Method of manufacturing scaffold-free three-dimensional cartilage tissue bodies, scaffold-free three-dimensional cartilage tissue bodies, and modular polyimide porous membranes for manufacturing scaffold-free three-dimensional cartilage tissue bodies

Abstract

The purpose of this invention is to provide a cartilage tissue body with sufficient mechanical strength and thickness, which does not contain scaffold components derived from other animals and has high safety. This invention provides a method for manufacturing a scaffold-free three-dimensional cartilage tissue body, comprising: suspending a modular polyimide porous membrane seeded with chondrocytes in a culture medium in a state not fixed within a cell culture container for suspension culture, while simultaneously applying shear stress to the modular polyimide porous membrane and performing a culture step. Furthermore, this invention provides a modular polyimide porous membrane for manufacturing scaffold-free three-dimensional cartilage tissue bodies and a scaffold-free three-dimensional cartilage tissue body.

Description

Technical Field

[0001] This invention relates to a method for manufacturing scaffold-free three-dimensional cartilage tissue, scaffold-free three-dimensional cartilage tissue, and a modular polyimide porous membrane for manufacturing scaffold-free three-dimensional cartilage tissue. Background Technology

[0002] Cartilage tissue is a nerveless and blood vessel-free tissue, composed of chondrocytes and extracellular matrix (type II collagen, proteoglycans, water, etc.). Furthermore, the supply of nutrients required for repair during injury is difficult, thus natural repair is considered challenging. Therefore, various treatment methods are employed, including high osteotomy and artificial joint replacement. In recent years, many regenerative medical methods have been developed, involving the encapsulation of cultured autologous cells into the injured area to heal the injury. Chondrocytes are easily dedifferentiated cells that lose their characteristics in conventional planar culture methods; various studies have yielded results in different culture methods to maintain their characteristics. Furthermore, it has been determined that encapsulating the chondrocyte-specific extracellular matrix (glycosaminoglycans, type II collagen) along with the cells into the injured area is important from a healing perspective; therefore, much research has been conducted on how to generate large quantities of this extracellular matrix during cell culture. As a result of these efforts, some products have been marketed as research and development achievements and are being used in medical settings. While these methods are beginning to be widely applied in practice, numerous methodological challenges have been identified, and their resolution remains highly anticipated. If we analyze the characteristics of various topics, we can say that the following types of topics are the focus.

[0003] (i) Composition (mixture of components other than human-derived components): In order to maintain cartilage properties and produce an appropriate extracellular matrix, some products employ a methodology of using culture media, but the culture media are essentially not human-derived components, but foreign substances.

[0004] (ii) Extracellular matrix: Encapsulating the cartilage-specific extracellular matrix along with the cells during treatment is an important element. Transplantation is highly necessary when these matrices are sufficiently rich in them.

[0005] (iii) Cell count: The main body of the cure is the transplanted chondrocytes, so it is important to embed a large number of cells in a defined area.

[0006] (iv) Strength and size: In order to securely embed and fix it to the affected area, strength and size that are not easily lost from the affected area and are easy to handle during the operation become important.

[0007] (v) Processability: In order to use live cells as raw materials for embedding, it is important to process the embedded cells easily, efficiently and with high reproducibility from the culture device before whole embedding.

[0008] (vi) Integrity of the embedded material: Since it is a raw material embedded in the human body, it is required to grow fully. On the other hand, it is important to carry out the culture process in a state free of foreign objects and xeno-free.

[0009] (vii) Ease of use: It is desirable to be able to supply embedded tissues easily in various locations, so that no special equipment is required in the culture process.

[0010] In response to the above issues, in the field of cartilage regenerative medicine, methods have been proposed to inject or transplant chondrocyte suspensions, spheroids (spherical cell aggregates where cells are aggregated together), chondrocyte sheets made using temperature-responsive polymers, and cultured cartilage made by culturing chondrocytes in a scaffold into the affected area.

[0011] (1) Cell suspension and spheroids

[0012] The method using cell suspension involves collecting a small amount of cartilage tissue from a non-weight-bearing area of ​​a patient with cartilage defects. After enzymatic treatment to separate chondrocytes, the tissue is cultured in a monolayer. The cells are then harvested after approximately three weeks and injected into the cartilage defect as a cell suspension. To retain the injected cells within the defect, the defect is covered with periosteum pre-harvested from the patient's tibia and sealed with fibrin glue before the cell suspension is injected. This method, published in 1994 by a Swedish orthopedic surgeon, was groundbreaking in its application of regenerative medicine in bone defect treatment. However, because the cells are injected in suspension, there are risks of postoperative leakage, cell settling due to gravity, and uneven tissue repair. Furthermore, Patent Document 1 proposes spherical bodies for cell transplantation therapy, but due to their small diameter (less than 1 mm), they also pose the same risk of leakage when applied to areas larger than 1 mm. Therefore, countermeasures such as fixing the spherical bodies to sheet-like structures before transplantation are employed. In other words, fluid transplant materials lack autonomy, thus posing a risk of cell leakage, requiring countermeasures. In addition, while the cell count can be ensured, no special measures have been taken to ensure the extracellular matrix; it can be said that the main focus is on methods for transferring large numbers of cells.

[0013] (2) Chondrocyte slices made using temperature-responsive polymers

[0014] As a transplantation material immobilized with cells, for example, Patent Document 2 proposes cell sheets prepared by culturing chondrocytes in a culture vessel treated with a temperature-responsive polymer. It describes how these sheets do not produce type II collagen, which is specific to cartilage, during culture, but produce type II collagen after transplantation into animals, yielding good results. However, as described in Table 1 of Patent Document 2, the method using temperature-responsive polymers results in cell sheets that are only tens of µm thick, thus exhibiting weak mechanical strength, and shrinkage when peeled from the culture vessel becomes a problem. Regarding this, for example, Patent Document 3 proposes a method to thicken the sheets by stacking them, making the culture sheets adhere tightly to the carrier for easy peeling. However, it describes that cell sheets stacked more than 10 times have difficulty reaching oxygen and nutrients, therefore, stacking times of 4 times or less are preferred. Thus, it can be said that it is difficult to form tissues with thickness using temperature-responsive polymers.

[0015] (3) Cultured cartilage made by culturing chondrocytes in a scaffold.

[0016] As a type of transplant material with fixed cells and thickness, transplant materials made using natural or artificial scaffold materials have been proposed. For example, Patent Documents 4 and 5 propose transplant materials made by seeding or embedding chondrocytes in a gel-like structure containing collagen and proteoglycans and then culturing them. This method can provide transplant materials with thicknesses depending on the size and amount of the scaffold. However, since the scaffolds use collagen derived from other animals, such as bovine or porcine collagen, the impact on the human body cannot be ignored. Furthermore, the collagen solution used has a high concentration and high viscosity, and the collagen gels immediately at 37°C, leading to problems with the uniformity of the produced transplant material.

[0017] <Polyimide porous membrane>

[0018] A cell culture method including the step of culturing cells on a polyimide porous membrane was reported (Patent Document 6).

[0019] Polyimides are a general term for polymers whose repeating units contain imide bonds. Aromatic polyimides are polymers composed of aromatic compounds directly linked by imide bonds. Because aromatic polyimides have a conjugated structure between aromatic compounds via imide bonds, they possess a rigid and robust molecular structure. Furthermore, due to the strong intermolecular forces inherent in imide bonds, they exhibit very high levels of thermal, mechanical, and chemical properties.

[0020] Polyimide porous membranes have been used prior to this application in applications such as filters, low dielectric constant films, and electrolyte membranes for fuel cells, particularly those centered on battery relationships. Patent documents 7-10 specifically describe a polyimide porous membrane with excellent permeability to gases and other substances, high porosity, excellent surface smoothness, and relatively high strength. Despite its high porosity, it possesses multiple large pores with excellent yield strength against compressive stress in the membrane thickness direction. These are all polyimide porous membranes made via ammonium acid.

[0021] Existing technical documents

[0022] Patent documents

[0023] Patent Document 1: Japanese Patent Application Publication No. 2011-41472

[0024] Patent Document 2: International Publication No. 2018 / 154813

[0025] Patent Document 3: Japanese Patent Application Publication No. 2014-155494

[0026] Patent Document 4: Japanese Patent Application Publication No. 2001-293081

[0027] Patent Document 5: International Publication No. 2006 / 011296

[0028] Patent Document 6: International Publication No. 2015 / 012415

[0029] Patent Document 7: International Publication No. 2010 / 038873

[0030] Patent Document 8: Japanese Patent Application Publication No. 2011-219585

[0031] Patent Document 9: Japanese Patent Application Publication No. 2011-219586

[0032] Patent Document 10: International Publication No. 2018 / 021368 Summary of the Invention

[0033] The problem the invention aims to solve

[0034] In the field of cartilage regenerative medicine, various technologies have been developed and put into practical use, but the challenges described above remain. Therefore, the objective of this invention is to provide a cartilage tissue body with sufficient mechanical strength and thickness, which does not contain scaffold components derived from other animals and exhibits high safety.

