Method for cryopreserving cells, method for culturing cells, and cryopreservation substrate

Abstract

The present invention provides a method for cryopreserving cells, the method being characterized in that the cells are cryopreserved for 30 days or longer in a state where the cells are seeded onto a porous membrane without using liquid nitrogen. Also provided is a method for culturing cells, the method including a step for cryopreserving cells seeded onto a porous membrane for 30 days or longer without using liquid nitrogen. Further provided is a cryopreservation substrate which is formed from a porous film and is used for the cryopreservation of seeded cells for 30 days or longer.

Description

Method for cryopreserving cells, cell culture method, and cryopreservation substrate

[0001] The present invention relates to a method for cryopreserving cells, a cell culture method, and a cryopreservation substrate, and in particular to a technique for cryopreserving cells in a state where the cells are seeded on a porous membrane.

[0002] In recent years, there has been a great deal of research using cells, such as cells that produce proteins in vivo, such as vaccines, enzymes, hormones, antibodies, and cytokines, cells that produce viruses, cells used in regenerative medicine, etc. In particular, cell culture substrates, which serve as scaffolds for culturing cells, are known to have various effects on cells, such as changing the ease of differentiation of stem cells depending on their hardness (Non-Patent Document 1), and have attracted much attention.

[0003] Proposed technologies relating to cell culture substrates include, for example, a technology using a nonwoven fabric with a special surface treatment as the substrate (Patent Document 1), a technology using a nanofiber substrate (Patent Document 2), a technology using a polyimide porous membrane as the substrate (Patent Document 3), and a technology using a hollow microfiber substrate (Patent Document 4). All of these technologies suggest that cells seeded on the cell culture substrate can be cryopreserved under cryogenic conditions (approximately −196° C.) using liquid nitrogen.

[0004] However, although the cryogenic conditions using liquid nitrogen disclosed in Patent Documents 1 to 4 have little effect on cells, they require special equipment and liquid nitrogen, which is easily volatile, and therefore are difficult to manage and procure, and furthermore, temperature control is complicated and costly.

[0005] In light of these problems, research is being conducted into techniques for cryopreservation at higher temperatures. For example, techniques for cryopreservation at temperatures around -80°C using ultra-low temperature freezers or dry ice are known. However, temperatures around -80°C have a greater impact on cells than cryogenic conditions using liquid nitrogen, so they are limited to short-term storage or short-distance transportation and are not suitable for long-term storage of cells.

[0006] Therefore, techniques related to the improvement of culture medium composition or cryoprotective solution have been proposed as techniques that enable long-term storage of cells even at relatively high temperatures around −80° C. (Non-patent Document 2, Patent Document 5).

[0007] Japanese Patent Application Laid-Open No. 6-209767 International Publication No. 2014 / 196549 International Publication No. 2016 / 121767 Japanese Patent Application Laid-Open No. 2020-171317 Japanese Patent Application Laid-Open No. 2020-039326

[0008] Cell 2006, 126, 677-689Scientific Reports 2016, 6, 1-13

[0009] The techniques disclosed in Non-Patent Document 2 and Patent Document 5 are performed in a suspension state, even for adhesive cells. This technique of cryopreserving cells in a suspension involves culturing cells in a dish or bottle, detaching them by trypsin treatment, recovering them by centrifugation, and then repeatedly washing and centrifuging them. The cells are then placed in a storage tube or the like, filled with a storage solution, frozen in an ultra-low temperature freezer, and then stored in liquid nitrogen. Not only is this procedure cumbersome, but trypsin treatment and washing also place stress on the cells.

[0010] In addition, when freezing and preserving cells in a suspended state, thawing and re-culturing them requires washing and centrifugation to remove the cell preservation solution and replace it with culture solution, which again places stress on the cells, posing a problem.

[0011] On the other hand, the techniques disclosed in Patent Documents 1 to 4 are focused on the adhesion between cells and cell culture substrates, and only suggest that cells can be cryopreserved even in an extremely low-temperature environment using liquid nitrogen. Therefore, the cryopreservation period is only short, ranging from a few days to 20 days. In other words, there has been no attempt to solve the issues related to the storage temperature and storage period for cryopreserved cells by focusing on cell culture substrates.

[0012] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for cryopreserving cells for a long period of time inexpensively and simply without impairing the function of the cells. Another object is to provide a cell culture method for cells cryopreserved for a long period of time. A further object is to provide a cryopreservation substrate for long-term cryopreservation.

[0013] As a result of extensive research, the present inventors have surprisingly discovered a relationship between cell culture substrates and cryopreserved cells. Specifically, they have discovered that, as an alternative to conventional cryopreservation using liquid nitrogen under extremely low temperature conditions, the use of a porous membrane as a cryopreservation substrate simplifies the freezing and thawing procedures and enables stable cryopreservation of cells for long periods of time even at higher temperatures without the use of liquid nitrogen, thereby completing the present invention. Furthermore, the present inventors have also completed an invention relating to a cell culture method, in which cells can be stably cultured without losing their function, even after long-term cryopreservation, by using a porous membrane as a cryopreservation substrate, and an invention relating to a cryopreservation substrate formed from a porous membrane.

[0014] That is, the present invention provides the following.

[0015] [1] A method for cryopreserving cells, characterized in that cells are cryopreserved for 30 days or more in a state in which the cells are seeded on a porous membrane without using liquid nitrogen.

[0016] [2] The method according to [1], wherein the cells are animal cells.

[0017] [3] The cell cryopreservation method described in [1] or [2], wherein the porous membrane is a polymer porous membrane.

[0018] [4] The method according to any one of [3], wherein the average pore size of the B side of the porous polymer membrane is larger than the average pore size of the A side.

[0019] [5] The method according to either [3] or [4], wherein the average pore size of the surface B is 10 μm or more.

[0020] [6] The method according to any one of [3] to [5], wherein the average pore size of the A-side is 0.01 μm or more. [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.

[0021] [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.

[0022] [9] The method according to any one of [3] to [8], wherein the porous membrane is provided on a cell culture insert.

[0023]

[10] The method according to any one of [2] to [9], wherein the animal cells are virus-producing cells or hybridomas.

[0024]

[11] The method according to any one of [2] to [9], wherein the animal cells are mesenchymal stem cells.

[0025]

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

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

[0026]

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

[0027] (1) Step: Seeding cells on a porous membrane; (2) Step: Freezing and preserving the cells seeded on the porous membrane for 30 days or more without using liquid nitrogen; (3) Step: Thawing the cells seeded on the porous membrane; and (4) Step: Culturing the thawed cells.

[0028]

[14] The method according to

[13] , wherein the porous membrane is a porous membrane provided on a cell culture insert.

[0029]

[15] A cryopreservation substrate formed of a porous membrane, used for cryopreserving seeded cells for 30 days or more.

[16] The cryopreservation substrate according to

[15] , wherein the porous membrane is a porous membrane provided on a cell culture insert.

[0030] According to the present invention, it is now possible to cryopreserve cells for an extremely long period of time without using liquid nitrogen, while maintaining their function without alteration. This makes long-distance transportation possible, both domestically and internationally. Furthermore, since expensive liquid nitrogen and special equipment are not required, costs can be significantly reduced compared to conventional cryogenic conditions. From another perspective, the operation is simplified because the cells can be frozen, stored, and thawed while still attached to the porous membrane. From yet another perspective, cryopreservation and cell culture are possible without exchanging the cell culture substrate and freezing substrate, making it possible to cryopreserve and culture cells simply and inexpensively without placing a burden on the cells.

[0031] Figure 1 is a graph showing the viral titer of cells cryopreserved in an ultra-low temperature freezer. Figure 2 is a graph showing the viral titer of cells cryopreserved using liquid nitrogen. Figure 3 shows the state of inserting a cell culture insert into a well.

[0032] In this specification, terms related to the present invention will be defined, and then each invention will be described in detail.

[0033] First, terms used in the present invention will be explained.

[0034] The term "porous membrane" in the present invention refers to a membrane (film) having numerous small voids inside or on the surface. The material forming this porous membrane is not particularly limited, and may be an inorganic substance, an organic substance, or a composite of these. However, the term "porous membrane" does not include so-called "woven fabric" or "nonwoven fabric" which are mostly made of fibers. On the other hand, the term "porous membrane" includes porous membranes containing fibers as a secondary component (for example, less than 30 wt% of the entire porous membrane). Naturally, a laminate of a porous membrane and a nonwoven fabric, etc., is also included in the present invention, since it uses a porous membrane.

[0035] In the present invention, "without using liquid nitrogen" means that liquid nitrogen is not used as a refrigerant during freezing and / or preservation, and the use of liquid nitrogen in other steps is not included in the concept of "without using liquid nitrogen." In particular, it is preferable not to use liquid nitrogen during any steps other than freezing and / or preservation.

[0036] In the present invention, "seeding" refers to a state in which cells are in contact with a substrate or the act of doing so, and preferably refers to a state in which cells are attached or cultured on a substrate (such as a porous membrane) or the act of doing so.

