluence, and the expression amounts of other miRNAs were similar.

[0160] Comparison of exosome production amount and miRNA analysis between Comparative Example 1 (Dish_Day 6) and Example 2 (Bottle (normal oxygen)_Day 27) A comparison of the amount of exosome production per cell between Comparative Example 1 (Dish_Day 6) and Example 2 (Bottle (normal oxygen)_Day 27) is shown in Figure 37. It was confirmed that the exosome production capacity was 2.5 times higher in Bottle (normal oxygen) culture using a polyimide porous membrane module than in Dish_Day 6, even in a long-term continuous stable culture state.

[0161] FIG. 38 shows a scatterplot of the miRNA array analysis of Comparative Example 1 (Dish_Day 6) and Example 2 (Bottle (normal oxygen)_Day 27), and Table 3 shows the miRNAs for which a difference in expression ratio of 10-fold or more was observed. [Table 3]

[0162] The results of miRNA array analysis of hsa-miR-146a, hsa-miR-210, hsa-miR-22, and hsa-miR-24, which are miRNAs effective in treating damaged or diseased cardiac tissue as described in Patent Document 2, are shown in Figure 39. Compared to Dish_Day6, Bottle (normal oxygen) culture using a polyimide porous membrane module showed 3.9-fold higher production of hsa-miR-24-3p, even in long-term continuous stable culture conditions, while other miRNAs showed similar expression levels.

[0163] The results of miRNA array analysis of hsa-miR-34b, hsa-miR-132, hsa-miR-203, hsa-miR-137, and hsa-miR-193, which are miRNAs effective in suppressing oral squamous cell carcinoma as described in Patent Document 3, are shown in Figure 40. Bottle (normal oxygen) culture using a polyimide porous membrane module showed the same level of production as Dish_Day6 even in long-term continuous stable culture conditions.

[0164] The results of miRNA array analysis of hsa-miR-26a / b, hsa-miR-23b, hsa-miR-27b, and hsa-miR-24-1, which are miRNAs effective in suppressing gastric cell carcinoma or prostate cancer cells as described in Non-Patent Documents 6 and 7, are shown in Figure 41. Bottle (normal oxygen) culture using a polyimide porous membrane module showed expression levels comparable to those of Dish_Day6, even in long-term continuous stable culture conditions.

[0165] Comparison of exosome production amount between Comparative Example 1 (Dish_Day 13) and Example 2 (Bottle (normal oxygen)_Day 27) and miRNA array analysis A comparison of the amount of exosome production per cell between Comparative Example 1 (Dish_Day 13) and Example 2 (Bottle (normal oxygen)_Day 27) is shown in Figure 42. It was confirmed that the exosome production capacity was 2.6 times higher in Bottle (normal oxygen) culture using a polyimide porous membrane module than in Dish_Day 13, even in a long-term continuous stable culture state.

[0166] FIG. 43 shows a scatterplot of the miRNA array analysis of Comparative Example 1 (Dish_Day 13) and Example 2 (Bottle (normal oxygen)_Day 27), and Table 4 shows the miRNAs for which a difference in expression ratio of 10-fold or more was observed. [Table 4]

[0167] The results of miRNA array analysis of hsa-miR-146a, hsa-miR-210, hsa-miR-22, and hsa-miR-24, which are miRNAs effective for treating damaged or diseased cardiac tissue as described in Patent Document 2, are shown in Figure 44. Compared to Dish_Day13 just before reaching confluence, Bottle (normal oxygen) culture using a polyimide porous membrane module showed 3.4-fold higher production of hsa-miR-24-3p, even in a long-term continuous stable culture state, while other miRNAs showed similar expression levels.

[0168] The results of miRNA array analysis of hsa-miR-34b, hsa-miR-132, hsa-miR-203, hsa-miR-137 and hsa-miR-193, which are miRNAs effective in suppressing oral squamous cell carcinoma described in Patent Document 3, are shown in Figure 45. Compared to the long-term continuous stable culture state of Bottle (normal oxygen) culture using a polyimide porous membrane module, Dish_Day13, just before reaching confluence, showed high production amounts of hsa-miR-193a-5p 7.0 times, hsa-miR-193b-5p 4.6 times and hsa-miR-193b-3p 4.4 times, and the expression amounts of other miRNAs were similar.

[0169] The results of the miRNA array analysis of hsa-miR-26a / b, hsa-miR-23b, hsa-miR-27b and hsa-miR-24-1, which are miRNAs effective in suppressing gastric cell carcinoma or prostate cancer cells as described in Non-Patent Documents 6 and 7, are shown in Figure 46. Even in a long-term continuous stable culture state, Bottle (normal oxygen) culture using a polyimide porous membrane module showed high production of hsa-miR-26a-5p 43.8 times, hsa-miR-23b-3p 9.7 times, hsa-miR-26a-3p 3.4 times, and hsa-miR-27b-3p 3.1 times, with other miRNAs showing similar expression levels.

[0170] Comparison of exosome production amount between Comparative Example 1 (Dish_Day 6) and Example 3 (Reactor (5% oxygen)_Day 27) and miRNA array analysis A comparison of the amount of exosome production per cell between Comparative Example 1 (Dish_Day 6) and Example 3 (Reactor (5% oxygen)_Day 27) is shown in Figure 47. It was confirmed that the exosome production capacity was 1.7 times higher in the Reactor (5% oxygen) culture using a polyimide porous membrane module than in the Dish_Day 6 culture, even in a long-term continuous stable culture state.

