Cell culture method, cell culture device and kit

Abstract

The present invention relates to a method for culturing cells, as well as a cell culture device and a kit used in the culturing method. The method for culturing cells of the present invention includes applying cells to a polyimide porous membrane and culturing them. In one embodiment of the method of the present invention, it includes a step of inoculating cells onto the surface of the above-mentioned polyimide porous membrane. Alternatively, in one embodiment, it includes the following steps: loading a cell suspension onto the dry surface of the above-mentioned polyimide porous membrane, placing the above-mentioned polyimide porous membrane, or moving the above-mentioned polyimide porous membrane to promote the outflow of liquid, or stimulating a part of the surface to suck the cell suspension into the above-mentioned membrane, and then accumulating the cells in the cell suspension into the above-mentioned membrane and allowing the water to flow out. Alternatively, in one embodiment, it includes the following steps: wetting one or both surfaces of the above-mentioned polyimide porous membrane with a cell culture solution or a sterilized liquid, loading a cell suspension onto the wetted polyimide porous membrane, and then accumulating the cells in the cell suspension into the above-mentioned membrane and allowing the water to flow out.

Description

Technical Field

[0001] This application claims priority based on Japanese Patent Application No. 2013-155550 filed on July 26, 2013, the entire content of which is incorporated into this specification.

[0002] The present invention relates to a method for culturing cells, a cell culture device, and a kit.

Background Art

[0003]

Cell Culture

[0004] Cells generally exist as three-dimensional aggregates in vivo, but in classical planar culture, they are cultured in a monolayer in the shape of cells spreading on the surface of a container. It has been reported that due to differences in the culture environment, the properties of cells are quite different. In addition, for suspension culture of cells in a liquid medium, there are cells suitable for suspension culture and cells not suitable for it.

[0005] The NanoCulture (registered trademark) Plate (NCP) developed by SCIVAX Corporation is an adhesive three-dimensional culture multi-well plate with patterns (micro rectangles and micro honeycombs) imitating the extracellular matrix implemented on the bottom surface by nanoimprint technology. The micro rectangle is a quadrilateral shape, and the micro honeycomb is a regular repeating structure with a hexagonal shape. By using this micro pattern as a scaffold by cells, spherical shapes (three-dimensional states formed by the aggregation of a large number of cells) are actively formed.

[0006] In addition, there are reports of cases where a cell culture sheet ("nanopillar cell culture sheet") with a large number of regularly arranged uniform cell protrusions (nanopillars) is used to culture a three-dimensional cell tissue body (spherical shape) close to the structure of living liver tissue (Takahashi et al., Tissue Engineering Part A. June 2012, Vol. 16, No. 6, p. 1983 - 1995).

[0007] JP-A-2009-213421 describes a method for manufacturing spherical shapes and a spherical shape manufacturing device using a honeycomb-shaped porous membrane (honeycomb membrane).

[0008] These cell culture methods are common in that they use a sheet (membrane) with a regular repeating pattern, and cells adhere to the surface of the sheet and proliferate, and the cells formed by cell culture are spherical shapes in which the same-shaped cells aggregate into a three-dimensional state, and are limited in this sense. There is a need to develop a method for culturing cells more simply, effectively, and stably.

[0009]

Polyimide Porous Membrane

[0010] Polyimide refers to the general term for polymers containing imide bonds in the repeating units. Usually, aromatic polyimides are directly connected by imide bonds in aromatic compounds. Aromatic polyimides have a covalent structure due to the aromatic groups and the imide bonds, with a rigid and strong molecular structure. Moreover, due to the strong intermolecular forces of the imide bonds, they have very high levels of thermal, mechanical, and chemical properties.

[0011] Polyimide porous membranes have been used for applications such as filters, low dielectric constant membranes, and electrolyte membranes for fuel cells, especially with a focus on battery-related uses, since before the present invention. International Publication WO2010 / 038873, Japanese Unexamined Patent Application Publication No. 2011-219585, and Japanese Unexamined Patent Application Publication No. 2011-219586 disclose polyimide porous membranes that are particularly excellent in the permeability of substances such as gases, have a high porosity, excellent smoothness on both surfaces, high relative strength, and do not involve a high porosity, and have large voids with excellent resistance to compressive stress in the thickness direction of a plurality of opposing membranes.

[0012]

Prior Art Documents

[0013]

Patent Documents

[0014] Patent Document 1: Japanese Unexamined Patent Application Publication No. 2009-213421

[0015] Patent Document 2: WO2010 / 038873

[0016] Patent Document 3: Japanese Unexamined Patent Application Publication No. 2011-219585

[0017] Patent Document 4: Japanese Unexamined Patent Application Publication No. 2011-219586

[0018]

Non-Patent Documents

[0019] Non-Patent Document 1: Takahashi et al., Tissue Engineering Part A. June 2012, Vol. 16, No. 6, p. 1983-1995

Summary of the Invention

[0020]

Technical Problem to be Solved by the Invention

[0021] The present invention relates to a method for culturing cells, and a cell culture device and a kit for use in the culturing method.

[0022]

Technical Solution for Solving the Problem

[0023] Although not limited thereto, the present invention preferably includes the following embodiments.

[0024]

Embodiment 1

[0025] A method for culturing cells, which comprises applying the cells to a polyimide porous membrane and culturing them.

[0026]

Embodiment 2

[0027] The method according to Embodiment 1, which comprises the step of inoculating cells on the surface of the above polyimide porous membrane.

[0028]

Embodiment 3

[0029] The method according to Embodiment 1, which comprises the following steps:

[0030] Loading a cell suspension on the dry surface of the above polyimide porous membrane,

[0031] Placing the above polyimide porous membrane, or moving the above polyimide porous membrane to promote the outflow of the liquid, or stimulating a part of the surface to suck the cell suspension into the above membrane, and then

[0032] Accumulating the cells in the cell suspension into the above membrane and allowing the water to flow out.

[0033]

Embodiment 4

[0034] The method according to Embodiment 1, which comprises the following steps:

[0035] Wetting one or both sides of the above polyimide porous membrane with a cell culture medium or a sterilized liquid,

[0036] Loading a cell suspension onto the above wet polyimide porous membrane, and then

[0037] Accumulating the cells in the cell suspension into the above membrane and allowing the water to flow out.

[0038]

Embodiment 5

[0039] The method according to Embodiment 4, wherein live cells remain in the above polyimide porous membrane, and dead cells flow out together with the water.

[0040]

Embodiment 6

[0041] The method according to Embodiment 4 or 5, wherein the above sterilized liquid is sterilized water or a sterilized buffer solution.

[0042]

Embodiment 7

[0043] The method according to any one of Embodiments 1 to 6, which comprises placing a cell culture medium, cells and one or more of the above polyimide porous membranes into a cell culture container, wherein the polyimide porous membrane is in a state of being suspended in the cell culture medium.

[0044]

Embodiment 8

[0045] The method according to Embodiment 7, characterized in that small pieces of two or more of the above polyimide porous membranes are used.

[0046]

Embodiment 9

[0047] The method according to Embodiment 7 or 8, wherein cells spontaneously adhere to the above polyimide porous membrane.

[0048]

Embodiment 10

[0049] The method according to any one of Embodiments 1 to 6, wherein the above polyimide porous membrane

[0050] (i) is folded,

[0051] (ii) is rolled into a roll,

[0052] (iii) the sheets or small pieces are connected with a filamentous structure, or

[0053] (iv) is formed into a rope shape

[0054] and is suspended or fixed in the cell culture medium in the cell culture container.