[0035] Solution for solving the problem

[0036] In view of the above-mentioned problems, the inventors conducted research and discovered that a safe cartilage tissue body with sufficient mechanical strength and thickness, and free of scaffold components derived from other animals, can be produced by a simpler method than before, thus completing the present invention. That is, the present invention includes the following invention.

[0037] [1] A method for manufacturing a scaffold-free three-dimensional cartilage tissue body, comprising:

[0038] A process involving suspending a modular polyimide porous membrane inoculated with chondrocytes in a culture medium in a state not fixed within a cell culture container for suspension culture, while simultaneously applying shear stress to the modular polyimide porous membrane, is performed during the culture process.

[0039] The modular polyimide porous membrane comprises a polyimide porous membrane and a housing containing the polyimide porous membrane. The polyimide porous membrane is fixed to the housing in a state that does not continuously fluctuate due to fluid.

[0040] The shell has one or more culture medium outflow inlets for exposing culture medium to at least a portion of the surface of the polyimide porous membrane.

[0041] The polyimide porous membrane has a three-layer structure, consisting of a surface layer A, a surface layer B, and a large-void layer sandwiched between the surface layers A and B. The surface layers A and B each have a plurality of pores, wherein the average pore diameter of the pores in the surface layer A is smaller than the average pore diameter of the pores in the surface layer B. The large-void layer has a partition wall bonded to the surface layers A and B and a plurality of large voids surrounded by the partition wall and the surface layers A and B. The pores in the surface layers A and B communicate with the large voids.

[0042] [2] According to the method of Project 1, wherein the modular polyimide porous membrane stack contains two or more of the polyimide porous membranes.

[0043] [3] According to the method of item 1 or 2, wherein the shear stress is generated by oscillation and / or stirring in a state of gyratory motion, reciprocating motion, up-and-down motion, wave-like oscillation motion, rotational motion or a combination of these motions.

[0044] [4] The method according to any one of items 1 to 3, wherein the polyimide porous membrane has a plurality of fine pores with an average pore size of 0.01 to 100 µm.

[0045] [5] The method according to any one of items 1 to 4, wherein the average pore size of the surface layer A is 0.01 to 50 µm.

[0046] [6] The method according to any one of items 1 to 5, wherein the average pore size of the surface layer B is 20 to 100 µm.

[0047] [7] The method according to any one of items 1 to 6, wherein the total thickness of the polyimide porous membrane is 5 to 500 µm.

[0048] [8] The method according to any one of items 1 to 7, wherein the polyimide porous membrane is a polyimide porous membrane comprising a polyimide obtained from a tetracarboxylic acid dianhydride and a diamine. [9]

[0050] According to any one of items 1 to 8, the polyimide porous membrane is a colored polyimide porous membrane obtained by heat-treating a polyamic acid solution composition comprising a polyamic acid solution obtained from tetracarboxylic dianhydride and diamine and a coloring precursor at a temperature above 250°C after molding.

[0051]

[10] A scaffold-free three-dimensional cartilage tissue obtained by any one of items 1 to 9.

[0052]

[11] A scaffold-free three-dimensional cartilage tissue body, characterized in that it comprises chondrocytes,

[0053] It contains no externally added scaffold components other than the extracellular matrix generated by the chondrocytes.

[0054] The scaffold-free three-dimensional cartilage tissue has an average thickness of 0.2 mm or more.

[0055] Each 1 mg wet weight contains more than 5 µg of glycosaminoglycans.

[0056]

[12] The scaffold-free three-dimensional cartilage tissue body according to item 11, wherein the scaffold-free three-dimensional cartilage tissue body has an average thickness of 0.5 mm or more.

[0057]

[13] A modular polyimide porous membrane, characterized in that it is a modular polyimide porous membrane for manufacturing scaffold-free three-dimensional cartilage tissue,

[0058] The modular polyimide porous membrane comprises a polyimide porous membrane and a housing containing the polyimide porous membrane. The polyimide porous membrane is fixed to the housing in a state that does not continuously fluctuate due to fluid.

[0059] The shell has one or more culture medium outflow inlets for exposing culture medium to at least a portion of the surface of the polyimide porous membrane.

[0060] The polyimide porous membrane has a three-layer structure comprising a surface layer A, a surface layer B, and a large-void layer sandwiched between surface layer A and surface layer B. Surface layer A and surface layer B each have multiple pores, wherein the average pore diameter of the pores in surface layer A is smaller than the average pore diameter of the pores in surface layer B. The large-void layer has a partition wall bonded to surface layers A and B and multiple large voids surrounded by the partition wall and surface layers A and B. The pores in surface layers A and B communicate with the large voids.

[0061] The modular polyimide porous membrane is used in cell culture containers, cell culture devices, or cell culture systems for suspension culture, but is not fixed within the culture section of the cell culture container, cell culture device, or cell culture system for suspension culture.

[0062]

[14] The modular polyimide porous membrane according to item 13 is stacked to house two or more of the polyimide porous membranes.

[0063]

[15] The modular polyimide porous membrane according to item 13 or 14, wherein the polyimide porous membrane has a plurality of fine pores with an average pore size of 0.01 to 100 µm.

[0064]

[16] A modular polyimide porous membrane according to any one of items 13 to 15, wherein the average pore size of the surface layer A is 0.01 to 50 µm.

[0065]

[17] A modular polyimide porous membrane according to any one of items 13 to 16, wherein the average pore size of the surface layer B is 20 to 100 µm.

[0066]

[18] A modular polyimide porous membrane according to any one of items 13 to 17, wherein the total thickness of the polyimide porous membrane is 5 to 500 µm.

[0067]

[19] A modular polyimide porous membrane according to any one of items 13 to 18, wherein the polyimide porous membrane is a polyimide porous membrane comprising a polyimide obtained from a tetracarboxylic dianhydride and a diamine.

[0068]

[20] A modular polyimide porous membrane according to any one of items 13 to 19, wherein the polyimide porous membrane is a colored polyimide porous membrane obtained by molding a polyamic acid solution composition comprising a polyamic acid solution obtained from a tetracarboxylic acid dianhydride and a diamine and a coloring precursor, and then heat-treating it at a temperature above 250°C.

[0069] The effects of the invention

[0070] According to the present invention, a three-dimensional cartilage tissue body with sufficient mechanical strength and thickness compared to three-dimensional cartilage tissue bodies obtained by conventional methods can be provided. Furthermore, the three-dimensional cartilage tissue body provided by the present invention has a higher cell count and extracellular matrix quality than conventional methods, exhibits superior strength, is easily retrievable during fabrication, and demonstrates excellent reproducibility. Additionally, the three-dimensional cartilage tissue body obtained by the present invention is primarily composed of chondrocytes and the extracellular matrix produced by these cells, containing no components derived from other animals, thus ensuring high safety for organisms. Attached Figure Description

[0071] Figure 1 This represents one embodiment of a modular polyimide porous membrane.

[0072] Figure 2 This represents one embodiment of a modular polyimide porous membrane. Figure 1 A top view of a modular polyimide porous membrane.

[0073] Figure 3 This represents one embodiment of a modular polyimide porous membrane. It is... Figure 2 A diagram of the A-A' cross section of the modular polyimide porous membrane.

[0074] Figure 4 A diagram illustrating a cell culture model using a polyimide porous membrane.

[0075] Figure 5 This indicates the modular polyimide porous membrane used in the examples.

[0076] Figure 6 These are photographs of the scaffold-free three-dimensional cartilage tissue obtained in the examples. Top section: scaffold-free three-dimensional cartilage tissue constructed on the polyimide porous membrane after the shell components were removed; Bottom section: scaffold-free three-dimensional cartilage tissue peeled off from the polyimide porous membrane.

[0077] Figure 7 This indicates the results of comparing the amount of DNA or glycosaminoglycans contained in three-dimensional cartilage tissues prepared by examples or comparative examples. Detailed Implementation

[0078] I. Methods for fabricating scaffold-free three-dimensional cartilage tissue bodies

[0079] In one embodiment, the present invention provides a method for manufacturing a scaffold-free three-dimensional cartilage tissue body, comprising:

[0080] A process involving suspending a modular polyimide porous membrane inoculated with chondrocytes in a culture medium in a state not fixed within a cell culture container for suspension culture, while simultaneously applying shear stress to the modular polyimide porous membrane, is performed during the culture process.

[0081] The modular polyimide porous membrane comprises a polyimide porous membrane and a housing containing the polyimide porous membrane. The polyimide porous membrane is fixed to the housing in a state that does not continuously fluctuate due to fluid.

[0082] The shell has one or more culture medium outflow inlets for exposing culture medium to at least a portion of the surface of the polyimide porous membrane.

[0083] The polyimide porous membrane has a three-layer structure, consisting of a surface layer A, a surface layer B, and a large-void layer sandwiched between the surface layers A and B. The surface layers A and B each have a plurality of pores, wherein the average pore diameter of the pores in the surface layer A is smaller than the average pore diameter of the pores in the surface layer B. The large-void layer has a partition wall bonded to the surface layers A and B and a plurality of large voids surrounded by the partition wall and the surface layers A and B. The pores in the surface layers A and B communicate with the large voids.

