Cryopreservation method for cells, cell culture method, and cryopreservation substrate

Abstract

Provided is a method for cryopreserving cells, which is characterized in that the cells are cryopreserved for 30 days or more in a state in which the cells are inoculated on a porous membrane without using liquid nitrogen. Also provided is a cell culture method comprising a step for cryopreserving cells inoculated on the porous membrane for 30 days or more without using liquid nitrogen. Further provided is a cryopreservation base material comprising a porous membrane for cryopreservation of inoculated cells for 30 days or more.

Description

Technical Field

[0001] This invention relates to methods for cryopreservation and cell culture, as well as cryopreservation substrates, and particularly to techniques for cryopreservation of cells seeded in a porous membrane. Background Technology

[0002] In recent years, research on the use of cells has been actively pursued, including cells that produce proteins in the body such as vaccines, enzymes, hormones, antibodies, and cytokines, cells that produce viruses, and cells used in regenerative medicine. In particular, cell culture media, which are known to be used as scaffolds for culturing cells, have various effects on cells; for example, their stiffness can lead to changes in the differentiation susceptibility of stem cells (Non-Patent Literature 1), and therefore have attracted much attention.

[0003] As for technologies related to cell culture media, the following technologies have been proposed: technologies using nonwoven fabrics with special surface treatments as substrates (Patent Document 1); technologies using nanofibers as substrates (Patent Document 2); technologies using polyimide porous membranes as substrates (Patent Document 3); and technologies using hollow microfibers as substrates (Patent Document 4), etc. These technologies all suggest that cells can be cryopreserved at extremely low temperatures (approximately -196°C) using liquid nitrogen while they are seeded on cell culture media.

[0004] However, although the extremely low temperature conditions using liquid nitrogen disclosed in these patent documents 1 to 4 have little impact on cells, they present difficulties in management and procurement due to the use of special equipment and easily volatile liquid nitrogen. Moreover, temperature management is complicated and costly.

[0005] Given these issues, techniques for cryopreservation at higher temperatures are being researched. For example, techniques using ultra-low temperature freezers or dry ice to cryopreserve cells at temperatures around -80°C are known. However, temperatures around -80°C have a greater impact on cells compared to extremely low temperatures using liquid nitrogen, thus limiting them to short-term preservation and short-distance transport, and making them unsuitable for long-term cell storage.

[0006] Therefore, as a technology that can preserve cells for a long time even in the relatively high temperature range of around -80°C, techniques for improving the composition of the culture medium or the cryoprotectant have been proposed (Non-Patent Document 2, Patent Document 5).

[0007] Existing technical documents

[0008] Patent documents

[0009] Patent Document 1: Japanese Patent Application Publication No. 6-209767

[0010] Patent Document 2: International Publication No. 2014 / 196549

[0011] Patent Document 3: International Publication No. 2016 / 121767

[0012] Patent Document 4: Japanese Patent Application Publication No. 2020-171317

[0013] Patent Document 5: Japanese Patent Application Publication No. 2020-039326

[0014] Non-patent literature

[0015] Non-patent literature 1: Cell 2006, 126, 677-689

[0016] Non-patent literature 2: Scientific Reports 2016, 6, 1-13 Summary of the Invention

[0017] The problem the invention aims to solve

[0018] The techniques disclosed in Non-Patent Document 2 and Patent Document 5 involve cryopreservation of even adhesive cells in a suspension state. In this technique of cryopreservation in suspension, cells cultured in culture dishes, flasks, etc., are detached by trypsin treatment, centrifuged, and recovered; then, washing and centrifugation are repeated. The cells are then placed in preservation tubes, filled with preservation solution, frozen in an ultra-low temperature freezer, and stored in liquid nitrogen. This not only makes the operation cumbersome but also presents the problem of stress on the cells caused by trypsin treatment and washing.

[0019] Furthermore, the method of cryopreservation in suspension also has the following problems: when thawing and reculturing, washing and centrifugation are required to remove the cell preservation solution and replace it with culture medium, which causes stress to the cells again.

[0020] On the other hand, the technologies disclosed in Patent Documents 1-4 consistently focus on the tight seal between cells and cell culture media, only suggesting that cells can be cryopreserved even at extremely low temperatures using liquid nitrogen. Therefore, the cryopreservation period is only a short time, ranging from a few days to about 20 days. In other words, regarding the issues related to the storage temperature and time for cryopreservation of cells, there are currently no reports of addressing solutions by focusing on cell culture media.

[0021] This invention was made in view of the following circumstances, and its object is to provide a method for long-term cryopreservation that is inexpensive and simple without impairing cell function. Another object is to provide a cell culture method for long-term cryopreserved cells. Furthermore, another object is to provide a cryopreservation substrate for long-term cryopreservation.

[0022] Solution for solving the problem

[0023] The inventors conducted in-depth research and surprisingly discovered a relationship between cell culture media and cryopreserved cells. Specifically, they found that, as an alternative to conventional cell cryopreservation methods using extremely low temperatures with liquid nitrogen, using a porous membrane as the cryopreservation substrate simplifies the freezing and thawing process and enables long-term stable cryopreservation of cells even at higher temperatures without the use of liquid nitrogen, thus completing the invention. Furthermore, the inventors also completed the following inventions: an invention relating to a cell culture method that allows stable cell culture even after long-term cryopreservation and thawing without impairing cell function, using a porous membrane as the cryopreservation substrate; and an invention relating to a cryopreservation substrate formed from a porous membrane.

[0024] That is, the present invention provides the following solution.

[0025] [1] A method for cryopreserving cells, characterized in that liquid nitrogen is not used, and the cells are cryopreserved for more than 30 days while they are seeded in a porous membrane.

[0026] [2] According to the method described in [1], wherein the cell is an animal cell.

[0027] [3] The cell cryopreservation method according to [1] or [2], wherein the porous membrane is a polymer porous membrane.

[0028] [4] The method according to any one of [3], wherein the average pore size of the B side of the polymer porous membrane is greater than the average pore size of the A side.

[0029] [5] The method according to any one of [3] or [4], wherein the average aperture of the B-side is 10 μm or more.

[0030] [6] The method according to any one of [3] to [5], wherein the average aperture of the A surface is 0.01 μm or more;

[0031] [7] The method according to any one of [1] to [6], wherein the average pore size of the porous membrane is 0.01 μm or more and 500 μm or less.

[0032] [8] The method according to any one of [3] to [7], wherein the polymer porous membrane is a polyimide porous membrane or a polyethersulfone porous membrane.

[0033] [9] According to the method described in [3] to [8], wherein the porous membrane is a porous membrane disposed in the cell culture insert.

[0034]

[10] The method according to any one of [2] to [9], wherein the animal cell is a virus-producing cell or a hybridoma.

[0035]

[11] The method according to any one of [2] to [9], wherein the animal cell is a mesenchymal stem cell.

[0036]

[12] The method according to any one of [1] to

[11] , wherein the temperature for cryopreservation is -150°C or higher.

[0037]

[13] A cell culture method comprising the following steps.

[0038] (1) Process: The process of seeding cells onto a porous membrane;

[0039] (2) Process: The process of freezing and storing cells inoculated on a porous membrane for more than 30 days without using liquid nitrogen;

[0040] (3) Process: The process of thawing the cells seeded on the porous membrane;

[0041] (4) Process: The process of culturing thawed cells.

[0042]

[14] According to the method described in

[13] , the porous membrane is a porous membrane disposed in a cell culture insert.

[0043]

[15] A cryopreservation substrate formed of a porous membrane for cryopreserving inoculated cells for more than 30 days.

[0044]

[16] The cryopreservation substrate according to

[15] , wherein the porous membrane is a porous membrane disposed in the cell culture insert.

[0045] The effects of the invention

[0046] According to the present invention, even without the use of liquid nitrogen, cells can be cryopreserved for an unprecedented period of time while maintaining cell function without deterioration. This enables long-distance transport both domestically and internationally. Furthermore, since expensive liquid nitrogen and specialized equipment are not required, costs can be significantly reduced compared to conventional extremely low-temperature conditions. From another perspective, freezing, storage, and thawing operations can be performed with cells attached to a porous membrane, simplifying the process. From yet another perspective, since cryopreservation and cell culture can be performed without changing the cell culture medium and cryopreservation substrate, cell cryopreservation and cell culture can be performed easily and inexpensively without subjecting the cells to stress. Attached Figure Description

[0047] Figure 1It is a chart about the viral titer of cells that have been cryopreserved in an ultra-low temperature freezer.

[0048] Figure 2 It is a chart about the viral titer of cells that have been cryopreserved using liquid nitrogen.