[0171] FIG. 48 shows a scatterplot of the miRNA array analysis of Comparative Example 1 (Dish_Day 6) and Example 3 (Reactor (5% oxygen)_Day 27), and Table 5 shows the miRNAs for which a difference in expression ratio of 10-fold or more was observed. [Table 5]

[0172] The results of miRNA array analysis of hsa-miR-146a, hsa-miR-210, hsa-miR-22, and hsa-miR-24, which are miRNAs effective in treating damaged or diseased cardiac tissue as described in Patent Document 2, are shown in Figure 49. Even in a long-term continuous stable culture state in the Reactor (5% oxygen) culture using a polyimide porous membrane module compared to Dish_Day6, hsa-miR-24a-3p showed a 10.3-fold higher production amount, and other miRNAs showed similar expression amounts.

[0173] The results of miRNA array analysis of hsa-miR-34b, hsa-miR-132, hsa-miR-203, hsa-miR-137, and hsa-miR-193, which are miRNAs effective in suppressing oral squamous cell carcinoma as described in Patent Document 3, are shown in Figure 50. Reactor (5% oxygen) culture using a polyimide porous membrane module showed production amounts comparable to those of Dish_Day6 even in long-term continuous stable culture conditions.

[0174] The results of miRNA array analysis of hsa-miR-26a / b, hsa-miR-23b, hsa-miR-27b, and hsa-miR-24-1, which are miRNAs effective in suppressing gastric cell carcinoma or prostate cancer cells as described in Non-Patent Documents 6 and 7, are shown in Figure 51. Even in a long-term continuous stable culture state, the Reactor (5% oxygen) culture using a polyimide porous membrane module showed a 5.0-fold higher production of hsa-miR-26a-5p and a 5.4-fold higher production of hsa-miR-23b-3p compared to Dish_Day6, and the expression levels of other miRNAs were comparable.

[0175] Comparison of exosome production amount between Comparative Example 1 (Dish_Day 13) and Example 3 (Reactor (5% oxygen)_Day 27) and miRNA array analysis A comparison of the amount of exosome production per cell between Comparative Example 1 (Dish_Day13) and Example 3 (Reactor (5% oxygen)_Day27) is shown in Figure 52. It was confirmed that the exosome production capacity was 1.8 times higher in the Reactor (5% oxygen) culture using a polyimide porous membrane module than in the Dish_Day13 culture, even in a long-term continuous stable culture state.

[0176] FIG. 53 shows a scatterplot of the miRNA array analysis of Comparative Example 1 (Dish_Day 13) and Example 3 (Reactor (5% oxygen)_Day 27), and Table 6 shows the miRNAs for which a difference in expression ratio of 10-fold or more was observed. [Table 6]

[0177] The results of miRNA array analysis of hsa-miR-146a, hsa-miR-210, hsa-miR-22, and hsa-miR-24, which are miRNAs effective in treating damaged or diseased cardiac tissue as described in Patent Document 2, are shown in Figure 54. Even in a long-term continuous stable culture state, Reactor (5% oxygen) culture using a polyimide porous membrane module showed a 3.4-fold higher production of hsa-miR-24-3p for Dish_Day13 just before reaching confluence, and the expression levels of other miRNAs were comparable.

[0178] The results of miRNA array analysis of hsa-miR-34b, hsa-miR-132, hsa-miR-203, hsa-miR-137 and hsa-miR-193, which are miRNAs effective in suppressing oral squamous cell carcinoma described in Patent Document 3, are shown in Figure 55. In the long-term continuous stable culture state of the Reactor (5% oxygen) culture using the polyimide porous membrane module, Dish_Day13, just before reaching confluence, showed high production amounts of hsa-miR-193a-5p 5.2 times, hsa-miR-193b-5p 2.9 times and hsa-miR-193b-3p 5.4 times, and the expression amounts of other miRNAs were similar.

[0179] The results of miRNA array analysis of hsa-miR-26a / b, hsa-miR-23b, hsa-miR-27b, and hsa-miR-24-1, which are miRNAs effective in suppressing gastric cell carcinoma or prostate cancer cells as described in Non-Patent Documents 6 and 7, are shown in Figure 56. Even in a long-term continuous stable culture state in the Reactor (5% oxygen) culture using a polyimide porous membrane module for Dish_Day13 just before reaching confluence, hsa-miR-26a-5p showed a 4.0-fold higher production amount, and other miRNAs showed similar expression amounts.

[0180] Comparison of exosome production amount between Comparative Example 1 (Dish_Day 6) and Example 4 (Reactor (normal oxygen)_Day 27) and miRNA array analysis A comparison of the amount of exosome production per cell between Comparative Example 1 (Dish_Day 6) and Example 4 (Reactor (normal oxygen)_Day 27) is shown in Figure 57. It was confirmed that the exosome production capacity was 2.6 times higher in the Reactor (normal oxygen) culture using a polyimide porous membrane module than in the Dish_Day 6 culture, even in a long-term continuous stable culture state.

[0181] A scatterplot of the miRNA array analysis of Comparative Example 1 (Dish_Day 6) and Example 4 (Reactor (normal oxygen)_Day 27) is shown in Figure 58. Table 7 shows miRNAs for which a difference in expression ratio of 10-fold or more was observed. [Table 7]

[0182] The results of miRNA array analysis of hsa-miR-146a, hsa-miR-210, hsa-miR-22, and hsa-miR-24, which are miRNAs effective in treating damaged or diseased cardiac tissue as described in Patent Document 2, are shown in Figure 59. In the Reactor (normal oxygen) culture using a polyimide porous membrane module, hsa-miR-24-3p showed a 10.1-fold increase in production in a long-term continuous stable culture state compared to Dish_Day6, and the expression levels of other miRNAs were comparable.

[0183] The results of miRNA array analysis of hsa-miR-34b, hsa-miR-132, hsa-miR-203, hsa-miR-137, and hsa-miR-193, which are miRNAs effective in suppressing oral squamous cell carcinoma as described in Patent Document 3, are shown in Figure 60. Reactor (normal oxygen) culture using a polyimide porous membrane module showed the same production amount even in long-term continuous stable culture conditions for Dish_Day6.