[0037] The terms "side A" and "side B" used in the present invention refer to one of the two main surfaces of the porous membrane as side A and the other as side B. In other words, "side A" and "side B" are numbers given to the main surfaces for convenience, and do not represent the front or back surfaces, etc.

[0038] Next, the cryopreservation method, cell culture method, and cryopreservation substrate of the present invention will be described in this order.

[0039] I. Cryopreservation Method The cryopreservation method of the present invention involves cryopreserving cells seeded on a porous membrane for 30 days or more without using liquid nitrogen. For convenience of explanation, the present specification will be described in the order of cells, porous membrane, and cryopreservation method.

[0040] 1. Cells The cells applicable to the present invention are not particularly limited. This is because the method of the present invention places a low burden on cells. Examples include animal cells, insect cells, plant cells, yeast cells, and bacteria.

[0041] Animal cells are broadly divided into cells derived from animals belonging to the phylum Vertebrate and cells derived from invertebrates (animals other than those belonging to the phylum Vertebrate), but in this specification, the origin of the animal cells is not particularly limited.

[0042] For example, the phylum Vertebrate includes the classes Agnathostomata and Gnathostomata, and examples of the Gnathostomata include the classes Mammalia, Aves, Amphibia, and Reptilia. Preferred examples of mammals include mice, rats, humans, monkeys, pigs, dogs, sheep, and goats.

[0043] The origin of the plant cells in this specification is not particularly limited, and includes, for example, cells of plants including mosses, ferns, and spermatophytes.

[0044] Plants from which seed plant cells are derived include both monocotyledonous and dicotyledonous plants. Examples of monocotyledonous plants include, but are not limited to, plants of the Orchidaceae family, grasses (rice, corn, barley, wheat, sorghum, etc.), and Cyperaceae family. Examples of dicotyledonous plants include plants belonging to many subclasses such as the Asteraceae, Magnolias, and Rosaceae.

[0045] Algae can be considered as cellular organisms, and include different groups, from the true bacteria, cyanobacteria (blue-green algae), to the eukaryotic, unicellular organisms (diatoms, xanthophytes, dinoflagellates, etc.) and the multicellular marine algae (red algae, brown algae, green algae).

[0046] The types of archaea and bacteria used herein are not particularly limited. Archaea are classified into the group consisting of methanogens, extreme halophiles, thermoacidophiles, and hyperthermophiles. Bacteria can be selected from the group consisting of lactic acid bacteria, Escherichia coli, Bacillus subtilis, and cyanobacteria.

[0047] As described above, the types of animal or plant cells that can be used in the methods of the present invention are not limited, but are preferably selected from the group consisting of hybridomas, pluripotent stem cells, tissue stem cells, somatic cells, and germ cells.

[0048] As used herein, the term "hybridoma" refers to antibody-producing cells obtained by fusing myeloma cells with B cells obtained by immunizing a non-human animal with an antigen. The organisms from which the B cells and myeloma cells are derived are not particularly limited, and include mice (including nude mice), rats, guinea pigs, rabbits, goats, sheep, chickens, etc.

[0049] As used herein, the term "pluripotent stem cells" refers collectively to stem cells that have the ability to differentiate into cells of any tissue (pluripotency). Pluripotent stem cells include, but are not limited to, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ stem cells (EG cells), and germ stem cells (GS cells). Preferred examples include ES cells and iPS cells. iPS cells are particularly preferred for reasons such as the absence of ethical issues. Any known pluripotent stem cells can be used, but for example, the pluripotent stem cells described in International Publication No. 2009 / 123349 (PCT / JP2009 / 057041) can be used.

[0050] As used herein, "tissue stem cells" refer to stem cells whose differentiable cell lineages are limited to specific tissues but which have the ability to differentiate into a variety of cell types (pluripotency). For example, hematopoietic stem cells in bone marrow become the source of blood cells, and neural stem cells differentiate into neurons. There are also various other types, such as hepatic stem cells that form the liver and skin stem cells that form skin tissue. Preferably, tissue stem cells can be selected from mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, neural stem cells, skin stem cells, or hematopoietic stem cells.

[0051] As used herein, the term "somatic cells" refers to cells other than germ cells that constitute a multicellular organism. Preferably, the somatic cells can 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, erythrocytes, leukocytes, monocytes, macrophages, and megakaryocytes.

[0052] As used herein, the term "germ cells" refers to cells that transmit genetic information to the next generation during reproduction. Examples of such cells include gametes for sexual reproduction, i.e., eggs, egg cells, sperm, and sperm cells, and spores for asexual reproduction.

[0053] The cells may also be selected from the group consisting of sarcoma cells, cell lines and transformed cells.

[0054] As used herein, "sarcoma" refers to cancer that develops in connective tissue cells derived from non-epithelial cells such as bone, cartilage, fat, muscle, and blood, and includes soft tissue sarcoma, malignant bone tumor, etc. Sarcoma cells are cells derived from sarcoma.

[0055] As used herein, the term "cell line" refers to cultured cells that have been maintained in vitro for a long period of time, have acquired certain stable properties, and can be subcultured semi-permanently. Cell lines derived from various tissues of various species, including humans, exist, 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 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 hepatoma), and porcine kidney cell lines (CPK), and these can be used. Virus-producing cells, such as cell lines infected with various viruses (virus-producing cell lines) and cell lines transfected with vectors that produce various viruses (virus-producing vectors), can also be cryopreserved for long periods of time.

[0056] As used herein, the term "transformed cells" refers to cells whose genetic properties have been altered by introducing nucleic acid (e.g., DNA) from outside the cell. Suitable methods for the transformation of animal cells, plant cells, and bacteria are known, and these methods can also be used for transformed cells.

[0057] The method of the present invention enables the long-term cryopreservation of any cell type. Among these, hybridomas, tissue stem cells, and established cell lines are preferably selected, and hybridomas, mesenchymal stem cells, established cell lines, and virus-producing cells are particularly preferred. Furthermore, in one aspect, the method of the present invention enables the long-term cryopreservation of a single layer of cells or tissue, or a multi-layer (e.g., 2 to 5, 2 to 4, or 2 to 3) cell layer or tissue. As used herein, the term "tissue" refers to a structure formed by the accumulation of multiple cells, such as skin tissue (e.g., skin tissue associated with hair roots), cardiac muscle tissue, skeletal muscle tissue, smooth muscle tissue, liver tissue, kidney tissue, gastrointestinal tissue, ocular tissue (e.g., corneal tissue), brain tissue, thymus tissue, testis tissue, pancreatic tissue, thyroid tissue, mammary gland tissue, salivary gland tissue, and lung tissue. The term "tissue" may be a sheet-like tissue (e.g., a cell sheet) formed by culturing cells derived from such tissue, or a sheet-like tissue isolated from a living tissue.

[0058] 2. Porous Membrane The porous membrane of the present invention primarily functions as a substrate for seeding cells during cryopreservation. This porous membrane refers to a membrane (film) with numerous small voids inside or on the surface. The material forming this porous membrane is not particularly limited, and may be an inorganic substance such as ceramics, an organic substance such as a polymer porous membrane, or a composite of these. Below, as one embodiment, a polymer porous membrane formed from a polymer will be described in detail.

[0059] 2-1. Polymer porous membrane In this specification, a "polymer porous membrane" has a surface layer A (hereinafter sometimes referred to as "side A" or "mesh side") and a surface layer B (hereinafter sometimes referred to as "side B" or "large hole side"). The pore shapes on sides A and B may be the same or different. Furthermore, the pore sizes (average pore sizes) on sides A and B are not particularly limited, but it is preferable that the average pore size of the pores present on side B is larger than the average pore size of the pores present on side A.

[0060] The average pore diameter of the pores present on side A (hereinafter sometimes referred to as "average pore diameter") is not particularly limited, but is, for example, preferably 0.01 μm or more, more preferably 0.05 μm or more, even more preferably 0.1 μm or more, and particularly preferably 0.5 μm or more. The upper limit is preferably less than 200 μm, more preferably 150 μm or less, even more preferably 100 μm or less, and particularly preferably 50 μm or less, 40 μm or less, 30 μm or less, 20 μm, or 15 μm or less.

[0061] The average pore size of the pores present on surface B is preferably larger than the average pore size of the pores present on surface layer A. For example, it is preferably greater than 5 μm, more preferably 20 μm or more, even more preferably 30 μm or more, particularly preferably 50 μm or more, or 60 μm or more. The upper limit is preferably 200 μm or less, more preferably 100 μm or less.

[0062] The average pore size of the pores present in the surface layer of the polymer porous membrane can be determined by measuring the pore areas of 200 or more open pores in a scanning electron microscope photograph of the porous membrane surface, and calculating the average diameter of the pores, assuming that the pores are perfectly circular, from the average pore area according to the following formula (1):

[0063]

[0064] (In the formula, Sa means the average value of the pore area.)

[0065] The thickness of surface layer A and surface layer B may be the same or different and is not particularly limited. For example, the thickness is preferably 0.01 μm or more, and the upper limit is preferably 50 μm or less, and more preferably 20 μm or less.