[0049] Figure 3 This is a diagram showing the state of the cell culture plug inserted into the well. Detailed Implementation

[0050] In this specification, the terms involved in the present invention are defined, and then the inventions are described in detail.

[0051] First, the terminology involved in this invention will be explained.

[0052] The "porous membrane" involved in this invention refers to a membrane (thin film) having multiple small pores inside or on its surface. The material forming the porous membrane is not particularly limited and can be inorganic, organic, or a combination thereof. The term "porous membrane" excludes so-called "woven fabrics" and "non-woven fabrics" that are primarily composed of fibers. On the other hand, it includes cases where the porous membrane contains fibers as a minor component (e.g., less than 30 wt% relative to the total porous membrane). Of course, structures formed by laminating porous membranes with non-woven fabrics are relevant to this invention because they utilize the porous membrane.

[0053] The "not using liquid nitrogen" in this invention refers to the absence of liquid nitrogen as a refrigerant during freezing and / or storage. The use of liquid nitrogen in other processes is not included in the concept of "not using liquid nitrogen." In particular, it is preferable to avoid using liquid nitrogen except during freezing and / or storage.

[0054] In this invention, "inoculation" refers to the state or behavior of cells in contact with a substrate, preferably the state or behavior of cells adhering to or being cultured on a substrate (such as a porous membrane).

[0055] Regarding the "A side" and "B side" of this invention, one of the two main surfaces of the porous membrane is referred to as the A side, and the other surface is referred to as the B side. That is, "A side" and "B side" are used for convenience to label the main surfaces and do not indicate the surface or back side, etc.

[0056] Next, the cryopreservation method, cell culture method, and cryopreservation substrate of the present invention will be described in sequence.

[0057] I. Cryopreservation Method

[0058] The cryopreservation method of the present invention does not use liquid nitrogen; the cells are cryopreserved for more than 30 days while seeded on a porous membrane. In this specification, for ease of explanation, the description is presented in the order of cells, porous membrane, and cryopreservation method.

[0059] 1. Cells

[0060] There are no particular limitations on the cells suitable for use in this invention. This is because the method of this invention places a low load on cells. Examples include animal cells, insect cells, plant cells, yeast, and bacteria.

[0061] Animal cells are broadly classified into cells derived from animals belonging to the phylum Vertebrata and cells derived from invertebrates (animals other than those belonging to the phylum Vertebrata). In this specification, the source of animal cells is not specifically limited.

[0062] For example, the phylum Vertebrata includes the superclasses Agnathus and Jawedoidus. Jawedoids can include mammals, birds, amphibians, reptiles, etc., and preferably, cells derived from animals generally belonging to the class Mammalia, which is called mammals. Examples of mammals preferably include mice, rats, humans, monkeys, pigs, dogs, sheep, goats, etc.

[0063] The source of plant cells in this specification is not specifically limited. For example, the subject is the cells of plants including bryophytes, ferns, and seed plants.

[0064] The source plants of seed plant cells include any of the monocotyledonous and dicotyledonous plants. While not limited, examples of monocotyledonous plants include: orchids, grasses (rice, corn, barley, wheat, sorghum, etc.), and sedges. Examples of dicotyledonous plants include plants belonging to multiple subclasses such as Asteridae, Magnoliidae, and Rosidae.

[0065] Algae can be considered as cell-derived organisms. For example, they include different groups, ranging from cyanobacteria (blue-green algae) that are eubacteria, to single-celled organisms (diatoms, yellow-green algae, dinoflagellates, etc.) that are eukaryotes, and seaweed (red algae, brown algae, green algae) that are multicellular organisms.

[0066] The types of archaea and bacteria mentioned in this specification are not specifically limited. Archaea consist of groups such as methanogens, highly halophilic bacteria, thermophilic acidophilic bacteria, and hyperthermophilic bacteria. Bacteria, for example, can be selected from groups such as lactic acid bacteria, Escherichia coli, Bacillus subtilis, and cyanobacteria.

[0067] As described above, the method of the present invention is not limited to the types of animal or plant cells that can be used, but preferably can be selected from the group consisting of hybridomas, pluripotent stem cells, tissue stem cells, somatic cells and germ cells.

[0068] In this specification, "hybridoma" refers to antibody-producing cells obtained by fusing B cells (obtained through immunization against animal antigens other than humans) with myeloma cells. The source organisms of B cells and myeloma cells are not specifically limited, and include mice (including nude mice), rats, guinea pigs, rabbits, goats, sheep, chickens, etc.

[0069] In this specification, "pluripotent stem cells" refers to a general term for stem cells that have the ability to differentiate into cells of all tissues (differentiation pluripotency). Although not limited, pluripotent stem cells include, for example, 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 preferably shown as examples. iPS cells are particularly preferred for reasons such as the absence of ethical concerns. As pluripotent stem cells, any known pluripotent stem cell can be used, such as the pluripotent stem cells described in International Publication No. 2009 / 123349 (PCT / JP2009 / 057041).

[0070] In this specification, "tissue stem cells" refers to stem cells whose cell lineage is limited to a specific tissue but possess the ability to differentiate into multiple cell types (differentiation pluripotency). For example, hematopoietic stem cells in bone marrow become the source of blood cells, and neural stem cells differentiate into nerve cells. In addition, there are various other types, such as liver stem cells for liver production and skin stem cells for skin tissue formation. Preferably, tissue stem cells can be selected from mesenchymal stem cells, liver stem cells, pancreatic stem cells, neural stem cells, skin stem cells, or hematopoietic stem cells, etc.

[0071] In this specification, "somatic cell" refers to a cell other than a germ cell among the cells that constitute a multicellular organism. Preferably, the somatic cell may be selected from hepatocytes, pancreatic cells, muscle cells, bone cells, osteoblasts, osteoclasts, chondrocytes, adipocytes, skin cells, fibroblasts, pancreatic cells, kidney cells, lung cells, or blood cells such as lymphocytes, red blood cells, white blood cells, monocytes, macrophages, or megakaryocytes.

[0072] In this specification, "germ cell" refers to a cell that plays a role in passing on genetic information to the next generation during reproduction. Examples include gametes used for sexual reproduction, i.e., ovum, egg cell, sperm, spermatocyte, etc., and spores used for asexual reproduction.

[0073] In addition, the cells can be selected from a group consisting of sarcoma cells, lineage cells, and transformed cells.

[0074] In this specification, "sarcoma" refers to cancer originating from connective tissue cells of non-epithelial cell origin, such as bone, cartilage, fat, muscle, and blood, including soft tissue sarcoma and malignant bone tumors. Sarcoma cells are cells derived from sarcomas.

[0075] In this specification, "cultured cells" refers to cultured cells that have been maintained in vitro for a long period of time, possess a certain degree of stability, and can be cultured semi-permanently. Cell lines derived from various organisms and tissues, including humans, are available, such as PC12 cells (derived from rat adrenal medulla), CHO cells (derived from Chinese hamster ovaries), HEK293 cells (derived from human fetal kidneys), HL-60 cells (derived from human leukocytes), HeLa cells (derived from human cervical cancer), Vero cells (derived from African green monkey kidney epithelial cells), MDCK cells (derived from canine renal tubular epithelial cells), HepG2 cells (derived from human liver cancer), and porcine kidney cultured cells (CPK). Even virus-producing cells, such as cells infected with various viruses (virus-producing cultured cells) or cultured cells introduced with vectors that produce various viruses (virus-producing vectors), can be cryopreserved for extended periods.

[0076] In this specification, "transformed cell" refers to a cell whose genetic properties are altered by the introduction of nucleic acids (DNA, etc.) from outside the cell. Suitable methods for the transformation of animal cells, plant cells, and bacteria are well known and can also be used for transformed cells.

[0077] In the method of this invention, all cells can be cryopreserved for a long period of time. Hybridomas, tissue stem cells, and lined cells are preferably selected, and in particular, hybridomas, mesenchymal stem cells, lined cells, and virus-producing cells can be selected. Additionally, in one embodiment, the method of this invention can cryopreserve a single layer of cells or tissue, or multiple layers (e.g., 2-5 layers, 2-4 layers, or 2-3 layers) of cells or tissue for a long period of time. In this specification, "tissue" refers to a structure composed of multiple aggregated cells, such as skin tissue (e.g., skin tissue with hair follicles), cardiac tissue, skeletal muscle tissue, smooth muscle tissue, liver tissue, kidney tissue, digestive tract tissue, eye tissue (e.g., corneal tissue), brain tissue, thymus tissue, testicular tissue, pancreatic tissue, thyroid tissue, breast tissue, salivary gland tissue, lung tissue, etc. It can be a sheet-like tissue (e.g., a cell sheet) formed by culturing cells derived from the tissue, or a sheet-like tissue isolated from a biological tissue.