[0055]

Embodiment 11

[0056] The method according to Embodiment 10, wherein cells spontaneously adhere to the above polyimide porous membrane.

[0057]

Embodiment 12

[0058] The method according to Embodiment 1, which includes laminating two or more polyimide porous membranes one above the other or side by side for use in the cell culture medium.

[0059]

Embodiment 13

[0060] The method according to Embodiment 1, wherein two or more methods of the methods according to any one of Embodiments 2 to 12 are used in combination.

[0061]

Embodiment 14

[0062] The method according to any one of Embodiments 1 to 13, wherein cells grow and proliferate on the surface and inside of the polyimide porous membrane.

[0063]

Embodiment 15

[0064] The method according to any one of Embodiments 1 to 14, wherein the cells are selected from animal cells, insect cells, plant cells, yeasts, and bacteria.

[0065]

Embodiment 16

[0066] The method according to Embodiment 15, wherein the animal cell is a cell derived from an animal belonging to the phylum Chordata.

[0067]

Embodiment 17

[0068] The method according to Embodiment 15, wherein the bacterium is selected from lactic acid bacteria, Escherichia coli, Bacillus subtilis, and cyanobacteria.

[0069]

Embodiment 18

[0070] The method according to any one of Embodiments 1 to 14, wherein the cell is selected from pluripotent stem cells, tissue stem cells, somatic cells, and germ cells.

[0071]

Embodiment 19

[0072] The method according to any one of Embodiments 1 to 13, wherein the cell is selected from sarcoma cells, established cells, and transformed cells.

[0073]

Embodiment 20

[0074] The method according to any one of Embodiments 1 to 19, wherein the polyimide porous membrane is a polyimide porous membrane containing a polyimide obtained from a tetracarboxylic dianhydride and a diamine.

[0075]

Embodiment 21

[0076] A cell culture device for use in the cell culture method according to any one of Embodiments 1 to 20, which contains a polyimide porous membrane.

[0077]

Embodiment 22

[0078] The cell culture device according to Embodiment 21, wherein two or more polyimide porous membranes are stacked one on top of the other or side by side.

[0079]

Embodiment 23

[0080] A kit for use in the cell culture method according to any one of Embodiments 1 to 20, which contains a polyimide porous membrane.

[0081]

Advantages of the Invention

[0082] Based on the discovery that when a cell is applied to a polyimide porous membrane, the cell grows. The cell preferably spontaneously adheres to the polyimide porous membrane and can proliferate on the surface and inside of the membrane. By the method of the present invention, it becomes possible to simply, effectively, and stably culture cells.

Brief Description of the Drawings

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[0102]

Figure 16-1

[0103]

Figure 16-2

[0104]

Figure 16-3

[0105]

Embodiment

[0106]

I. Cell culture method

[0107] The present invention relates to a method for culturing cells.

[0108] The cell culture method of the present invention includes applying cells to a polyimide porous membrane and culturing them. The present inventors found that a polyimide porous membrane is suitable for cell adhesion and culture, and thus came up with the present invention. The method of the present invention is characterized by including applying cells to the polyimide porous membrane and culturing the cells on the surface or inside of the polyimide membrane.

[0109]

Application of cells to polyimide porous membrane

[0110] The specific process of applying cells to the polyimide porous membrane is not particularly limited. The processes described in this specification or any method suitable for applying cells to a membrane-like carrier can be used. Without aiming to limit, the method of the present invention, for example, includes the following embodiments.

[0111] (A) Embodiment, which includes the process of inoculating cells on the surface of the above polyimide porous membrane;

[0112] (B) Embodiment, which includes the following processes:

[0113] Placing a cell suspension on the dry surface of the above polyimide porous membrane, or moving the above polyimide porous membrane to promote the outflow of the liquid, or stimulating a part of the surface to suck the cell suspension into the above membrane, and then

[0114] accumulating the cells in the cell suspension into the above membrane and allowing the water to flow out; and,

[0115] (C) Embodiment, which includes the following processes:

[0116] Wetting one or both sides of the above polyimide porous membrane with a cell culture solution or a sterilized liquid,

[0117] Load a cell suspension onto the above-mentioned wetted polyimide porous membrane, and then

[0118] Accumulate the cells in the cell suspension into the above-mentioned membrane and let the water flow out.

[0119] The embodiment of (A) includes directly inoculating cells and cell aggregates on the surface of the polyimide porous membrane. Alternatively, putting the polyimide porous membrane into a cell suspension also includes an embodiment in which the cell culture solution infiltrates from the surface of the membrane.

[0120] The cells inoculated on the surface of the polyimide porous membrane adhere to the polyimide porous membrane and enter the interior of the pores. Preferably, even without particularly applying physical or chemical forces from the outside, the cells spontaneously adhere to the polyimide porous membrane. The cells inoculated on the surface of the polyimide porous membrane can stably grow and proliferate on the surface and / or inside of the membrane. Depending on the position of the membrane where growth and proliferation occur, the cells can take various different forms.

[0121] In the embodiment of (B), load the cell suspension on the dry surface of the polyimide porous membrane. Place the polyimide porous membrane, or move the above-mentioned polyimide porous membrane to promote the outflow of the liquid, or stimulate a part of the surface. By sucking the cell suspension into the above-mentioned membrane, the cell suspension penetrates into the membrane. Without being bound by theory, this is considered to be due to the properties derived from the surface shape, etc. of the polyimide porous membrane. By this embodiment, the cells are sucked and inoculated at the position of the cell suspension filled in the membrane.

[0122] Alternatively, as in the embodiment of (C), after wetting a part or all of one or both sides of the above-mentioned polyimide porous membrane with a cell culture solution or a sterilized liquid, a cell suspension can also be loaded onto the wetted polyimide porous membrane. At this time, the passing speed of the cell suspension increases greatly.

[0123] For example, the one-point wet method described in Example 4 of this specification is a method of wetting only a very small part of the membrane mainly for the purpose of preventing the membrane from flying, and is substantially close to the dry method ((B) embodiment) in which the membrane is hardly wetted. However, it is considered that the permeation of the cell solution through the small wetted part becomes rapid. In addition, the wet membrane method described in Example 3 of this specification is a method of loading a cell suspension onto a polyimide porous membrane that is fully wetted on one or both sides (hereinafter referred to as "wet membrane"). At this time, in the whole polyimide porous membrane, the passing speed of the cell suspension increases greatly.

[0124] In the embodiments of (B) and (C), accumulate the cells in the cell suspension into the above-mentioned membrane and let the water flow out. Thereby, it becomes possible to concentrate the cell concentration in the cell suspension and to perform treatments such as letting the unnecessary components other than the cells flow out together with the water.

[0125] The embodiment of (A) is sometimes referred to as "natural inoculation", and the embodiments of (B) and (C) are sometimes referred to as "inhalation inoculation".

[0126] Although not intended to be limiting, preferably, viable cells selectively stay in the polyimide porous membrane. Thus, in a preferred embodiment of the present invention, viable cells stay within the above polyimide porous membrane, and dead cells preferentially flow out together with moisture. In Example 3 of the present invention, the viable cell rate in the cell culture medium (cell suspension) before applying the above polyimide porous membrane applicable to the present invention is 90%, but when applied to the polyimide porous membrane, the viable cell rate in the liquid that flows out is 65%. Comparing the viability and death of cells, the adsorption rate of viable cells to the membrane is 88% and the exudation rate is 12%, and the adsorption rate of dead cells to the membrane is 40% and the exudation rate is 60%. It is considered that viable cells are selectively adsorbed in the above polyimide porous membrane of the present invention.