[0066] The polymer porous membrane used in the present invention may have an intermediate layer between the A surface and the B surface. This intermediate layer may be a solid layer without pores, or a porous layer with pores. If the intermediate layer has pores, it may be the same as or different from the surface layer of the polymer porous membrane. Preferably, the intermediate layer is a porous layer, and this porous layer forms a macrovoid layer.

[0067] A macrovoid layer in which the intermediate layer is a porous layer will be described as an example. In this specification, "macrobioid layer" refers to a film having an average pore size of 10 μm or more in the film plane direction. The average pore size of the macrovoids in the macrovoid layer in the film plane direction is not particularly limited, but for example, the upper limit of the 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 partition walls (frames forming the pores in the macrovoid layer) in the macrovoid layer is not particularly limited, but for example, it is preferably 0.01 μm or more, with the upper limit being preferably 50 μm or less, more preferably 20 μm or less.

[0068] In one embodiment, at least one partition wall in the macrovoid layer may have pores that connect adjacent macrovoids. The average pore size of these connecting pores is preferably 0.01 or more, with the upper limit being preferably 100 μm or less, more preferably 50 μm or less. The macrovoid layer may have one or more connecting pores. In another embodiment, the partition wall in the macrovoid layer may not have any pores.

[0069] The total thickness of the polymeric porous membrane (total thickness of the A and B sides and, if present, the intermediate layer) is not particularly limited, but may be 5 μm or more, 10 μm or more, 20 μm or more, or 25 μm or more, or 500 μm or less, 300 μm or less, 100 μm or less, 75 μm or less, or 50 μm or less. It is preferably 5 to 500 μm, and more preferably 10 to 100 μm.

[0070] In this specification, the thickness of the porous polymer membrane can be measured using a contact type thickness meter.

[0071] In this specification, the porosity of the polymer porous membrane is not particularly limited, but is, for example, preferably 40% or more, more preferably 50% or more, and even more preferably 60% or more, and the upper limit thereof is preferably less than 95%, more preferably 90% or less, and even more preferably 85% or less.

[0072] In this specification, the porosity of a porous polymer membrane can be determined by measuring the thickness and mass of a porous polymer membrane cut to a predetermined size and calculating the porosity from the basis weight according to the following formula (2).

[0073]

[0074] (In the formula, S is the area of ​​the polymer porous membrane, d is the total membrane thickness, w is the measured mass, and D is the density of the polymer. When the polymer is polyimide, the density is 1.34 g / cm ３ (Assuming that.)

[0075] In this specification, the polymer porous membrane is preferably a three-layer structure having surface layers A and B each having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B. The average pore size of the pores in the 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 surface layer B is preferably 20 μm or more and 100 μm or less. The macrovoid layer has partition walls bonded to the surface layers A and B and a plurality of macrovoids surrounded by the partition walls and the surface layers A and B. The thicknesses of the partition walls of the macrovoid layer and the surface layers A and B are preferably 0.01 μm or more and 20 μm or less. The pores in the surface layers A and B are connected to the macrovoids, and the total 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 partition wall in the macrovoid layer has one or more pores that connect adjacent macrovoids and have an average pore size of 0.01 to 100 μm, preferably 0.01 to 50 μm. In another embodiment, the partition wall does not have such pores.

[0076] In this specification, the shape of the polymeric porous membrane is not particularly limited, and the size can be adjusted to match the shape of the culture vessel or cryopreservation vessel to be used, for example.

[0077] In this specification, the polymer porous membrane can be used in a state where it is placed in a culture dish or the like in a state where it is a single membrane, but it may also be used in a state where a part of the membrane is fixed to a resin or metal, or in a state where it is housed in an appropriate casing such as that described in WO2018 / 021368.

[0078] In this specification, the polymer porous membrane is preferably sterilized. The sterilization treatment is not particularly limited, and any sterilization treatment can be selected from dry heat sterilization, ethylene oxide gas sterilization, high-pressure steam sterilization, sterilization with a disinfectant such as ethanol, and sterilization by electromagnetic waves such as ultraviolet rays and gamma rays and / or radiation. It is also possible to select a plurality of these sterilization methods and combine them as appropriate.

[0079] In this specification, the polymer porous membrane preferably has the structural characteristics described above, and particularly preferably, a polyimide porous membrane formed from a polyimide porous material or a polyethersulfone porous membrane formed from polyethersulfone (PES) can be used.

[0080] By using the porous membrane as described above as a substrate for cryopreservation, the effects of the cryopreservation method of the present invention can be exerted.

[0081] 3. Cell Culture Insert In one aspect of the present invention, a cell culture insert comprising a cylindrical container and the above-described porous membrane disposed at the bottom of the cylindrical container can be used as a cryopreservation substrate. Hereinafter, this cell culture insert will also be referred to as the "cell culture insert of the present invention." In this specification, the term "cell culture insert" can also be simply referred to as "insert," and even if interchangeable, they mean the same thing.

[0082] As used herein, the term "cell culture insert" refers to a cell culture insert commonly used for cell culture, inserted into each well of a dish or multi-well plate. A typical insert is a cylindrical container sized to fit into a dish or well, with a bottom and sides to ensure a certain volume for cell culture. According to the present invention, a cell culture insert is provided by placing the above-described porous membrane on the bottom of a cylindrical container. The cylindrical container has a space for cell culture inside the cell culture insert and is sized to fit into a typical dish or well. The top surface of the cylindrical container preferably has an arm structure that can be hung on the top surface of the dish or well to avoid contact with the bottom of the dish or well. The cylindrical container is not limited as long as it has the above-described shape, and any commonly used, general-purpose, or commercially available container can be used. A cell culture insert is provided by adhering a porous membrane to the bottom of the cylindrical container. Alternatively, a commonly used, general-purpose, or commercially available insert with a membrane may be used, with the porous membrane applied in accordance with the present invention replaced. The porous membrane used in the present invention can be easily molded to fit the size and shape of the bottom of the insert to be used. Although not particularly limited, a schematic diagram of a cell culture insert inserted into a well is shown in Figure 3.

[0083] The bottom of the insert generally has a circular or elliptical shape, and if circular, it is equal to or smaller than the diameter of the upper opening of the dish or well. Therefore, the diameter of the porous membrane placed at the bottom is appropriately adjusted to be equal to or smaller than the opening diameter of the dish (e.g., nominal diameter 35 mm, nominal diameter 60 mm, nominal diameter 100 mm, nominal diameter 150 mm, etc.) or well (e.g., 6-well, 12-well, 24-well, 96-well, etc.) into which the cell culture insert is inserted. The nominal diameter of the dish is a general value; for a nominal diameter of 100 mm, the actual diameter of the dish is approximately 80 mm, and does not represent an exact diameter. For example, the diameter of the porous membrane is typically 3 to 130 mm, which is the size that fits into the dish or well.

[0084] The insert used in the present invention is preferably sterilized. The sterilization method is not particularly limited, and any sterilization method can be selected from dry heat sterilization, ethylene oxide gas sterilization, high-pressure steam sterilization, sterilization with a disinfectant such as ethanol, and sterilization with electromagnetic waves such as ultraviolet rays and gamma rays and / or radiation. It is also possible to select and combine multiple sterilization methods from these methods as appropriate.

[0085] 4. Cryopreservation Method The cryopreservation method of the present invention can be carried out in a manner similar to conventional methods, except that it uses a porous membrane (or a cell culture insert provided with a porous membrane) with cells still attached and supported thereon, and does not require cell detachment or centrifugation. For example, the porous membrane (or cell culture insert provided with a porous membrane) with cells still attached and supported thereon can be transferred from the culture medium into a cryopreservation container containing a cryoprotectant, slowly frozen in an ultra-low temperature freezer (e.g., a deep freezer at approximately -80°C), and then cryopreserved at the desired temperature. Alternatively, the culture medium can be removed from the container, a cryoprotectant solution added, slowly frozen in an ultra-low temperature freezer, and then cryopreserved at the desired temperature. Furthermore, washing with, for example, PBS, may be performed as appropriate during these procedures. In one embodiment, when a cell culture insert carrying cells or tissues is cryopreserved, a cryoprotectant solution may be placed only on the inside or on both the inside and outside of the cell culture insert, and the insert may be frozen in an ultra-low temperature freezer (e.g., a deep freezer at approximately −80° C.), and then further cryopreserved at a desired temperature.

[0086] As used herein, "under higher temperature conditions without using liquid nitrogen" refers to temperature conditions that are higher than those under liquid phase storage or vapor phase storage using liquid nitrogen, and the lower limit is -150°C or higher, preferably -120°C or higher, and more preferably -90°C or higher.

[0087] To achieve such a temperature, for example, the temperature setting of the ultra-low temperature freezer may be set to -150°C or higher, -120°C or higher, -100°C or higher, or -80°C.

[0088] Under "higher temperature conditions without using liquid nitrogen," the upper limit may be, for example, -10°C or lower, -20°C or lower, -30°C or lower, -40°C or lower, -50°C or lower, -60°C or lower, or -70°C or lower.