[0078] 2.Porous membrane

[0079] The porous membrane of this invention primarily functions as a substrate for inoculating cells during cryopreservation. This porous membrane refers to a membrane (thin film) having multiple small pores internally or on its surface. The material forming the porous membrane is not particularly limited; it can be inorganic materials such as ceramics, organic materials such as polymer porous membranes, or composites thereof. The following detailed description uses a polymer porous membrane formed from polymers as an example of an embodiment.

[0080] 2-1. Polymer porous membrane

[0081] In this specification, the "polymer porous membrane" has a surface layer A (hereinafter sometimes referred to as "A-side" or "mesh side") and a surface layer B (hereinafter sometimes referred to as "B-side" or "macropore side"). The shapes of the pores on the A-side and B-side can be the same or different. Furthermore, the size of the pores (average pore diameter) on the A-side and B-side is not particularly limited, but it is preferable that the average pore diameter of the pores present on the B-side is larger than the average pore diameter of the pores present on the A-side.

[0082] The average pore diameter (hereinafter sometimes referred to as "average fine pore diameter") of the holes present on surface A is not particularly limited, but is preferably 0.01 μm or more, more preferably 0.05 μm or more, further preferably 0.1 μm or more, and particularly preferably 0.5 μm or more. Its upper limit is preferably less than 200 μm, more preferably less than 150 μm, further preferably less than 100 μm, and particularly preferably less than 50 μm, less than 40 μm, less than 30 μm, less than 20 μm, or less than 15 μm.

[0083] The average pore diameter of the pores present in surface layer B is preferably greater than the average pore diameter of the pores present in surface layer A. For example, it is preferably more than 5 μm, more preferably more than 20 μm, even more preferably more than 30 μm, and particularly preferably more than 50 μm or more, or more than 60 μm. The upper limit is preferably less than 200 μm, and more preferably less than 100 μm.

[0084] The average pore diameter of the pores existing in the surface layer of the polymer porous membrane 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 pores when the shape is a perfect circle is calculated from the average value of the pore area according to the following formula (1).

[0085] [Mathematical Expression 1]

[0086]

[0087] (In the formula, Sa refers to the average area of ​​the holes.)

[0088] The thicknesses of surface layer A and surface layer B can be the same or different, without particular restrictions. For example, their thicknesses are independent, preferably 0.01 μm or more, and their upper limit is preferably 50 μm or less, more preferably 20 μm or less.

[0089] The porous polymer membrane used in this invention may have an intermediate layer between surface A and surface B. This intermediate layer can be a solid layer without pores or a porous layer with pores. If it has pores, it may be the same as or different from the surface layer of the porous polymer membrane. Preferably, the intermediate layer is a porous layer that forms a macroporous layer.

[0090] The following description uses a macroporous layer, which is a porous layer, as an example. In this specification, a "macroporous layer" refers to a membrane with an average pore size of 10 μm or more in the membrane plane direction. The average pore size in the membrane plane direction of the macropores in the macroporous layer is not particularly limited; for example, the upper limit of its average pore size is preferably 500 μm or less, more preferably 100 μm or less, and particularly preferably 80 μm or less. Furthermore, the thickness of the spacers (frames forming the pores in the macroporous layer) in this macroporous layer is not particularly limited; for example, it is preferably 0.01 μm or more, and its upper limit is preferably 50 μm or less, more preferably 20 μm or less.

[0091] In one embodiment, at least one spacer wall in the macroporous layer may have a pore that connects adjacent macropores to each other. The average pore diameter of the connecting pore is preferably 0.01 μm or more, and its upper limit is preferably 100 μm or less, more preferably 50 μm or less. There may be one or more connecting pores. In another embodiment, the spacer wall in the macroporous layer may not have a pore.

[0092] The total thickness of the polymer porous membrane (the combined thickness of surface A and surface B, and including the intermediate layer if present) is not particularly limited, and can be 5 μm or more, 10 μm or more, 20 μm or more, or 25 μm or more; and can be 500 μm or less, 300 μm or less, 100 μm or less, 75 μm or less, or 50 μm or less. Preferably, it is 5 to 500 μm, more preferably 10 to 100 μm.

[0093] In this specification, the thickness of the polymer porous membrane can be measured using a contact thickness gauge.

[0094] In this specification, the porosity of the polymer porous membrane is not particularly limited, but is preferably 40% or more, more preferably 50% or more, and even more preferably 60% or more. Its upper limit is preferably less than 95%, more preferably less than 90%, and even more preferably less than 85%.

[0095] In this specification, the porosity of the polymer porous membrane can be determined by measuring the membrane thickness and mass of the polymer porous membrane cut to a specified size, and the mass per unit area can be calculated according to the following formula (2).

[0096] [Mathematical Expression 2]

[0097]

[0098] (In the formula, S represents the area of ​​the porous polymer 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 。

[0099] In this specification, the polymer porous membrane is preferably a three-layer polymer porous membrane, comprising a surface layer A and a surface layer B having multiple pores, and a macroporous layer sandwiched between the aforementioned surface layer A and surface layer B. Here, the average pore size of the pores present in the aforementioned surface layer A is preferably 0.01 μm or more and 15 μm or less, and the average pore size of the pores present in the aforementioned surface layer B is preferably 20 μm or more and 100 μm or less. The aforementioned macroporous layer has spacer walls bonded to the aforementioned surface layers A and B, and multiple macropores surrounded by the spacer walls and the aforementioned surface layers A and B. The thickness of the spacer walls of the aforementioned macroporous layer, and the aforementioned surface layers A and B, is preferably 0.01 μm or more and 20 μm or less. The pores in the aforementioned surface layers A and B are in communication with the macropores, and the total membrane thickness is preferably 5 μm or more and 500 μm or less. The porosity of this polymer porous membrane is preferably 40% or more and less than 95%. In one embodiment, at least one spacer wall in the macroporous layer has one or more pores with an average pore diameter of 0.01 to 100 μm, preferably 0.01 to 50 μm, that connect adjacent macropores to each other. In another embodiment, the spacer wall does not have such pores.

[0100] In this specification, the shape of the polymer porous membrane is not particularly limited; for example, the size can be adjusted according to the shape of the culture container or cryopreservation container used.

[0101] In this specification, the polymer porous membrane can also be used in the form of a membrane monomer placed in a petri dish or the like, for example, it can also be used in the form of a portion of the membrane fixed to a resin or metal, or in the form of being housed in a suitable shell as described in WO2018 / 021368.

[0102] In this specification, the polymer porous membrane is preferably sterilized. There are no particular limitations on the sterilization process; any sterilization treatment can be selected from dry heat sterilization, ethylene oxide gas sterilization, autoclaving, sterilization using disinfectants such as ethanol, sterilization using electromagnetic waves such as ultraviolet light or gamma rays, and / or radiation. Alternatively, multiple sterilization methods can be selected and appropriately combined.

[0103] In this specification, the polymer porous membrane preferably has the above-described structural features, and it is particularly preferred to use a polyimide porous membrane formed of polyimide porous material or a polyethersulfone porous membrane formed of polyethersulfone (PES).

[0104] By using the porous membrane described above as the substrate for cryopreservation, the cryopreservation method of the present invention can achieve its intended effect.

[0105] 3. Cell Culture Plug-in

[0106] In one aspect of the present invention, a cell culture insert comprising a cylindrical container and the aforementioned porous membrane located at the bottom of the cylindrical container can be used as the substrate for cryopreservation. Hereinafter, this cell culture insert will also be referred to as the "cell culture insert of the present invention." It should be noted that in this specification, the term "cell culture insert" can be simply referred to as "insert," and the meaning remains the same even if the terms are interchanged.

[0107] In this specification, "cell culture plug" refers to a cell culture plug commonly used in cell culture, inserted into the inside of the wells of a culture dish or multi-well plate. A typical plug is a cylindrical container of a size capable of being inserted into a culture dish or well, having a bottom and sides with a certain volume to ensure cell culture can be performed. According to the invention, the cell culture plug is provided by disposing the aforementioned porous membrane at the bottom of the cylindrical container. Here, the cylindrical container has space for cell culture to be performed inside the cell culture plug, and is sized to be inserted into a typical culture dish or well. To avoid contact with the bottom of the culture dish or well, it is preferable to have an arm structure on the upper surface of the cylindrical container that hangs on the upper surface of the culture dish or well. The cylindrical container is not limited to the aforementioned shape; a commonly used, general-purpose, or commercially available container can be used, and the cell culture plug is provided by adhering the porous membrane to the bottom surface of the cylindrical container. Alternatively, a cell culture plug using a commonly used, general-purpose, or commercially available plug with a membrane disposed thereon and replaced with the porous membrane used in this invention can also be applied. The porous membrane used in this invention can be easily molded to match the size and shape of the bottom of the insert used. While not particularly limited, a schematic diagram of inserting the cell culture insert into the well is shown. Figure 3 .