[0084] 1. Chondrocytes

[0085] In this specification, "chondrocyte" refers to cells that produce the extracellular matrix of cartilage tissue, such as collagen and proteoglycans, or their precursor cells (e.g., mesenchymal stem cells, osteoblasts, or chondrocytes). Chondrocytes applicable to this invention can be cells expressing chondrocyte markers. Examples of chondrocyte markers include, but are not limited to, type II collagen (COL2A1) or SOX9. COL2A1 can be exemplified by, for example, as an NCBI accession number, in the human case, a gene having the nucleotide sequence described in NM_001844 or NM_033150 and the protein encoded by that gene, and a naturally occurring mutant having these functions; in the mouse case, a gene having the nucleotide sequence described in NM_001113515 or NM_031163 and the protein encoded by that gene, and a naturally occurring mutant having these functions, but is not limited to these. Additionally, SOX9 can be exemplified by, for example, as an NCBI accession number, in the case of humans, a gene having the nucleotide sequence described in NM_000346 and the protein encoded by that gene, and naturally occurring mutants having those functions; in the case of mice, a gene having the nucleotide sequence described in NM_011448 and the protein encoded by that gene, and naturally occurring mutants having those functions, but is not limited thereto.

[0086] The chondrocytes used in this invention can be isolated from biological tissues, such as articular cartilage, auricular cartilage, or nasal cartilage, or collected from the extra fingers of a subject with polydactyly. Furthermore, the chondrocytes can be autologous cells collected from a transplant recipient, or allogeneic cells collected from a subject other than a transplant recipient.

[0087] The chondrocytes used in this invention can be chondrocytes induced from the differentiation of pluripotent stem cells. Pluripotent stem cells refer to a general term for stem cells capable of differentiating into cells of all tissues (differentiation pluripotency). Pluripotent stem cells include, but are not limited to, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germline stem cells (EG cells), and germline stem cells (GS cells). ES cells or iPS cells are preferred. iPS cells are particularly preferred due to the absence of ethical concerns. Any known pluripotent stem cell can be used, such as the pluripotent stem cells described in International Publication WO2009 / 123349 (PCT / JP2009 / 057041). The method for inducing chondrocytes applicable to this invention from pluripotent stem cells is not particularly limited; known methods can be referenced, such as International Publication No. 2016 / 133208.

[0088] 2. Scaffold-free three-dimensional cartilage tissue

[0089] In this specification, "scaffold-free three-dimensional cartilage tissue" refers to a three-dimensional tissue containing chondrocytes but without externally added scaffold components (such as determinated collagen, matrix gel, or gelatin) other than the extracellular matrix generated by the chondrocytes themselves. Specifically, the "scaffold-free three-dimensional cartilage tissue" provided by this invention is a tissue generated by self-three-dimensionalization of the extracellular matrix generated by the chondrocytes themselves, having an average thickness of 0.2 mm or more, preferably 0.5 mm or more. Furthermore, the "scaffold-free three-dimensional cartilage tissue" provided by this invention contains at least 5 µg of glycosaminoglycans per 1 mg wet weight. The scaffold-free three-dimensional cartilage tissue provided by this invention has a higher cell count and extracellular matrix quality than previous methods, excellent strength, and is easy to recycle after fabrication. Therefore, it is easy to handle. In addition, the scaffold-free three-dimensional cartilage tissue provided by this invention is mainly composed of chondrocytes and the extracellular matrix generated by these cells themselves; therefore, it does not contain components derived from other animals, and therefore contains almost no substances that may cause allergic reactions, thus exhibiting high safety.

[0090] 3. Modular polyimide porous membrane

[0091] The "modular polyimide porous membrane" used in or provided by this invention comprises a polyimide porous membrane and a housing containing the polyimide porous membrane, wherein the polyimide porous membrane is fixed to the housing in a state that does not continuously fluctuate due to fluid.

[0092] The shell has one or more culture medium outflow inlets for exposing culture medium to at least a portion of the surface of the polyimide porous membrane.

[0093] The polyimide porous membrane has a three-layer structure comprising a surface layer A, a surface layer B, and a large-void layer sandwiched between the surface layers A and B. Surface layers A and B each have a plurality of pores, wherein the average pore diameter of the pores in surface layer A is smaller than the average pore diameter of the pores in surface layer B. The large-void layer has a partition wall bonded to the surface layers A and B and a plurality of large voids surrounded by the partition wall and the surface layers A and B. The pores in the surface layers A and B communicate with the large voids (e.g., see reference). Figure 1 ).

[0094] Chondrocytes grow on the surface of a modular polyimide porous membrane, specifically the portion of the membrane that opens as the culture medium flows out through the inlet. In this state, the modular polyimide porous membrane is suspended in the culture medium and cultured while being subjected to shear stress. This promotes the generation of the chondrocyte's own extracellular matrix and facilitates chondrocyte proliferation, resulting in a scaffold-free three-dimensional cartilage tissue mass that is thickened in the vertical direction relative to the plane of the polyimide porous membrane.

[0095] The shape of the resulting scaffold-free three-dimensional cartilage tissue body is altered according to the shape of the culture medium inlet and outlet of the modular polyimide porous membrane. Therefore, for the intended use, such as transplantation, the shape of the culture medium inlet and outlet of the modular polyimide porous membrane can be designed according to the shape and size of the transplantation site, and its shape and size are not particularly limited. For example, the shape and size of the opening of the culture medium inlet and outlet can be designed as any shape and size, such as rectangle, circle, ellipse, triangle, pentagon, or hexagon.

[0096] In one embodiment, the modular polyimide porous membrane may have two or more culture medium effluent inlets / outlets. For example, multiple culture medium effluent inlets / outlets may be provided on the same side of the housing of the modular polyimide porous membrane, or multiple inlets / outlets may be provided on both sides of the housing of the polyimide porous membrane housed within the housing (see reference). Figure 3 ).

[0097] The thickness of the opening at the culture medium outflow inlet of the modular polyimide porous membrane, i.e., the thickness of the shell housing the polyimide porous membrane, can be varied to change the thickness of the scaffold-free three-dimensional cartilage tissue. For example, if the shell thickness is 0.2 mm, the culture medium outflow inlet becomes an opening with a depth of 0.2 mm from the surface of the shell to the surface of the polyimide porous membrane (hereinafter referred to as the "thickness of the culture medium outflow inlet") (see reference). Figure 3 On the surface of the polyimide porous membrane forming the concave portion, chondrocytes proliferate in a vertical direction, forming a scaffold-free three-dimensional cartilage tissue body with its own thickness. The thickness of the obtained scaffold-free three-dimensional cartilage tissue body can be adjusted by changing the thickness of the culture medium inlet / outlet; for example, shells with thicknesses of 0.3 mm or more, 0.4 mm or more, 0.5 mm or more, 0.6 mm or more, 0.7 mm or more, 0.8 mm or more, 0.9 mm or more, 1.0 mm or more, or greater can be used.

[0098] In one embodiment of the modular polyimide porous membrane, the surface of the polyimide porous membrane exposed to the culture medium is preferably surface layer B. If the modular polyimide porous membrane is configured such that the surface layer B of the polyimide porous membrane is in contact with the culture medium outflow inlet, the scaffold-free three-dimensional cartilage tissue formed at the culture medium outflow inlet can be easily peeled off, thus facilitating recycling.

[0099] In one embodiment, the modular polyimide porous membrane may have a shell composed of decomposable components. For example, such as Figures 1-3 As shown, the modular polyimide porous membrane can also have a rectangular culture medium outflow inlet at the center. Two or more stacked polyimide porous membranes are clamped from both sides using a flat, plate-shaped shell component that is substantially the same shape as the polyimide porous membrane, and their ends are clamped by two or more clamps for fixation. If the shell of the modular polyimide porous membrane can be disassembled, the fabricated scaffold-free three-dimensional cartilage tissue can be easily recovered. In one embodiment, when multiple polyimide porous membranes are stacked, the outermost polyimide porous membrane can be configured such that its surface layer B is in contact with the culture medium outflow inlet.

[0100] In this specification, the term "modular polyimide porous membrane" may also be abbreviated as "module," and the two terms have the same meaning even if used interchangeably.

[0101] In this specification, "modular polyimide porous membrane" refers to a cell culture medium suitable for cell culture containers, cell culture apparatuses, and cell culture systems, particularly for suspension culture. The modular polyimide porous membrane (cell culture module) described in International Publication WO2018 / 021368 is also applicable to this invention. As an example, the modular polyimide porous membrane of this invention can be used via a WAVE reactor. Alternatively, it can be used via an Erlenmeyer reactor.

[0102] The modular polyimide porous membrane of the present invention, by housing the polyimide porous membrane within a shell, prevents the membrane-like polyimide porous membrane from continuously deforming within the shell. This prevents stress on cells growing inside the polyimide porous membrane, inhibits apoptosis, and allows for stable and large-scale cell culture. Furthermore, the modular polyimide porous membrane used in this invention can be cultured in suspension under conditions of strong shear stress, including rotary motion, reciprocating motion, up-and-down motion, undulating motion, or combinations thereof, thereby forming scaffold-free three-dimensional cartilage tissue.