[0127] The sterilized liquid used in embodiment (C) is not particularly limited and may be a sterilized buffer solution or sterilized water. The buffer solution, for example, is (+) and (-) Dulbecco's PBS, (+) and (-) Hank's balanced salt solution, etc. Examples of the buffer solution are shown in Table 1 below.

[0128]

Table 1

[0129] Component Concentration (mmol / L) Concentration (g / L) NaCl 137 8.00 KCl 2.7 0.20 <![CDATA[Sodium 2 HPO 4 > 10 1.44 <![CDATA[KH 2 PO 4 > 1.76 0.24 pH (-) 7.4 7.4

[0130] The present invention also includes an embodiment (immersion) in which adhesive cells in a suspended state are allowed to coexist with a polyimide porous membrane in suspension so that the cells adhere to the membrane. For example, in the cell culture method of the present invention, in order to apply cells to the polyimide porous membrane, a cell culture medium, cells, and one or more of the above polyimide porous membranes may be placed in a cell culture container. When the cell culture medium is a liquid, the polyimide porous membrane is in a state of being suspended in the cell culture medium. Due to the properties of the polyimide porous membrane, cells can adhere to the polyimide porous membrane. Thus, even cells that are inherently unsuitable for suspension culture can be cultured in a state of being suspended in the cell culture medium by the polyimide porous membrane. Preferably, the cells spontaneously adhere to the polyimide porous membrane. "Spontaneous adhesion" means that even without particularly applying physical or chemical forces from the outside, the cells stay on the surface or inside of the polyimide porous membrane.

[0131] Cell culture: Depending on the morphological state in cell culture, cultured cells can be classified into adherent culture system cells and suspension culture system cells. Adherent culture system cells are cultured cells that attach to the culture vessel and proliferate, and the culture medium is changed during subculture. Suspension culture system cells are cultured cells that proliferate in a suspended state in the culture medium. Generally, the culture medium is not changed during subculture, and dilution culture is performed. Since suspension culture can be carried out in a suspended state, i.e., in a liquid, a large number of cells can be cultured. Compared with adherent cells that only grow on the surface of the culture vessel, since it is a three-dimensional culture, there is an advantage that the number of culturable cells per unit space is large.

[0132] According to the present invention, conceptually, it becomes possible to cultivate cells in a form similar to suspension culture regardless of the cell type, and a method for simply culturing a large number of cells is provided. In the cell culture method of the present invention, when a polyimide porous membrane is used in a state of being suspended in a cell culture medium, small pieces of two or more of the above polyimide porous membranes can also be used. Since the polyimide porous membrane is a flexible thin film, for example, by using the small pieces suspended in the culture solution, it becomes possible to hold a polyimide porous membrane with a large surface area in a certain volume of cell culture medium. In the case of normal culture, the bottom area of the container becomes the upper limit of the area where cells can be cultured. However, in the cell culture using the polyimide porous membrane of the present invention, the entire large surface area of the previously held polyimide porous membrane becomes the area where cells can be cultured. Since the polyimide porous membrane allows the cell culture solution to pass through, it becomes possible to supply nutrients, oxygen, etc. even inside the wrinkled membrane.

[0133] The size and shape of the small pieces of the polyimide porous membrane are not particularly limited. The shape can be any shape such as circular, oval, square, triangular, polygonal, cord-like, etc. For example, a quadrilateral (square, rectangle, etc.), triangle, etc. having a side length of about 0.1 mm to about 20 mm, preferably about 0.2 mm to about 10 mm, and more preferably about 1 mm to about 5 mm. Or, for example, a circle having a diameter of preferably about 0.1 mm to about 20 mm, more preferably about 0.5 mm to about 10 mm. By dispersing these small pieces into the liquid, a form similar to suspension culture is formed.

[0134] Due to the flexibility of the polyimide porous membrane of the present invention, it can be used with shape changes. The polyimide porous membrane can also be processed into a three-dimensional shape rather than a planar shape for use. For example, the polyimide porous membrane can be (i) folded, (ii) rolled into a roll, (iii) connected with a filamentous structure for sheets or small pieces, or (iv) formed into a rope shape and suspended or fixed in the cell culture medium in a cell culture container. By processing the shape as in (i)-(iv), similar to when using small pieces, a variety of polyimide porous membranes can be placed in a cell culture medium of a certain volume. Furthermore, since each small piece can be treated as an aggregate, it becomes possible to aggregate and move cell bodies, and the comprehensive applicability is high.

[0135] As the same idea as the small piece aggregate, two or more polyimide porous membranes can also be stacked up and down or left and right in the cell culture medium for use. Stacking means an embodiment in which a part of the polyimide porous membrane also overlaps. By stacked culture, it becomes possible to culture cells at high density in a narrow space. It is also possible to stack membranes on a membrane that has already been cell-cultured to form a multi-layer system with different types of cells. Furthermore, it is also possible to utilize it for drug development, such as verifying cell-cell interactions in a three-dimensional environment or a stress-free cell culture method. The number of stacked polyimide porous membranes is not particularly limited.

[0136] The above-described cell culture method of the present invention can also be used in combination of two or more methods. For example, using any of the methods of Embodiments (A)-(C) to apply cells to the polyimide porous membrane first, and then, the polyimide porous membrane to which the cells are adhered can be cultured in suspension. Alternatively, as a step of applying to the polyimide porous membrane, two or more of any of the above-described methods of Embodiments (A)-(C) can be used in combination.

[0137] In the method of the present invention, preferably, cells grow and proliferate on the surface and inside of the polyimide porous membrane. There is no reported example showing that cells grow and proliferate inside a three-dimensional structure. By using the polyimide porous membrane in the present invention, continuous three-dimensional culture of cells becomes possible. Without being limited thereto, by the method of the present invention, cells proliferate continuously for 2 days or more, more preferably 4 days or more, and still more preferably 6 days or more. In Examples 1 and 4 described in this specification, an increase in cells was observed for 21 days.

[0138] 【2. Cells】

[0139] The type of cells that can be used in the method of the present invention is not particularly limited, and can be used in the proliferation of any cells.

[0140] For example, the cells are selected from animal cells, insect cells, plant cells, yeasts, and bacteria. Animal cells are broadly classified into cells derived from animals belonging to the phylum Chordata and cells derived from invertebrates (animals other than those belonging to the phylum Chordata). In the present specification, the origin of the animal cells is not particularly limited. Preferably, it refers to cells derived from animals belonging to the phylum Chordata. The phylum Chordata includes agnathans and gnathostomes, and the gnathostomes include Mammalia, Aves, Amphibia, Reptilia, etc. Preferably, generally, it is cells derived from animals belonging to the class Mammalia, which are called mammals. The mammals are not particularly limited, but preferably include mice, rats, humans, monkeys, pigs, dogs, sheep, goats, etc.

[0141] The origin of the plant cells in the present specification is not particularly limited. Cells of plants including bryophytes, pteridophytes, and spermatophytes are targeted.