[0089] In one embodiment of the present invention, the "higher temperature condition without using liquid nitrogen" may be a range obtained by combining any of the above lower and upper limits, for example, -150°C to -10°C, -150°C to -20°C, -150°C to -30°C, -150°C to -40°C, -150°C to -50°C, -150°C to -60°C, or -150°C to -70°C. Alternatively, the temperature may be, for example, -120°C to -10°C, -120°C to -20°C, -120°C to -30°C, -120°C to -40°C, -120°C to -50°C, -120°C to -60°C, or -120°C to -70°C. Alternatively, the temperature may be, for example, −100° C. to −10° C., −100° C. to −20° C., −100° C. to −30° C., −100° C. to −40° C., −100° C. to −50° C., −100° C. to −60° C., or −100° C. to −70° C. Alternatively, the temperature may be, for example, −90° C. to −10° C., −90° C. to −20° C., −90° C. to −30° C., −90° C. to −40° C., −90° C. to −50° C., −90° C. to −60° C., or −90° C. to −70° C.

[0090] As used herein, a "long-term" period for cryopreservation of cells refers to 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). Furthermore, as used herein, a "long-term" period for cryopreservation of cells may refer to 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).

[0091] As described above, the cryopreservation method of the present invention does not use liquid nitrogen, and therefore is extremely simple to perform, reduces the burden on the cells being cryopreserved, and is advantageous in that it can be applied to all types of cryopreservation methods. In particular, although a special protective solution such as that described in Non-Patent Document 2 or Patent Document 5 may be used, the method has the advantage of being able to cryopreservate cells without using a protective solution with a special composition, for example, by using a commercially available cryoprotective solution (cell freezing solution) to maintain cell function and prevent cell deterioration.

[0092] II. Cell Culture Method The cell culture method of the present invention is a method comprising the following steps:

[0093] (1) Step: Seeding cells on a porous membrane; (2) Step: Freezing and preserving the cells seeded on the porous membrane for 30 days or more without using liquid nitrogen; (3) Step: Thawing the cells seeded on the porous membrane; and (4) Step: Culturing the thawed cells.

[0094] Step (1) is a step of seeding cells onto a porous membrane before cryopreservation. Preferably, the porous membrane can be used as a substrate to culture the cells to be cryopreserved. Here, the cells may simply be attached to the porous membrane, or the cells may be cultured. This can be selected appropriately depending on the type and amount of cells to be cryopreserved.

[0095] Known cell culture methods can be used, and the culture conditions can be appropriately determined depending on the type of cells, etc. Culture methods suitable for animal cells, plant cells, and bacterial cells are known, and those skilled in the art can culture cells on a polymeric porous membrane using any known method. The cell culture medium can also be appropriately prepared depending on the type of cells.

[0096] Methods for culturing animal cells and cell culture media are described, for example, in the Lonza Cell Culture Media Catalog. Methods for culturing plant cells and cell culture media are described, for example, in the WAKO Plant Tissue Culture Medium Series. Methods for culturing bacterial cells and cell culture media are described, for example, in the BD General Bacteria Media Catalog. The cell culture medium used in the method of the present invention may be in any form, such as a liquid medium, a semi-solid medium, or a solid medium. Alternatively, droplets of liquid medium may be sprayed into a cell culture vessel so that the medium comes into contact with the polymeric porous membrane carrying the cells.

[0097] Step (2) is the process of cryopreserving cells seeded on a porous membrane, and details are as described in the cryopreservation method. To freeze cells, so-called slow cooling can be employed. Slow cooling is preferred because it freezes extracellular water before ice forms inside the cells. While the techniques described in Non-Patent Document 2 or Patent Document 5 may be used in this step, the present invention is advantageous in that it maintains cell function and allows cryopreservation without alteration, even without using these techniques.

[0098] Step (3) is a step of thawing the cryopreserved cells, which can be carried out in a manner similar to conventional methods, except that washing procedures using a centrifuge are not required.

[0099] Step (4) is a step of culturing the thawed cells (hereinafter also referred to as "re-culturing"). This step can be carried out in a manner similar to conventional methods, except that washing using a centrifuge or cell seeding is not required. For example, without particular limitation, after thawing the cryoprotectant, the polymer porous membrane with the cells attached and supported thereon can be moved from the cryoprotectant to a culture medium, and culturing can be resumed. Conversely, the cryoprotectant can be removed after thawing, and the culture medium can be introduced to resume culturing. Furthermore, washing with, for example, PBS, can be carried out as appropriate during these operations.

[0100] III. Cryopreservation substrate The cryopreservation substrate of the present invention is significant in that it uses a porous membrane as the substrate. Details of the porous membrane, particularly the polymer porous membrane, are as described above. Here, polyimide porous membranes and polyethersulfone porous membranes are used as examples of polymer porous membranes, and their manufacturing methods are described in detail.

[0101] III-1. Polyimide Porous Film In this specification, "polyimide" is a general term for polymers containing imide bonds in repeating units, preferably those containing 50 mol% or more of all repeating units, and usually refers to aromatic polyimides in which aromatic compounds are directly linked by imide bonds. Aromatic polyimides have a rigid and strong molecular structure because aromatics have a conjugated structure via imide bonds, and the imide bonds have strong intermolecular forces, resulting in extremely high levels of thermal, mechanical, and chemical properties.

[0102] The polyimide porous film that can be used in the present invention is preferably a polyimide porous film containing, as a main component, a polyimide obtained from a tetracarboxylic dianhydride and a diamine, more preferably a polyimide porous film consisting of a polyimide obtained from a tetracarboxylic dianhydride and a diamine. "Containing as a main component" means that the polyimide porous film does not essentially contain any components other than the polyimide obtained from a tetracarboxylic dianhydride and a diamine as constituent components, or may contain additional components that do not affect the properties of the polyimide obtained from a tetracarboxylic dianhydride and a diamine.

[0103] Polyamic acid is obtained by polymerizing a tetracarboxylic acid component and a diamine component. Polyamic acid is a precursor for forming polyimide, which can be ring-closed by thermal or chemical imidization to form polyimide.

[0104] The polyamic acid may be partially imidized as long as the imidization does not affect the present invention. That is, the polyamic acid may be partially thermally or chemically imidized.

[0105] When polyamic acid is thermally imidized, additives such as an imidization catalyst, an organic phosphorus-containing compound, or fine particles such as inorganic or organic fine particles can be added to a solution in which polyamic acid is dissolved (hereinafter also referred to as a "polyamic acid solution") as needed. When polyamic acid is chemically imidized, additives such as a chemical imidization agent, a dehydrating agent, or fine particles such as inorganic or organic fine particles can be added to the polyamic acid solution as needed.

[0106] In one embodiment, the polyimide porous film that can be used in the present invention also includes a colored polyimide porous film obtained by molding a polyamic acid solution composition containing a polyamic acid solution obtained from a tetracarboxylic acid component and a diamine component and a colored precursor, followed by heat treatment at 250°C or higher.

[0107] In this specification, the term "colored precursor" refers to a precursor that is partially or completely carbonized by heat treatment at 250°C or higher to produce a colored product.

[0108] The colored precursor that can be used in the production of the polyimide porous film is preferably one that is uniformly dissolved or dispersed in a polyamic acid solution or a polyimide solution, and is thermally decomposed by heat treatment at 250°C or higher, preferably 260°C or higher, more preferably 280°C or higher, and more preferably 300°C or higher, preferably in the presence of oxygen such as air, at 250°C or higher, preferably 260°C or higher, more preferably 280°C or higher, and more preferably 300°C or higher, and carbonized to produce a colored product, more preferably one that produces a black colored product, and more preferably a carbon-based colored precursor.

[0109] When heated, the colored precursors become what at first glance appear to be carbonized products, but structurally they contain elements other than carbon, and include layered structures, aromatic cross-linked structures, and disordered structures including tetrahedral carbon.

[0110] The carbonaceous color precursor is not particularly limited, and examples thereof include tar or pitch such as petroleum tar, petroleum pitch, coal tar, and coal pitch, coke, a polymer obtained from a monomer containing acrylonitrile, a ferrocene compound (ferrocene and ferrocene derivatives), etc. Among these, a polymer and / or a ferrocene compound obtained from a monomer containing acrylonitrile is preferred, and polyacrylonitrile is preferred as a polymer obtained from a monomer containing acrylonitrile.

[0111] When a color precursor is used, the above-mentioned additives may be used. In this case, it is preferable to carry out the process under conditions in which the color precursor does not precipitate even when the additives are added to the polyamic acid solution.

[0112] In another embodiment, the polyimide porous film that can be used in the present invention also includes a polyimide porous film that can be obtained by molding a polyamic acid solution obtained from a tetracarboxylic acid component and a diamine component, followed by heat treatment, without using the above-mentioned colored precursor.