[0108] The bottom of the aforementioned inserts is typically circular or elliptical in shape. If circular, its diameter is less than or equal to the upper opening diameter of the culture dish or well. Therefore, the diameter of the porous membrane placed at the bottom is appropriately adjusted to be less than or equal to the opening diameter of the culture dish (e.g., nominal diameter 35mm, 60mm, 100mm, 150mm, etc.) or well (e.g., 6-well, 12-well, 24-well, 96-well, etc.) used to insert the cell culture insert. The nominal diameter of the culture dish is a general nominal diameter; for example, a nominal diameter of 100mm means the actual diameter of the culture dish is approximately 80mm, and does not represent an exact diameter. For instance, the diameter of the porous membrane is typically 3–130mm, which is the size that allows it to enter the culture dish or well.

[0109] The plug-in used in this invention is preferably sterilized. There are no particular limitations on the sterilization process; any sterilization treatment can be selected from dry heat sterilization, ethylene oxide gas sterilization, autoclaving, sterilization using disinfectants such as ethanol, sterilization using electromagnetic waves such as ultraviolet light or gamma rays, and / or radiation sterilization. Alternatively, multiple sterilization methods can be selected and appropriately combined.

[0110] 4. Freezing preservation method

[0111] The cryopreservation method of the present invention uses a porous membrane (or a cell culture insert with a porous membrane) with cells attached and loaded, eliminating the need for cell removal and centrifugation. Otherwise, it can be implemented using methods similar to existing methods. For example, the porous membrane (or cell culture insert with a porous membrane) with attached and loaded cells can be transferred from the culture medium to a cryopreservation container containing cryoprotectant, slowly frozen in an ultra-low temperature freezer (e.g., an ultra-low temperature freezer at approximately -80°C), and then cryopreserved at a target temperature. Alternatively, the culture medium can be removed from the container, cryoprotectant placed inside, slowly frozen in an ultra-low temperature freezer, and then cryopreserved at a target temperature. Washing with PBS or similar methods can also be appropriately performed during these operations. In one embodiment, when cryopreserving a cell culture insert loaded with cells or tissue, cryoprotectant can be placed only on the inside of the cell culture insert, or on both the inside and outside, frozen in an ultra-low temperature freezer (e.g., an ultra-low temperature freezer at approximately -80°C), and then further cryopreserved at a target temperature.

[0112] In this specification, "higher temperature conditions without the use of liquid nitrogen" refers to temperature conditions higher than those for liquid-phase preservation and gas-phase preservation using liquid nitrogen, with a lower limit of -150°C or higher, preferably -120°C or higher, and more preferably -90°C or higher.

[0113] To set such a temperature, for example, the setting temperature of the ultra-low temperature freezer can be set to above -150℃, above -120℃, above -100℃, or -80℃.

[0114] In “higher temperature conditions without the use of liquid nitrogen”, the upper limit can be, for example, below -10°C, below -20°C, below -30°C, below -40°C, below -50°C, below -60°C, or below -70°C.

[0115] In one embodiment of the present invention, "a higher temperature condition without the use of liquid nitrogen" can be a range formed by any combination of the aforementioned lower and upper limits, for example, -150℃ to -10℃, -150℃ to -20℃, -150℃ to -30℃, -150℃ to -40℃, -150℃ to -50℃, -150℃ to -60℃, or -150℃ to -70℃. Alternatively, it can be, for example, -120℃ to -10℃, -120℃ to -20℃, -120℃ to -30℃, -120℃ to -40℃, -120℃ to -50℃, -120℃ to -60℃, or -120℃ to -70℃. Alternatively, it could be -100℃~-10℃, -100℃~-20℃, -100℃~-30℃, -100℃~-40℃, -100℃~-50℃, -100℃~-60℃, or -100℃~-70℃. Alternatively, it could be -90℃~-10℃, -90℃~-20℃, -90℃~-30℃, -90℃~-40℃, -90℃~-50℃, -90℃~-60℃, or -90℃~-70℃.

[0116] In this specification, "long-term" in cell cryopreservation time means a period of more than one month (or 30 days), preferably more than three months (or 90 days), more preferably more than six months (or 180 days), and more preferably more than 12 months (or 365 days). Additionally, in this specification, "long-term" in cell cryopreservation time can mean a period of at least one month (or at least 30 days), preferably at least three months (or at least 90 days), more preferably at least six months (or at least 180 days), and more preferably at least 12 months (or at least 365 days).

[0117] Thus, the cryopreservation method of the present invention, since it does not use liquid nitrogen, can be performed very simply, reducing the load on the cryopreserved cells and being applicable to all cryopreservation methods, which can be considered an advantage. In particular, special cryoprotectants such as those in Non-Patent Document 2 or Patent Document 5 can be used, but even without using cryoprotectants with special compositions, for example, by using commercially available cryoprotectants (cell cryoprotectants), cell function can be maintained and cryopreservation can be performed without causing deterioration, which is excellent.

[0118] II. Cell Culture Methods

[0119] The cell culture method of the present invention comprises the following steps.

[0120] (1) Process: The process of seeding cells onto a porous membrane;

[0121] (2) Process: The process of freezing and storing cells inoculated on a porous membrane for more than 30 days without using liquid nitrogen;

[0122] (3) Process: The process of thawing the cells seeded on the porous membrane;

[0123] (4) Process: The process of culturing thawed cells.

[0124] (1) The process involves seeding cells onto a porous membrane before cryopreservation. Preferably, the cells to be cryopreserved can be cultured using the porous membrane as a substrate. Here, cells can simply adhere to the porous membrane or be cultured. The appropriate method can be chosen depending on the type and quantity of cells to be cryopreserved.

[0125] Cell culture methods can employ well-known techniques, and the culture conditions can be appropriately determined based on the cell type, etc. Suitable cell culture methods for animal cells, plant cells, and bacteria are well-known, and those skilled in the art can use any well-known method to culture cells in a polymer porous membrane. The culture medium for cell culture can also be appropriately prepared according to the cell type.

[0126] Methods for culturing animal cells and culture media for cell culture are described, for example, in the Lonza Company's catalog of cell culture media. Methods for culturing plant cells and culture media for cell culture are described, for example, in WAKO Company's plant tissue culture media series. Methods for culturing bacteria and culture media for cell culture are described, for example, in BD Company's catalog of general bacterial culture media. The culture media used in the method of the present invention can be any form, such as liquid culture medium, semi-solid culture medium, or solid culture medium. Alternatively, liquid culture medium in droplet form can be sprayed into the cell culture container, allowing the culture medium to contact the cell-loaded polymer porous membrane.

[0127] (2) The process involves cryopreserving cells seeded onto a porous membrane, as detailed in the cryopreservation method. Slow cooling is employed to freeze the cells. Slow cooling is preferred because it freezes the extracellular water before ice forms inside the cells. While techniques from Non-Patent Document 2 or Patent Document 5 can also be used in this process, the present invention excels in its ability to maintain cell function and prevent deterioration even without employing these techniques.

[0128] (3) The process is to thaw the frozen cells. Except for washing operations that do not require the use of a centrifuge, this process can be carried out using methods similar to existing methods.

[0129] (4) The process involves culturing the thawed cells (hereinafter also referred to as "re-culturing"). This process can be performed in a manner similar to conventional methods, except that washing with a centrifuge and cell seeding are not required. While not particularly limited, for example, after thawing the cryoprotectant, a porous polymer membrane with attached, cell-loaded cells can be moved from the cryoprotectant to the culture medium, and culturing can begin again. Conversely, the cryoprotectant can be removed after thawing, and the cells can be placed in culture medium and culturing can begin again. Additionally, washing with, for example, PBS can be appropriately performed during these operations.

[0130] III. Cryopreservation substrate

[0131] The cryopreservation substrate of the present invention is significant in its choice of using porous membranes as the substrate. Details of the porous membranes, particularly polymeric porous membranes, are described above. Here, as examples of polymeric porous membranes, the manufacturing methods of polyimide porous membranes and polyethersulfone porous membranes are described in detail.

[0132] III-1. Polyimide porous membrane

[0133] In this specification, "polyimide" refers to a polymer containing imide bonds in repeating units, preferably containing more than 50 mol% of polymers in all repeating units. It typically refers to aromatic polyimides formed by aromatic compounds directly linked by imide bonds. Aromatic polyimides possess a rigid and robust molecular structure due to the conjugated structure of aromatic compounds via imide bonds, and because imide bonds exhibit strong intermolecular forces, they possess very high levels of thermal, mechanical, and chemical properties.