[0142] The plants from which the spermatophyte cells are derived include any of monocotyledonous plants and dicotyledonous plants. It is not intended to be limiting, but among the monocotyledonous plants, it includes plants of the Orchidaceae family, plants of the Poaceae family (rice, corn, barley, wheat, sorghum, etc.), plants of the Cyperaceae family, etc. Among the dicotyledonous plants, it includes plants belonging to many subclasses such as Asteridae, Magnoliidae, Rosidae, etc.

[0143] Algae can also be regarded as cell source organisms. It includes different groups such as cyanobacteria (blue-green algae) which are true bacteria, eukaryotes, and unicellular organisms (diatoms, yellow-green algae, dinoflagellates, etc.) and multicellular organisms such as seaweeds (red algae, brown algae, green algae).

[0144] The types of archaea and bacteria in the present specification are also not particularly limited. Archaea are composed of methanogens - extremely halophilic archaea - thermoacidophilic archaea - hyperthermophilic archaea, etc. Bacteria, for example, are selected from lactic acid bacteria, Escherichia coli, Bacillus subtilis, and cyanobacteria, etc.

[0145] The types of animal cells or plant cells that can be used in the method of the present invention are not intended to be limited, but preferably are selected from pluripotent stem cells, tissue stem cells, somatic cells, and germ cells.

[0146] In the present invention, "pluripotent stem cells" refers to the general term for stem cells having the ability to differentiate into cells of all tissues (differentiation pluripotency). It is not intended to be limiting, but pluripotent stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ stem cells (EG cells), germline stem cells (GS cells), etc. Preferably, they are ES cells or iPS cells. iPS cells are particularly preferred for reasons such as no ethical issues. Any well-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.

[0147] "Tissue stem cells" refer to stem cells whose differentiable cell lineages are limited to specific tissues but have the ability to differentiate into diverse cell types (pluripotency). For example, hematopoietic stem cells in the bone marrow give rise to blood cells, and neural stem cells differentiate into nerve cells. In addition, there are various types such as hepatic stem cells that form the liver and skin stem cells that form 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, or hematopoietic stem cells.

[0148] "Somatic cells" refer to cells other than germ cells among the cells that make up multicellular organisms. They are not inherited to the second generation in sexual reproduction. Preferably, the somatic cells are selected from liver cells, 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, red blood cells, white blood cells, monocytes, macrophages, or megakaryocytes.

[0149] "Germ cells" refer to cells that have the function of transmitting genetic information to the second generation during reproduction. For example, it includes gametes for sexual reproduction, namely eggs, oocytes, sperm, spermatids, and spores for asexual reproduction.

[0150] Cells can also be selected from sarcoma cells, immortalized cells, and transformed cells. "Sarcoma" refers to cancer that occurs in connective tissue cells derived from non-epithelial cells such as bone, cartilage, fat, muscle, and blood, including soft tissue sarcoma, malignant bone tumors, etc. Sarcoma cells are cells derived from sarcoma. "Immortalized cells" refer to cultured cells that have been maintained in vitro for a long time until they have certain stable properties and can be subcultured semi-permanently. There are various cell lines from various tissue sources of various biological species including 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), etc. "Transformed cells" refer to cells whose genetic properties have been changed by introducing nucleic acids (such as DNA) from outside the cell. Appropriate methods for the transformation of animal cells, plant cells, and bacteria are well-known.

[0151] 【3. Polyimide Porous Membrane】

[0152] Polyimide refers to the general term for polymers containing imide bonds in the repeating units, and usually refers to aromatic polyimides in which aromatic compounds are directly connected by imide bonds. Since aromatic polyimides have a covalent structure with aromatic groups connected by imide bonds, they have a rigid and strong molecular structure, and because the imide bond has strong intermolecular forces, they have very high levels of thermal, mechanical, and chemical properties.

[0153] The polyimide porous film of 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 containing a polyimide obtained from a tetracarboxylic dianhydride and a diamine. "Containing as a main component" means that, as a constituent of the polyimide porous film, components other than the polyimide obtained from the tetracarboxylic dianhydride and the diamine are substantially not contained, or may be contained, but are additional components that do not affect the properties of the polyimide obtained from the tetracarboxylic dianhydride and the diamine.

[0154] There is also a colored polyimide porous film obtained by heat treatment at 250 °C or higher after forming a polyamic acid solution composition containing a polyamic acid obtained from a tetracarboxylic acid component and a diamine component and a coloring precursor.

[0155]

Polyamic acid

[0156] The polyamic acid is obtained by polymerizing a tetracarboxylic acid component and a diamine component. The polyamic acid is a polyimide precursor that can be cyclized to a polyimide by thermal imidization or chemical imidization.

[0157] The polyamic acid can be used as long as a part of the amic acid is imidized without affecting the present invention. That is, the polyamic acid can also be partially thermally imidized or chemically imidized.

[0158] When thermally imidizing the polyamic acid, if necessary, fine particles such as an imidization catalyst, a compound containing organic phosphorus, inorganic fine particles, and organic fine particles can be added to the polyamic acid solution. In addition, when chemically imidizing the polyamic acid, if necessary, a chemical imidizing agent, a dehydrating agent, inorganic fine particles, organic fine particles and other fine particles can be added to the polyamic acid solution. Even if the above components are added to the polyamic acid solution, it is preferably carried out under conditions where the coloring precursor does not precipitate.

[0159]

Coloring precursor

[0160] In the present invention, the coloring precursor refers to a precursor that is partially or completely carbonized by heat treatment at 250 °C or higher to form a colored product.

[0161] As the coloring precursor used in the present invention, it is uniformly dissolved or dispersed in the polyamic acid solution or the 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 still more preferably 300 °C or higher, preferably heat treatment at 250 °C or higher, preferably 260 °C or higher, more preferably 280 °C or higher, and still more preferably 300 °C or higher in the presence of oxygen such as air, and preferably carbonized to form a colored product, more preferably a black-colored colored product, and still more preferably a carbon-based coloring precursor.

[0162] When the coloring precursor is heated, it becomes a substance that seems to be a carbide, but contains heteroelements other than carbon in its structure, and has a layered structure, an aromatic crosslinked structure, and a disordered structure containing tetrahedral carbon.

[0163] The carbon-based coloring precursor is not particularly limited. For example, it may include tars or pitches such as petroleum tar, petroleum asphalt, coal tar, and coal asphalt, coke, polymers obtained from acrylonitrile-containing monomers, ferrocene compounds (ferrocene and ferrocene derivatives), etc. Among these, polymers obtained from acrylonitrile-containing monomers and / or ferrocene compounds are preferred, and polyacrylic resin nitrile, which is a polymer obtained from acrylonitrile-containing monomers, is more preferred.

[0164] Any tetracarboxylic dianhydride can be used for the tetracarboxylic dianhydride, and it can be appropriately selected according to desired properties, etc. Specific examples of the tetracarboxylic dianhydride include biphenyltetracarboxylic dianhydrides such as pyromellitic dianhydride, 3,3',4,4'-biphenyltetracarboxylic dianhydride (s-BPDA), 2,3,3',4'-biphenyltetracarboxylic dianhydride (a-BPDA), 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-phenylene bis(trimellitic monoester anhydride), p-biphenylene bis(trimellitic monoester anhydride), m-terphenyl-3,4,3',4'-tetracarboxylic dianhydride, p-terphenyl-3,4,3',4'-tetracarboxylic 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-naphthalenetetracarboxylic dianhydride, 1,4,5,8-naphthalenetetracarboxylic dianhydride, 4,4'-(2,2-hexafluoroisopropylidene)diphthalic dianhydride, etc. In addition, aromatic tetracarboxylic acids such as 2,3,3',4'-diphenylsulfone tetracarboxylic acid are also preferably used. These can be used alone or in combination of two or more.