[0113] The polyimide porous film, whether or not a colored precursor is used, may be produced by, for example, casting a polyamic acid solution comprising 3 to 60% by weight of a polyamic acid having an intrinsic viscosity of 1.0 to 3.0 and 40 to 97% by weight of an organic polar solvent into a film, immersing or contacting the film with a coagulation solvent containing water as an essential component to produce a porous polyamic acid film, and then heat-treating the polyamic acid porous film to imidize it. In this method, the coagulation solvent containing water as an essential component may be water or a mixture of 5% to less than 100% by weight of water and more than 0% to 95% by weight of an organic polar solvent. Furthermore, after the imidization, at least one surface of the resulting porous polyimide film may be subjected to a plasma treatment.

[0114] The tetracarboxylic dianhydride that can be used in the production of the polyimide porous film may be any tetracarboxylic dianhydride, and can be appropriately selected depending on the desired properties, etc. Specific examples of tetracarboxylic dianhydrides include pyromellitic dianhydride, biphenyltetracarboxylic dianhydrides such as 3,3',4,4'-biphenyltetracarboxylic dianhydride (s-BPDA) and 2,3,3',4'-biphenyltetracarboxylic dianhydride (a-BPDA), oxydiphthalic dianhydride, diphenylsulfone-3,4,3',4'-tetracarboxylic dianhydride, bis(3,4-dicarboxyphenyl)sulfide dianhydride, 2,2-bis(3,4-dicarboxyphenyl)-1,1,1,3,3,3-hexafluoropropane dianhydride, 2,3,3',4'-benzophenonetetracarboxylic dianhydride, 3,3',4,4'-benzophenonetetracarboxylic dianhydride, bis(3,4-dicarboxyphenyl)methane dianhydride, and 2,2-bis(3,4-dicarboxyphenyl)propane. dianhydride, p-phenylene bis(trimellitic acid monoester acid anhydride), p-biphenylene bis(trimellitic acid monoester acid anhydride), m-terphenyl-3,4,3',4'-tetracarboxylic acid dianhydride, p-terphenyl-3,4,3',4'-tetracarboxylic acid dianhydride, 1,3-bis(3,4-dicarboxyphenoxy)benzene dianhydride, 1,4-bis(3,4-dicarboxyphenoxy)benzene dianhydride, 1,4-bis(3,4-dicarboxyphenoxy)biphenyl dianhydride, 2,2-bis[(3,4-dicarboxyphenoxy)phenyl]propane dianhydride, 2,3,6,7-naphthalenetetracarboxylic acid dianhydride, 1,4,5,8-naphthalenetetracarboxylic acid dianhydride, 4,4'-(2,2-hexafluoroisopropylidene)diphthalic acid dianhydride, and the like. It is also preferable to use aromatic tetracarboxylic acids such as 2,3,3',4'-diphenylsulfonetetracarboxylic acid, which may be used alone or in combination of two or more.

[0115] Among these, at least one aromatic tetracarboxylic dianhydride selected from the group consisting of biphenyltetracarboxylic dianhydride and pyromellitic dianhydride is particularly preferred. As the biphenyltetracarboxylic dianhydride, those containing 3,3',4,4'-biphenyltetracarboxylic dianhydride can be suitably used.

[0116] Any diamine can be used in the production of the porous polyimide film. Specific examples of the diamine include the following:

[0117] 1) Benzenediamines with one benzene nucleus, such as 1,4-diaminobenzene (paraphenylenediamine), 1,3-diaminobenzene, 2,4-diaminotoluene, and 2,6-diaminotoluene; 2) Diaminodiphenyl ethers such as 4,4'-diaminodiphenyl ether and 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenylmethane, 3,3'-dimethyl-4,4'-diaminobiphenyl, 2,2'-dimethyl-4,4'-diaminobiphenyl, 2,2'-bis(trifluoromethyl)-4,4'-diaminobiphenyl, 3,3'-dimethyl-4,4'-diaminodiphenylmethane, 3,3'-dicarboxy-4,4'-diaminodiphenylmethane, 3 , 3',5,5'-tetramethyl-4,4'-diaminodiphenylmethane, bis(4-aminophenyl)sulfide, 4,4'-diaminobenzanilide, 3,3'-dichlorobenzidine, 3,3'-dimethylbenzidine, 2,2'-dimethylbenzidine, 3,3'-dimethoxybenzidine, 2,2'-dimethoxybenzidine, 3,3'-diaminodiphenyl ether, 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenyl ether, 3,3'-diaminodiphenyl Sulfide, 3,4'-diaminodiphenyl sulfide, 4,4'-diaminodiphenyl sulfide, 3,3'-diaminodiphenyl sulfone, 3,4'-diaminodiphenyl sulfone, 4,4'-diaminodiphenyl sulfone, 3,3'-diaminobenzophenone, 3,3'-diamino-4,4'-dichlorobenzophenone, 3,3'-diamino-4,4'-dimethoxybenzophenone, 3,3'-diaminodiphenylmethane, 3,4'-diaminodiphenylmethane, 4,4'-di diamines having two benzene nuclei such as aminodiphenylmethane, 2,2-bis(3-aminophenyl)propane, 2,2-bis(4-aminophenyl)propane, 2,2-bis(3-aminophenyl)-1,1,1,3,3,3-hexafluoropropane, 2,2-bis(4-aminophenyl)-1,1,1,3,3,3-hexafluoropropane, 3,3'-diaminodiphenyl sulfoxide, 3,4'-diaminodiphenyl sulfoxide, and 4,4'-diaminodiphenyl sulfoxide;3) 1,3-bis(3-aminophenyl)benzene, 1,3-bis(4-aminophenyl)benzene, 1,4-bis(3-aminophenyl)benzene, 1,4-bis(4-aminophenyl)benzene, 1,3-bis(4-aminophenoxy)benzene, 1,4-bis(3-aminophenoxy)benzene, 1,4-bis(4-aminophenoxy)benzene, 1,3-bis(3-aminophenoxy)-4-trifluoromethylbenzene, 3,3'-diamino-4-(4-phenyl)phenoxybenzophenone, 3,3'-diamino-4,4'-di(4-phenylphenoxy)benzophenone, 1,3 -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, and other diamines having three benzene nuclei;4) 3,3'-bis(3-aminophenoxy)biphenyl, 3,3'-bis(4-aminophenoxy)biphenyl, 4,4'-bis(3-aminophenoxy)biphenyl, 4,4'-bis(4-aminophenoxy)biphenyl, bis[3-(3-aminophenoxy)phenyl]ether, bis[3-(4-aminophenoxy)phenyl]ether, bis[4-(3-aminophenoxy)phenyl]ether, bis[4-(4-aminophenoxy)phenyl]ether, bis[3-(3-aminophenoxy)phenyl]ketone, bis[3-(4-aminophenoxy)phenyl]ketone bis[4-(3-aminophenoxy)phenyl]ketone, bis[4-(4-aminophenoxy)phenyl]ketone, bis[4-(4-aminophenoxy)phenyl]ketone, bis[3-(3-aminophenoxy)phenyl]sulfide, bis[3-(4-aminophenoxy)phenyl]sulfide, bis[4-(3-aminophenoxy)phenyl]sulfide, bis[4-(4-aminophenoxy)phenyl]sulfide, bis[3-(3-aminophenoxy)phenyl]sulfone, bis[3-(4-aminophenoxy)phenyl]sulfone, bis[4-(3-aminophenoxy)phenyl]sulfone bis[4-(4-aminophenoxy)phenyl]sulfone, bis[3-(3-aminophenoxy)phenyl]methane, bis[3-(4-aminophenoxy)phenyl]methane, bis[4-(3-aminophenoxy)phenyl]methane, bis[4-(4-aminophenoxy)phenyl]methane, 2,2-bis[3-(3-aminophenoxy)phenyl]propane, 2,2-bis[3-(4-aminophenoxy)phenyl]propane, 2,2-bis[4-(3-aminophenoxy)phenyl]propane, 2,2- Diamines with four benzene nuclei, such as bis[4-(4-aminophenoxy)phenyl]propane, 2,2-bis[3-(3-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, 2,2-bis[3-(4-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, 2,2-bis[4-(3-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, and 2,2-bis[4-(4-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane;

[0118] These may be used alone or in combination of two or more. The diamine to be used may be appropriately selected depending on the desired properties.

[0119] Among these, aromatic diamine compounds are preferred, and 3,3'-diaminodiphenyl ether, 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenyl ether, paraphenylenediamine, 1,3-bis(3-aminophenyl)benzene, 1,3-bis(4-aminophenyl)benzene, 1,4-bis(3-aminophenyl)benzene, 1,4-bis(4-aminophenyl)benzene, 1,3-bis(4-aminophenoxy)benzene, and 1,4-bis(3-aminophenoxy)benzene can be suitably used. In particular, at least one diamine selected from the group consisting of benzenediamine, diaminodiphenyl ether, and bis(aminophenoxy)phenyl is preferred.

[0120] From the viewpoints of heat resistance and dimensional stability at high temperatures, the polyimide porous film that can be used in the present invention is preferably formed from a polyimide obtained by combining a tetracarboxylic dianhydride and a diamine, which has a glass transition temperature of 240°C or higher or has no clear transition point at 300°C or higher.