[0184] The results of miRNA array analysis of hsa-miR-26a / b, hsa-miR-23b, hsa-miR-27b, and hsa-miR-24-1, which are miRNAs effective in suppressing gastric cell carcinoma or prostate cancer cells as described in Non-Patent Documents 6 and 7, are shown in Figure 61. Even in a long-term continuous stable culture state, the Reactor (normal oxygen) culture using a polyimide porous membrane module showed high production of hsa-miR-26a-5p by 55.0 times, hsa-miR-23b-3p by 24.2 times, and hsa-miR-27b-3p by 4.2 times compared to Dish_Day6, and the expression levels of other miRNAs were comparable.

[0185] Comparison of exosome production amount between Comparative Example 1 (Dish_Day 13) and Example 4 (Reactor (normal oxygen)_Day 27) and miRNA array analysis A comparison of the amount of exosome production per cell between Comparative Example 1 (Dish_Day13) and Example 4 (Reactor (normal oxygen)_Day27) is shown in Figure 62. It was confirmed that the exosome production capacity was 2.7 times higher in the Reactor (normal oxygen) culture using a polyimide porous membrane module than in the Dish_Day13 culture, even in a long-term continuous stable culture state.

[0186] FIG. 63 shows a scatterplot of the miRNA array analysis of Comparative Example 1 (Dish_Day 13) and Example 4 (Reactor (normal oxygen)_Day 27), and Table 8 shows the miRNAs for which a difference in expression ratio of 10-fold or more was observed. [Table 8]

[0187] The results of miRNA array analysis of hsa-miR-146a, hsa-miR-210, hsa-miR-22, and hsa-miR-24, which are miRNAs effective in treating damaged or diseased cardiac tissue as described in Patent Document 2, are shown in Figure 64. In the Reactor (normal oxygen) culture using a polyimide porous membrane module, hsa-miR-24-3p showed 3.4-fold higher production than Dish_Day13 in a long-term continuous stable culture state, and other miRNAs showed similar expression levels.

[0188] The results of miRNA array analysis of hsa-miR-34b, hsa-miR-132, hsa-miR-203, hsa-miR-137 and hsa-miR-193, which are miRNAs effective in suppressing oral squamous cell carcinoma described in Patent Document 3, are shown in Figure 65. In the long-term continuous stable culture state of Reactor (normal oxygen) culture using a polyimide porous membrane module for Dish_Day13 just before reaching confluence, hsa-miR-193a-5p was 7.0 times, hsa-miR-193b-5p was 4.6 times, and hsa-miR-193b-3p was 4.4 times, showing high production, and other miRNAs were expressed at the same level.

[0189] The results of miRNA array analysis of hsa-miR-26a / b, hsa-miR-23b, hsa-miR-27b and hsa-miR-24-1, which are miRNAs effective in suppressing gastric cell carcinoma or prostate cancer cells described in Non-Patent Documents 6 and 7, are shown in Figure 66. Even in a long-term continuous stable culture state, the Reactor (normal oxygen) culture using a polyimide porous membrane module showed high production of hsa-miR-26a-5p 43.8 times, hsa-miR-23b-3p 9.7 times, hsa-miR-26b-3p 3.4 times, and hsa-miR-27b-3p 3.2 times, with other miRNAs showing similar expression levels.

[0190] Example 6: Cell culture using modules in a WAVE reactor In a similar manner to Example 5, 60 modules that had been moistened in advance with medium (Ham's F-12 medium; Fujifilm Wako Pure Chemical Industries, Ltd., containing glutamine and phenol red) for at least 2 days were placed in a GE WAVE culture bag (CELLBAG DISPOSABLE BIOREACTOR Part# CB0002L11-33) and 250 mL of medium (Kohjin Bio's xeno-free medium; KBM ADSC-4R) was poured into the bag, which had been placed in a GE WAVE reactor (ReadyToProcess WAVE25). The modules were then shaken for approximately 1 hour under conditions of 5% CO2, 37°C temperature, 15 rpm vibration speed, and 9° vibration angle.

[0191] Mesenchymal stem cells (21Y Male; ATCC Lot 70011721) at the fourth passage cultured in a dish were detached using Gibco Trypsin-EDTA (0.05%) and phenol red, and suspended in 11 mL of culture medium (Kohjin Bio Xeno-free medium; KBM ADSC-4R). The area of ​​the culture substrate polyimide porous membrane inside the module was 1 cm 2 1.0×10 per 4 cells (Total: 1.1 × 10 7 A cell suspension of 1000 cells was seeded on the module, and the cells were allowed to adhere to the module under the same vibration conditions as when the module was wetted, and long-term culture was then initiated.

[0192] After continuing the vibration culture under the same conditions for 3 days, the cell culture medium was extracted from the GE WAVE culture bag at a rate of 110 mL / day via the Harvest tube pump using the reactor's perfusion function (bag weight management function), and the same volume of medium (glucose-adjusted KBM ADSC-4R) was sent to the GE WAVE culture bag via the Feed tube pump. Since the liquid sending speed of the harvest pump attached to the WAVE25 was too high, the medium was collected using the AS ONE microtube pump set FP100-1. The collected medium was observed for cell growth behavior by changes in each metabolic parameter using Roche CedexBio, and stable cell proliferation and growth were observed over a long period of time. In addition, when necessary, the total number of cells growing in the module in the bag was measured using a color reaction using Dojindo Cell Counting Kit-8 to confirm the cell growth status. The time-dependent changes in glucose consumption and lactate production are shown in Figure 67. When a shortage was predicted due to an increase in glucose consumption, the glucose concentration was adjusted by adding glucose solution to avoid depletion.