[0165] Among these, in particular, at least one aromatic tetracarboxylic dianhydride selected from biphenyltetracarboxylic dianhydride and pyromellitic dianhydride is preferred. As the biphenyltetracarboxylic dianhydride, 3,3',4,4'-biphenyltetracarboxylic dianhydride can be suitably used.

[0166] Any diamine can be used for the diamine. Specific examples of the diamine are as follows.

[0167] (1) Benzenediamines having one benzene nucleus, such as 1,4-diaminobenzene (p-phenylenediamine), 1,3-diaminobenzene, 2,4-diaminotoluene, 2,6-diaminotoluene;

[0168] (2) Diamines having two benzene nuclei, such as 4,4'-diaminodiphenyl ether, 3,4'-diaminodiphenyl ether, etc., 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'-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, 4,4'-diaminodiphenyl sulfoxide;

[0169] (3) Diamines with three benzene nuclei such as 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)phenoxydibenzophenone, 3,3'-diamino-4,4'-bis(4-phenylphenoxy)dibenzophenone, 1,3-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, 1,4-bis[2-(4-aminophenyl)isopropyl]benzene;

[0170] (4) Diamines having four benzene nuclei such as 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-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, 2,2-bis[4-(4-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, etc.

[0171] These can be used alone or in combination of two or more. The diamines to be used can be appropriately selected according to desired properties, etc.

[0172] Among these, aromatic diamine compounds are preferred, and 3,3'-diaminodiphenyl ether, 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenyl ether, 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, 1,4-bis(3-aminophenoxy)benzene can be suitably used. In particular, at least one diamine selected from phenylenediamine, diaminodiphenyl ether and bis(aminophenoxy)phenyl is preferred.

[0173] The polyimide porous membrane is preferably formed from a self-polyimide obtained by combining a tetracarboxylic dianhydride having a glass transition temperature of 240 °C or higher or no distinct transition point of 300 °C or higher and a diamine from the viewpoints of heat resistance and dimensional stability at high temperatures.

[0174] The polyimide porous membrane of the present invention is preferably a polyimide porous membrane containing the following aromatic polyimide from the viewpoints of heat resistance and dimensional stability at high temperatures.

[0175] (i) An aromatic polyimide composed of at least one tetracarboxylic acid unit selected from a biphenyltetracarboxylic acid unit and a pyromellitic acid unit and an aromatic diamine unit

[0176] (ii) An aromatic polyimide composed of a tetracarboxylic acid unit and at least one aromatic diamine unit selected from a phenylenediamine unit, a diaminodiphenyl ether unit, and a bis(aminophenoxy)phenyl unit

[0177] and / or

[0178] (iii) An aromatic polyimide composed of at least one tetracarboxylic acid unit selected from a biphenyltetracarboxylic acid unit and a pyromellitic acid unit and at least one aromatic diamine unit selected from a phenylenediamine unit, a diaminodiphenyl ether unit, and a bis(aminophenoxy)phenyl unit.

[0179] Although not intended to be limiting, as the polyimide porous membrane, a polyimide porous membrane having a multilayer structure with at least two surface layers (A surface and B surface) and a large void layer sandwiched between the two surface layers can be used in the method of the present invention. Preferably, the polyimide porous membrane has partitions in which the large void layer is combined with the above surface layers (A surface and B surface), and a plurality of large voids having an average pore diameter in the film plane direction of 10 to 500 μm surrounded by the partitions and the above surface layers (A surface and B surface). The partitions of the above large void layer and the above surface layers (A surface and B surface) each have a thickness of 0.01 to 20 μm and a plurality of pores having an average pore diameter of 0.01 to 100 μm, and the pores can communicate with each other. Furthermore, they are partially or entirely in a multilayer structure in communication with the above large voids, and the total film thickness is 5 to 500 μm, and the porosity is less than 40% and 95% or more.

[0180] The total film thickness is not intended to be limiting, but as one embodiment, it can also be 25 - 75 μm. Due to the difference in film thickness, differences in cell proliferation rate, cell morphology, in-plane cell saturation, etc. can be observed.

[0181] In one embodiment, the A side of the polyimide porous membrane has a network structure with small pores having an average pore diameter of 15 μm or less, and the B side has a macroporous structure with an average pore diameter of 20 μm or more.

[0182] For example, the polyimide porous membranes described in International Publication WO2010 / 038873, JP-A-2011-219585, or JP-A-2011-219586 can also be used in the method of the present invention.

[0183] Cells inoculated on the surface of the polyimide porous membrane can stably grow and proliferate on the surface and / or inside of the membrane. Depending on the position of growth and proliferation in the membrane, the cells can take various different forms. In one embodiment of the present invention, depending on the cell type, there are also cases where the cells move on the surface and inside of the polyimide porous membrane and proliferate while changing their shape.

[0184] 【4. Cell culture method, cell culture medium】

[0185] In the method of the present invention, after applying cells to the polyimide porous membrane, the cells can be cultured using any known method. Culturing methods suitable for animal cells, plant cells, and bacteria are known, and those skilled in the art can use any known method to culture cells on the polyimide porous membrane. The cell culture medium can also be appropriately prepared according to the cell type.

[0186] Cell culture methods and cell culture media for animal cells are described, for example, in the cell culture medium catalog of LONZA. Cell culture methods and cell culture media for plant cells are described, for example, in the plant tissue culture medium series of WAKO. Cell culture methods and cell culture media for bacteria are described, for example, in the general bacteria culture medium catalog of BD.

[0187]

II. Cell culture device

[0188] The present invention further relates to a cell culture device containing a polyimide porous membrane for use in the culture method of the present invention. In the cell culture device of the present invention, the polyimide porous membrane can be used in a fixed manner or can also be suspended in the cell culture medium. In the cell culture device, two or more polyimide porous membranes can also be stacked vertically or horizontally.

[0189] The cell culture device of the present invention can use a known cell culture device as long as it satisfies the requirement of including a polyimide porous membrane. The shape, scale, etc. of the culture device are not particularly limited, and it can be appropriately used from a culture dish, a test tube to a large box. For example, it includes a cell culture dish manufactured by BD Falcon or a Nunc cell factory manufactured by Thermo Scientific. Furthermore, by using the polyimide porous membrane in the present invention, it becomes possible to culture cells that are inherently impossible to be cultured in suspension in a state similar to suspension culture using a suspension culture device. As a device for suspension culture, for example, a spinner flask manufactured by Corning can be used.

[0190]

III. Kit for use in a method for culturing cells

[0191] The present invention also relates to a kit for use in a method for culturing cells, which contains a polyimide porous membrane.

[0192] The kit of the present invention may appropriately contain components necessary for cell culture in addition to the polyimide porous membrane. For example, it includes cells suitable for the polyimide porous membrane, a cell culture medium, a cell culture device, an instruction manual for using the kit, etc.

[0193] Not intended to be limiting, but as one embodiment, it includes a kit in which a sterilized polyimide porous membrane is stored alone or in multiple in a transparent bag, with a packaging in a form that can be directly used in cell culture, or a membrane-liquid integrated form kit in which a sterilized liquid is sealed in the same bag together with the polyimide porous membrane to enable efficient aspiration inoculation.

