Graft method, culture carrier substrate, package, culture carrier substrate equipped with cell sheet, method for producing culture carrier substrate equipped with cell sheet, cryopreservation method, and frozen product of culture carrier substrate equipped with cell sheet

Abstract

A graft method according to the present invention comprises: a culture step in which a cell sheet is cultured on a culture surface of a culture carrier substrate; and a graft step in which the cell sheet formed on the culture surface of the culture carrier substrate is attached to a recipient site, after which the culture carrier substrate is detached from the cell sheet.

Description

Transplantation method, culture carrier substrate, package, culture carrier substrate with cell sheet, method for producing culture carrier substrate with cell sheet, cryopreservation method, and frozen culture carrier substrate with cell sheet

[0001] The present invention relates to a transplantation method, a culture carrier substrate, a package, a culture carrier substrate with a cell sheet, a method for producing a culture carrier substrate with a cell sheet, a cryopreservation method, and a frozen culture carrier substrate with a cell sheet.

[0002] Various developments have been made so far regarding the "cultivation" and "transportation" of cell sheets, which constitute the technical elements of transplantation technology. One example of this type of technology is known, for example, from Patent Document 1. The background art of Patent Document 1 describes that transplanting a cell sheet to a target affected area requires a series of operations, including detaching a cell sheet cultured on a temperature-responsive material by changing the temperature, covering the resulting cell sheet with a separately prepared sheet, removing the cell sheet from a container with the cell sheet attached to the sheet, transporting it to the affected area, and transplanting (applying) it to the affected area.

[0003] In contrast, claim 1 and paragraph 0007 of Patent Document 1 describe a technique for transporting a cell sheet cultured in a cell culture vessel having a temperature-responsive polymer layer, in which the bottom of the cell culture vessel is peeled off with the cell sheet still attached, and the peeled bottom and the cell sheet attached to the bottom are transported.

[0004] JP 2016-013111 A

[0005] However, as a result of investigations by the present inventors, it has been found that in the transplantation method utilizing the technology of Patent Document 1, it is necessary to release the adhesion between the cell sheet and the substrate by low-temperature treatment when detaching the cell sheet from the temperature-responsive polymer layer, and that low-temperature treatment reduces the adhesion between the substrate and the cell sheet, making it difficult to freeze and store a cell sheet cultured in a cell culture vessel having a temperature-responsive polymer layer while it is attached to the cell culture vessel having a temperature-responsive polymer layer.

[0006] After extensive research, the inventors came up with a new concept that integrates the "cultivation" and "transportation" of cell sheets, which had previously been considered separately, and developed a transplantation method and a culture carrier substrate, etc., thereby completing the present invention.

[0007] According to one aspect of the present invention, the following transplantation method, culture carrier substrate, package, cell sheet-attached culture carrier substrate, method for manufacturing a cell sheet-attached culture carrier substrate, cryopreservation method, and frozen product of cell sheet-attached culture carrier substrate are provided. 1. A transplantation method comprising: a culturing step of culturing a cell sheet on the culture surface of a culture carrier substrate; and a transplanting step of attaching the cell sheet formed on the culture surface of the culture carrier substrate to a transplantation site and then peeling the culture carrier substrate from the cell sheet. 2. The transplantation method described in 1., wherein the culture carrier substrate has a substrate thickness of 5 μm to 250 μm, the culture carrier substrate is a polyether ether ketone film or a polyethylene terephthalate film, and the culture surface of the culture carrier substrate has been subjected to a hydrophilization treatment. 3. The transplantation method described in 1. or 2., wherein the culture carrier substrate has a substrate thickness of more than 10 μm, and in the culturing step, the culture carrier substrate functions as a free-standing membrane without being fixed to the inside of a culture vessel. 4. 1. to 3. 5. The transplantation method according to any one of 1. to 4., wherein in the transplantation step, the time from attachment to detachment is 10 minutes or less. 6. The transplantation method according to any one of 1. to 4., wherein the area of ​​the cell sheet obtained by the culture step is 0.3 cm ２A transplantation method as described above. 6. A culture carrier substrate used for transporting a cell sheet cultured on a culture surface, wherein the culture carrier substrate has a substrate thickness of 5 μm or more and 250 μm or less. 7. The culture carrier substrate according to 6., wherein the culture carrier substrate has a substrate thickness of more than 10 μm and functions as a free-standing membrane without being fixed to the inside of a culture vessel during culture of the cell sheet. 8. The culture carrier substrate according to 6. or 7., wherein the culture surface has been subjected to a hydrophilization treatment. 9. The culture carrier substrate according to 8., wherein the hydrophilization treatment is a plasma treatment. 10. The culture carrier substrate according to any one of 6. to 9., wherein the culture carrier substrate is a polyether ether ketone film or a polyethylene terephthalate film. 11. The culture carrier substrate according to any one of 6. to 10., wherein the culture surface does not contain a temperature-responsive polymer. 12. 6. A package obtained by packaging the culture carrier substrate according to any one of items 6. to 11. in a packaging material. 13. A culture carrier substrate with a cell sheet, comprising a laminate of the culture carrier substrate according to any one of items 6. to 11. and a cell sheet, wherein the cell sheet covers at least 50% of the surface on the culture side of the culture carrier substrate. 14. A culture carrier substrate with a cell sheet according to item 13., wherein the area of ​​the cell sheet is 0.3 cm ２A cell sheet-attached culture carrier substrate as described above. 15. A method for producing a cell sheet-attached culture carrier substrate, comprising: a culturing step of introducing a plurality of cells, a medium, and the culture carrier substrate described in any one of 6. to 11. into a culture vessel, and culturing a cell sheet on the culture surface of the culture carrier substrate; and a removal step of removing the culture carrier substrate with the cell sheet attached from the culture vessel. 16. A method for producing a cell sheet-attached culture carrier substrate described in 15., wherein, in the culturing step, the cell sheet is cultured without fixing the culture carrier substrate to the inside of the culture vessel. 17. A method for producing a cell sheet-attached culture carrier substrate described in 15. or 16., wherein, in the removal step, an operation of physically separating the culture carrier substrate from the culture vessel by a means of physical means is not required. 18. A cryopreservation method, comprising a cooling step of cooling a cell sheet-attached culture carrier substrate, which comprises a culture carrier substrate and a cell sheet formed on the surface of the culture carrier substrate, in a cryopreservation solution. 19. 18. The cryopreservation method according to item 18, wherein, in the cooling step, the cell sheet-attached culture carrier substrate is cooled in a cryopreservation solution using a non-throughflow cooling device. 20. The cryopreservation method according to item 18 or 19, wherein, in the cooling step, the cooling rate at 0 to -5°C is 0.1°C / min or more and 15°C / min or less. 21. The cryopreservation method according to any one of items 18 to 20, comprising, after the cooling step, a cryopreservation step of storing the cell sheet-attached culture carrier substrate at -196 to -60°C. 22. A frozen product of a cell sheet-attached culture carrier substrate, comprising: a culture carrier substrate; and a cell sheet formed on the surface of the culture carrier substrate, wherein the culture carrier substrate and the cell sheet are in a frozen state. 23. A frozen product of the cell sheet-attached culture carrier substrate according to item 22, wherein the cell sheet-attached culture carrier substrate is in a frozen state at -196 to -60°C.24. A frozen product of the cell sheet-attached culture carrier substrate according to 22., which is stored frozen at -196°C to -135°C. 25. A frozen product of the cell sheet-attached culture carrier substrate according to 22., which is stored frozen at -134°C to -60°C. 26. A package obtained by packaging the frozen product according to any one of 22. to 25. with a packaging material.