[0193] To measure the number of exosomes produced per day in the collected cell culture fluid, the collected media from approximately every 20 days was combined into one lot to create five collected fluids. After removing foreign matter with a 0.2 μm filter, each fluid was measured using the procedure described above in "Measurement of exosome quantity using a zeta potential / particle size distribution analyzer," and the results are shown in Table 9. It was confirmed that stable exosome production continued in the five collected fluids representing 100 days of culture. [Table 9]

[0194] Furthermore, these five samples (culture recovery fluid) were concentrated 30 to 50 times using the tangential flow filtration method (VIVAFLOW 200 100,000MWCO HY manufactured by Sartorius), and then half the amount of the concentrated fluid, ThermoFisher's Total Exosome Isolation Reagent (from cell culture media), was added, and the precipitate was separated by stirring, refrigerating overnight, and centrifuging (10,000G / 60min). The precipitate was suspended and dissolved in DPBS in an amount equal to the amount of the removed fluid, and foreign matter was removed using a 0.2μm filter to obtain the exosome fluid. In this way, five exosome fluid samples were obtained for each culture period. These five samples were measured using the procedure described above for "Exosome amount measurement using a zeta potential / particle size distribution measurement device." In addition, the amount of exosomes was quantified as the amount of CD63 protein using a CD9 / CD63 ELISA kit for quantifying human-derived exosomes (manufactured by Cosmo Bio Co., Ltd.; product number EXH0102EL). The results of the exosome amount evaluation are shown in Table 10, and the results of the exosome particle size distribution are shown in Figure 68. [Table 10]

[0195] For each of the exosome samples, miRNA array analysis was performed using the GeneChip™ miRNA 4.0 Array shown in Example 5. Comparative gene expression analysis confirmed stable gene expression in the exosomes regardless of the culture period ( FIG. 76 ).

[0196] The exosomes obtained in the same manner as in the above examples were purified using a gel filtration column (HiLoad 16 / 600 Superdex 200 prep grade), concentrated using an ultrafiltration membrane, and then observed with a TEM (transmission electron microscope). The sample solution was adsorbed onto a Cu mesh with a carbon support membrane, and then washed, stained, washed, and dried to prepare a sample for TEM observation. Images were taken using a JEM-2100F field emission transmission electron microscope manufactured by JEOL. An example of the measurement is shown in Figure 69.

[0197] Example 7: Cell culture using modules in a WAVE reactor In a similar manner to Example 5, 70 modules that had been moistened in advance with medium (Ham's F-12 medium; Fujifilm Wako Pure Chemical Industries, Ltd., containing glutamine and phenol red) for at least 2 days were placed in a GE WAVE culture bag (CELLBAG DISPOSABLE BIOREACTOR Part# CB0002L11-03) and 250 mL of medium (Kohjin Bio's xeno-free medium; KBM ADSC-4R) was poured into the bag, which had been placed in a GE WAVE reactor (Xuri Cell Expansion System W5). The modules were then shaken for approximately 1 hour under conditions of 5% CO2, 37°C temperature, 11 rpm vibration speed, and 9° vibration angle.

[0198] Mesenchymal stem cells (21Y Male; ATCC Lot 70011721) at the second passage cultured in a dish were detached using Gibco Trypsin-EDTA (0.05%) and phenol red, and suspended in 11 mL of culture medium (Kohjin Bio Xeno-free medium; KBM ADSC-4R). The area of ​​the culture substrate polyimide porous membrane inside the module was 1 cm 2 1.0×10 per 4 cells (Total: 1.3 × 10 7 A cell suspension of 1000 cells was seeded, and the cells were attached under the same vibration conditions as when the module was wetted, and long-term culture was started. In addition, at the same time as the mesenchymal stem cells were seeded on the module, subculture seeding was also performed on IWAKI collagen type 1 coated dishes, and culture was carried out for a comparative experiment of exosome production.

[0199] After continuing the vibration culture under the same conditions for 3 days, approximately 20 mL of cell culture medium was extracted from the GE WAVE culture bag via the Harvest tube pump at approximately 3-hour intervals using the perfusion function (bag weight management function) of the reactor accessory (Xuri Cell Expansion System W5 Perfusion Controller), and the same volume of medium (glucose-adjusted KBM ADSC-4R) was pumped into the GE WAVE culture bag via the Feed tube pump, performing a liquid exchange of 150 mL / day. The collected medium was used to observe the cell growth behavior by changes in each metabolic parameter using Roche CedexBio, and stable cell proliferation and growth over a long period of time was observed. In addition, on the 14th, 21st, 28th, and 50th days, 1 cm of human mesenchymal stem cells growing in the module in the bag were counted using a color reaction using Dojindo Cell Counting Kit-8. 2 The cell density per bag and the total number of cells in the bag were measured to confirm the cell growth status. The measurement results are shown in Table 11. [Table 11]

[0200] To measure the number of exosomes produced per day in the collected cell culture fluid, samples were taken periodically and foreign matter was removed using a 0.2 μm filter, after which each fluid was measured using the procedure described above for "Measurement of exosome quantity using a zeta potential / particle size distribution analyzer." The measurement results are shown in Figure 70, along with the results of operation with other cells. The human mesenchymal stem cell WAVE reactor (1) in the figure shows the results of the experiment. As is clear from the results, it was confirmed that stable exosome production continued over the course of 50 days of culture using this culture method.

[0201] In addition, for exosome comparison, a culture supernatant collected on the 14th day of culture from the same mesenchymal stem cells cultured in a Kohjin Bio xeno-free medium KBM ADSC-4R on an IWAKI collagen type 1 coated dish, and a medium collected on the 50th day of the continuous culture system using the WAVE reactor were used to carry out miRNA array analysis using the GeneChip™ miRNA 4.0 Array shown in Example 5. The results are shown in Figure 71.