Examples

[0194] Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples. Those skilled in the art can easily modify and change the present invention based on the description of this specification, and they are included in the technical scope of the present invention. Hereinafter, unless otherwise specified, "polyimide porous membrane" refers to a polyimide porous membrane with a membrane thickness of 25 μm.

[0195]

Example 1: Natural inoculation of human mesenchymal stem cells onto a polyimide porous membrane

[0196] In this example, human mesenchymal stem cells were used for inoculation onto the polyimide porous membrane.

[0197] Add 0.5 ml of cell culture medium to a sterilized 2 cm × 2 cm square container, and immerse a sterilized 1.4 cm square polyimide porous membrane with the A side of the mesh structure or the B side of the macroporous structure facing up. Prepare separately a medium with 3.6 × 10 5A suspension of human mesenchymal stem cells (among which, the number of live cells is 3.4×10 5 and the number of dead cells is 2.0×10 4 , with a live cell rate of 94%). 60 μl of each cell suspension was added to the cell culture medium in the above-mentioned square container.

[0198] It was cultured using a cell culture device. After 1 hour, 5 hours, 24 hours, 48 hours, 4 days, 7 days, 14 days, and 21 days, it was fixed and stained (with DAPI, or actin + DAPI) to confirm cell growth and proliferation. The staining of actin was performed with phalloidin. The proliferation of cells over time and the morphology specific to the inoculation surface were observed. The results are shown in Figure 3 . The results showed that human mesenchymal stem cells, one of the representative stem cells, could also be cultured by the method of the present invention.

[0199]

Example 2: Natural Inoculation of Human Dermal Fibroblasts onto Polyimide Porous Membrane

[0200] In this example, human dermal fibroblasts were used, and the application of cells onto the polyimide porous membrane was carried out by natural inoculation.

[0201] 0.5 ml of cell culture medium was added to a sterilized 2 cm × 2 cm square container, and a sterilized 1.4 cm square polyimide porous membrane was immersed with the A side of the mesh structure or the B side of the macroporous structure facing upward. Separately, a cell suspension of human dermal fibroblasts with 8.3×10 5 cells per 1 ml of medium (among which, the number of live cells is 8.1×10 5 and the number of dead cells is 2.0×10 4 , with a live cell rate of 98%) was prepared. 50 μl of each cell suspension was added to the cell culture medium in the above-mentioned square container.

[0202] It was cultured using a cell culture device. After 1 hour, 5 hours, 24 hours, 48 hours, 4 days, 7 days, 14 days, and 21 days, it was fixed and stained (with DAPI, or actin + DAPI) to confirm cell growth and proliferation. The proliferation of cells over time and the morphology specific to the inoculation surface and the observation surface were observed. The results are shown in Figure 4 . From these results, it was shown that human dermal fibroblasts, one of the representative fibroblasts, could also be cultured by the method of the present invention.

[0203]

Example 3: Inhalation Inoculation (Wet Membrane Method) of Human Dermal Fibroblasts onto Polyimide Porous Membrane

[0204] In this example, human skin fibroblasts were used, and the cells were applied to the polyimide porous membrane by inhalation inoculation into a wet membrane (a sufficiently wetted membrane; details will be described later).

[0205] Prepare a culture medium with 1.0×10 6 cells suspended per 1 ml (among them, the number of live cells is 9.1×10 5 cells, the number of dead cells is 1.0×10 5 cells, and the live cell rate is 90%). Suspend human skin fibroblast cells. Without overlapping, arrange 25 square polyimide porous membranes with a side length of 1.4 cm, which have been pre-wetted with 1 ml of cell culture medium, side by side on a 10 cm×14 cm rectangular plate. Add 20 μl of the cell suspension to the B side of each polyimide porous membrane. Remove all the exuded liquid, transfer the polyimide porous membrane to a 75 cm 2 petri dish and add 12 ml of culture medium. Cultivate directly under normal culture conditions and fix the samples the next day. Figure 5 Show fluorescence microscope photos and stereoscopic fluorescence microscope photos of the samples fixed - fluorescence stained (DAPI, or actin + DAPI) after 24 hours.

[0206] Furthermore, during the inoculation process, collect the exuded liquid, and also wash the above-mentioned rectangular plate with 2 ml of culture medium. When counting the remaining cells on the plate, the total number of cells is 6.0×10 4 cells, among which the number of viable cells is 5.5×10 4 cells, the number of dead cells is 3.0×10 4 cells, and the live cell rate is 65%. Comparing the survival and death of the cells, the adsorption rate of live cells to the membrane is 88%, and the exudation rate is 12%; the adsorption rate of dead cells to the membrane is 40%, and the exudation rate is 60%.

[0207]

Example 4: Inhalation inoculation (one - point wet method) of human mesenchymal stem cells onto a polyimide porous membrane

[0208] In this example, human mesenchymal stem cells were used, and the cells were applied to the polyimide porous membrane by inhalation inoculation into a one - point wet membrane (a membrane simply fixed by infiltrating only a central liquid droplet; details will be described later).

[0209] This example shows an example of applying human mesenchymal stem cells to a polyimide porous membrane by inhalation inoculation onto only a central part of a wetted membrane material.

[0210] Form a liquid droplet of about 10 μl at the predetermined central part of the placed membrane. Place the polyimide porous membrane on it to fix the position of the membrane and create an easy - to - inoculate site. At the same time, by using wetting, it aims to accelerate the permeation of the cell liquid through the membrane.

[0211] Prepare a culture medium containing 1.0×10 6 cells per 1 ml in suspension (among which, the number of live cells is 9.6×10 5 cells, the number of dead cells is 4.0×10 4 cells, and the live cell rate is 96%) to obtain a suspension of human mesenchymal stem cells. Arrange 10 sterilized and dried square polyimide porous membranes with a side length of 1.4 cm side by side without overlapping on the A and B sides of a 10 cm×14 cm angular plate, and add 40 μl of the cell suspension to each polyimide porous membrane. Remove the exuded liquid completely, and transfer each polyimide porous membrane with the inoculated surface to a 10 cm 2 culture dish, and add 2 ml of the culture medium. Directly culture under normal culture conditions. After 0.5 hours, 3 days, 7 days, and 12 days, fix and stain (DAPI, actin + DAPI, or actin), and confirm the cell growth and proliferation. The results are shown in Figure 6 and 7 .

[0212] Furthermore, during the inoculation process, when the exuded liquid is recovered and the above-mentioned angular plate is washed with 4 ml of the culture medium, and the cells remaining on the plate are counted, when inoculating on the A side, the total number of cells is 6.5×10 4 cells, among which the number of live cells is 4.0×10 4 cells, the number of dead cells is 2.5×10 4 cells, and the live cell rate is 62%. Comparing the survival and death of the cells, the adsorption rate of the live cells to the membrane is 89%, and the exudation rate is 11%. It is considered that almost no dead cells are adsorbed and flow out. In addition, in the case of inoculating on the B side, the total number of recovered cells is 6.5×10 4 cells, among which the number of live cells is 8.0×10 4 cells, the number of dead cells is 2.0×10 4 cells, and the live cell rate is 80%. Comparing the survival and death of the cells, the adsorption rate of the live cells to the membrane is 79%, and the exudation rate is 21%. It is considered that almost no dead cells are adsorbed and flow out.

[0213]

Example 5: Immersion Inoculation of Human Mesenchymal Stem Cells onto Polyimide Porous Membrane

[0214] In this example, human mesenchymal stem cells are used, and the cells are applied to the polyimide porous membrane by immersion from the cell suspension.