[0008] According to the present invention, there are provided a transplantation method that enables both cell sheet cultivation and transportation, a culture carrier substrate, a package, a culture carrier substrate with a cell sheet, a method for manufacturing a culture carrier substrate with a cell sheet, a cryopreservation method, and a frozen culture carrier substrate with a cell sheet.

[0009] Fig. 1 is a cross-sectional view showing a process of a transplantation method according to an embodiment of the present invention; Fig. 2 is a cross-sectional view showing a process of a modified example of a freezing method according to an embodiment of the present invention; Fig. 3 is a cross-sectional view showing a process of a modified example of a freezing method according to an embodiment of the present invention;

[0010] Hereinafter, embodiments of the present invention will be described with reference to the drawings. In all drawings, similar components are designated by similar reference numerals, and descriptions thereof will be omitted as appropriate. Furthermore, the drawings are schematic diagrams and do not correspond to actual dimensional proportions.

[0011] <Transplantation method> The transplantation method of this embodiment includes a culture step of culturing a cell sheet on the culture surface of a culture carrier substrate, and a transplantation step of attaching the cell sheet formed on the culture surface of the culture carrier substrate to the transplantation site, and then peeling the culture carrier substrate from the cell sheet.

[0012] The transplantation site may be at least a portion of the body of a recipient, such as a mammal, bird, amphibian, fish, insect, plant, or microorganism. Specific examples of mammals and birds include humans, monkeys, chimpanzees, cows, horses, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, and chickens. However, the transplantation site may exclude the interior and exterior of the human body. Examples of transplantation sites other than the recipient include other cell sheets, medical devices, and tissues or organs isolated from the recipient. Examples of tissues or organs isolated from the recipient include skin, oral tissue, esophagus, trachea, bronchi, lungs, lung lobes, stomach, duodenum, pancreas, spleen, small intestine, large intestine, muscle tissue, and bone (excluding tissues that are to be returned to the same recipient for treatment).

[0013] The transplantation method of this embodiment will be specifically described using Figure 1. Figure 1 is a cross-sectional view showing a schematic example of the steps of the transplantation method. In Figure 1, (a) shows the processes of cell seeding, (b) shows cell culture, (c) shows cryopreservation and thawing, (d) shows removal, and (e) shows attachment. The cryopreservation and thawing process in (c) is an optional process. Here, a transplantation method in which (c) is not performed will be described, and details of (c) will be described later in <Cryopreservation Method>.

[0014] The culture process may include a cell seeding process shown in FIG. 1( a) and a cell culture process shown in FIG. 1( b). FIG. 2 shows a cross-sectional schematic diagram of an example of a cell sheet-attached culture carrier substrate 50 obtained in the culture process. In the cell seeding process shown in FIG. 1( a), a culture solution 30 containing cells is brought into contact with a culture carrier substrate 10 placed in a culture vessel 20. The cells may be pre-suspended in the culture solution 30, or a cell suspension containing cells may be added to the culture solution 30. The culture carrier substrate 10 may be partially or entirely immersed in the culture solution 30 during the culture process. Specifically, in the cell seeding process, the culture solution 30 may be dropped onto a portion of the surface of the culture carrier substrate 10, or the culture carrier substrate 10 may be immersed in the culture solution 30 so that at least a portion of the culture surface 12 and the side surface of the culture carrier substrate 10 are in contact with the culture solution 30. Compared to the former dripping method, the latter immersion method makes it possible to maintain a stable culture environment during the subsequent cell culture process, and / or to increase the area of ​​the culture surface 12 of the culture carrier substrate 10.