[0134] The polyimide porous membrane that can be used in this invention is preferably a polyimide porous membrane containing polyimide obtained from tetracarboxylic dianhydride and diamine as a main component, and more preferably a polyimide porous membrane composed of polyimide obtained from tetracarboxylic dianhydride and diamine. "Containing...as a main component" means that the polyimide porous membrane substantially does not contain any components other than polyimide obtained from tetracarboxylic dianhydride and diamine, or may contain additional components that do not affect the properties of polyimide obtained from tetracarboxylic dianhydride and diamine.

[0135] Polyamic acid can be obtained by polymerizing a tetracarboxylic acid component with a diamine component. Polyamic acid is a precursor for the formation of polyimides, which can be cyclically closed by thermal imidization or chemical imidization.

[0136] Regarding polyamic acid, even if a portion of the amic acid is imidized, it can be used as long as it does not affect the scope of the present invention. That is, polyamic acid can be partially thermally imidized or chemically imidized.

[0137] When performing thermal imidization of polyamic acid, imidization catalysts, additives containing organophosphorus compounds, inorganic particles, organic particles, etc., can be added to the solution containing dissolved polyamic acid (hereinafter also referred to as "polyamic acid solution") as needed. Alternatively, in the case of chemical imidization of polyamic acid, chemical imidizing agents, dehydrating agents, inorganic particles, organic particles, etc., can be added to the polyamic acid solution as needed.

[0138] 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.

[0139] 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 product.

[0140] 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 thermally decomposed and carbonized by heat treatment at 250°C or above, preferably 260°C or above, more preferably 280°C or above, more preferably 300°C or above, preferably in the presence of oxygen such as air, to generate a coloring product, more preferably a black coloring product, and more preferably a carbon-based coloring precursor.

[0141] The coloring precursor appears as a carbide when heated, but it contains different elements other than carbon in its structure, and includes layered structures, aromatic cross-linked structures, and disordered structures containing tetrahedral carbon.

[0142] There are no particular limitations on carbon-based coloring precursors. Examples include polymers obtained from monomers containing petroleum tar, petroleum pitch, coal tar, coal pitch, or other tar or pitch, coke, acrylonitrile, and ferrocene compounds (ferrocene and ferrocene derivatives). Among these, polymers and / or ferrocene compounds obtained from monomers containing acrylonitrile are preferred, and polyacrylonitrile is preferred as a polymer obtained from monomers containing acrylonitrile.

[0143] When using a coloring precursor, the above-mentioned additives can also be used. In this case, it is preferable to carry out the process under conditions where the coloring precursor does not precipitate even when the additive is mixed in a polyamic acid solution.

[0144] 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 made from a tetracarboxylic acid component and a diamine component without using the above-mentioned coloring precursor.

[0145] Polyimide porous membranes can be manufactured, for example, with or without a coloring precursor, by casting a polyamic acid solution comprising 3-60% by mass of polyamic acid with an intrinsic viscosity of 1.0-3.0 and 40-97% by mass of an organic polar solvent into a thin film, immersing it in or in contact with a coagulation solvent containing water as an essential component to form a porous polyamic acid membrane, and then subjecting the porous polyamic acid membrane to heat treatment to imidize it. In this method, the coagulation solvent containing water as an essential component can be water, or a mixture of 5% by mass or more but less than 100% by mass of water and 0% by mass or less 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.

[0146] In the manufacture of the above-mentioned polyimide porous membrane, any tetracarboxylic acid dianhydride can be used, and appropriate selection can be made 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.; oxo-diphthalic 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 Examples of dianhydrides include: 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, and 4,4'-(2,2-hexafluoroisopropylidene)diphthalic acid dianhydride. In addition, aromatic tetracarboxylic acids such as 2,3,3',4'-diphenylsulfone tetracarboxylic acid are preferred. They can be used alone or in combination of two or more.

[0147] Of these, at least one aromatic tetracarboxylic dianhydride from the group consisting of biphenyltetracarboxylic dianhydride and pyromellitic dianhydride is particularly preferred. As a biphenyltetracarboxylic dianhydride, a substance containing 3,3',4,4'-biphenyltetracarboxylic dianhydride is preferred.

[0148] The diamine that can be used in the manufacture of the above-mentioned polyimide porous membrane can be any diamine. Specific examples of diamines include the following.

[0149] 1) Phenylenediamines with one benzene ring, such as 1,4-diaminobenzene (p-phenylenediamine), 1,3-diaminobenzene, 2,4-diaminotoluene, and 2,6-diaminotoluene;

[0150] 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'-diaminobenzophenone, 3,3'-diamino-4,4'-dichlorobenzophenone, 3,3'-diamino-4,4'-dimethoxybenzophenone, 3,3'-diaminodiphenylmethane, 3,4'-diaminodiphenylmethane Alkane, 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, etc., are diamines with two benzene rings;

[0151] 3) 1,3-bis(3-aminophenyl)benzene, 1,3-bis(4-aminophenyl)benzene, 1,4-bis(3-aminophenyl)benzene, 1,4-bis(4-aminophenyl)benzene, 1,3-bis(4-aminophenoxy)benzene, 1,4-bis(3-aminophenoxy)benzene, 1,4-bis(4-aminophenoxy)benzene, 1,3-bis(3-aminophenoxy)-4-trifluoromethylbenzene, 3,3'-diamino-4-(4-phenyl)phenoxybenzophenone, 3,3'-diamino-4,4'-di(4-phenylphenoxy) Diamines with three benzene rings, including benzophenone, 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.

[0152] 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)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.

[0153] 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.

[0154] Among these, 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)phenyl is especially preferred.

[0155] 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 with a glass transition temperature of 240°C or higher, or a polyimide obtained by combining a tetracarboxylic acid dianhydride with a diamine that does not have a definite transition point above 300°C.

[0156] 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.

[0157] (i) An aromatic polyimide comprising at least one tetracarboxylic acid unit selected from the group consisting of biphenyl tetracarboxylic acid units and pyromellitic acid units, and an aromatic diamine unit;

[0158] (ii) An aromatic polyimide comprising 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)phenyl unit;

[0159] and / or

[0160] (iii) An aromatic polyimide comprising at least one tetracarboxylic acid unit selected from the group consisting of a biphenyltetracarboxylic acid unit and a pyromellitic 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)phenyl unit.

[0161] The polyimide porous membrane used in this invention is preferably a three-layer polyimide porous membrane, comprising a surface layer A and a surface layer B having multiple pores, and a macroporous layer sandwiched between the aforementioned surface layer A and surface layer B. Here, the average pore size of the pores in the aforementioned surface layer A is preferably 0.01 μm or more and 15 μm or less, and the average pore size of the pores in the aforementioned surface layer B is preferably 20 μm or more and 100 μm or less. The aforementioned macroporous layer preferably has a spacer wall bonded to the aforementioned surface layers A and B, and multiple macropores surrounded by the spacer wall and the aforementioned surface layers A and B. The thickness of the spacer wall of the aforementioned macroporous layer, and the aforementioned surface layers A and B, is preferably 0.01 μm or more and 20 μm or less. The pores in the aforementioned surface layers A and B are in communication with the macropores, and the total membrane thickness is preferably 5 μm or more and 500 μm or less. Furthermore, the porosity of the polyimide porous membrane is preferably 40% or more and less than 95%. Here, at least one spacer wall in the macroporous layer may have one or more pores with an average pore diameter of 0.01 μm or more and 100 μm or less, preferably 0.01 μm or more and 50 μm or less, that connect adjacent macropores to each other.

[0162] 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.

[0163] III-2. Polyethersulfone (PES) porous membrane

[0164] The PES porous membranes that can be used in this invention comprise polyethersulfone, and are typically substantially composed of polyethersulfone. Polyethersulfone can be synthesized by methods known to those skilled in the art, such as by polycondensation of a diphenol, an alkali metal compound, and a dihalodiphenyl compound in an organic polar solvent, or by pre-synthesizing an alkali metal disalt of a diphenol and then polycondensing it with a dihalodiphenyl compound in an organic polar solvent.

[0165] Examples of alkali metal compounds include alkali metal carbonates, alkali metal hydroxides, alkali metal hydrides, and alkali metal alkoxides. Sodium carbonate and potassium carbonate are particularly preferred.