[0121] The polyimide porous film that can be used in the present invention is preferably a polyimide porous film made of the following aromatic polyimides, from the viewpoints of heat resistance and dimensional stability at high temperatures.

[0122] (i) an aromatic polyimide comprising at least one tetracarboxylic acid unit selected from the group consisting of biphenyltetracarboxylic acid units and pyromellitic acid units, and an aromatic diamine unit; (ii) an aromatic polyimide comprising a tetracarboxylic acid unit and at least one aromatic diamine unit selected from the group consisting of benzenediamine units, diaminodiphenyl ether units, and bis(aminophenoxy)phenyl units; and / or (iii) an aromatic polyimide comprising at least one tetracarboxylic acid unit selected from the group consisting of biphenyltetracarboxylic acid units and pyromellitic acid units, and at least one aromatic diamine unit selected from the group consisting of benzenediamine units, diaminodiphenyl ether units, and bis(aminophenoxy)phenyl units.

[0123] The polyimide porous membrane that can be used in the present invention is preferably a three-layer polyimide porous membrane having surface layers A and B each having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B. The average pore size of the pores in the 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 surface layer B is preferably 20 μm or more and 100 μm or less. The macrovoid layer preferably has partition walls bonded to the surface layers A and B and a plurality of macrovoids surrounded by the partition walls and the surface layers A and B. The thickness of the partition walls of the macrovoid layer and the surface layers A and B is preferably 0.01 μm or more and 20 μm or less. The pores in the surface layers A and B are connected to the macrovoids, and the total thickness is preferably 5 μm or more and 500 μm or less. The porosity of the polyimide porous membrane is preferably 40% or more and less than 95%. At least one partition wall in the macrovoid layer may have one or more pores that connect adjacent macrovoids and have an average pore size of 0.01 μm or more and 100 μm or less, preferably 0.01 μm or more and 50 μm or less.

[0124] For example, the polyimide porous films described in International Publication No. 2010 / 038873, Japanese Patent Application Laid-Open No. 2011-219585, or Japanese Patent Application Laid-Open No. 2011-219586 can also be used in the present invention.

[0125] III-2. Polyethersulfone (PES) Porous Membrane The PES porous membrane that can be used in the present invention contains polyethersulfone, and typically consists essentially of polyethersulfone. The polyethersulfone may be synthesized by a method known to those skilled in the art, for example, by subjecting a dihydric phenol, an alkali metal compound, and a dihalogenodiphenyl compound to a polycondensation reaction in an organic polar solvent, or by pre-synthesizing an alkali metal disalt of a dihydric phenol and then subjecting it to a polycondensation reaction with a dihalogenodiphenyl compound in an organic polar solvent.

[0126] Examples of the alkali metal compound include alkali metal carbonates, alkali metal hydroxides, alkali metal hydrides, alkali metal alkoxides, etc. Sodium carbonate and potassium carbonate are particularly preferred.

[0127] Examples of dihydric phenol compounds include hydroquinone, catechol, resorcinol, 4,4'-biphenol, bis(hydroxyphenyl)alkanes (for example, 2,2-bis(hydroxyphenyl)propane and 2,2-bis(hydroxyphenyl)methane), dihydroxydiphenyl sulfones, dihydroxydiphenyl ethers, and compounds in which at least one hydrogen atom on the benzene ring of these compounds has been substituted with a lower alkyl group such as a methyl group, an ethyl group, or a propyl group, or a lower alkoxy group such as a methoxy group or an ethoxy group. Two or more of the above compounds can be mixed and used as the dihydric phenol compound.

[0128] The polyethersulfone may be a commercially available product, and examples of the commercially available product include Sumikaexcel 7600P and Sumikaexcel 5900P (both manufactured by Sumitomo Chemical Co., Ltd.).

[0129] The logarithmic viscosity of the polyethersulfone is preferably 0.5 or more, more preferably 0.55 or more, from the viewpoint of favorable formation of macrovoids in the PES porous membrane, and is preferably 1.0 or less, more preferably 0.9 or less, even more preferably 0.8 or less, and particularly preferably 0.75 or less, from the viewpoint of ease of production of the PES porous membrane.

[0130] Furthermore, from the viewpoint of heat resistance and dimensional stability at high temperatures, the PES porous membrane or the polyethersulfone used as its raw material preferably has a glass transition temperature of 200°C or higher, or no clear glass transition temperature is observed.

[0131] The method for producing the PES porous membrane that can be used in the present invention is not particularly limited, and the membrane may be produced by a method comprising, for example, the steps of: casting a polyethersulfone solution containing 0.3% by mass to 60% by mass of polyethersulfone having an inherent viscosity of 0.5 to 1.0 and 40% by mass to 99.7% by mass of an organic polar solvent into a film; and immersing or contacting the film with a coagulation solvent containing, as an essential component, a poor solvent or a non-solvent for polyethersulfone, to produce a coagulated membrane having pores; and heat-treating the coagulated membrane having pores obtained in the step to coarsen the pores, thereby obtaining a PES porous membrane, wherein the heat treatment comprises raising the temperature of the coagulated membrane having pores to a temperature equal to or higher than the glass transition temperature of the polyethersulfone, or to 240° C. or higher.

[0132] The PES porous membrane that can be used in the present invention is preferably a PES porous membrane having a surface layer A, a surface layer B, and a macrovoid layer sandwiched between the surface layer A and the surface layer B, wherein the macrovoid layer has partition walls bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition walls and the surface layers A and B and having an average pore size of 10 μm to 500 μm in the membrane plane direction, the partition walls of the macrovoid layer have a thickness of 0.1 μm to 50 μm, and each of the surface layers A and B has a thickness of 0.1 μm to 50 μm, one of the surface layers A and B has a plurality of pores with an average pore size of more than 5 μm and not more than 200 μm, and the other has a plurality of pores with an average pore size of 0.01 μm or more and less than 200 μm, one of the surface layers A and B has a surface opening ratio of 15% or more, and the other surface layer has a surface opening ratio of 10% or more, The pores of the surface layer A and the surface layer B communicate with the macrovoids, and the PES porous membrane has a total thickness of 5 μm to 500 μm and a porosity of 50% to 95%.

[0133] In this way, a cryopreservation substrate made of a porous membrane can be produced. The cryopreservation substrate according to the present invention may be a porous membrane as it is, or may be a laminated porous membrane made by laminating a plurality of porous membranes, or may be a laminated porous membrane with another layer such as a mesh.

[0134] The cryopreservation substrate thus obtained can be used as a substrate for cell culture in addition to cryopreservation, thereby reducing the burden on the cells being cryopreserved.

[0135] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples. Those skilled in the art can easily modify and alter the present invention based on the description in this specification, and such modifications and alterations are within the technical scope of the present invention.

[0136] [Experiment 1] <Porous polyimide membrane> A porous polyimide membrane with an average thickness of 25 μm and a porosity of 73% was prepared. The porous polyimide membrane had two different surface layers (side A and side B) and a macrovoid layer sandwiched between the two surface layers. The average pore diameter of the holes present on side A was 6 μm, and the average pore diameter of the holes present on side B was 46 μm.

[0137] <Modularized polymer porous membrane> The modularized polymer porous membrane (hereinafter also referred to as "module") was obtained by alternately stacking the following laminated polyimide porous membrane (A) and mesh (B) in the order A, B, A, B, A, B, A, B, C ...

[0138] Laminated polyimide porous membrane (A): Six 1.0 x 1.0 cm polyimide porous membranes were laminated in the following order: A side / B side / / A side / B side / / A side / B side / / A side / B side / / A side / / B side / A side / / B side / A side.

[0139] Mesh (B): A 1.0 x 1.0 cm insole (polypropylene / polyethylene) (manufactured by NBC Meshtec, product number: ESP10TC) was used.

[0140] 5 wt% FBS-Eagle medium: Eagle medium to which FBS (fetal bovine serum) (hereinafter also referred to as "FBS") has been added so that the concentration is 5 wt%. 2 wt% FBS-Eagle medium: Eagle medium to which FBS has been added so that the FBS concentration is 2 wt%. 10 wt% FBS / 10 wt% DMSO-Eagle medium: Eagle medium to which FBS and DMSO have been added so that the FBS concentration is 10 wt% and the DMSO concentration is 10 wt%.

[0141] The evaluation method in this example is as follows: <Virus titer> The culture supernatant was centrifuged at 3,000 rpm at 4°C for 15 minutes, and the supernatant was recovered and serially diluted 10-fold with 5 wt% FBS-Eagle medium to obtain diluted solutions.

[0142] A porcine kidney-derived cell line (1.5 × 10) previously suspended in 5% FBS-Eagle medium ５Each dilution was inoculated into four wells at 0.05 mL / well, and the plate was incubated at 37°C in 5% CO ２ On day 7 of culture, 0.05 mL of the supernatant from each well was transferred to a new 96-well V-bottom plate, and 0.05 mL of a 0.5 vol% guinea pig red blood cell suspension was added to each well. The plate was then left to stand overnight at 4°C, and the virus titer was calculated by the Behrens-Kärber method using the presence or absence of hemagglutination as an indicator.