[0015] 1(b), the cells are cultured under appropriate culture conditions to form a cell sheet 40 on the culture surface 12 of the culture carrier substrate 10. A lid (not shown) may be placed over the cells during the culture.

[0016] In the cell culture process, the cell sheet 40 can be cultured without adhering the culture carrier substrate 10 to the inside of the culture vessel 20. In this case, the culture carrier substrate 10 functions as a free-standing membrane during cell culture. Because the culture carrier substrate 10 is not fixed, the cell sheet-attached culture carrier substrate 50 can be easily removed from the culture vessel 20 in the removal process. Furthermore, damage to the cell sheet 40 during removal can be suppressed. Here, "adhesion" refers to a state in which the cell sheet is in close contact with the culture carrier substrate 10 to the extent that physical means are required for removal. Fixation means that hold the cell sheet down with a load other than its own weight to prevent movement are not included in the "adhesion" means used here. In this specification, physical means include, for example, a method of gripping the film and peeling it off, or a method of scratching the film with a knife or the like to remove it. As a method for immersing the culture carrier substrate 10 in the culture solution 30 without fixing it, one or more of the following may be employed: a method for making the specific gravity of the culture carrier substrate 10 greater than that of the culture solution 30; a method for fixing a load to a part of the culture surface 12 of the culture carrier substrate 10 by using a weight; or a method for fixing the position of the culture carrier substrate 10 by using a tool. That is, during the cell culture step, cells can be cultured in a state where the culture carrier substrate 10 is immersed in the culture solution 30 by fixing the load and / or the position.

[0017] The transplantation step may include a removal step of the cell sheet-attached culture carrier substrate 50 shown in Fig. 1(d) and a step of attaching the cell sheet 40 shown in Fig. 1(e). In the removal step shown in Fig. 1(d), the cell sheet-attached culture carrier substrate 50 is removed from the culture vessel 20 and transported to a desired location, such as a transplantation site. In the removal step of this embodiment, the removal method is not particularly limited, and examples include a method of pinching and removing the culture carrier substrate 10 constituting the cell sheet-attached culture carrier substrate 50, a method of bringing the culture carrier substrate 10 into contact with a suction nozzle and lifting it while suctioning, and a method of pulling up the culture carrier substrate 10 by tying it with a string.

[0018] In the attaching step shown in FIG. 1( e ), the cell sheet 40 of the cell sheet-attached culture carrier substrate 50 is attached to the transplantation site 70 , and then the culture carrier substrate 10 is peeled off from the cell sheet 40 .

[0019] In this embodiment, the time from attachment to detachment during the attachment process can be shortened. By shortening the time, the cell sheet is not exposed to the substrate film as a foreign body for a long period of time, reducing the risk of immune rejection of the substrate film, reducing the possibility of cell death, and simplifying the operation. This method can also be used in surgeries such as thoracotomy to attach a cell sheet to the body. The time from attachment to detachment is preferably 10 minutes or less, more preferably 5 minutes or less, even more preferably 3 minutes or less, and even more preferably 1 minute or less. Although the detailed mechanism is unclear, it is presumed that the adhesion between the culture surface 12 of the culture carrier substrate 10 and the surface of the cell sheet 40 is moderately strong, making it possible to detach the culture carrier substrate 10 from the cell sheet 40 even before the cell sheet 40 is biologically bound to the transplantation site 70.

[0020] Furthermore, in this embodiment, it is possible to prevent the cell sheet 40 from remaining on the culture carrier substrate 10 after peeling. The remaining rate of the cell sheet 40 on the culture carrier substrate 10 after peeling is preferably 50% or less, more preferably 30% or less, and even more preferably 10% or less, in terms of area ratio.

[0021] Cell transplantation therapy suppresses or prevents the onset and recurrence of symptoms associated with cell, tissue, or organ loss, dysfunction, or dysfunction. Examples of diseases that can be treated with cell transplantation therapy include spinal cord injury, knee articular cartilage injury, ischemic heart disease, age-related macular degeneration, corneal epithelial stem cell deficiency, aplastic anemia, severe limb ischemia, refractory skin ulcers, and the prevention of postoperative complications (e.g., various organ suture failures, bronchial stump fistulas, pancreatic fistulas, and bile leaks), burns, and the like. Furthermore, cells used in cell transplantation therapy may be autologous, allogeneic non-autologous, or xenogeneic. Autologous cells are preferred from the standpoints of clinical application and safety, while non-autologous cells are preferred from the standpoints of clinical application and productivity.

[0022] The elements of each step of the transplantation method are described below.

[0023] (Culture Vessel) The culture vessel 20 is a vessel for culturing cells in a culture medium. It is not particularly limited as long as it is suitable for the type of cells to be cultured and the intended use. Examples of the culture vessel 20 include dishes, Petri dishes, tissue culture dishes, multi-dishes, flasks, tissue culture flasks, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, Petri dishes, tubes, trays, culture bags, and roller bottles. The culture vessel 20 may be a surface-treated culture vessel with cell adhesive properties (for adherent cells) or an untreated culture vessel (for suspension cells). The presence or absence of a surface treatment on the culture vessel 20 does not affect the culture carrier substrate 10. Hereinafter, unless otherwise specified, either type can be used.