[0202] Comparison of miRNA expression levels under two different culture conditions is shown as a distribution level, normalized between the same amount of RNA. The left side shows the overall distribution of human miRNAs, and the right side shows the partial distribution limited to miR21-24. As is clear from the comparison of the figures on the left, even if the amount of RNA is the same, the expression level of miRNAs is generally higher in the module culture system. Conversely, the expression level of miR-22-3p was confirmed to be higher in plate culture than in module culture. miR-22 is a miRNA whose expression level is known to increase with cell aging. Although the period of module culture was more than three times longer than that of the plate culture used for comparison, the expression level of the same aging-related miRNA was found to be more than three times higher in the plate culture system. It was confirmed that module culture can be used to culture cells for a long period of time while maintaining their characteristics well.

[0203] Example 8: Cell culture using modules in a WAVE reactor In a similar manner to Example 5, 250 mL of medium (Kohjin Bio's xeno-free medium; KBM ADSC-4R) was poured into two GE WAVE culture bags (CELLBAG DISPOSABLE BIOREACTOR Part# CB0002L11-03) containing 55 modules and 55 type 2 modules that had been previously wetted with medium (Ham's F-12 medium; Fujifilm Wako Pure Chemical Industries, Ltd., containing glutamine and phenol red) for more than 2 days, and the bags were then placed in a GE WAVE reactor (Xuri Cell Expansion System W5). The modules were then shaken for about 1 hour under conditions of 5% CO2, 37°C temperature, 13 rpm vibration speed, and 9° vibration angle.

[0204] Second-passage mesenchymal stem cells (25Y Male; Lonza Lot 00451491) cultured in a dish were detached using Gibco Trypsin-EDTA (0.05%) and phenol red, and suspended in 11 mL of culture medium (Kohjin Bio Xeno-free medium; KBM ADSC-4R). The area of ​​the culture substrate polyimide porous membrane inside the module was 1 cm. 2 1.0×10 per 4 cells (Total: 1.3 × 10 7 A cell suspension of 1000 cells was seeded on the module, and the cells were allowed to adhere to the module under the same vibration conditions as when the module was wetted, and long-term culture was then initiated.

[0205] After continuing the vibration culture under the same conditions for 3 days, approximately 20 mL of cell culture medium was extracted from the GE WAVE culture bag via the Harvest tube pump at approximately 3-hour intervals using the perfusion function (bag weight management function) of the reactor accessory (Xuri Cell Expansion System W5 Perfusion Controller), and the same volume of medium (glucose-adjusted KBM ADSC-4R) as the extracted medium was pumped into the GE WAVE culture bag via the Feed tube pump, performing a liquid exchange of 150 mL / day. The collected medium was used to observe the cell growth behavior based on changes in each metabolic parameter using Roche's CedexBio, and stable cell proliferation and growth over a long period of time was observed. In addition, 1 cm of cells growing in the module in the bag were counted using a color reaction using Dojindo Cell Counting Kit-8 on the 7th, 14th, 21st, 29th, and 48th days. 2 The number of cells grown per unit area was expressed as cell density, and the number of exosome particles released into the harvest medium at the time of measurement was measured to confirm the cell growth status and continuous exosome production behavior. The measurement results are shown in Table 12. [Table 12]

[0206] To confirm consistency with other experiments, the behavior of exosome production is shown in Figure 70. The results of the normal module experiment are shown as human mesenchymal stem cell WAVE reactor (2), and the results of the type 2 module experiment are shown as human mesenchymal stem cell WAVE reactor (3) in the figure.

[0207] Example 9: Long-term modular culture of osteoblasts in an overflow reactor 100 mL of medium (Kohjin Bio's xeno-free medium; KBM ADSC-4) was poured into an overflow reactor culture vessel containing 50 modules that had been previously moistened with medium (Ham's F-12 medium; Fujifilm Wako Pure Chemical Industries, Ltd., containing glutamine and phenol red) for more than two days, and the vessel was then placed in a CO2 incubator (aniCell) equipped with a humidified shaker manufactured by N-BIOTEK, set at 5% CO2 and 37°C. The modules were then stirred and shaken at a rotation speed of 80 rpm for approximately 1 hour.

[0208] Human osteoblasts (Y30 female_439Z037.2) at the 6th passage, cultured in a Corning cell culture FALCON dish, were detached using Gibco Trypsin-EDTA (0.05%) and phenol red, centrifuged to recover the cells, and then suspended in 9 ml of the above-mentioned medium (KBM ADSC-4). The area of ​​the culture substrate polyimide porous membrane inside the module was 1 cm 2 1.0×10 per 4 cells (Total; 9.0 × 10 6 After pouring the cell suspension of 1000 cells into the overflow reactor culture vessel, cell attachment and culture were initiated under stirring conditions at a rotation speed of 80 rpm.

[0209] After cell adsorption, the medium (glucose-adjusted KBM ADSC-4) was pumped into the overflow reactor culture vessel at a rate of 100 mL / day using a tube pump, and the medium overflowing from the medium collection port installed on the wall of the culture vessel was collected in a collection bottle. The collected medium was observed using Roche's CedexBio to observe the cell growth behavior by changing each metabolic parameter of the collected medium, and the liquid supply amount and rotation speed were appropriately changed according to the consumption status of glucose, etc. of the cells, and long-term cell culture was performed. The time-dependent changes in glucose consumption and lactate production are shown in Figure 72. Stable glucose consumption and lactate production were observed over a long period of 200 days.

[0210] To measure the amount of exosomes produced in the collection medium, the procedure described above for "Exosome amount measurement using a zeta potential / particle size distribution analyzer" was used, and the results are shown in Table 13. As with the metabolic behavior, stable exosome production behavior over a long period of more than 100 days was confirmed.