[0103] The modular polyimide porous membrane of the present invention preferably has a shell with strength that prevents deformation under normal culture conditions, such as the movement of the culture medium under stirred or oscillating conditions. Furthermore, the shell is preferably formed from raw materials that do not affect cell growth during cell culture. Examples of such materials include polymers such as polyethylene, polypropylene, nylon, polyester, polystyrene, polycarbonate, polymethyl methacrylate, and polyethylene terephthalate; and metals such as stainless steel and titanium, but are not limited thereto. By giving the shell a certain degree of strength, continuous deformation of the polyimide porous membrane inside the shell can be prevented, further enhancing the effects of the present invention. In this specification, "shell does not deform" means that it does not deform under loads subjected to normal culture conditions, and does not mean absolutely no deformation.

[0104] In one embodiment, the modular polyimide porous membrane used or provided in this invention houses two or more of the polyimide porous membranes stacked within a housing. In this case, a gasket can be provided between the polyimide porous membranes. By providing the gasket, culture medium can be efficiently supplied between the stacked polyimide porous membranes. The gasket is not particularly limited as long as it has the function of forming arbitrary spaces between the stacked polyimide porous membranes and efficiently supplying culture medium; for example, a planar structure with a mesh structure can be used. The material of the gasket can be, for example, polystyrene, polycarbonate, polymethyl methacrylate, polyethylene terephthalate, or stainless steel mesh, but is not limited to these. When a gasket with a mesh structure is provided, the mesh size can be appropriately selected as long as it is large enough to supply culture medium between the stacked polyimide porous membranes.

[0105] In this specification, "folded polyimide porous membrane" refers to a polyimide porous membrane that, by folding within the housing, becomes stationary within the housing due to frictional forces between the membrane and its surfaces and / or the surfaces inside the housing. In this specification, "folded" can refer to the polymer porous membrane having creases or not having creases.

[0106] In this specification, "the polyimide porous membrane remaining stationary within the housing" refers to the state in which the modular polyimide porous membrane is housed within the housing in a way that prevents continuous morphological changes when culturing the membrane in a cell culture medium. In other words, the polyimide porous membrane itself is in a state where it does not continuously fluctuate due to fluid. Because the polyimide porous membrane remains stationary within the housing, stress on the cells growing within it can be prevented, thus preventing cell death due to apoptosis and other reasons, and ensuring stable cell culture.

[0107] In one embodiment, the modular polyimide porous membrane used in this invention is suspended in a culture medium in a state not fixed within a cell culture container for suspension culture, and cultured under conditions that impart shear stress to the modular polyimide porous membrane. This promotes the proliferation of chondrocytes growing within the polyimide porous membrane at the culture medium outflow inlet, resulting in a thickened, scaffold-free three-dimensional cartilage tissue.

[0108] Shear stress can be imparted, for example, by applying the modular polyimide porous membrane to commercially available culture containers, culture devices, and systems capable of stirring in rotational, reciprocating, up-and-down, oscillating, or combined motions. For example, it can also be applied to culture devices where the culture container is made of a flexible bag, allowing for use while suspended within the culture container. Furthermore, the modular polyimide porous membrane of the present invention can be applied, for example, to stirred reactors such as rotary flasks for culturing. Moreover, as a culture container, both open and closed containers can be used. For example, culture dishes, flasks, plastic bags, test tubes, and large culture jars for cell culture can all be appropriately utilized. Examples include cell culture dishes manufactured by BD Falcon and Nunc cell factories manufactured by Thermo Scientific.

[0109] 4. Application of modular polyimide porous membranes in cell culture devices

[0110] In this specification, "cell culture apparatus" is generally used synonymously with cell culture system, bioreactor or reactor, and the two terms have the same meaning even when used interchangeably.

[0111] 5. Polyimide porous membrane

[0112] The average pore size of the pores present in the surface layer A (hereinafter also referred to as "A surface" or "mesh surface") of the polyimide porous membrane used in this invention is not particularly limited, for example, it is 0.01µm or more and less than 200µm, 0.01~150µm, 0.01~100µm, 0.01~50µm, 0.01µm~40µm, 0.01µm~30µm, 0.01µm~20µm, or 0.01µm~15µm, preferably 0.01µm~15µm.

[0113] The average pore diameter of the pores present in the surface layer B (hereinafter also referred to as "B surface" or "macropore surface") of the polyimide porous membrane used in this invention is not particularly limited as long as it is greater than the average pore diameter of the pores present in the surface layer A. For example, it can be more than 5µm and less than 200µm, 20µm~100µm, 30µm~100µm, 40µm~100µm, 50µm~100µm, or 60µm~100µm, preferably 20µm~100µm.

[0114] The average pore size of the polyimide porous membrane surface can be calculated as follows: Based on the scanning electron microscope image of the porous membrane surface, the pore area of ​​more than 200 openings is measured, and the average diameter of the pore when the shape of the pore is a perfect circle is calculated from the average value of the pore area according to the following formula (1).

[0115] [Number 1]

[0116]

[0117] (In the formula, Sa refers to the average area of ​​the pores.)

[0118] The thickness of surface layers A and B is not particularly limited, for example, it is 0.01~50µm, preferably 0.01~20µm.

[0119] The average pore size in the membrane plane of the large-pore layer in the polyimide porous membrane is not particularly limited, but is, for example, 10-500 µm, preferably 10-100 µm, and more preferably 10-80 µm. Furthermore, the thickness of the partition walls in the large-pore layer is not particularly limited, but is, for example, 0.01-50 µm, preferably 0.01-20 µm. In one embodiment, at least one partition wall in the large-pore layer has one or more pores with an average pore size of 0.01-100 µm, preferably 0.01-50 µm, connecting adjacent large-pores to each other. In another embodiment, the partition walls in the large-pore layer do not have pores.

[0120] The total thickness of the polyimide porous membrane surface used in this invention is not particularly limited, and can be 5µm or more, 10µm or more, 20µm or more, or 25µm or more, and can be less than 500µm, less than 300µm, less than 100µm, less than 75µm, or less than 50µm. Preferably, it is 5 to 500µm, more preferably 25 to 75µm.

[0121] The thickness of the polyimide porous membrane used in this invention can be measured using a contact thickness gauge.

[0122] The porosity of the polyimide porous membrane used in this invention is not particularly limited, for example, it is 40% or more and less than 95%.

[0123] The porosity of the polyimide porous membrane used in this invention can be determined by measuring the membrane thickness and mass of the porous membrane cut to a specified size, and is calculated from the mass per unit area according to the following formula (2).

[0124] [Number 2]

[0125]

[0126] (In the formula, S represents the area of ​​the porous membrane, d represents the total membrane thickness, w represents the measured mass, and D represents the density of the polymer. When the polymer is polyimide, the density is 1.34 g / cm³.) 3 。 )

[0127] The polyimide porous membrane used in this invention preferably has a three-layer structure comprising a surface layer A, a surface layer B, and a large-pore layer sandwiched between the surface layers A and B. The surface layers A and B have multiple pores, wherein the average pore diameter of the pores in the surface layer A is 0.01µm to 15µm, and the average pore diameter of the pores in the surface layer B is 20µm to 100µm. The large-pore layer has a partition wall bonded to the surface layers A and B and multiple large pores surrounded by the partition wall and the surface layers A and B. The thickness of the partition wall of the large-pore layer and the surface layers A and B is 0.01 to 20µm. The pores in the surface layers A and B are connected to the large pores. The total membrane thickness is 5 to 500µm, and the porosity is 40% or more and less than 95%. In one embodiment, at least one partition wall in the large void layer has one or more pores with an average pore size of 0.01 to 100 µm, preferably 0.01 to 50 µm, that connect adjacent large voids to each other. In another embodiment, the partition wall does not have such pores.

[0128] The polyimide porous membrane used in this invention is preferably sterilized. The sterilization process is not particularly limited and can include any sterilization method such as dry heat sterilization, steam sterilization, sterilization using disinfectants such as ethanol, or electromagnetic wave sterilization such as ultraviolet light or gamma rays.

[0129] Polyimide is a general term for polymers whose repeating units contain imide bonds. It usually refers to aromatic polyimides formed by aromatic compounds directly linked by imide bonds. Aromatic polyimides have a rigid and strong molecular structure because aromatic compounds are conjugated by imide bonds. Furthermore, due to the strong intermolecular forces of the imide bonds, they possess very high levels of thermal, mechanical, and chemical properties.

[0130] The polyimide porous membrane used in this invention is preferably a polyimide porous membrane comprising a polyimide obtained from tetracarboxylic dianhydride and diamine (as a main component), more preferably a polyimide porous membrane composed of a polyimide obtained from tetracarboxylic dianhydride and diamine. "Comprising as a main component" means that, as a constituent component of the polyimide porous membrane, it essentially does not contain any components other than the polyimide obtained from tetracarboxylic dianhydride and diamine, or it may contain additional components that do not affect the properties of the polyimide obtained from tetracarboxylic dianhydride and diamine.