[0024] The material of the culture vessel 20 is not particularly limited as long as it is impermeable to the culture solution 30, and examples thereof include polystyrene, polyethylene, polypropylene, polyvinyl alcohol, polyethylene terephthalate, polyacetal, polyvinyl chloride, acrylic resin, polycarbonate, polyether ether ketone, polyether sulfone, polytetrafluoroethylene, polyimide, polyamide, cellulose, silicone, nylon 6,6, glass, and metals such as stainless steel and aluminum.

[0025] The area of ​​the culture vessel 20 is not particularly limited, but any commercially available culture vessel can be used for culture without any problems. For example, a size of 0.3 cm ２ More than 1000cm ２ The lower limit is more preferably 0.35 cm. ２ More preferably, 1.0 cm or more ２ More than 1.9 cm, particularly preferably ２ On the other hand, the upper limit is more preferably 900 cm ２ More preferably, 800 cm or less ２ Particularly preferably 500 cm ２ The following is the result.

[0026] (Cells) The cells are not particularly limited as long as they are clinically useful cells for treating or preventing symptoms associated with cell, tissue, or organ deficiency, dysfunction, or dysfunction, or culturable cells for use in non-clinical trials, and are cells isolated from a living organism. Examples of cells include biological tissue cells, mesenchymal stem cells capable of differentiating into cells belonging to mesenchymal tissue, pluripotent stem cells capable of differentiating into various biological tissues, and stem cells and progenitor cells that can be induced to differentiate. The cells may be adherent cells or suspension cells.

[0027] Specific examples of biological tissue cells include fibroblasts, myofibroblasts, corneal epithelial cells, retinal cells, nerve cells, muscle cells, cardiac muscle cells, myoblasts, bone cells, osteoblasts, chondrocytes, adipocytes, hepatocytes, pancreatic cells, kidney cells, gingival cells, periosteal cells, skin cells, and endothelial cells. Specific examples of mesenchymal stem cells include adipose tissue-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, and umbilical cord-derived mesenchymal stem cells. Specific examples of pluripotent stem cells include induced pluripotent stem cells, embryonic stem cells, nuclear transfer embryonic stem cells, embryonic tumor cells, and embryonic germ cells. These cells may be cultured alone or in combination of two or more types. These cells may be appropriately selected from known types depending on the intended use of the cells.

[0028] The origin of the cells is not particularly limited, and examples thereof include mammals, birds, amphibians, fish, insects, plants, microorganisms, etc. Specific examples of mammals and birds include humans, monkeys, chimpanzees, cows, horses, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, chickens, etc.

[0029] (Culture conditions) The cell culture is not particularly limited, and conventional means used in technical fields such as medical care, pharmaceuticals, quasi-drugs, cosmetics, food, and veterinary drugs, as well as in basic technical fields such as regenerative medicine and bioengineering, can be used.

[0030] The culture conditions are not particularly limited as long as they can bring the cultured cells into the desired state. Typical culture conditions include, for example, culture at 37°C, 5% CO using a prepared basal culture medium. ２ The cell culture period is not particularly limited as long as the cultured cells reach the desired state. The cell culture period is, for example, within 28 days, within 21 days, within 14 days, within 7 days, within 5 days, or within 3 days. When culturing for a long period of time, the medium may be replaced. The frequency and method of medium replacement are not particularly limited. In general, it is preferable to replace the medium every 1 to 7 days. It is particularly preferable to replace the medium every 1 to 5 days. At this time, the entire medium may be replaced, or a portion of the medium may be left and new medium may be added.

[0031] The density of the cells to be cultured is not particularly limited as long as it is suitable for the cells to be cultured, the culture vessel, and the intended use of the cultured cells. For example, ２ cells / cm ２ 1x10 or more ９ cells / cm ２ The lower limit of the cell density is more preferably 1 × 10 ３ cells / cm ２ More preferably, 5×10 ３ cells / cm ２ More preferably, 5 × 10 ４ cells / cm ２ On the other hand, the upper limit of the cell density is more preferably 1 × 10 ８ cells / cm ２ More preferably, 5 × 10 ７ cells / cm ２ Below, particularly preferably 1 × 10 ７ cells / cm ２ The density of the cells to be cultured is, for example, 5 × 10 ２ cells / cm ２ 1x10 or more ９ cells / cm ２ Below, 5 x 10 ２ cells / cm ２ 1x10 or more ８ cells / cm ２ Below, 5 x 10 ２ cells / cm ２Above 5×10 ７ Cells / cm ２ Below, 5×10 ２ Cells / cm ２ Above 1×10 ７ Cells / cm ２ Below, 1×10 ３ Cells / cm ２ Above 1×10 ９ Cells / cm ２ Below, 1×10 ３ Cells / cm ２ Above 1×10 ８ Cells / cm ２ Below, 1×10 ３ Cells / cm ２ Above 5×10 ７ Cells / cm ２ Below, 1×10 ３ Cells / cm ２ Above 1×10 ７ Cells / cm ２ Below, 5×10 ３ Cells / cm ２ Above 1×10 ９ Cells / cm ２ Below, 5×10 ３ Cells / cm ２ Above 1×10 ８ Cells / cm ２ Below, 5×10 ３ Cells / cm ２ Above 5×10 ７ Cells / cm ２ Below, 5×10 ３ Cells / cm ２ Above 1×10 ７ Cells / cm ２ Below, 5×10 ４ Cells / cm ２ Above 1×10 ９ Cells / cm ２ Below, 5×10 ４ Cells / cm ２ Above 1×10 ８ Cells / cm ２ Below, 5×10 ４ Cells / cm ２ Above 5×10 ７ Cells / cm ２ Below, 5×10 ４ Cells / cm ２ Above 1×10 ７ Cells / cm２ The following is the result.