[0211] The medium collected throughout the culture period was collected by period, filtered, and concentrated using the tangential flow filtration method (VIVAFLOW 200 100,000MWCO HY, manufactured by Sartorius). ThermoFisher Total Exosome Isolation Reagent (from cell culture media) was added, and the mixture was stirred, refrigerated, and centrifuged (10,000G / 60min) to obtain exosome precursors as precipitates. An appropriate amount of DPBS was added to this, and the exosome solution was obtained by removing foreign matter using a 0.2μm filter. In this way, exosome solution was obtained for each culture period.

[0212] From these, two samples, one from Day 72 to Day 93 and the other from Day 108 to Day 122, were selected and the expression levels of each miRNA were compared by miRNA array measurement. The results are shown in FIG. [Table 13]

[0213] As is clear from the test results, there was almost no difference in the amount of expressed miRNA between the two exosome samples, demonstrating that exosomes of stable quality were obtained over a long period of time.

[0214] Example 10: Long-term modular culture of osteoblasts in a WAVE reactor 100 mL of medium (Kohjin Bio xeno-free medium; KBM ADSC-4R) was poured into a WAVE reactor culture vessel containing 70 modules that had been moistened with medium (Ham's F-12 medium; Fujifilm Wako Pure Chemical Industries, Ltd., containing glutamine and phenol red) for more than 2 days in advance. 250 mL of medium (Kohjin Bio xeno-free medium; KBM ADSC-4R) was poured into a GE WAVE culture bag (CELLBAG DISPOSABLE BIOREACTOR Part# CB0002L11-03). After placing the bag in a GE WAVE reactor (Xuri Cell Expansion System W5), the modules were shaken for about 1 hour under conditions of 5% CO2, 37°C temperature, 15 rpm vibration speed, and 9° vibration angle.

[0215] PromoCell's human osteoblasts (Y30 female_439Z037.2) at the third passage, cultured in a Corning cell culture FALCON dish, were detached using Gibco's Trypsin-EDTA (0.05%) and phenol red, centrifuged to recover the cells, and then suspended in 13 ml of the above-mentioned medium (KBM ADSC-4R). The area of ​​the culture substrate polyimide porous membrane inside the module was 1 cm 2 1.0×10 per 4 cells (Total; 1.3 × 10 7 After pouring the cell suspension of 1000 cells into the WAVE reactor culture vessel, cell attachment and culture were initiated under vibration conditions of a vibration speed of 15 rpm and a vibration angle of 9°.

[0216] After continuing the vibration culture under the same conditions for 3 days, approximately 20 mL of cell culture medium was extracted from the GE WAVE culture bag via the Harvest tube pump at approximately 3-hour intervals using the perfusion function (bag weight management function) of the reactor accessory (Xuri Cell Expansion System W5 Perfusion Controller), and the same volume of medium (glucose-adjusted KBM ADSC-4R) was pumped into the GE WAVE culture bag via the Feed tube pump, performing a liquid exchange of 150 mL / day. The collected medium was used to observe the cell growth behavior by changes in each metabolic parameter using Roche CedexBio, and stable cell proliferation and growth over a long period of time was observed. In addition, 1 cm of human osteoblasts growing in the module in the bag was counted using a color reaction with Dojindo Cell Counting Kit-8 on the 8th, 22nd, 36th, and 58th days. 2 The cell density per bag and the total number of cells in the bag were measured to confirm the cell growth status. The results are shown in Table 14. In addition, to measure the amount of exosomes produced in the collection medium, the procedure described above for "Exosome amount measurement using a zeta potential / particle size distribution analyzer" was used, and the results are shown in Table 15. As with the metabolic behavior, stable exosome production behavior over a long period of more than 130 days was confirmed. [Table 14] [Table 15]

[0217] Example 11: Long-term modular culture of fetal cardiomyocytes in an overflow reactor 100 mL of medium (Kohjin Bio Co., Ltd. xeno-free medium; KBM ADSC-4) was poured into an overflow reactor culture vessel containing 40 modules that had been previously moistened with medium (Ham's F-12 medium; Fujifilm Wako Pure Chemical Industries, Ltd., containing glutamine and phenol red) for more than two days, and the vessel was then placed in a CO2 incubator (aniCell) equipped with a humidified shaker manufactured by N-BIOTEK, set at 5% CO2 and 37°C. The modules were then stirred and shaken at a rotation speed of 80 rpm for approximately 1 hour.

[0218] First-passage human fetal cardiomyocytes (Lot. No. 21757) (SicenCell) cultured in a Corning cell culture FALCON dish were detached using Gibco Trypsin-EDTA (0.05%) and phenol red, centrifuged to recover the cells, and then suspended in 7.2 ml of the above-mentioned medium (KBM ADSC-4). The area of ​​the culture substrate polyimide porous membrane inside the module was 1 cm 2 1.0×10 per 4 cells (Total; 7.2 × 10 6 After pouring the cell suspension of 1000 cells into the overflow reactor culture vessel, cell attachment and culture were initiated under stirring conditions at a rotation speed of 80 rpm.

[0219] After 24 hours, the medium (glucose-adjusted KBM ADSC-4) was pumped into the overflow reactor culture vessel at a rate of 100 mL / day using a tube pump, and the medium overflowing from the medium collection port installed on the wall of the culture vessel was collected in a collection bottle. The collected medium was observed for cell growth behavior by changes in each metabolic parameter of the collected medium using Roche's CedexBio, and the liquid supply volume and rotation speed were appropriately changed according to the consumption status of glucose, etc. of the cells, and long-term cell culture was performed. The time-dependent changes in glucose consumption and lactate production are shown in Figure 74. Stable glucose consumption and lactate production were observed over a long period of 80 days.

[0220] On days 14, 38, 93, and 114, 1 cm of human fetal cardiomyocytes growing in the module in the bag were counted using a color reaction using Cell Counting Kit-8 (Dojindo Laboratories). 2 The cell density per unit area and the total number of cells in the reactor were measured to confirm the cell growth status. The results are shown in Table 16. Since extremely high proliferation was observed from the beginning, the measurements showed that the cell density was maintained at approximately the same level throughout the culture period. Similar behavior was also expected from glucose consumption and lactate production.