[0131] In one embodiment, the polyimide porous membrane that can be used in the present invention also includes a colored polyimide porous membrane obtained by molding a polyamic acid solution composition comprising a polyamic acid solution obtained from a tetracarboxylic acid component and a diamine component and a coloring precursor, and then heat-treating it at a temperature above 250°C.

[0132] Polyamic acid can be obtained by polymerizing a tetracarboxylic acid component with a diamine component. Polyamic acid is a polyimide precursor that can be ring-closed to form polyimides through thermal imidization or chemical imidization.

[0133] Even if polyamic acid is partially imidized, it can still be used as long as it does not affect the scope of this invention. That is, polyamic acid can be partially thermally imidized or chemically imidized.

[0134] When performing thermal imidization of polyamic acid, imidization catalysts, organophosphorus compounds, inorganic microparticles, organic microparticles, etc., can be added to the polyamic acid solution as needed. Similarly, when performing chemical imidization of polyamic acid, chemical imidizing agents, dehydrating agents, inorganic microparticles, organic microparticles, etc., can be added to the polyamic acid solution as needed. Even when the aforementioned components are mixed in the polyamic acid solution, it is preferable to carry out the process under conditions where the coloring precursor does not precipitate.

[0135] In this specification, "coloring precursor" refers to a precursor that is partially or completely carbonized by heat treatment at 250°C or above to generate a coloring compound.

[0136] As a coloring precursor that can be used in the manufacture of the above-mentioned polyimide porous membrane, it is preferably uniformly dissolved or dispersed in a polyamic acid solution or a polyimide solution, and then subjected to heat treatment at 250°C or above, preferably 260°C or above, more preferably 280°C or above, and more preferably 300°C or above. It is also preferred to generate a coloring compound by thermal decomposition and carbonization in the presence of oxygen such as air at 250°C or above, preferably 260°C or above, more preferably 280°C or above, and more preferably 300°C or above. It is even more preferred to generate a black coloring compound, and more preferably a carbon-based coloring precursor.

[0137] The coloring precursor appears as a carbide when heated, but contains different elements other than carbon in its structure, including layered structures, aromatic cross-linked structures, and disordered structures containing tetrahedral carbon.

[0138] There are no particular limitations on carbon-based coloring precursors, such as petroleum tar, petroleum pitch, coal tar, coal pitch, etc.; coke; polymers obtained from monomers containing acrylonitrile; ferrocene compounds (ferrocene and ferrocene derivatives); etc. Among these, polymers obtained from monomers containing acrylonitrile and / or ferrocene compounds are preferred, and polyacrylonitrile is preferred as a polymer obtained from monomers containing acrylonitrile.

[0139] In another embodiment, the polyimide porous membrane that can be used in the present invention also includes a polyimide porous membrane obtained by heat treatment after molding a polyamic acid solution obtained from a tetracarboxylic acid component and a diamine component without using the above-mentioned coloring precursor.

[0140] A porous polyimide membrane manufactured without using a coloring precursor can be produced, for example, as follows: A polyamic acid solution consisting of 3-60% by mass of polyamic acid with an intrinsic viscosity number of 1.0-3.0 and 40-97% by mass of an organic polar solvent is cast into a film, which is then immersed in or contacted with a coagulation solvent in which water is an essential component to form a porous polyamic acid membrane. The porous polyamic acid membrane is then heat-treated to imidize it. In this method, the coagulation solvent in which water is an essential component can be water, or a mixture of 5% by mass or more but less than 100% by mass of water and more than 0% by mass but less than 95% by mass of an organic polar solvent. Furthermore, after the above imidization, at least one side of the obtained porous polyimide membrane can be subjected to plasma treatment.

[0141] In the manufacture of the above-mentioned polyimide porous membrane, any tetracarboxylic acid dianhydride can be used, and it can be appropriately selected according to the desired properties, etc. Specific examples of tetracarboxylic dianhydrides include pyromellitic dianhydride, 3,3',4,4'-biphenyltetracarboxylic dianhydride (s-BPDA), 2,3,3',4'-biphenyltetracarboxylic dianhydride (a-BPDA), etc.; oxydiphthalic dianhydride, diphenyl sulfone-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, 2,2-bis(3,4- Dicarboxyphenyl)propane dianhydride, p-phenylenebis(triphenyltriacrylic acid monoester anhydride), p-biphenylenebis(triphenyltriacrylic acid monoester 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)phenyl dianhydride, 1,4-bis(3,4-dicarboxyphenoxy)phenyl dianhydride, 1,4-bis(3,4-dicarboxyphenoxy)biphenyl dianhydride, 2,2-bis[(3,4-dicarboxyphenoxy)phenyl]propane dianhydride, 2,3,6,7-naphthalenetetracarboxylic acid dianhydride, 1,4,5,8-naphthalenetetracarboxylic acid dianhydride, 4,4'-(2,2-hexafluoroisopropylidene)diphthalic acid dianhydride, etc. In addition, aromatic tetracarboxylic acids such as 2,3,3',4'-diphenyl sulfone tetracarboxylic acid are preferred. They can be used alone or in combination of two or more.

[0142] Of particular preference is at least one aromatic tetracarboxylic dianhydride selected from the group consisting of biphenyltetracarboxylic dianhydride and pyromellitic dianhydride. 3,3',4,4'-biphenyltetracarboxylic dianhydride is preferably used as the biphenyltetracarboxylic dianhydride.

[0143] The diamine used in the manufacture of the aforementioned polyimide porous membrane can be any diamine. Specific examples of diamines include the following.

[0144] 1) Phenylenediamines with one benzene ring, such as 1,4-diaminobenzene (p-phenylenediamine), 1,3-diaminobenzene, 2,4-diaminotoluene, and 2,6-diaminotoluene;

[0145] 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'-diaminobenzoylaniline, 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'-diaminodiphenyl ketone, 3,3'-diamino-4,4'-dichlorobenzophenone, 3,3'-diamino-4,4'-dimethoxybenzophenone, 3,3'-diaminodiphenylmethane, 3,4'-diaminodiphenylmethane, Diamines with two benzene rings, including 4,4'-diaminodiphenylmethane, 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;

[0146] 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-phenyl) Diamines with three benzene rings, including phenoxybenzophenone, 1,3-bis(3-aminophenyl sulfone)benzene, 1,3-bis(4-aminophenyl sulfone)benzene, 1,4-bis(4-aminophenyl sulfone)benzene, 1,3-bis(3-aminophenyl sulfone)benzene, 1,4-bis(4-aminophenyl sulfone)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.

[0147] 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 ...3-aminophenoxy)phenyl] ketone, bis[3-(4-aminophenoxy)phenyl] ketone, bis[3-(3-aminophenoxy)phenyl] ketone, bis[3-(4-aminophenoxy)phenyl] ketone, bis[3-(3-aminophenoxy)phenyl] ketone, bis[3-(3-aminophenoxy)phenyl] ketone, bis[3-(3-aminophenoxy)phenyl] ketone, bis[3-(3-aminophenoxy)phenyl] ketone, bis[3-(3-aminophenoxy)phenyl] ketone, bis[3-(3-aminophenoxy) [Phenoxy]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-bis[4-( Diamines with four benzene rings, including 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.

[0148] They can be used alone or in combination of two or more. The diamine used can be selected appropriately based on the desired properties, etc.

[0149] Among them, aromatic diamine compounds are preferred, and 3,3'-diaminodiphenyl ether, 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenyl ether, and p-phenylenediamine, 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 are particularly preferred. At least one diamine selected from the group consisting of phenylenediamine, diaminodiphenyl ether, and bis(aminophenoxy)benzene is especially preferred.

[0150] From the viewpoint of heat resistance and dimensional stability at high temperatures, the polyimide porous membrane that can be used in this invention is preferably formed from a polyimide obtained by combining a tetracarboxylic acid dianhydride with a glass transition temperature of 240°C or higher, or one with no obvious transition point at 300°C or higher, with a diamine.

[0151] From the viewpoint of heat resistance and dimensional stability at high temperatures, the polyimide porous membrane that can be used in this invention is preferably a polyimide porous membrane composed of aromatic polyimides.

[0152] (i) An aromatic polyimide consisting of at least one tetracarboxylic acid unit selected from the group consisting of biphenyl tetracarboxylic acid units and pyromellitic tetracarboxylic acid units and an aromatic diamine unit;

[0153] (ii) An aromatic polyimide consisting of a tetracarboxylic acid unit and at least one aromatic diamine unit selected from the group consisting of a phenylenediamine unit, a diaminodiphenyl ether unit and a bis(aminophenoxy)benzene unit;

[0154] and / or

[0155] (iii) An aromatic polyimide consisting of at least one tetracarboxylic acid unit selected from the group consisting of biphenyltetracarboxylic acid unit and pyromellitic acid unit, and at least one aromatic diamine unit selected from the group consisting of phenylenediamine unit, diaminodiphenyl ether unit and bis(aminophenoxy)benzene unit.