[0032] (Culture Solution) The culture solution 30 is not particularly limited as long as it is suitable for the cells to be cultured. Examples of culture solution components include sugars, amino acids, vitamins, inorganic salts, trace metals, additives, etc. These culture solution components may be blended alone or in combination of two or more. These culture solution components may be appropriately selected from known components depending on the cells to be cultured, etc.

[0033] Specific examples of sugars include monosaccharides such as glucose, fructose, mannose, and galactose; disaccharides such as sucrose, sucralose, trehalose, maltose, and lactose; trisaccharides such as glucosylsucrose, lactosucrose, and raffinose; tetrasaccharides such as acarbose and maltotetraose; cyclodextrins; and oligosaccharides.

[0034] Specific examples of amino acids include L-glutamic acid, L-glutamine, L-arginine, L-cystine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-alanine, L-asparagine, L-aspartic acid, L-cysteine, and L-hydroxyproline.

[0035] Specific examples of vitamins include sodium L-ascorbate, L-ascorbic acid-diphosphate, choline, folic acid, niacin, biotin, pantothenic acid, pyridoxine, riboflavin, thiamine, thymidine, and vitamin B12.

[0036] Specific examples of inorganic salts include sodium chloride, sodium hydroxide, sodium sulfate, sodium phosphate, disodium hydrogen phosphate, sodium carbonate, sodium bicarbonate, potassium chloride, potassium hydroxide, potassium sulfate, potassium phosphate, dipotassium hydrogen phosphate, potassium carbonate, potassium bicarbonate, calcium chloride, calcium sulfate, calcium nitrate, calcium phosphate, calcium carbonate, magnesium chloride, magnesium sulfate, magnesium nitrate, magnesium phosphate, magnesium carbonate, etc. Specific examples of trace metals include iron sulfate, iron nitrate, copper sulfate, copper nitrate, zinc sulfate, etc.

[0037] Specific examples of additives include serum such as bovine serum, horse serum, and human serum; growth factors such as FGF2, EGF, HGF, VEGF, and PDGF; proteins such as albumin; antioxidants such as glutathione, ascorbic acid, and ascorbic acid derivatives; antibiotics such as penicillin and streptomycin; pH adjusters such as HEPES; organic acids such as lactic acid and propionic acid; lipids such as cholesterol; fatty acids such as linolenic acid; amines such as ethanolamine and putrescine; reducing agents such as mercaptoethanol and 3-mercapto-1,2-propanediol; thickeners such as sodium alginate, polyvinylpyrrolidone, carboxymethylcellulose, and pullulan; and pH indicators such as phenol red.

[0038] Examples of the culture medium 30 containing the above-mentioned culture medium components include AIM V medium, HFDM-1 Medium, equilibrium buffers such as Dulbecco's phosphate buffered saline (D-PBS) and Hank's balanced salt solution (HBSS), DMEM (Dulbecco's Modified Eagle Medium), EMEM (Eagle's Minimum Essential Medium), α-MEM (Minimum Essential Medium alpha Modification), IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), and Ham's F-10 medium, Ham's F-12 medium, Ham's F-12K medium, RPMI medium 1640, M-199 medium, L-15 medium, McCoy's 5A Medium, MCDB105 medium, MCDB107 medium, MCDB131 medium, MCDB153 medium, MCDB201 medium, NCTC109 medium, NCTC135 medium, Waymouth's MB752 / 1 medium, CMRL-1066 Examples of basal culture media include basal culture media such as BMOC-3 medium, Williams' medium E, Brinster's BMOC-3 medium, and E8 medium. These basal culture media may be used alone or in combination of two or more. Furthermore, culture media components may be added, removed, increased, or decreased in volume depending on the type and condition of the cells. These basal culture media may be appropriately selected from known media depending on the cells to be cultured, etc.

[0039] (Cell Sheet) The cell sheet 40 has a sheet structure in which cells are physically and functionally connected to one another via adhesion molecules, extracellular matrix, etc. The cell sheet 40 may have a single-layer structure consisting of one cell layer, or a laminated structure consisting of two or more cell layers. The laminated structure is not particularly limited, but examples include multi-layer structures such as two-layer, three-layer, four-layer, and five-layer structures.

[0040] When the cell sheet 40 has a multilayer structure, the multilayer structure may be obtained when cultured on the culture carrier substrate 10, or it may be obtained by stacking cell sheets with a single layer structure. In particular, a cell sheet with a multilayer structure can be obtained by preparing a plurality of the culture carrier substrates 50 with cell sheets of the present invention, overlaying one cell sheet on another cell sheet, and peeling off the culture carrier substrate from the other cell sheet.