[0221] To measure the amount of exosomes produced in the collection medium, the procedure described above for "Exosome amount measurement using a zeta potential / particle size distribution analyzer" was used, and the results are shown in Table 17. As with the metabolic behavior, stable exosome production behavior over a long period of more than 100 days was confirmed. [Table 16] [Table 17]

[0222] Example 12: Long-term modular culture of fetal cardiomyocytes in a WAVE reactor 250 mL of medium (Kohjin Bio's xeno-free medium; KBM ADSC-4R) was poured into a GE WAVE culture bag (CELLBAG DISPOSABLE BIOREACTOR Part# CB0002L11-03) containing 50 type 2 modules that had been moistened in advance with DPBS (Fujifilm Wako Pure Chemical Industries, Ltd.) for at least 2 days, and the bag was placed in a GE WAVE reactor (Xuri Cell Expansion System W5). The bag was then stirred for approximately 1 hour under conditions of 5% CO2, 37°C temperature, 15 rpm vibration speed, and 9° vibration angle to shake the modules.

[0223] Human fetal cardiomyocytes (Lot. No. 21757) at the fourth passage, cultured in a Corning cell culture FALCON dish, were detached using Gibco Trypsin-EDTA (0.05%) and phenol red, and after centrifugation and cell recovery, were suspended in 10 ml of the above-mentioned medium (KBM ADSC-4R). The area of ​​the culture substrate polyimide porous membrane inside the module was 1 cm 2 1.0×10 per 4 cells(Total;1.0×10 7 After pouring the cell suspension of 1000 cells into the WAVE reactor culture vessel, cell adhesion and culture were initiated under vibration conditions of a vibration speed of 11 rpm and a vibration angle of 7°.

[0224] After continuing the vibration culture under the same conditions for 3 days, approximately 20 mL of cell culture medium was extracted from the GE WAVE culture bag via the Harvest tube pump at approximately 3-hour intervals using the perfusion function (bag weight management function) of the reactor accessory (Xuri Cell Expansion System W5 Perfusion Controller), and the same volume of medium (glucose-adjusted KBM ADSC-4R) was pumped into the GE WAVE culture bag via the Feed tube pump, performing a liquid exchange of 150 mL / day. The collected medium was used to observe the cell growth behavior by changes in each metabolic parameter using Roche CedexBio, and stable cell proliferation and growth over a long period of time was observed. In addition, 1 cm of human fetal cardiomyocytes growing in the module in the bag was counted using a color reaction with Dojindo Cell Counting Kit-8 on the 7th, 14th, 21st, and 35th days. 2 The cell density per bag and the total number of cells in the bag were measured, and the favorable cell growth conditions specific to fetal cardiomyocytes were confirmed. The results are shown in Table 18. [Table 18]

[0225] In addition, to measure the amount of exosomes produced in the collected medium, approximately 20 days' worth of discharged medium was collected and three collected stock solution lots were prepared. After filtering these, they were measured using the procedure for "Measurement of exosome amount using a zeta potential / particle size distribution analyzer" as in Example 6, and the amount of exosomes produced was measured as the protein amount of CD63 using the ELISA method. The results are shown in Table 19. Stable exosome production behavior was confirmed except at the start of the culture. [Table 19]

[0226] Example 13: Chondrocyte module culture on Erlenmeyer and small-piece porous membranes Thermo Fisher Scientific's 125mL and 250mL Erlenmeyer tubes were placed inside, each containing five Type 2 modules that had been pre-wetted with DPBS (Fujifilm Wako Pure Chemical Industries, Ltd.) for at least two days, and 120cm of sterile polyimide porous membrane pieces (1mm x 1mm). 2 Prepare a Corning 125 ml square PET storage bottle (with a 45 mm cap) containing the above material. Add 20 ml of medium (Kohjin Bio xeno-free medium; KBM ADSC-4R) with a glucose level adjusted to 3000 mg / L to the Erlenmeyer flask, and add 30 ml of the same medium to the Corning square bottle containing the small porous membrane.

[0227] Human chondrocytes (Y30 female_439Z037.3) at the fourth passage, cultured on IWAKI collagen I-coated dishes, were detached using Gibco Trypsin-EDTA (0.05%) and phenol red, and then suspended in medium (glucose-adjusted KBM ADSC-4R) after centrifugation and cell recovery. The polyimide porous membrane culture substrate inside the module and the polyimide porous membrane pieces with an area of ​​1 cm 2 1.0×10 per 4 cells (Erlenmeyer flask: Total; 1.0 × 10 6 cells, and the small porous membrane had a total of 1.2×106 The cell suspension was adjusted to 1.0 × 10 cells per ml. 6 The cells were added at a suspension cell density of 1000 cells. The Erlenmeyer flask culture was performed using the shaking incubator of the aniCell described above, and the chondrocyte culture on the small piece porous membrane was performed using static culture in a CO2 incubator.

[0228] The day after the culture, 10 ml of glucose-adjusted KBM ADSC-4R medium was added to the Erlenmeyer flask, and the aforementioned aniCell was used for rotary shaking culture at 80 rpm. Culture was continued with medium changes about twice a week. Module culture in Erlenmeyer flask (five type 2 modules) and small piece porous membrane culture (polyimide porous membrane small piece 120 cm 2 ) were cultured in 30 ml of medium. On days 8, 15, 20, and 35, the total number of cells growing in the modules and porous membrane pieces in each reactor was measured using a color reaction with Cell Counting Kit-8 (Dojindo Laboratories). The favorable cell growth conditions of human chondrocytes were confirmed by the unique proliferation behavior of each reactor. The cell density and total cell number are shown in Figure 75. It was demonstrated that it is possible to easily culture large amounts of cells in various forms stably and for long periods using small culture vessels.