[0156] The polyimide porous membrane used in this invention preferably has a three-layer structure comprising a surface layer A, a surface layer B, and a large-pore layer sandwiched between the surface layers A and B. The surface layers A and B have multiple pores, wherein the average pore diameter of the pores in the surface layer A is 0.01µm to 15µm, and the average pore diameter of the pores in the surface layer B is 20µm to 100µm. The large-pore layer has a partition wall bonded to the surface layers A and B and multiple large pores surrounded by the partition wall and the surface layers A and B. The thickness of the partition wall of the large-pore layer and the surface layers A and B is 0.01 to 20µm. The pores in the surface layers A and B are connected to the large pores. The total membrane thickness is 5 to 500µm, and the porosity is 40% or more and less than 95%. Here, at least one partition wall in the large void layer has one or more pores with an average pore size of 0.01 to 100 µm, preferably 0.01 to 50 µm, that connect adjacent large voids to each other.

[0157] For example, the polyimide porous membrane described in International Publication No. 2010 / 038873, Japanese Patent Application Publication No. 2011-219585, or Japanese Patent Application Publication No. 2011-219586 can also be used in this invention.

[0158] 6. Application of chondrocytes to modular polyimide porous membranes

[0159] The specific steps for applying cells to the modular polyimide porous membrane are not particularly limited. The steps described in this specification, or any method suitable for applying chondrocytes to a membrane-like carrier, may be used. In the method of the present invention, the application (seeding) of chondrocytes to the modular polyimide porous membrane may include, for example, the following methods, but is not limited thereto.

[0160] Includes: (1) the process of applying a first culture medium containing suspended chondrocytes to a modular polyimide porous membrane;

[0161] (2) The step of maintaining the modular polyimide porous membrane at a temperature suitable for cell culture, so that the cells are adsorbed onto the polyimide porous membrane; and

[0162] (3) The modular polyimide porous membrane with the adsorbed cells is suspended in the culture medium in a state where it is not fixed in the cell culture container for suspension culture, and the modular polyimide porous membrane is oscillated and / or stirred in a second culture medium while being oscillated in a state of rotational motion, reciprocating motion, up and down motion, wave-like oscillation motion, rotational motion or a combination of these motions.

[0163] A cell culture model using modular polyimide porous membranes is illustrated in [the diagram]. Figure 4 . Figure 4 This is an illustrative diagram; the elements are not actual dimensions. In the cell culture method of the present invention, by applying and culturing cells to a modular polyimide porous membrane, a large number of cells grow on the surface of the polyimide porous membrane, which has multiple interconnected porous portions inside, thus enabling the easy cultivation of large numbers of cells. Furthermore, compared to conventional methods, the cell culture method of the present invention can significantly reduce the amount of culture medium used for cell culture and can cultivate large numbers of cells. For example, even when part or all of the polyimide porous membrane is not in contact with the liquid phase of the cell culture medium, large numbers of cells can be cultured for a long period. Additionally, the total volume of the cell culture medium contained in the cell culture container can also be significantly reduced relative to the total volume of the polyimide porous membrane containing the cell survival region.

[0164] In this specification, the volume occupied by the cell-free polyimide porous membrane, including its internal interstitial spaces, in the intrinsic space is referred to as the "apparent polyimide porous membrane volume" (see reference). Figure 4Furthermore, when cells are applied to a polyimide porous membrane, the total volume occupied by the polyimide porous membrane, cells, and culture medium infiltrating the interior of the polyimide porous membrane, with cells loaded on its surface and interior, is called the "polyimide porous membrane volume including the cell viability area" (see reference). Figure 4 In the case of a 25µm thick polyimide porous membrane, the volume of the polyimide porous membrane containing the cell viability region is approximately 50% larger than the apparent volume of the polyimide porous membrane. The method of this invention allows for the culture of multiple polyimide porous membranes in a single cell culture vessel. In this case, the sum of the volumes of the polyimide porous membranes containing the cell viability region of each of the multiple polyimide porous membranes carrying cells is sometimes simply referred to as the "sum of the volumes of the polyimide porous membranes containing the cell viability region."

[0165] By using the modular polyimide porous membrane, cells can be cultured well for extended periods even when the total volume of the cell culture medium in the cell culture vessel is 10,000 times or less than the total volume of the polyimide porous membrane containing the cell viability region. Furthermore, cells can be cultured well for extended periods even when the total volume of the cell culture medium in the cell culture vessel is 1,000 times or less than the total volume of the polyimide porous membrane containing the cell viability region. Moreover, cells can be cultured well for extended periods even when the total volume of the cell culture medium in the cell culture vessel is 100 times or less than the total volume of the polyimide porous membrane containing the cell viability region. And, cells can be cultured well for extended periods even when the total volume of the cell culture medium in the cell culture vessel is 10 times or less than the total volume of the polyimide porous membrane containing the cell viability region.

[0166] That is, according to the present invention, compared with conventional cell culture apparatuses for two-dimensional culture, the space (container) for cell culture can be miniaturized to the extreme. Furthermore, when it is desired to increase the number of cells cultured, the volume of cell culture can be flexibly increased by simple operations such as increasing the number of stacked polyimide porous membrane sheets. If a cell culture apparatus is equipped with the polyimide porous membrane used in the present invention, the space (container) for culturing cells can be separated from the space (container) for storing the cell culture medium, and the required amount of cell culture medium can be prepared according to the number of cells cultured. The space (container) for storing the cell culture medium can be enlarged or miniaturized depending on the purpose, or it can be a replaceable container; there is no particular limitation.

[0167] In the method of the present invention, for example, after culturing using a polyimide porous membrane, the number of cells contained in the cell culture vessel, expressed as the number of cells completely and uniformly dispersed in the cell culture medium contained in the cell culture vessel, can be cultured to a concentration of 1.0 × 10⁻⁶ cells per 1 ml of medium. 5 More than 1.0 × 10 6 More than 2.0 × 10 6 More than 5.0 × 10 6 More than one, 1.0 × 10 7 More than 2.0 × 10 7 More than 5.0 × 10 7 More than 1.0 × 10 8 More than 2.0 × 10 8 More than 5.0 × 10 8 More than one, 1.0 × 10 9 More than 2.0 × 10 9 More than one, or 5.0 × 10 9 More than one.

[0168] In this specification, "culture medium" refers to a culture medium used for culturing cells, particularly animal cells. The meaning of culture medium is the same as that of cell culture medium. Therefore, the culture medium used in this invention refers to a liquid culture medium. The type of culture medium can be any commonly used medium, appropriately determined according to the type of cells being cultured.

[0169] In the cell culture of the present invention, the first culture medium used in step (1) is not particularly limited as long as it is a culture medium capable of culturing cells. For example, in the case of culturing CHO cells, BalanCD (trademark) CHO GROWTH A manufactured by Irvine Scientific can be used.

[0170] In the cell culture of the present invention, the temperature at which cell culture can be carried out in step (2) is only required to allow the cells to adsorb onto the polyimide porous membrane, which 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. Furthermore, in the cell culture method of the present invention, the adsorption time for 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.

[0171] In the cell culture of the present invention, step (2) can involve oscillation and / or stirring while allowing cells to adhere to the modular polyimide porous membrane, or it can be left to stand while allowing cells to adhere to the modular polyimide porous membrane. The oscillation method is not particularly limited; for example, a culture container containing the modular polyimide porous membrane and cells of the present invention can be placed on a commercially available oscillation device and oscillated. Oscillation can be continuous or intermittent; for example, oscillation and standing can be alternated and adjusted as needed. The stirring method is not particularly limited; for example, the modular polyimide porous membrane and cells of the present invention can be placed in a commercially available rotary flask and stirred by rotating a stirrer. Stirring can be continuous or intermittent; for example, stirring and standing can be alternated and adjusted as needed.

[0172] In the cell culture of the present invention, the second culture medium used in step (3) is selected from the culture medium for adhering cells, such as D-MEM, E-MEM, IMDM, Ham's F-12, etc., but is not limited thereto. The second culture medium is preferably a culture medium that does not contain components that hinder cell adhesion to the substrate, such as surfactants. The second culture medium used is appropriately selected according to the type of cell. In step (3), by culturing in the second culture medium, the cells adsorbed on the polyimide porous membrane in step (2) are promoted to adhere to the polymer porous membrane. As a result, the cells will not detach from the polyimide porous membrane and can be cultured stably. In addition, the cell culture method of the present invention uses a modular polyimide porous membrane having the aforementioned polyimide porous membrane. The modular polyimide porous membrane used in the cell culture method of the present invention prevents the membrane-like polyimide porous membrane from continuously deforming within the shell by housing the polyimide porous membrane in the shell. Therefore, it is possible to prevent stress on cells growing in polyimide porous membranes, inhibit cell apoptosis, and stabilize and culture large numbers of cells.

[0173] In the cell culture of the present invention, step (3) can be performed using commercially available cell culture containers, devices, and systems. For example, the culture container can be a flexible bag-type culture container, or it can be cultured in a stirred reactor, such as a rotary flask. Furthermore, both open and closed containers can be used as culture containers. For example, cell culture dishes, flasks, plastic bags, test tubes, and large culture jars can all be used appropriately. For example, cell culture dishes manufactured by BD Falcon and Nunc cell factories manufactured by Thermo Scientific are examples. In addition, in the cell culture method of the present invention, by using a modular polyimide porous membrane, cells that cannot be cultured in suspension can be cultured in a state similar to suspension culture using a device for suspension culture. As a device for suspension culture, a stirred reactor such as a rotary flask manufactured by Corning can be used, for example. In addition, step (3) can be performed using the cell culture apparatus described in this specification.