[0041] The thickness of the cell sheet 40 is not particularly limited, but is, for example, 0.001 mm or more and 2.0 mm or less. The lower limit of the thickness of the cell sheet 40 is more preferably 0.01 mm or more, even more preferably 0.03 mm or more, and particularly preferably 0.05 mm or more. On the other hand, the upper limit of the thickness of the cell sheet 40 is more preferably 1.5 mm or less, even more preferably 1.2 mm or less, and particularly preferably 1.0 mm or less. The thickness of the cell sheet 40 may be, for example, 0.001 mm to 2.0 mm, 0.001 mm to 1.5 mm, 0.001 mm to 1.2 mm, 0.001 mm to 1.0 mm, 0.01 mm to 2.0 mm, 0.01 mm to 1.5 mm, 0.01 mm to 1.2 mm, 0.01 mm to 1.0 mm, 0.03 mm to 2.0 mm, 0.03 mm to 1.2 mm, 0.03 mm to 1.0 mm, 0.05 mm to 2.0 mm, 0.05 mm to 1.5 mm, 0.05 mm to 1.2 mm, or 0.05 mm to 1.0 mm. By keeping the thickness of the cell sheet 40 within the above ranges, high cell activity within the cell sheet 40 and excellent shape retention ability, which are advantageous for cell transplantation, can be achieved.

[0042] The area of ​​the cell sheet 40 is not particularly limited, but may be, for example, 0.3 cm ２ More than 1000cm ２ The lower limit of the area of ​​the cell sheet 40 is more preferably 0.6 cm ２ More preferably, 1.6 cm or more ２ More than 8 cm, especially preferred ２ More than 3 cm ２ More than 6cm ２ or more or 10 cm ２On the other hand, the upper limit of the area of ​​the cell sheet 40 is more preferably 900 cm ２ More preferably, 800 cm or less ２ Particularly preferably 500 cm ２ is less than or equal to 100 cm ２ Below, 50cm ２ Less than or equal to 20cm ２ For example, the area of ​​the cell sheet 40 may be 0.6 cm ２ More than 900cm ２ Below, 1.6cm ２ More than 800cm ２ Below, 3cm ２ More than 500cm ２ Below, 6cm ２ More than 100cm ２ Below, 8cm ２ More than 50cm ２ Less than or equal to 10 cm ２ 20cm or more ２ Conventionally, when a cell sheet is transplanted alone, the strength of the cell sheet is low, and therefore there is a high possibility that the cell sheet will break during transportation if it is large in area. However, in the present disclosure, the cell sheet 40 is supported on a culture carrier substrate, and therefore the cell sheet can be prevented from breaking during transplantation. Therefore, the size of the cell sheet 40 can be reduced to 8 cm. ２ The cell sheet can be made larger than this. Alternatively, a larger cell sheet can be prepared and adjusted to the size of the affected area as needed. Although the shape of the culture carrier substrate is shown as a circle in FIG. 1, it may be any other suitable shape, such as a regular polygon such as a square, a regular pentagon, a regular hexagon, or a regular octagon, or an oval or rectangle.

[0043] <Culture carrier substrate> The culture carrier substrate 10 of this embodiment is used both as a cell sheet scaffold for culturing cells to form a cell sheet 40 and as a cell sheet support for transporting the cultured cell sheet 40, and is a substrate that is detached from the cell sheet after being attached to the transplantation site of the cell sheet.

[0044] The area of ​​the culture carrier substrate 10 is not particularly limited, but is preferably smaller than the culture vessel and equal to or larger than the cell sheet.２ More than 1000cm ２ The lower limit of the area of ​​the culture carrier substrate 10 is preferably 0.6 cm or less. ２ More preferably, 1.6 cm ２ More than 8 cm, especially preferred ２ More than 3 cm ２ More than 6cm ２ or more or 10 cm ２ On the other hand, the upper limit of the area of ​​the culture carrier substrate 10 is more preferably 900 cm ２ More preferably, 800 cm or less ２ Particularly preferably 500 cm ２ is less than or equal to 100 cm ２ Below, 50cm ２ Less than or equal to 20cm ２ If the area of ​​the culture carrier substrate 10 is larger than the culture vessel, it may be impossible to obtain a cell sheet. For example, the area of ​​the culture carrier substrate 10 may be 0.6 cm or less. ２ More than 900cm ２ Below, 1.6cm ２ More than 800cm ２ Below, 3cm ２ More than 500cm ２ Below, 6cm ２ More than 100cm ２ Below, 8cm ２ More than 50cm ２ Less than or equal to 10 cm ２ 20cm or more ２ The following is the result.

[0045] The substrate thickness of the culture carrier substrate 10 is not particularly limited, but is preferably 5 μm or more and 250 μm or less. The lower limit of the substrate thickness is more preferably 6 μm or more, even more preferably 8 μm or more, 10 μm or more, or 12 μm or more. On the other hand, the upper limit of the substrate thickness is more preferably 50 μm or less, 30 μm or less, 25 μm or less, and even more preferably 20 μm or less. For example, the substrate thickness of the culture carrier substrate 10 is 6 μm or more and 50 μm or less, 8 μm or more and 30 μm or less, or 10 μm or more and 20 μm or less. By setting the substrate thickness at or above the lower limit, the culture carrier substrate 10 can be prevented from breaking or curling during transportation, improving handleability. By setting the substrate thickness at or below the upper limit, the conformability to the affected area during transplantation can be improved. In particular, a substrate thickness of 20 μm or less can improve conformability to affected areas with higher curvatures, and can be applied to, for example, areas where internal organs have been sutured during surgery.