[0166] Examples of diphenol compounds include hydroquinone, catechol, resorcinol, 4,4'-biphenol, bis(hydroxyphenyl)alkanes (e.g., 2,2-bis(hydroxyphenyl)propane and 2,2-bis(hydroxyphenyl)methane), dihydroxydiphenyl sulfones, dihydroxydiphenyl ethers, or compounds in which at least one hydrogen atom in the benzene ring is substituted by a lower alkyl group such as methyl, ethyl, or propyl, or a lower alkoxy group such as methoxy or ethoxy. Two or more of the above compounds may be used in combination as diphenol compounds.

[0167] Polyethersulfone can be commercially available. Examples of commercially available products include SUMIKAEXCEL 7600P and SUMIKAEXCEL 5900P (both manufactured by Sumitomo Chemical Co., Ltd.).

[0168] From the viewpoint of forming large pores in a good PES porous membrane, the specific logarithmic viscosity of polyethersulfone is preferably 0.5 or more, more preferably 0.55 or more, and from the viewpoint of ease of manufacturing PES porous membrane, it is preferably 1.0 or less, more preferably 0.9 or less, further preferably 0.8 or less, and particularly preferably 0.75 or less.

[0169] In addition, from the viewpoint of heat resistance and dimensional stability at high temperatures, PES porous membranes or polyethersulfone used as their raw materials preferably have a glass transition temperature of 200°C or higher, or no definite glass transition temperature can be observed.

[0170] The manufacturing method of the PES porous membrane used in this invention is not particularly limited. For example, it can be manufactured by the following method, which includes the following steps:

[0171] The process of casting a polyethersulfone solution containing 0.3% to 60% by mass of polyethersulfone with a specific viscosity of 0.5 to 1.0 and 40% to 99.7% by mass of an organic polar solvent into a thin film, and then impregnating or contacting it with a coagulation solvent in which polyethersulfone is an undesirable solvent or a non-solvent is an essential component, to produce a porous coagulated film; and

[0172] The process of heat-treating the porous solidified membrane obtained in the aforementioned steps to coarsen the pores, thereby obtaining a PES porous membrane, wherein...

[0173] The aforementioned heat treatment includes heating the solidified film having the aforementioned pores to above the glass transition temperature of the aforementioned polyethersulfone or above 240°C.

[0174] The PES porous membrane that can be used in this invention is preferably a PES porous membrane having a surface layer A, a surface layer B, and a macroporous layer sandwiched between the aforementioned surface layer A and the aforementioned surface layer B.

[0175] The aforementioned macroporous layer comprises: a spacer wall bonded to the aforementioned surface layers A and B, and a plurality of macropores with an average pore size of 10 μm to 500 μm in the film plane direction, which are surrounded by the spacer wall and the aforementioned surface layers A and B.

[0176] The thickness of the spacer walls in the macroporous layer is 0.1 μm to 50 μm.

[0177] The thicknesses of the aforementioned surface layers A and B are each between 0.1 μm and 50 μm.

[0178] One of the aforementioned surface layers A and B has multiple pores with an average pore size greater than 5 μm and less than 200 μm, and the other has multiple pores with an average pore size greater than 0.01 μm and less than 200 μm.

[0179] One of surface layer A and surface layer B has a surface aperture ratio of 15% or more, and the other surface layer has a surface aperture ratio of 10% or more.

[0180] The aforementioned micropores in surface layer A and surface layer B are connected to the aforementioned macropores.

[0181] The total thickness of the aforementioned PES porous membrane is 5 μm to 500 μm, and the porosity is 50% to 95%.

[0182] This allows for the manufacture of cryopreservation substrates containing porous membranes. The cryopreservation substrates of the present invention can use porous membranes directly, or they can be fabricated as stacked porous membranes with multiple layers of porous membranes, or the stacked porous membranes can be stacked with other layers such as sieves.

[0183] In addition to cryopreservation, the cryopreservation substrate obtained in this way can also be used as a substrate for cell culture, thus reducing the load on cryopreserved cells.

[0184] Example

[0185] The present invention will now be described in more detail with reference to specific embodiments. It should be noted that 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 protection of the present invention.

[0186] [Experiment 1]

[0187] <Polyimide porous membrane>

[0188] A polyimide porous membrane with an average thickness of 25 μm and a porosity of 73% was prepared. The polyimide porous membrane has two different surface layers (A side and B side) and a macroporous layer sandwiched between the two surface layers. The average pore size of the pores on the A side is 6 μm, and the average pore size of the pores on the B side is 46 μm.

[0189] Modular polymer porous membranes

[0190] Modular polymer porous membranes (hereinafter also referred to as "modules") are obtained by stacking the following polyimide porous membranes (A) and screens (B) alternately in the order ABABA, housing the stack in a polyethylene shell with 16 2mm×2mm culture medium outflow inlets on one side, and then sterilizing it with γ-rays.

[0191] Laminated polyimide porous membrane (A): A membrane formed by stacking six 1.0×1.0cm polyimide porous membranes in the following order: A side / B side / / A side / B side / / A side / B side / / B side / A side / / B side / A side / / B side / A side / / B side / A side / / B side / A side.

[0192] Screen (B): Use a 1.0×1.0cm backing plate (polypropylene / polyethylene) (manufactured by NBC Meshtec, model: ESP10TC).

[0193] 5wt% FBS-Igor medium: A medium prepared by adding FBS to Igor medium at a concentration of 5wt% fetal bovine serum (hereinafter also referred to as "FBS").

[0194] 2wt% FBS-Igor medium: A medium prepared by adding FBS to Igor medium at a concentration of 2wt%.

[0195] 10wt% FBS·10wt% DMSO-Igor medium: A medium prepared by adding FBS and DMSO to Igor medium at a concentration of 10wt% each.

[0196] The evaluation method in this embodiment is as follows.

[0197] Viral titer

[0198] After centrifuging the culture supernatant at 3,000 rpm for 15 minutes at 4°C, the supernatant was recovered and diluted 10 times with 5wt% FBS-Igor medium to obtain the diluted solution.

[0199] Porcine kidney-derived cell lines (1.5 × 10⁻⁶) pre-suspended in 5% FBS-Igor medium were used to prepare the cells. 5 Virus titer was calculated by seeding 0.15 mL of the diluted solution into each well of a 96-well plate (cells / mL) at 0.05 mL / well, and then seeding 4 wells with each dilution. The plates were incubated at 37°C and 5% CO2. On day 7 of incubation, 0.05 mL of the supernatant was transferred from each well to a new 96-well V plate, and 0.05 mL of 0.5 vol% guinea pig erythrocyte suspension was added to each plate. The plates were incubated overnight at 4°C. The presence or absence of erythrocyte agglutination was used as an indicator, and the viral titer was calculated using the Behrens-Karber method.

[0200] <Viral Genome Base Sequence Analysis>

[0201] The culture supernatant stored in an ultra-low temperature freezer was thawed, and viral nucleic acid was extracted using the QIAmp (registered trademark) DNA Mini Kit (manufactured by QIAGEN) according to the accompanying instruction manual. The extracted nucleic acid was used as a template to prepare the PCR reaction solution as shown in Table 1. Primer sequences are shown in Table 2, using combinations of No. 1 (sequence number 1) and No. 2 (sequence number 2), and No. 3 (sequence number 3) and No. 4 (sequence number 4).

[0202] [Table 1]

[0203]

[0204] [Table 2]

[0205]

[0206] PCR reactions were performed using a thermal cycler according to the procedure in Table 3.

[0207] It should be noted that in the table below, the program of 98℃ for 10 seconds to 72℃ for 1 minute refers to performing 98℃ for 10 seconds, 55℃ for 30 seconds, and 72℃ for 1 minute in sequence as a group, and performing this group for a total of 30 cycles.

[0208] [Table 3]

[0209]

[0210] The PCR products were subjected to 2% agarose gel electrophoresis to confirm the detection of bands of the target size shown in Table 2. The PCR products were purified using NucleoSpin (registered trademark) Gel and PCR Clean-up (manufactured by TaKaRa) according to the accompanying operating procedure manual.

[0211] The base sequence was determined from the purified product using direct sequencing, and mutation analysis was performed. The resolution range was set to bases 2131–2868, corresponding to "NS1 gene downstream + intergenic sequence + VP1 / VP2 gene upstream", and bases 3365–4549, corresponding to "VP1 / VP2 gene downstream", in the sequence “ACCESSION: NC_001718, Porcine parvovirus complete genome”.

[0212] (Example 1)

[0213] Cryopreservation of porcine parvovirus-infected porcine kidney-derived strain cells

[0214] (1) Cell culture of porcine kidney-derived strain cells and inoculation with porcine parvovirus

[0215] Porcine kidney-derived cell lines (2.0 × 10⁻⁶) were added to 5% FBS-Igor medium. 4 45 mL of the solution obtained by distilling cells / mL and 5 modules obtained by pre-soaking in PBS and shaking for 24 hours were placed in a 50 mL tube and cultured at 37°C and 5% CO2, while rotating the tube at 5 rpm for 24 hours.