[0143] <Analysis of viral genome sequence> Culture supernatant stored in an ultra-low temperature freezer was thawed, and viral nucleic acid was extracted using a QIAmp (registered trademark) DNA Mini Kit (manufactured by QIAGEN) according to the attached operating instructions. Using the extracted nucleic acid as a template, a PCR reaction solution was prepared as shown in Table 1. The primer sequences used were combinations of No. 1 (SEQ ID NO: 1) and No. 2 (SEQ ID NO: 2), and No. 3 (SEQ ID NO: 3) and No. 4 (SEQ ID NO: 4), as shown in Table 2.

[0144]

[0145]

[0146] PCR reaction was carried out using a thermal cycler according to the program in Table 3.

[0147] In the table below, the program from 98°C for 10 seconds to 72°C for 1 minute means that a set of 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 1 minute, in that order, was repeated 30 times in total.

[0148] The PCR products were subjected to 2% agarose gel electrophoresis to confirm that bands of the target sizes shown in Table 2 were detected. The PCR products were then purified using NucleoSpin (registered trademark) Gel and PCR Clean-up (manufactured by TaKaRa) according to the accompanying operating instructions.

[0149] The purified product was sequenced by direct sequencing and subjected to mutation analysis. The analysis range was 2131 to 2868 bases, which correspond to the "NS1 gene downstream + intergenic sequence + VP1 / VP2 gene upstream" of "ACCESSION: NC_001718, Porcine parvovirus complete genome," and 3365 to 4549, which correspond to the "VP1 / VP2 gene downstream."

[0150] Example 1: Cryopreservation of porcine parvovirus-infected porcine kidney-derived cell line

[0151] (1) Cell culture of porcine kidney-derived cell line and inoculation with porcine parvovirus. ４ 45 mL of a solution containing 100 μg of PBS (100 μg / mL) and five modules that had been previously immersed in PBS and shaken for 24 hours were placed in a 50 mL tube and incubated at 37°C and 5% CO ２ Below, the tube was rotated at 5 rotations / min for 24 hours during rotation culture.

[0152] After 24 hours of rotational culture, the entire contents of the 50 mL tube were transferred to a 75 cm ２ The mixture was transferred to a flask and incubated at 37°C and 5% CO ２ After a further 6 days of static incubation under 50°C, the modules were washed with PBS.

[0153] The module washed with PBS was placed in a 75 cm tissue culture tube containing 20 mL of porcine parvovirus solution (hereinafter also referred to as "virus solution") prepared to a multiplicity of infection of approximately 0.01. ２ The mixture was transferred to a flask and incubated at 37°C and 5% CO ２ The mixture was left standing under vacuum for 60 minutes.

[0154] The module was then removed from the virus solution and placed in a new 75 cm tissue culture container containing 30 mL of 2% FBS-Eagle medium. ２ The mixture was transferred to a flask and incubated at 37°C and 5% CO ２ Static culture was initiated under

[0155] On the 7th day after the virus inoculation, the culture supernatant was removed by suction, and 30 mL of 2% FBS-Eagle medium was added, followed by further culturing for 7 days.

[0156] In this manner, the porcine parvovirus-infected porcine kidney-derived cell line was cultured for 14 days after virus infection (a total of 14 days of culture after virus inoculation).

[0157] (2) Cryopreservation The porcine parvovirus-infected porcine kidney-derived cell line obtained in (1) was cryopreserved in a 75 cm tissue culture tube without detaching it from the module. ２ The cells were removed from the flask and placed in a cryopreservation tube containing 10 wt% FBS / 10 wt% DMSO-Eagle medium. The storage tube was then placed in a Bicell freezing container (manufactured by Nippon Freezer Co., Ltd.), which was then placed in an ultra-low temperature freezer set at -80°C and frozen by slow cooling.

[0158] After freezing overnight, the samples were stored frozen in an ultra-low temperature freezer at the same set temperature for one month.

[0159] Similarly, porcine parvovirus-infected porcine kidney-derived cell lines were cryopreserved for 3, 6, 9, or 12 months instead of 1 month.

[0160] In this way, five types of porcine parvovirus-infected porcine kidney-derived cell lines with different frozen storage periods were cryopreserved.

[0161] Reference Example 1 Cultivation and cryopreservation were carried out for the same cryopreservation period as in Example 1, except that cryopreservation was carried out in liquid nitrogen instead of in an ultra-low temperature freezer.

[0162] (Example 2) Cultivation of porcine parvovirus-infected porcine kidney-derived cell line after cryopreservation (3) Thawing, reculture, and supernatant recovery After the cryopreservation period ended, the storage tubes were removed from the ultra-low temperature freezer or liquid nitrogen and immediately thawed in a water bath at 37°C.

[0163] After thawing, the module was removed from the storage tube, immersed in PBS to wash it, and then placed in a 75 cm tissue culture container containing 30 mL of Eagle's medium supplemented with 2% FBS. ２ The mixture was transferred to a flask and incubated at 37°C and 5% CO ２ Static culture was resumed under

[0164] On days 3, 7, 10, 14, 21, and 28 of this replenishment period (17, 21, 24, 28, 35, and 42 days in total of the culture period), 2 mL of each culture supernatant was collected.

[0165] During this entire period, once a week after the supernatant was collected, the remaining culture supernatant was completely removed, and 30 mL of Eagle's medium supplemented with 2% FBS was newly added and cultured.

[0166] The collected culture supernatant was used to evaluate the porcine parvovirus-infected porcine kidney-derived cell line, and the results are shown in Tables 4 to 11 and FIGS.

[0167] Reference Example 2 A porcine parvovirus-infected porcine kidney-derived cell line was cultured for 42 days in the same manner as in Example 1, except that the cells were not cryopreserved. 2 mL of culture supernatant was recovered on days 21, 28, 35, and 42 of the culture period. The results of evaluation of the porcine parvovirus-infected porcine kidney-derived cell line using the recovered supernatant are shown in Tables 4 to 11 and FIGS. 1 and 2.

[0168] (Reference Example 3) A porcine parvovirus-infected porcine kidney-derived cell line was cultured using a 75 cm flask for tissue culture. (1) Initial inoculation ２ The flask was filled with 5% FBS-Eagle medium and 2.0 × 10 ５ A porcine kidney-derived cell line (20 mL) adjusted to 100 cells / mL was added, and a porcine parvovirus solution was added to give a multiplicity of infection of 0.01. The cells were then incubated at 37°C, 5% CO ２ were cultured under

[0169] The next day, the culture supernatant was removed by suction, and 30 mL of 2% FBS-Eagle medium was added, and the cells were incubated at 37°C and 5% CO ２ The mixture was further cultured under the same conditions for 6 days (total of 7 days of culture after inoculation).

[0170] (2) Passage of virus and recovery of supernatant 100 μL of the culture supernatant of the porcine parvovirus-inoculated porcine kidney-derived cell line cultured for 7 days was collected and diluted to 2.0 × 10 ５ The solution was added to a new porcine kidney-derived cell line (20 mL) that had been adjusted to 1000 cells / mL, and incubated at 37°C in 5% CO ２ The cells were cultured under 5% CO₂ for 7 days. After that, the cells were inoculated with a new porcine kidney-derived cell line every 7 days of culture.

[0171] Two mL of each culture supernatant was collected and used to evaluate the porcine parvovirus-infected porcine kidney-derived cell line. The evaluation results are shown in Tables 4 to 11 and Figures 1 and 2.

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180] Table 4 and Figures 1 and 2 show that compared with cells cryopreserved in liquid nitrogen and cells that were not cryopreserved, the cells cryopreserved in an ultra-low temperature freezer for 12 months showed no change in virus titer over the 4-week period, indicating that the cell function was maintained.

[0181] Furthermore, Tables 5 to 11 show that compared with cells cryopreserved in liquid nitrogen and cells that were not cryopreserved, cells cryopreserved in an ultra-low temperature freezer for 12 months showed no significant difference in viral genome mutations over a 4-week period.

[0182] [Experiment 2] The following 6-well inserts were used in this example. The existing membrane was peeled off from a 6-well cell culture insert (Greiner ThinCert®, 6-well, product number 657641), and a polyimide porous membrane was attached to prepare the insert. The "polyimide porous membrane" used refers to a polyimide porous membrane with a total thickness of 25 μm and a porosity of 73%. The polyimide porous membrane had two different surface layers (sides A and B) and a macrovoid layer sandwiched between the two surface layers. The average pore size of the holes on side A was 6 μm, and the average pore size of the holes on side B was 30 μm. The following 24-well inserts were used in this example. The existing membrane was peeled off from a 24-well cell culture insert (Greiner ThinCert®, 24-well, product number 662640), and a polyimide porous membrane was attached to prepare the insert. The "polyimide porous membrane" used herein refers to a polyimide porous membrane with a total thickness of 25 μm and a porosity of 73%. The polyimide porous membrane had two different surface layers (side A and side B) and a macrovoid layer sandwiched between the two surface layers. The average pore size of the holes present on side A was 6 μm, and the average pore size of the holes present on side B was 30 μm.