[0046] Furthermore, from the viewpoint of functioning as a free-standing membrane during culture, the lower limit of the substrate thickness of the culture carrier substrate 10 is preferably greater than 10 μm, more preferably 11 μm or greater, and even more preferably 12 μm or greater. Thus, by using a culture carrier substrate 10 with a substrate thickness greater than 10 μm, it is possible to produce a cell sheet 40 during the cell culture process without fixing or adhering the culture carrier substrate 10 to the inside of the culture vessel 20. A free-standing membrane is preferably a membrane that maintains the planar state of the culture surface 12 of the culture carrier substrate 10 even in the culture solution 30, and more preferably maintains the planar state even after the cell sheet 40 is formed on the culture surface 12. Because the planar state of the culture surface 12 is maintained, the culture carrier substrate 10 does not curl, improving cell culture characteristics in the culture solution 30 without adhering. Furthermore, this free-standing membrane may be capable of retaining a certain degree of sheet shape when one end is pinched and lifted with tweezers. In this case, the area of ​​the culture carrier substrate 10 is 1.6 cm. ２ It is preferable that this is equal to or greater than this.

[0047] The culture surface 12 of the culture carrier substrate 10 is preferably subjected to a hydrophilic treatment. This improves cell adhesion. Examples of hydrophilic treatment include UV ozone treatment and plasma treatment. Among these, it is preferable to use a culture carrier substrate 10 whose culture surface 12 is plasma-treated. In this specification, a hydrophilized surface refers to a surface whose upper limit of the water droplet contact angle θ measured by the θ / 2 method is 70° or less. On the other hand, the lower limit of the water droplet contact angle θ on the hydrophilized surface is preferably 3° or more, but is not limited thereto. The water droplet contact angle θ is measured by placing 2 μl of pure water on the substrate at 25°C and measuring the angle between the water droplet and the substrate using a contact angle meter using the θ / 2 method in accordance with ISO 19403-2:2017. Furthermore, the lower limit of the surface roughness Ra of the hydrophilized culture surface is 0.3 nm or more, preferably 0.5 nm or more, and the upper limit is 100 nm or less, preferably 10 nm or less, and more preferably 2 nm or less. The surface roughness Ra here refers to the arithmetic mean roughness in a square area with sides of 100 nm, obtained by using surface shape data measured with an atomic force microscope (AFM).

[0048] Examples of materials constituting the culture carrier substrate 10 include resin materials such as polyether ether ketone (PEEK), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polystyrene (PS), polycarbonate (PC), modified polyphenylene ether (mPPE), polyphenylene sulfide (PPS), polysulfone (PSU), polyarylate (PAR), liquid crystal polymer (LCP), polyethylene (PE), and polypropylene (PP). An example of the culture carrier substrate 10 may be a film containing at least one of these resin materials as a primary component, preferably a polyether ether ketone film or a polyethylene terephthalate film, and more preferably a polyether ether ketone film. The culture carrier substrate 10 preferably includes one or more resin layers made of the above resin materials. The two or more resin layers may contain different materials. The culture carrier substrate 10 may be composed of a resin substrate including a resin layer made of the above material, or a laminate substrate including a resin layer made of the above material and an inorganic layer made of another material. However, the resin layer does not need to include a layer formed by chemical vapor deposition such as parylene or a wet coating layer. Furthermore, the inorganic layer in the laminate substrate preferably does not include a glass layer, and may include a metal foil. Furthermore, the culture carrier substrate 10 preferably does not include a nonwoven fabric or a fiber substrate on the culture surface 12 side. Considering the low linear expansion coefficient, solvent resistance, and heat resistance, a culture carrier substrate 10 containing PEEK is preferable compared to one containing PET. Furthermore, the culture carrier substrate 10 is preferably made of a material with a higher specific gravity than the culture solution 30.

[0049] When the culture surface 12 of the culture carrier substrate 10 is measured with a laser microscope, there are three or fewer holes with a diameter of 1 μm to 100 μm and a depth of 0.5 μm to 100 μm within a 100 μm square area. The diameter of the holes is, for example, 1 μm to 100 μm, preferably 2 μm to 50 μm, and more preferably 3 μm to 30 μm. The depth of the holes is, for example, 0.5 μm to 100 μm, preferably 1 μm to 50 μm, and more preferably 2 μm to 20 μm. The combinations of the ranges of the hole diameter and the hole depth are, for example, a hole diameter of 1 μm or more and 100 μm or less and a hole depth of 0.5 μm or more and 100 μm or less, a hole diameter of 1 μm or more and 100 μm or less and a hole depth of 1 μm or more and 50 μm or less, a hole diameter of 1 μm or more and 100 μm or less and a hole depth of 2 μm or more and 20 μm or less, a hole diameter of 2 μm or more and 50 μm or less and a hole depth of 0.5 μm or more and 100 μm or less, a hole diameter of 1 μm or more and 10 ... The hole diameter is 2 μm to 50 μm and the hole depth is 1 μm to 50 μm, the hole diameter is 2 μm to 50 μm and the hole depth is 2 μm to 20 μm, the hole diameter is 3 μm to 30 μm and the hole depth is 0.5 μm to 100 μm, the hole diameter is 3 μm to 30 μm and the hole depth is 1 μm to 50 μm, or the hole diameter is 3 μm to 30 μm and the hole depth is 2 μm to 20 μm. The upper limit of the porosity of the culture carrier substrate 10 is, for example, 15% or less, preferably 10% or less, and more preferably 5% or less. The lower limit of the porosity of the culture carrier substrate 10 is not particularly limited, but may be 0% or more. By limiting the number of holes in the culture surface 12 to three or less and / or by limiting the porosity of the culture carrier substrate 10 to the upper limit, the adhesion to the cell sheet 40 can be made appropriate. The presence or absence of holes may be measured by measuring the surface of the resin layer formed on the culture surface 12 side of the culture carrier substrate 10. The porosity is calculated from the theoretical density and the measured density. Specifically, it is calculated using the formula: porosity = {1 - (actual density / theoretical density)} × 100. Furthermore, the culture surface 12 of the culture carrier substrate 10 may be configured without including a temperature-responsive polymer. This prevents a decrease in adhesion between the cell sheet 40 and the culture carrier substrate 10 in low-temperature environments, such as during cryopreservation.