[0174] In the cell culture of the present invention, this step (3) can also be performed using a continuous circulation device that continuously adds and recovers culture medium to a culture vessel containing the modular polyimide porous membrane.

[0175] In the cell culture of the present invention, step (3) can be a system in which cell culture medium is continuously or intermittently supplied to the cell culture container from a cell culture medium supply unit disposed outside the culture container containing the modular polyimide porous membrane. In this case, a system in which the cell culture medium circulates between the cell culture medium supply unit and the cell culture container can be formed.

[0176] 7. Culture conditions for chondrocytes

[0177] In the method of the present invention, any commercially available product can be used, such as a culture container, culture apparatus, or system that can suspend a modular polyimide porous membrane in a culture medium without it being fixed in a cell culture container for suspension culture, and that can agitate the modular polyimide porous membrane in a state of rotational motion, reciprocating motion, up-and-down motion, oscillating motion, rotational motion, or a combination of these motions. For example, the culture container can be a flexible bag-type culture container, or it can be cultured in a stirred reactor, such as a rotary flask. Furthermore, both open and closed containers can be used as culture containers. For example, culture dishes, flasks, plastic bags, test tubes, and large culture jars for cell culture can be appropriately used. Examples include cell culture dishes manufactured by BD Falcon and Nunc cell factories manufactured by ThermoScientific. In addition, in the cell culture method of the present invention, by using a modular polyimide porous membrane, cells that cannot be cultured in suspension under conditions similar to suspension culture can be cultured using a device for suspension culture. For example, stirred reactors such as rotary flasks manufactured by Corning can be used as devices for suspension culture.

[0178] In one embodiment, the cell culture container that can be used in the present invention can be a culture container selected from the group consisting of flasks, culture bottles, culture dishes, culture bags, and stirred reactors.

[0179] In this specification, "rotational motion" refers to the motion that rotates the culture vessel on a plane parallel to the horizontal plane, thereby agitating the liquid within the culture vessel. When applying rotational motion to this invention, a commercially available culture shaker, such as a rotary shaker, can be used. The rotational motion applied in this invention can, for example, be implemented at 40 rpm to 150 rpm. The rotational motion applied in this invention can also be implemented with a diameter of 0.5 to 10 cm.

[0180] In this specification, "reciprocating motion" refers to the movement of a culture container to stir the liquid within it, either by moving it left and right or back and forth. When applying reciprocating motion to this invention, a commercially available culture shaker, such as a reciprocating shaker, can be used. The reciprocating motion used in this invention can, for example, be implemented at a rate of 40 to 150 reciprocations per minute. The travel distance of the reciprocating motion used in this invention can, for example, be 0.5 to 10 cm.

[0181] In this specification, "up-and-down movement" refers to the movement of the culture container vertically up and down to agitate the liquid inside the container. When using up-and-down movement, a commercially available culture shaker, such as an up-and-down oscillator, can be used. The up-and-down movement used in this invention can, for example, be implemented at a rate of 40 to 150 reciprocations per minute. The reciprocating movement used in this invention can, for example, cover a distance of 0.5 to 10 cm.

[0182] In this specification, "wave-like oscillation motion" refers to a stirring motion that creates oscillations in the liquid within a culture vessel through a seesaw motion, moving back and forth and / or left and right. To implement wave-like oscillation motion, a commercially available culture shaker, such as a WAVE reactor, can be used. The wave-like oscillation motion used in this invention can also be achieved by tilting the bottom of the cell culture vessel within a range of 4 to 15 degrees. Alternatively, the wave-like oscillation motion used in this invention can be implemented at a speed of 5 to 25 rpm.

[0183] In this specification, "rotational motion" refers to the movement of a liquid within a culture vessel by rotating it using a physical stirring method. To implement rotational motion, a stirred reactor, such as a rotary flask or a stationary stirred reactor with stirring blades, can be used. The rotational motion used in this invention can also be implemented at, for example, 10 rpm to 300 rpm.

[0184] In this invention, cells are cultured using a modular polyimide porous membrane that is oscillated and / or stirred during rotational, reciprocating, up-and-down, undulating, or combined movements. In one embodiment, a culture bag is used as the culture container, and culture is performed by stirring the modular polyimide porous membrane while it is subjected to rotational and undulating movements using a culture shaker. This allows for the stable and efficient fabrication of scaffold-free three-dimensional cartilage tissue.

[0185] In one embodiment of the present invention, long-term culture can also be achieved by intermittently changing the culture medium.

[0186] In this invention, chondrocytes can be cultured continuously. For example, the culture in the method of this invention can be performed by continuously adding and recycling culture medium into a modular polyimide porous membrane, or by using an open device to expose the polyimide porous membrane to air.

[0187] In this invention, chondrocyte culture can be carried out in a system in which cell culture medium is continuously or intermittently supplied to the cell culture container from a cell culture medium supply unit located outside the cell culture container. In this case, a system in which the cell culture medium circulates between the cell culture medium supply unit and the cell culture container can be used.

[0188] This invention includes a method of culturing chondrocytes by placing a cell culture apparatus within an incubator. Additionally, this invention includes a method of culturing cells in a stand-alone cell culture apparatus such as a benchtop reactor. In the case of a system that continuously or intermittently supplies cell culture medium to a cell culture container from a cell culture medium supply unit located outside the cell culture container, the system may be a cell culture apparatus comprising a culture unit as the cell culture container and a culture medium supply unit as the cell culture medium supply unit. Here, the culture unit is a culture unit housing one or more modular polyimide porous membranes for carrying cells, and is a culture unit having a culture medium supply port and a culture medium discharge port. The culture medium supply unit includes a culture medium receiving container, a culture medium supply line, and a pump for delivering culture medium via the culture medium supply line. Here, a first end of the culture medium supply line is in contact with the culture medium in the culture medium receiving container, and a second end of the culture medium supply line is connected to the inside of the culture unit via the culture medium supply port of the culture unit.

[0189] Furthermore, in the aforementioned cell culture apparatus, the culture unit may be a culture unit without a culture medium supply line, a liquid delivery pump, an air supply port, and an air exhaust port; alternatively, it may be a culture unit with a culture medium supply line, a liquid delivery pump, an air supply port, and an air exhaust port. The culture unit may also lack an air supply port and an air exhaust port. Moreover, in the aforementioned cell culture apparatus, the culture unit may also include a culture medium discharge line. Here, the first end of the culture medium discharge line is connected to a culture medium receiving container, and the second end of the culture medium discharge line communicates with the interior of the culture unit via the culture medium discharge port, allowing the culture medium to circulate between the culture medium supply unit and the culture unit.

[0190] II. Reagent Kit

[0191] This invention also relates to a kit for the method of the invention comprising a modular polyimide porous membrane. In addition to the modular polyimide porous membrane, the kit of the invention may suitably include components required for chondrocyte culture. For example, it may include cells applied to the modular polyimide porous membrane, cell culture medium, a continuous culture medium supply device, a continuous culture medium circulation device, a cell culture apparatus, an evaluation unit for confirming cartilage tissue, and instructions for use of the kit.

[0192] III. Uses

[0193] The present invention also includes the use of modular polyimide porous membranes in the methods of the present invention described above.

[0194] The present invention will now be described in detail with reference to embodiments, but the present invention is not limited to these embodiments. Those skilled in the art can readily make modifications and alterations to the present invention based on the description herein, and these modifications are included within the scope of the present invention.

[0195] Example

[0196] The polymer porous membrane used in the following examples is a polyimide porous membrane, which is prepared by heat-treating a polyamic acid solution containing a polyamic acid solution of 3,3',4,4'-biphenyltetracarboxylic acid dianhydride (s-BPDA) as a tetracarboxylic acid component and 4,4'-diaminodiphenyl ether (ODA) as a diamine component, and polyacrylamide as a coloring precursor, after molding. The resulting polyimide porous membrane has a three-layer structure of surface layer A, surface layer B, and a large-pore layer sandwiched between surface layer A and surface layer B. Surface layer A and surface layer B have multiple pores, with an average pore size of 19 µm in surface layer A and 42 µm in surface layer B. The membrane thickness is 25 µm, and the porosity is 74%.