[0215] Cut a 10 cm square polyimide porous membrane into small pieces approximately 2 - 3 mm square using forceps, sterilize at 160°C for 10 minutes, then let it cool, and moisten it with 2 ml of sterilized culture medium. Then, take out the polyimide porous membrane with forceps and transfer it to a Falcon tube. Add a culture medium containing 3.6×10 per 1 ml to the membrane in the Falcon tube5 cells (among them, the viable cells are 3.4×10 5 cells, the dead cells are 2.0×10 4 cells, and the viable cell rate is 94%), a suspension of human mesenchymal stem cells of 1.6 ml (among the total cell number of 5.7×10 5 cells, the viable cells are 5.4×10 5 cells, the dead cells are 3.2×10 4 cells). While constantly vibrating and mixing, it is placed into the culture device for 2 hours, and the cell amount in the liquid part is measured, then 2.0×10 5 cells are observed (among them, the viable cells are 1.2×10 5 cells, the dead cells are 8.0×10 4 cells, and the viable cell rate is 60%). A small piece of polyimide porous membrane is taken out and transferred to a 20 cm 2 culture dish containing 4 ml of culture medium, and it is continuously cultured in the culture device. After 48 hours, 7 days, 14 days, 21 days, and 28 days of the small piece, it is fixed - stained (DAPI, or actin + DAPI), and the cell growth and proliferation are confirmed. The results are shown in Figure 8 and Figure 9 .

[0216]

Example 6: Inhalation Inoculation of Established Cells onto Small Pieces of Polyimide Porous Membrane

[0217] In this example, PC12 cells of established cells are used, and the cells are applied onto small pieces of polyimide porous membrane by inhalation.

[0218] On a 10 cm×14 cm square plate, it is pre - moistened with 5 ml of culture medium, and 15 square polyimide porous membranes with a side length of 1.4 cm, after removing the extra liquid, are randomly arranged side by side with some parts overlapping vertically and horizontally. Each 1 ml of the culture medium suspends 9.0×10 5 cells (among them, the viable cells are 7.4×10 5 cells, and the dead cells are 1.7×10 5 cells, and the viable cell rate is 82%), and a cell suspension of rat adrenal pheochromocytoma PC12 is prepared separately. While slightly tilting the plate to move the exudate downward with 2 ml of the cell suspension, it is slowly added to the membrane in sequence. After 5 minutes, the exudate of the polyimide porous membrane is removed completely, and it is transferred into a 20 cm 2 culture dish containing 4 ml of culture medium, and it is cultured in the culture device.

[0219] After 48 hours, 4 days, 6 days, and 9 days, the polyimide porous membrane was fixed and stained to confirm cell proliferation. Staining was performed using the membrane fluorescent stain CellMask (CellMask (trademark) Orange plasma membrane stain (Life Technologies)) + DAPI. The results are shown in Figure 10 . In these cells, it was also observed that a pattern of clustered proliferation formed within the polyimide porous membrane, confirming the possibility of applicability. The results showed that PC12 cells, one of the representative cell lines, could also be cultured by the method of the present invention.

[0220]

Example 7: Measurement of the number of cultured cells

[0221] In this example, human skin fibroblasts were cultured by the method of the present invention using a polyimide porous membrane, and the number of cultured cells was measured.

[0222] 【1. Measurement of the number of cells using CCK8 for normal cell culture】

[0223] First, using the following reagents and methods, the number of cells in culture was normally measured to obtain the correlation coefficient between absorbance and the actual number of cells.

[0224] [Reagents] Cell Counting Kit-8; solution reagent manufactured by Dojindo Laboratories (hereinafter referred to as "CCK8").

[0225] [Method] Human skin fibroblasts cultured on a 5 cm 2 chamber-type culture dish were prepared for a certain period. The culture supernatant was removed, replaced with a certain amount of medium containing 2% CCK8, and stored in an incubator for 2 hours. Thereafter, the colored supernatant was removed, and the absorbance was measured at a wavelength of 480 nm (using the condition of measuring only with the medium as the blank). Thereafter, after removing the same supernatant, the cells were washed twice with phosphate buffer, treated with 0.05% trypsin-EDTA solution, and the number of cells was counted.

[0226] The correlation coefficient between absorbance and the actual number of cells under the culture conditions and the CCK8 concentration conditions was obtained by this method.

[0227] 【2. Measurement of the number of cells proliferated on the polyimide porous membrane】

[0228] In the same manner as in 1, a 2 cm 2 (1.4 cm * 1.4 cm) polyimide porous membrane with cultured cells was transferred to a 5 cm 2For the chamber-type culture dish, add a certain amount of medium containing 5% CCK8, store it in an incubator for 1 - 3 hours, remove the supernatant, and measure the absorbance at a wavelength of 480 nm. At this time, use 2.5 times the amount of CCK8 used. On the other hand, since the area of the polyimide porous membrane is only 2.5 times smaller, it is possible to directly compare with the reading value in 1. (When changing conditions such as concentration or area, conversion becomes necessary.) Calculate the number of cells surviving on the component using the conversion coefficient obtained in 1, then wash it twice with the medium and return it to the incubator for continued culture. Repeat this operation to quantitatively analyze the proliferation pattern of cells on the polyimide porous membrane over time. Repeat the number of experiments and also verify the reproducibility.

[0229] The results of 3 natural inoculations and inhalation inoculations each are shown in Figure 11 . As Figure 11 shown, the cell proliferation rate varies depending on the inoculation method, but the difference is eliminated by culturing for about 1 month, reaching a similar number of cells per unit area. The dotted line in the figure shows the upper limit of the number of cultured cells in normal adherent culture as demonstrated by culture dishes and the like in comparative experiments. When comparing the number of cells based on area, more cells can be cultured per unit area compared to normal cell culture.

[0230]

Example 8: Culture of human skin keratinocytes

[0231] In this example, human skin keratinocytes were cultured by the method of the present invention using a polyimide porous membrane and observed with a confocal laser microscope and a stereofluorescence microscope.

[0232] Add 0.5 ml of cell culture medium (KGM-Gold keratinocyte proliferation medium BulletKit (manufactured by LONZA)) to a 2 cm × 2 cm sterilized square container, and immerse a sterilized 1.4 cm square polyimide porous membrane with the A side of the mesh structure facing up. Add 4×10 4 human skin keratinocytes to the cell culture medium in the above square container. That is, inoculate 4×10 4 human skin keratinocytes per 1.4 cm square polyimide porous membrane by natural inoculation.

[0233] Culture with a cell culture device, and fix and stain after 1 day, 3 days, and 6 days. The staining is performed with CellMask + DAPI or only CellMask. Then, observe the proliferation and morphology of cells over time using a confocal laser microscope (LSM700 (manufactured by Carl Zeiss)) and a stereofluorescence microscope (Leica M165FC (manufactured by Leica)).

[0234] The results are shown inFigure 12 The results showed that human skin keratinocytes, which are primary cultured cells of humans (primary cultured cells), can also be cultured by the method of the present invention.

[0235]

Example 9: Culture of human umbilical vein endothelial cells

[0236] In this example, human umbilical vein endothelial cells were cultured by the method of the present invention using a polyimide porous membrane, and observed with a confocal laser microscope and a stereoscopic fluorescence microscope.