[0229] Furthermore, when the amount of exosomes produced in the harvested medium on day 19 of culture was measured as the amount of CD63 protein using the ELISA method, after three days of storage, 218 pg / ml of exosomes were confirmed in the module using the 125 ml Erlenmeyer vessel, 141 pg / ml in the module using the 250 ml vessel, and 117 pg / ml in the culture system using the small piece porous membrane. As with the continuous culture system described above, efficient and stable exosome production was confirmed in the intermittent culture system.

Claims

1. A method for stably producing exosomes for a period of three months or more using cells, comprising: applying the cells to the cell culture module and culturing the cells; and Producing exosomes by cells; wherein the cell culture module comprises: A polymer porous membrane; A casing having two or more medium inlets and an inlet port, the polymer porous membrane being housed therein; The present invention provides The polymer porous membrane is a three-layer structure polymer porous membrane having surface layers A and B having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B, wherein the average pore size of the pores present in the surface layer A is smaller than the average pore size of the pores present in the surface layer B, the macrovoid layer has partition walls bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition walls and the surface layers A and B, and the pores in the surface layers A and B communicate with the macrovoids, Here, in the casing (i) Two or more independent porous polymer membranes are aggregated together, (ii) the polymeric porous membrane is folded, (iii) the polymer porous membrane is wound into a roll, and / or (iv) the polymeric porous membrane is knotted into a rope; It is housed, The polymer porous membrane is a polyimide porous membrane. The method.

2. The method according to claim 1 , wherein the diameter of the medium inlet is larger than the diameter of the cells and smaller than the diameter of the medium outlet of the porous polymer membrane.

3. The method according to any one of claims 1 to 2, wherein the casing has a mesh-like structure.

4. The method according to any one of claims 1 to 3, wherein the casing is made of a non-flexible material.

5. The method according to any one of claims 1 to 4, wherein the polymeric porous membrane has a plurality of pores having an average pore size of 0.01 to 100 µm.

6. The method according to any one of claims 1 to 5, wherein the average pore size of the surface layer A is 0.01 to 50 µm.

7. The method according to any one of claims 1 to 6, wherein the average pore size of the surface layer B is 20 to 100 µm.

8. The method according to any one of claims 1 to 7, wherein the total thickness of the polymer porous membrane is 5 to 500 µm.

9. The method according to any one of claims 1 to 8, wherein the polyimide porous film is a polyimide porous film containing a polyimide obtained from a tetracarboxylic dianhydride and a diamine.

10. The method according to any one of claims 1 to 9, wherein the polyimide porous film is a colored polyimide porous film obtained by molding a polyamic acid solution composition containing a polyamic acid solution obtained from a tetracarboxylic dianhydride and a diamine and a colored precursor, and then heat-treating the resulting product at 250°C or higher.

11. The method according to any one of claims 1 to 10, wherein the step of culturing the cells is carried out under static culture conditions.

12. The method according to any one of claims 1 to 11, wherein the step of culturing the cells is carried out under rotary or agitation conditions.

13. The method according to any one of claims 1 to 12, wherein the step of culturing the cells is carried out continuously.

14. the step of culturing the cells is carried out in a cell culture device installed in an incubator, wherein the cell culture device includes a culture unit housing the cell culture module supporting the cells, the culture unit having a culture medium inlet and a culture medium outlet; a culture medium supply unit comprising a culture medium storage container, a culture medium supply line, and a liquid delivery pump for delivering the culture medium through the culture medium supply line, wherein a first end of the culture medium supply line contacts the culture medium in the culture medium storage container, and a second end of the culture medium supply line communicates with the inside of the culture unit through the culture medium supply port of the culture medium supply unit; The method according to any one of claims 1 to 13, comprising:

15. The method according to claim 14, wherein the cell culture device does not have a medium supply line, a liquid pump, an air supply port, an air exhaust port, or an oxygen-permeable membrane.

16. The method according to any one of claims 1 to 15, wherein the cells are selected from the group consisting of pluripotent stem cells, tissue stem cells, somatic cells, germ cells, sarcoma cells, established cell lines, and transformed cells.

17. The method according to any one of claims 1 to 16, wherein the cells are selected from the group consisting of human mesenchymal stem cells, osteoblasts, chondrocytes and cardiomyocytes.

18. The method according to any one of claims 1 to 17, wherein the step of producing exosomes overlaps with at least a part of the step of culturing the cells.

19. The method according to any one of claims 1 to 18, wherein the process of producing exosomes continues for a period of six months or more.

20. Use of a cell culture module for the method according to any one of claims 1 to 19, comprising: The cell culture module comprises: A polymer porous membrane; A casing having two or more medium inlets and an inlet port, the polymer porous membrane being housed therein; The present invention provides The polymer porous membrane is a three-layer structure polymer porous membrane having surface layers A and B having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B, wherein the average pore size of the pores present in the surface layer A is smaller than the average pore size of the pores present in the surface layer B, the macrovoid layer has partition walls bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition walls and the surface layers A and B, and the pores in the surface layers A and B communicate with the macrovoids, Here, in the casing (i) Two or more independent porous polymer membranes are aggregated together, (ii) the polymeric porous membrane is folded, (iii) the polymer porous membrane is wound into a roll, and / or (iv) the polymeric porous membrane is knotted into a rope; It is housed, The polymer porous membrane is a polyimide porous membrane.

21. Exosomes obtained by the method according to any one of claims 1 to 20, The exosomes are characterized in that they have a higher expression of miRNA that is effective in suppressing cancer than exosomes obtained by a plate culture method. Exosomes.

22. The exosome described in claim 21, wherein the cancer is selected from the group consisting of oral squamous cell carcinoma, gastric cell carcinoma, and prostate cancer cells.

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