[0216] After 24 hours of rotation culture, the entire contents of the 50mL tube were transferred to a 75cm tube for tissue culture. 2 After further static incubation at 37°C and 5% CO2 for 6 days in the culture flask, the module was washed with PBS.

[0217] The module, washed with PBS, was transferred to a 75 cm tissue culture vessel containing 20 mL of porcine parvovirus solution (hereinafter referred to as "virus solution") prepared at a multiplicity of infection of approximately 0.01. 2 In the culture flask, let it stand at 37°C and 5% CO2 for 60 minutes.

[0218] Then, the module was removed from the virus solution and transferred to a new 75cm tissue culture medium containing 30mL of 2% FBS-Igor medium.2 In the culture flask, static incubation was started at 37°C and 5% CO2.

[0219] On day 7 after virus inoculation, the culture supernatant was aspirated and removed, and 30 mL of 2% FBS-Igor medium was added. The culture was then continued for another 7 days.

[0220] In this way, porcine parvovirus-infected porcine kidney-derived cells were cultured for 14 days after virus infection. (A total of 14 days of culture was conducted after virus inoculation).

[0221] (2) Frozen storage

[0222] The porcine parvovirus-infected porcine kidney-derived cell line obtained in (1) was extracted from the tissue culture without being detached from the module using a 75 cm⁻¹ PCR machine. 2 The culture flask was removed and placed into a cryopreservation tube containing 10 wt% FBS·10 wt% DMSO-Igor medium. The cryopreservation tube was then placed in a Vi-CELL cryopreservation container (manufactured by NIHON FREEZER CO., LTD.), and the Vi-CELL cryopreservation container was placed in an ultra-low temperature freezer set at -80°C for slow freezing.

[0223] After freezing overnight, store in an ultra-low temperature freezer for one month at the set temperature.

[0224] Similarly, the cryopreservation period was changed from 1 month to 3 months, 6 months, 9 months, and 12 months for cryopreservation of porcine parvovirus-infected porcine kidney-derived strains.

[0225] Thus, five strains of porcine parvovirus-infected porcine kidney-derived cells with different cryopreservation times were cryopreserved.

[0226] (See Example 1 for reference)

[0227] Instead of freezing in an ultra-low temperature freezer, the cells were frozen in liquid nitrogen, except that they were cultured / frozen for the same duration as in Example 1.

[0228] (Example 2)

[0229] Culture of porcine parvovirus-infected porcine kidney-derived cell lines after cryopreservation

[0230] (3) Thawing / re-culturing and supernatant recovery

[0231] After the freezing period is over, remove the storage tubes from the ultra-low temperature freezer or liquid nitrogen and thaw them quickly in a 37°C water bath.

[0232] After thawing, remove the module from the preservation tube, wash it in PBS, and then transfer it to a 75cm tissue culture container containing 30mL of Igor medium with 2% FBS. 2 In the culture flask, static incubation was started again at 37°C and 5% CO2.

[0233] Two mL of the culture supernatant was collected on days 3, 7, 10, 14, 21, and 28 (total culture days 17, 21, 24, 28, 35, and 42).

[0234] It should be noted that during this entire period, once a week after the supernatant was recovered, all the remaining culture supernatant was removed, and 30 mL of Igor medium containing 2% FBS was added for culturing.

[0235] The evaluation results of porcine parvovirus infection of porcine kidney-derived cell lines using the recovered culture supernatant are shown in Tables 4–11. Figure 1 and 2 .

[0236] (See Example 2 for reference)

[0237] Except for not freezing, porcine parvovirus-infected porcine kidney-derived cell lines were cultured for 42 days, similar to Example 1. 2 mL of culture supernatant was collected on days 21, 28, 35, and 42 of culture. The evaluation results of porcine parvovirus infection of porcine kidney-derived cell lines using the collected supernatant are shown in Tables 4–11. Figure 1 and 2 .

[0238] (See Example 3 for reference)

[0239] Culture flasks were used to culture porcine parvovirus-infected pig kidney-derived strains.

[0240] (1) Initial vaccination

[0241] 75cm in tissue culture 2 Add 5% FBS-Igor medium to the culture flask and adjust the temperature to 2.0 × 10⁻⁶. 5 20 mL of porcine kidney-derived lineage cells were cultured at 37°C and 5% CO2 with a multiplicity of infection of 0.01 in a porcine parvovirus solution.

[0242] On the second day, the culture supernatant was aspirated and removed, and 30 mL of 2% FBS-Igor medium was added. The culture was then incubated for another 6 days at 37°C and 5% CO2 (a total of 7 days of incubation after inoculation).

[0243] (2) Virus passage and inoculation, supernatant recovery

[0244] 100 μL of the culture supernatant from porcine parvovirus-inoculated porcine kidney-derived strains cultured for 7 days was collected and added to a medium pre-adjusted to 2.0 × 10⁻⁶ ppm using 5% FBS-Igor medium. 5 New porcine kidney-derived cell lines were cultured in 20 mL of fresh cells per mL at 37°C and 5% CO2 for 7 days. This process was repeated every 7 days thereafter, with the cells being inoculated into fresh porcine kidney-derived cell lines.

[0245] Two mL of each culture supernatant was collected and used to evaluate porcine parvovirus infection of porcine kidney-derived cell lines. The evaluation results are shown in Tables 4–11. Figure 1 and 2 .

[0246] [Table 4]

[0247]

[0248] [Table 5]

[0249]

[0250] [Table 6]

[0251]

[0252] [Table 7]

[0253]

[0254] [Table 8]

[0255]

[0256] [Table 9]

[0257]

[0258] [Table 10]

[0259]

[0260] [Table 11]

[0261]

[0262] From Table 4, Figure 1 and 2 It can be seen that, compared with cells that were cryopreserved with liquid nitrogen and cells that were not cryopreserved, cells that were cryopreserved in an ultra-low temperature freezer for 12 months did not show any change in viral titer within 4 weeks, thus maintaining cell function.

[0263] Furthermore, as shown in Tables 5-11, compared with cells that were cryopreserved in liquid nitrogen and cells that were not cryopreserved, cells that were cryopreserved in an ultra-low temperature freezer for 12 months did not show significant differences in viral genome mutations within 4 weeks.

[0264] [Experiment 2]

[0265] The 6-hole plug used in this embodiment uses the following material.

[0266] The insert is fabricated by peeling off the pre-set membrane from a 6-well cell culture insert (Greiner ThinCert (registered trademark), 6-well, product number 657641) and attaching a polyimide porous membrane. The "polyimide porous membrane" used refers to a polyimide porous membrane with a total thickness of 25 μm and a porosity of 73%. This polyimide porous membrane has two distinct surface layers (surface A and surface B) and a macroporous layer sandwiched between these two surface layers. The average pore size of the pores on surface A is 6 μm, and the average pore size of the pores on surface B is 30 μm.

[0267] The 24-hole plug used in this embodiment uses the following material.

[0268] The insert is fabricated by peeling off the pre-set membrane from a 24-well cell culture insert (Greiner ThinCert (registered trademark), 24-well, product number 662640) and attaching a polyimide porous membrane. The "polyimide porous membrane" used refers to a polyimide porous membrane with a total thickness of 25 μm and a porosity of 73%. This polyimide porous membrane has two distinct surface layers (surface A and surface B) and a macroporous layer sandwiched between these two surface layers. The average pore size of the pores on surface A is 6 μm, and the average pore size of the pores on surface B is 30 μm.

[0269] <Cultivation and Cryopreservation of Human Fibroblast Cell Lines>

[0270] <Experimental Materials and Methods>

[0271] (1) Large-scale culture of human fibroblasts

[0272] Add 12 mL of FGM-2 Fibroblast Growth Medium-2 Bullet Kit (trademark, product number: CC-3132, LONZA) to Falcon Dish (trademark, untreated, Corning) to achieve a seeding density of 5,000 cells / cm². 2 Human fibroblasts were inoculated using a method that allowed them to be statically cultured for 7 days at 37°C in the presence of 5% CO2.