[0237] Add 0.5 ml of cell culture medium (EGM-2 BulletKit (Lonza)) to a 2 cm × 2 cm sterilized square container, and immerse a sterilized 1.4 cm square polyimide porous membrane with the A side of the mesh structure facing up. Add 4×10 4 human umbilical vein endothelial cells to the cell culture medium in the above square container. That is, 4×10 4 human umbilical vein endothelial cells are naturally inoculated per 1.4 cm square polyimide porous membrane.

[0238] Culture with a cell culture device, and after 3 days, 6 days, and 10 days, fix and stain (CellMask + DAPI and CellMask). Observation with staining, a confocal laser microscope, and a stereoscopic fluorescence microscope was carried out in the same manner as in Example 8.

[0239] The results are shown in Figure 13 The results showed that human umbilical vein endothelial cells, which are primary cultured cells of humans (primary cultured cells), can also be cultured by the method of the present invention.

[0240]

Example 10: Culture of Vero cells

[0241] In this example, Vero cells were cultured by the method of the present invention using a polyimide porous membrane, and observed with a confocal laser microscope and a stereoscopic fluorescence microscope. Three types of polyimide porous membranes with 25 μm, 40 μm, and 75 μm were used. The culture period was 1 day - 15 days.

[0242] Add 0.5 ml of cell culture medium (DMEM supplemented with 10% FBS and antibiotics) to a 2 cm × 2 cm sterilized square container, and immerse a sterilized 1.4 cm square polyimide porous membrane with the A side of the mesh structure facing up. Add 4×10 4 Vero cells to the cell culture medium in the above square container. That is, 4×10 4 Vero cells are naturally inoculated per 1.4 cm square polyimide porous membrane.

[0243] Cultured using a cell culture device, fixed and stained after 1 day, 3 days, 7 days, 10 days, and 15 days. Staining was performed with actin + DAPI, and detection of actin was performed with phalloidin. Observation with a confocal laser microscope and a stereoscopic fluorescence microscope was carried out in the same manner as in Example 8.

[0244] The results are shown in Figure 14-1 to 14-3 . The results show that even for Vero cells, which are one of the representative established cell lines, they can be cultured by the method of the present invention.

[0245]

Example 11: Cultivation of HeLa cells

[0246] In this example, HeLa cells were cultured by the method of the present invention using a polyimide porous membrane, and observed with a confocal laser microscope and a stereoscopic fluorescence microscope. Three types of polyimide porous membranes with 25 μm, 40 μm, and 75 μm were used. The culture period, the microscope used, the specific procedures, etc. were as described in Example 10.

[0247] The results are shown in Figure 15-1 to Figure 15-3 . The results show that even for HeLa cells, which are one of the representative established cell lines, they can be cultured by the method of the present invention.

[0248]

Example 12: Cultivation of CHO cells

[0249] In this example, CHO cells were cultured by the method of the present invention using a polyimide porous membrane, and observed with a confocal laser microscope and a stereoscopic fluorescence microscope. Three types of polyimide porous membranes with 25 μm, 40 μm, and 75 μm were used. During the culture period from 1 day to 7 days, the microscope used, the specific procedures, etc. were as described in Example 10.

[0250] The results are shown in Figure 16-1 to Figure 16-3 . The results show that even for CHO cells, which are one of the representative established cell lines, they can be cultured by the method of the present invention.

Claims

1. A method for culturing cells, which comprises culturing the cells by applying them to a polyimide porous membrane, wherein the polyimide porous membrane is a polyimide porous membrane having a three-layer structure with the following layers: a surface layer A and a surface layer B each having a plurality of pores, and a large void layer sandwiched between the surface layer A and the surface layer B, the surface layer A is a network structure having small pores with an average pore diameter of 15 μm or less, the surface layer B is a large pore structure with an average pore diameter of 20 μm or more, and the large void layer has: partitions bonded to the surface layers A and B, and a plurality of large voids surrounded by the partitions and the surface layers A and B, wherein the plurality of large voids have a plurality of pores with an average pore diameter of 10 to 500 μm; wherein the total membrane thickness of the polyimide porous membrane is 5 to 500 μm, and the porosity is more than 40% and less than 95%; and wherein in the method for culturing cells, an uncoated polyimide porous membrane is not used, and wherein cells grow and proliferate on the surface and inside of the polyimide porous membrane.

2. The method according to claim 1, which comprises the step of inoculating cells onto the surface of the above polyimide porous membrane.

3. The method according to claim 1, which comprises the following steps: loading a cell suspension onto the dry surface of the above polyimide porous membrane, placing the above polyimide porous membrane, or moving the above polyimide porous membrane to promote the outflow of the liquid, or stimulating a part of the surface to suck the cell suspension into the above membrane, and then accumulating the cells in the cell suspension into the above membrane and allowing the water to flow out.

4. The method according to claim 1, which comprises the following steps: wetting one or both sides of the above polyimide porous membrane with a cell culture medium or a sterilized liquid, loading a cell suspension onto the wetted polyimide porous membrane, and then accumulating the cells in the cell suspension into the above membrane and allowing the water to flow out.

5. The method according to claim 4, wherein live cells remain in the above polyimide porous membrane, and dead cells flow out together with the water.

6. The method according to claim 4 or 5, wherein the above sterilized liquid is sterilized water or a sterilized buffer solution.

7. The method according to any one of claims 1 to 6, which comprises placing a cell culture medium, cells, and one or more of the above polyimide porous membranes into a cell culture container, wherein, the polyimide porous membrane is in a state of being suspended in the cell culture medium.

8. The method according to claim 7, characterized in that, small pieces of two or more of the above polyimide porous membranes are used.

9. The method according to claim 7 or 8, wherein cells spontaneously adhere to the above polyimide porous membrane.

10. The method according to any one of claims 1 to 6, wherein the above polyimide porous membrane (i) is folded, (ii) is rolled into a roll, (iii) the sheet or small piece is connected with a filamentous structure, or (iv) is formed into a rope shape and is suspended or fixed in the cell culture medium in the cell culture container.

11. The method according to claim 10, wherein cells spontaneously adhere to the above polyimide porous membrane.

12. The method according to claim 1, which comprises using two or more polyimide porous membranes stacked one above the other or side by side in a cell culture medium.

13. The method according to claim 1, wherein two or more of the methods according to any one of claims 2 to 12 are used in combination.

14. The method according to any one of claims 1 to 13, wherein the cells are selected from animal cells, insect cells, plant cells, yeasts, and bacteria.

15. The method according to claim 14, wherein the animal cells are cells derived from an animal belonging to the phylum Chordata.

16. The method according to claim 14, wherein the bacteria are selected from lactic acid bacteria, Escherichia coli, Bacillus subtilis, and cyanobacteria.

17. The method according to any one of claims 1 to 13, wherein the cells are selected from pluripotent stem cells, tissue stem cells, somatic cells, and germ cells.

18. The method according to any one of claims 1 to 12, wherein the cells are selected from sarcoma cells, established cells, and transformed cells.

19. The method according to any one of claims 1 to 18, wherein the polyimide porous membrane is a polyimide porous membrane containing a polyimide obtained from a tetracarboxylic dianhydride and a diamine.

20. A cell culture device for use in the method for culturing cells according to any one of claims 1 to 19, which contains a polyimide porous membrane.

21. The cell culture device according to claim 20, wherein two or more polyimide porous membranes are stacked one above the other or side by side.

22. A kit for use in the method for culturing cells according to any one of claims 1 to 19, which contains a polyimide porous membrane.

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