[0197] 1. Experimental Methods

[0198] 1-1. Culture Methods

[0199] <Example>

[0200] Second-generation Articular Engineering human chondrocytes (Y39_) cultured in IWAKI type I collagen-coated culture dishes were resuspended in Gibco-made Trypsin-EDTA (0.05%) and phenol red. The cells were then suspended in medium (7.51 × 10⁻⁶) by adding glucose to PromoCell chondrocyte growth medium adjusted to 3000 mg / L. 5 (cells / mL)

[0201] Prepare four 150mL storage bottles manufactured by Corning Incorporated, and add one moistened, sterilized modular polyimide porous membrane to each bottle. Figure 5Hereinafter referred to as "Apparatus 1" in this embodiment) and 20 mL of culture medium (adjusted to 3000 mg / L by injecting glucose into KBM ADSC-4 culture medium without heterologous components manufactured by Kohjin Bio Co., Ltd.) were placed in an N-BIOTEK humidified CO2 incubator (aniCell) equipped with a shaker, set to 37°C, and shaken at 80 rpm for 1 hour to complete the inoculation preparation. 719 µL of the above cell suspension (7.51 × 10⁻⁶) was added to each bottle. 5 Cells / mL), cultured with shaking for 1 day (inoculum: 5.4 × 10⁶ cells / mL per bottle). 5 (Number of cells). The next day, 10 mL of glucose-adjusted KBM ADSC-4 medium was added to each bottle. Then, two bottles were directly cultured with shaking at 80 rpm, and the remaining two bottles were transferred to an ASTEC CO2 incubator (APM-30D) for static culture. The medium was changed with glucose-adjusted KBM ADSC-4 medium twice a week.

[0202] <Comparative Example: Tert-Depleted Peptide Collagen Encapsulation>

[0203] The above cell suspension (7.51×10) 5 Centrifuge at 220×g for 3 minutes to prepare cell pellet, then resuspend in culture medium to prepare cell suspension (4.0×10⁻⁶ cells / mL). 6 Cells / mL). Mix the aforementioned cell suspension and 3% determinated peptide collagen solution (Gaoyan) at a 1:1 ratio on ice. Prepare two FALCON culture dishes, and add 270 µL of determinated peptide collagen solution (2.0 × 10⁻⁶ cells / mL) containing chondrocytes to the center of each dish. 6 (cells / mL) (seed number: 5.4 × 10⁴ cells / mL per culture dish) 5 (cells). Place the culture dishes in an ASTEC CO2 incubator (APM-30D) and let them stand for 1-2 hours to allow gelation. Then, pour 12 mL of glucose-adjusted KBM ADSC-4 medium into each culture dish. Replace the medium with glucose-adjusted KBM ADSC-4 medium twice a week.

[0204] 1-2. Quantification of DNA

[0205] Cartilage autogenous tissue (Example) or telopeptide-free collagen gel (Comparative Example) cultured for 50-52 days were recovered, washed with 4 mL of PBS, transferred to 15 mL tubes, and homogenized with 800 µL of lysis buffer. 80 µL of proteinase K was added to each tube and mixed, then incubated overnight at 55°C in a constant-temperature shaker. The next day, 293 µL of 5M NaCl was added, and the mixture was centrifuged at 15000 × g for 10 minutes. The supernatant was collected in a new tube. 587 µL of isopropanol was added to the supernatant, and the mixture was centrifuged at 15000 × g for 3 minutes to collect the precipitate. 880 µL of sterile water was added to the precipitate, and the mixture was incubated in a 37°C water bath for 2 hours to dissolve the precipitate. Then, 8 µL of RNase (10 mg / mL) was added, and the mixture was incubated at 37 °C for 1 hour. Next, 293 µL of 5 M NaCl was added, and the mixture was centrifuged at 15000 × g for 10 minutes. The supernatant was collected into a new test tube. 587 µL of isopropanol was added to the collected supernatant, and the mixture was centrifuged at 15000 × g for 3 minutes to collect the precipitate. The precipitate was washed twice with 1 mL of 70% ethanol. After air-drying the precipitate, 100 µL of sterile water was added, and the precipitate was dissolved at 37 °C. The concentration of DNA in the resulting aqueous solution was determined using a nanodropper.

[0206] 1-3. Quantitative analysis of glycosaminoglycans

[0207] After washing the sample with PBS and homogenizing it, 2 mL of papain extraction reagent was added. The mixture was reacted at 65°C for 27 hours and then centrifuged at 10000×g for 10 minutes. The GAG ​​in the supernatant was quantified using the Blyscan glycosaminoglycan assay kit.

[0208] 2. Experimental Results

[0209] The above results yielded cartilage tissue with the following properties ( Figure 6 and 7 ).

[0210] [Table 1]

[0211]

Claims

1. A method for manufacturing a scaffold-free three-dimensional cartilage tissue body, comprising: A process involving suspending a modular polyimide porous membrane inoculated with chondrocytes in a culture medium in a state not fixed within a cell culture container for suspension culture, while simultaneously applying shear stress to the modular polyimide porous membrane, is performed during the culture process. The modular polyimide porous membrane comprises a polyimide porous membrane and a housing containing the polyimide porous membrane. The polyimide porous membrane is fixed to the housing in a state that does not continuously fluctuate due to fluid. The shell has one or more culture medium outflow inlets for exposing culture medium to at least a portion of the surface of the polyimide porous membrane. The polyimide porous membrane has a three-layer structure, consisting of a surface layer A, a surface layer B, and a large-void layer sandwiched between the surface layers A and B. The surface layers A and B each have multiple pores, wherein the average pore diameter of the pores in the surface layer A is smaller than the average pore diameter of the pores in the surface layer B. The large-void layer has a partition wall bonded to the surface layers A and B and multiple large voids surrounded by the partition wall and the surface layers A and B. The pores in the surface layers A and B communicate with the large voids.

2. The method according to claim 1, wherein, The modular polyimide porous membrane stack houses two or more of the polyimide porous membranes.

3. The method according to claim 1, wherein, The shear stress is generated by oscillation and / or stirring under conditions of rotational motion, reciprocating motion, up-and-down motion, wave-like oscillation, rotational motion, or a combination of these motions.

4. The method according to claim 1, wherein, The polyimide porous membrane has multiple fine pores with an average pore size of 0.01~100µm.

5. The method according to claim 1, wherein, The average pore size of the surface layer A is 0.01~50µm.

6. The method according to claim 1, wherein, The average pore size of the surface layer B is 20~100µm.

7. The method according to claim 1, wherein, The total thickness of the polyimide porous membrane is 5~500µm.

8. The method according to claim 1, wherein, The polyimide porous membrane is a polyimide porous membrane containing polyimide obtained from tetracarboxylic dianhydride and diamine.

9. The method according to claim 1, wherein, The polyimide porous membrane is a colored polyimide porous membrane obtained by molding a polyamic acid solution containing a polyamic acid solution obtained from tetracarboxylic dianhydride and diamine and a coloring precursor, and then heat-treating it at a temperature above 250°C.

10. A scaffold-free three-dimensional cartilage tissue obtained by the method of claim 1.

11. A scaffold-free three-dimensional cartilage tissue body, characterized in that, It contains chondrocytes, It contains no externally added scaffold components other than the extracellular matrix generated by the chondrocytes. The scaffold-free three-dimensional cartilage tissue has an average thickness of 0.2 mm or more. Each 1 mg wet weight contains more than 5 µg of glycosaminoglycans.

12. The scaffold-free three-dimensional cartilage tissue body according to claim 11, wherein, The scaffold-free three-dimensional cartilage tissue has an average thickness of more than 0.5 mm.

13. A modular polyimide porous membrane, characterized in that, It is a modular polyimide porous membrane used to manufacture scaffold-free three-dimensional cartilage tissue. The modular polyimide porous membrane comprises a polyimide porous membrane and a housing containing the polyimide porous membrane. The polyimide porous membrane is fixed to the housing in a state that does not continuously fluctuate due to fluid. The shell has one or more culture medium outflow inlets for exposing culture medium to at least a portion of the surface of the polyimide porous membrane. The polyimide porous membrane has a three-layer structure comprising a surface layer A, a surface layer B, and a large-void layer sandwiched between surface layer A and surface layer B. Surface layer A and surface layer B each have multiple pores, wherein the average pore diameter of the pores in surface layer A is smaller than the average pore diameter of the pores in surface layer B. The large-void layer has a partition wall bonded to surface layers A and B and multiple large voids surrounded by the partition wall and surface layers A and B. The pores in surface layers A and B communicate with the large voids. The modular polyimide porous membrane is used in cell culture containers, cell culture devices, or cell culture systems for suspension culture, but is not fixed within the culture section of the cell culture container, cell culture device, or cell culture system for suspension culture.

14. The modular polyimide porous membrane according to claim 13, wherein two or more of the polyimide porous membranes are stacked together.

15. The modular polyimide porous membrane according to claim 13, wherein, The polyimide porous membrane has multiple fine pores with an average pore size of 0.01~100µm.

16. The modular polyimide porous membrane according to claim 13, wherein, The average pore size of the surface layer A is 0.01~50µm.

17. The modular polyimide porous membrane according to claim 13, wherein, The average pore size of the surface layer B is 20~100µm.

18. The modular polyimide porous membrane according to claim 13, wherein, The total thickness of the polyimide porous membrane is 5~500µm.

19. The modular polyimide porous membrane according to claim 13, wherein, The polyimide porous membrane is a polyimide porous membrane containing polyimide obtained from tetracarboxylic dianhydride and diamine.

20. The modular polyimide porous membrane according to claim 13, wherein, The polyimide porous membrane is a colored polyimide porous membrane obtained by molding a polyamic acid solution containing a polyamic acid solution obtained from tetracarboxylic dianhydride and diamine and a coloring precursor, and then heat-treating it at a temperature above 250°C.

Images