[0183] <Cultivation and cryopreservation of human fibroblast cell lines> <Experimental materials and methods> (1) Expansion culture of human fibroblasts 12 mL of FGM™-2 Fibroblast Growth Medium-2 Bullet Kit (trademark, product number: CC-3132, LONZA) was added to a Falcon™ Dish (trademark, untreated, Corning) to a seeding density of 5,000 cells / cm. ２ Human fibroblasts were seeded at 37°C and 5% CO ２ The mixture was subjected to static culture in the presence of β-glucan for 7 days.

[0184] (2) Cultivation of human fibroblasts using 6-well cell culture inserts (Comparative Example 1) 2.5 mL of medium was added per well to a 6-well plate (Tissue Culture Plate, product number: 353046, FALCON). The 6-well cell culture inserts were placed in the wells of the 6-well plate and incubated at 37°C, 5% CO ２ The inserts were then moistened in an incubator in the presence of PBS. After the moistening, 2 mL of medium was added per well to the inside of the inserts. The human fibroblasts expanded by the method (1) above were detached from a Falcon (registered trademark) Dish (untreated, Corning Inc.), and the cell suspension was then placed in a dish with a cell density of 20,000 cells / cm. ２ The cells were seeded inside the insert so that the temperature was 37°C and 5% CO ２ Static culture was performed for 118 days in the presence of HCl. The medium was changed twice a week, and 0.5 to 1 mL of the medium was collected at each change to obtain the culture supernatant. On days 9 and 118 of culture, the cells were cryopreserved without detaching from the inserts.

[0185] (3) Cultivation of human fibroblasts using 24-well cell culture inserts (Comparative Example 2) 0.5 mL of medium was added per well to a 24-well plate (Tissue Culture Plate, product number: 353047, FALCON). The 24-well cell culture inserts were placed in the wells of the plate and incubated at 37°C, 5% CO ２ The inserts were then moistened in an incubator in the presence of 0.5 mL of medium per well. After the inserts were removed from the incubator, 0.5 mL of medium was added to the inside of the moistened inserts. The human fibroblasts expanded by the method (1) above were detached from a Falcon (registered trademark) Dish (untreated, Corning Inc.), and the cell suspension was then transferred to a cell density of 20,000 cells / cm. ２ The cells were seeded inside the insert so that the temperature was 37°C and 5% CO ２ Static culture was performed for 118 days in the presence of HCl. The medium was changed twice a week, and 0.5 to 1 mL of medium was added at each change to obtain a culture supernatant. On days 9 and 118 of culture, the cells were cryopreserved without detaching from the inserts.

[0186] (4) Freezing of human fibroblasts using 6-well cell culture inserts. A cell freezing reagent (CELLBANKER1plus, product number: 11912, Nippon Zenyaku Kogyo) was added to a 6-well plate (Tissue Culture Plate, product number: 353046, FALCON) at 2.5 mL per well. The medium inside and outside of the inserts, which had been statically cultured for 9 and 118 days using the method described in (2) above, was collected and removed, and the inserts were placed in wells of a 6-well plate to which the cell freezing reagent had been added in advance. 2 mL of the freezing reagent was added per well to the inside of the insert. The 6-well plate was wrapped around the periphery with parafilm (product number PM-996, Bemis), placed in a zipper bag (zipper bag, thickness 0.04 mm, product number 4J, Monotaro), and placed in an ultra-low temperature freezer (MyBio, product number VT-78HC, Nippon Freezer) set at -80°C, where it was frozen and stored for approximately 9 months and 5 months.

[0187] (5) Freezing of human fibroblasts using 24-well cell culture inserts. A cell freezing reagent was added to a 24-well plate (Tissue Culture Plate, product number: 353047, FALCON) at 0.5 mL per well. After static culture for 9 days and 118 days using the method described in (3) above, the media inside and outside the inserts were collected and removed, and the inserts were placed in the wells of a 24-well plate to which the cell freezing reagent had been added. A cell freezing reagent of 0.5 mL per well was added to the inside of the insert. The 24-well plate was wrapped around the periphery with parafilm and then placed in a ziplock bag (ziplock bag, thickness 0.04 mm, product number: 4J, Monotaro), placed in an ultra-low temperature freezer set at -80°C, and frozen for approximately 9 and 5 months.

[0188] <Thawing and Reculturing of Human Fibroblast Cell Lines> (6) Thawing and Reculturing of Human Fibroblast Cells Using 6-Well Cell Culture Inserts (Example 1) 2.5 mL of PBS (D-PBS(-), product number: 045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) was added per well to a 6-well plate (Tissue Culture Plate, product number: 353046, FALCON). After the frozen storage period, the 6-well plate was removed from the ultra-low temperature freezer (-80°C) and immediately thawed in a water bath at 37°C. After removing the 6-well plate from the water bath, the insert was removed from the thawed frozen solution and placed in a well of a 6-well plate to which PBS had been added in advance. 2 mL of PBS was added to the inside of the insert. After removing the PBS, 4.5 mL of PBS was added.

[0189] 2.5 mL of medium was added per well to a 6-well plate (Tissue Culture Plate, product number: 353046, FALCON). The inserts immersed in PBS for washing were removed and placed in the 6-well plate containing the medium. 2 mL of medium was added. The plate was incubated at 37°C and 5% CO ２ The medium was changed twice a week, and 0.5 to 1 mL of the medium was collected to obtain the culture supernatant.

[0190] (7) Thawing and Reculturing of Human Fibroblasts Using 24-Well Cell Culture Inserts (Example 2) 0.5 mL of PBS was added per well to a 24-well plate (Tissue Culture Plate, Product Number: 353047, FALCON). After the frozen storage period, the well plate was removed from the ultra-low temperature freezer (-80°C) and immediately thawed in a water bath at 37°C. After removing the 24-well plate from the water bath, the insert was removed from the thawed frozen solution and placed in a well of a 24-well plate to which PBS had been added in advance. 0.5 mL of PBS was added to the inside of the insert. After removing the PBS, 1 mL of PBS was added.

[0191] 0.5 mL of medium was added per well to a 24-well plate (Tissue Culture Plate, product number: 353047, FALCON). The inserts immersed in PBS for washing were removed and placed in the 24-well plate to which medium had been added. 0.5 mL of medium was added to the inside of the insert. The incubation was continued at 37°C and 5% CO ２ The medium was changed twice a week, and 0.5 to 1 mL of the medium was collected to obtain the culture supernatant.

[0192] <Evaluation of Culture Supernatant> (11) Measurement of Cell Products The concentrations of fibronectin contained in the culture supernatants collected in Example 1, Example 2, Comparative Example 1, and Comparative Example 2 were measured using an ELISA fibronectin measurement kit (Fibronectin EIA Kit, product number: MK115, manufactured by Takara Bio Inc.). The results are shown in Table 12.

[0193]

[0194] Table 12 shows that, compared with the cells before freezing, the cells cryopreserved in an ultra-low temperature freezer for 5 or 9 months maintained the fibronectin concentration in the culture supernatant, indicating that the fibronectin production function was maintained.

[0195] According to the present invention, cells can be cryopreserved for long periods without changing the substrate, simplifying the freezing and thawing process and enabling the cells to maintain their function for long periods without using liquid nitrogen. This significantly reduces the costs of long-term storage and transportation of frozen cells. The present invention can make a significant contribution to the field of biology, particularly regenerative medicine.

[0196] 1 Cell culture insert 2 Cylindrical container having an arm structure on the upper surface of the cylindrical container 3 Porous membrane arranged on the bottom surface of the cylindrical container 4 Well

Claims

1. A method for cryopreserving cells, comprising freezing and preserving cells for 30 days or more in a state in which the cells are seeded on a porous membrane without using liquid nitrogen.

2. The method of claim 1, wherein the cell is an animal cell.

3. The method of claim 1, wherein the porous membrane is a polymeric porous membrane.

4. The method of claim 3, wherein the average pore size of side B of the polymeric porous membrane is greater than the average pore size of side A.

5. The method of claim 4, wherein the average pore size of said side B is 10 μm or greater.

6. The method according to claim 4, wherein the average pore size of the A-side is 0.01 μm or more.

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

8. The method of claim 3, wherein the polymeric porous membrane is a polyimide porous membrane or a polyethersulfone porous membrane.

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

10. The method of claim 2, wherein the animal cell is a virus-producing cell or a hybridoma.

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

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

13. A cell culture method comprising the following steps: (1) step: seeding cells on a porous membrane; (2) step: freezing and storing the cells seeded on the porous membrane for 30 days or more without using liquid nitrogen; (3) step: thawing the cells seeded on the porous membrane; and (4) step: culturing the thawed cells.

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

15. A cryopreservation matrix formed of a porous membrane used to freeze-preserve seeded cells for more than 30 days.

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

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