[0273] (2) Culture of human fibroblasts using a 6-well cell culture adapter (Comparative Example 1)

[0274] Add 2.5 mL of culture medium to each well of a 6-well Tissue Culture Plate (product number: 353046, FALCON). Place the 6-well cell culture insert into the wells of the 6-well plate and wet it in an incubator at 37°C in the presence of 5% CO2. Add 2 mL of culture medium to each well of the wetted insert after removing it from the incubator. The cell suspension obtained by peeling human fibroblasts cultured using the method described above (1) from Falcon (registered trademark) Dish (trademark, untreated, Corning) was prepared at a cell density of 20,000 cells / cm³. 2 The cells were inoculated into the inside of the insert and cultured statically for 118 days at 37°C in the presence of 5% CO2. The culture medium was changed twice a week, with 0.5–1 mL of medium being recovered each time to obtain the culture supernatant. The cells were cryopreserved on days 9 and 118 without being removed from the insert.

[0275] (3) Culture of human fibroblasts using a 24-well cell culture plug-in (Comparative Example 2)

[0276] Add 0.5 mL of culture medium to each well of a 24-well Tissue Culture Plate (product number: 353047, FALCON). Place the 24-well cell culture insert into the wells of the plate and wet it in an incubator at 37°C in the presence of 5% CO2. Add 0.5 mL of culture medium to the inside of each well of the wetted insert after removing it from the incubator. The cell suspension obtained by peeling human fibroblasts cultured using the method described above from Falcon (registered trademark) Dish (trademark, untreated, Corning) was prepared at a cell density of 20,000 cells / cm³. 2 The cells were inoculated into the inside of the insert and statically cultured for 118 days at 37°C and in the presence of 5% CO2. The culture medium was changed twice a week, with the supernatant obtained by reducing the medium volume to 0.5–1 mL. The cells were cryopreserved on days 9 and 118 without being removed from the insert.

[0277] (4) Freezing of human fibroblasts using a 6-well cell culture plug

[0278] Add 2.5 mL of cell freezing reagent (CELLBANKER1plus, product number: 11912, Nippon Zenya Kogyo) to each well of a 6-well plate (Tissue Culture Plate, product number: 353046, FALCON). The culture medium from the inner and outer sides of the inserts, which had been statically cultured for 9 days and 118 days using the method described in (2) above, was recovered and removed, and then placed in the wells of a 6-well plate pre-filled with cell freezing reagent. Add 2 mL of freezing reagent to each well inside the insert. Wrap the 6-well plate around the perimeter with sealing film (product number: PM-996, Bemis) and place it in a card-headed bag (0.04 mm thick, product number: 4J, MonotaRO). Place the bag in an ultra-low temperature freezer (Mybio, product number: VT-78HC, NIHON FREEZERCO., LTD.) at a set temperature of -80 degrees Celsius for cryopreservation for approximately 9 months and 5 months, respectively.

[0279] (5) Freezing of human fibroblasts using a 24-well cell culture plug

[0280] Add 0.5 mL of cell freezing reagent to each well of a 24-well Tissue Culture Plate (product number: 353047, FALCON). The culture medium on the inside and outside of the inserts, which had been statically cultured for 9 days and 118 days using the method described in (3) above, was recovered and removed. The inserts were then placed in the wells of the 24-well plate pre-filled with cell freezing reagent. Add 0.5 mL of freezing reagent to each well inside the insert. The 24-well plate was then sealed with a sealing film and placed in a card-headed bag (0.04 mm thick, product number: 4J, MonotaRO). The plate was then placed in an ultra-low temperature freezer at a set temperature of -80 degrees Celsius for freezing storage for approximately 9 months and 5 months, respectively.

[0281] Thawing and Reculturing of Human Fibroblast Cell Lines

[0282] (6) Thawing and reculturing of human fibroblasts using a 6-well cell culture plug-in (Example 1)

[0283] Add 2.5 mL of PBS (D-PBS(-), product number: 045-29795, FUJIFILM Wako Chemicals) to each well of a 6-well plate (Tissue Culture Plate, product number: 353046, FALCON). After the cryopreservation period, remove the 6-well plate from the ultra-low temperature freezer (-80°C) and thaw it rapidly in a 37°C water bath. After removing the 6-well plate from the water bath, remove the insert from the thawed cryoprotectant and place it into the wells of the 6-well plate pre-filled with PBS. Add 2 mL of PBS to the inside of the insert. After removing the PBS, add 4.5 mL of PBS.

[0284] Add 2.5 mL of culture medium to each well of a 6-well Tissue Culture Plate (product number: 353046, FALCON). Remove the insert previously soaked in PBS for washing and place it in the 6-well plate containing the culture medium. Add 2 mL of culture medium. Incubate again at 37°C in the presence of 5% CO2. Change the culture medium twice a week, recovering 0.5–1 mL of the degraded medium to obtain the culture supernatant.

[0285] (7) Thawing and reculturing of human fibroblasts using a 24-well cell culture plug-in (Example 2)

[0286] Add 0.5 mL of PBS to each well of a 24-well Tissue Culture Plate (product number: 353047, FALCON). After the cryopreservation period, remove the plate from the ultra-low temperature freezer (-80°C) and thaw it rapidly in a 37°C water bath. After removing the 24-well plate from the water bath, remove the insert from the thawed cryoprotectant and place it into the wells of the 24-well plate pre-filled with PBS. Add 0.5 mL of PBS to the inside of the insert. Remove the PBS and add 1 mL of PBS.

[0287] Add 0.5 mL of culture medium to each well of a 24-well Tissue Culture Plate (product number: 353047, FALCON). Remove the insert previously soaked in PBS for washing and place it in the 24-well plate containing the culture medium. Add 0.5 mL of culture medium to the inside of the insert. Incubate again at 37°C in the presence of 5% CO2. Change the culture medium twice a week, collecting 0.5–1 mL of the recycled medium to obtain the culture supernatant.

[0288] <Evaluation of Culture Supernatant>

[0289] (11) Determination of cell products

[0290] The concentration of fibronectin in the culture supernatants recovered from Examples 1, 2, Comparative Examples 1, and 2 was determined using an ELISA fibronectin assay kit (Fibronectin EIAK it, product number: MK115, manufactured by TakaraBio Inc.). The results are shown in Table 12.

[0291] [Table 12]

[0292]

[0293] As shown in Table 12, compared with the cells before freezing, the cells that were frozen and stored in an ultra-low temperature freezer for 5 or 9 months maintained the concentration of fibronectin in the culture supernatant and maintained the function of fibronectin production.

[0294] Industrial availability

[0295] According to the present invention, long-term cryopreservation is possible without altering the substrate for cryopreservation, thus simplifying cell freezing / thawing operations and enabling long-term maintenance of cell function even without the use of liquid nitrogen. This significantly reduces the costs of long-term preservation and transportation of frozen cells. The present invention makes a particularly significant contribution to the biological field, including regenerative medicine.

[0296] Explanation of reference numerals in the attached figures

[0297] 1. Cell Culture Plug-in

[0298] 2. A cylindrical container with an arm structure on its upper surface.

[0299] 3. A porous membrane disposed on the bottom surface of a cylindrical container

[0300] 4 holes

Claims

1. A method for cryopreservation of cells, characterized in that, The cells are cryopreserved for 30 days or more without using liquid nitrogen in a state where the cells are inoculated to a porous membrane.

2. The method of claim 1, wherein, The cells are animal cells.

3. The method of claim 1, wherein, The porous membrane is a high molecular porous membrane.

4. The method of claim 3, wherein, The average pore diameter of the B face of the high molecular porous membrane is larger than that of the A face.

5. The method of claim 4, wherein, The average pore diameter of the B face is 10 μm or more.

6. The method of claim 4, wherein, The average pore diameter of the A face is 0.01 μm or more.

7. The method of claim 1, wherein, The average pore diameter of the porous membrane is 0.01 μm or more and 500 μm or less.

8. The method of claim 3, wherein, The high molecular porous membrane is a polyimide porous membrane or a polyether sulfone porous membrane.

9. The method of claim 3, wherein, The porous membrane is a porous membrane provided to a cell culture insert.

10. The method of claim 2, wherein, The animal cells are virus production cells or hybridomas.

11. The method of claim 2, wherein, The animal cells are mesenchymal stem cells.

12. The method of any one of claims 1-11, wherein, The temperature of the cryopreservation is -150°C or more.

13. A cell culture method comprising the following steps, (1) a step of inoculating cells to a porous membrane; (2) a step of cryopreserving the cells inoculated to the porous membrane for 30 days or more without using liquid nitrogen; (3) a step of thawing the cells inoculated to the porous membrane; (4) a step of culturing the cells after thawing.

14. The method of claim 13, wherein, The porous membrane is a porous membrane provided to a cell culture insert.

15. A cryopreservation substrate formed of a porous membrane for cryopreserving inoculated cells for 30 days or more.

16. The cryopreservation substrate of claim 15, wherein, The porous membrane is a porous membrane provided to a cell culture insert.

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