[0050] A temperature-responsive polymer is a material that exhibits cell adhesiveness at the temperature used for cell culture, and exhibits cell non-adhesiveness by changing the temperature from that temperature, making it possible to easily detach a cell sheet. The temperature range in which the temperature-responsive polymer exhibits cell adhesiveness is preferably 10°C to 45°C, particularly 33°C to 40°C, because this allows stable cell culture. Furthermore, the temperature range in which the temperature-responsive polymer exhibits cell non-adhesiveness is preferably 1°C to 36°C, particularly 4°C to 32°C, because this reduces damage to the detachment of the cell sheet. Specific examples of materials constituting the temperature-responsive polymer include temperature-responsive polymers such as poly-N-isopropylacrylamide (PNIPAAm), poly-N-n-propylacrylamide, poly-N-n-propylmethacrylamide, poly-N-ethoxyethylacrylamide, poly-N-tetrahydrofurfurylacrylamide, poly-N-tetrahydrofurfurylmethacrylamide, and poly-N,N-diethylacrylamide, and among these, PNIPAAm, poly-N-n-propylmethacrylamide, and poly-N,N-diethylacrylamide are preferred.

[0051] Before being used for culturing a cell sheet, the culture carrier substrate 10 can be packaged in a packaging material. By using a sealed package, the culture carrier substrate 10 can be stored and distributed without soiling the culture surface. This package can also be sterilized. Sterilization methods include electron beam sterilization, gamma ray sterilization, EOG sterilization, and high-pressure steam sterilization.

[0052] Examples of packaging materials include aluminum, polyethylene terephthalate, ionomer, polyethylene, polyvinyl chloride, polyvinylidene chloride, polyvinyl alcohol, polypropylene, polyester, polycarbonate, polystyrene, polyacrylonitrile, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, ethylene-methacrylic acid copolymer, perfluoroalkoxy fluororesin, nylon, cellophane, and paper. These may be used alone or in combination of two or more. When sterilization requiring gas permeability, such as EOG sterilization or high-pressure steam sterilization, is performed, packaging materials combining paper and the above materials are preferably used. The packaging material may be multi-packaged, and additional packaging may be added after sterilization. Preferably, the outermost portion of the package is a resin film-like packaging material.

[0053] Culturing a cell sheet 40 on a culture carrier substrate 10 results in a cell sheet-attached culture carrier substrate 50 comprising a laminate of the cell sheet 40 and the culture carrier substrate 10. In the cell sheet-attached culture carrier substrate 50, the cell sheet 40 covers, for example, 50% to 100% of the surface on the culture surface 12 side where cells can be deposited. When a method of fixing a portion of the culture surface 12 of the culture carrier substrate 10 with a weight or a method of fixing the position of the culture carrier substrate 10 with an instrument is employed, the cell deposition area refers to an area where cells can be deposited and where there is no contact with a weight or instrument. Furthermore, when the culture surface 12 of the culture carrier substrate 10 is hydrophilized, the cell deposition area refers to the hydrophilized area. The lower limit of the coverage area of ​​the cell sheet 40 is preferably 70% or more, more preferably 90% or more. The upper limit of the coverage area of ​​the cell sheet 40 may be 100% or less, 99% or less, or 98% or less. The coverage area of ​​the cell sheet 40 is, for example, 50% to 100%, 50% to 99%, 50% to 98%, 70% to 100%, 70% to 99%, 70% to 98%, 90% to 100%, 90% to 99%, or 90% to 98%. The area of ​​the culture carrier substrate 10 is preferably equal to or larger than the area of ​​the cell-accumulating region of the cell sheet 40. By setting the coverage area to be equal to or larger than the lower limit, the healing effect can be improved.

[0054] Furthermore, by forming a cell sheet on a culture carrier substrate, the possibility of the cell sheet being torn when removed from a culture vessel or cryopreservation container can be reduced, making it easier to remove. Furthermore, after attaching the cell sheet to the transplantation site, the cell sheet can be easily peeled off from the culture carrier substrate. Because the cell sheet is easier to handle, it becomes possible to produce large-sized cell sheets.

[0055] <Method for manufacturing a culture carrier substrate with a cell sheet> An example of a method for manufacturing a culture carrier substrate with a cell sheet 50 of this embodiment may include, for example, a culture step in which a plurality of cells, a culture medium (culture solution 30), and a culture carrier substrate 10 are introduced into a culture vessel 20, and a cell sheet 40 is cultured on the culture surface 12 of the culture carrier substrate 10, and a removal step in which the culture carrier substrate with a cell sheet 50 is removed from the culture solution 30.

[0056] The cell sheet 40 of this embodiment is detached from the cell sheet-attached culture carrier substrate 50 and can be used for treatments such as cell transplantation therapy, prevention, etc. The detachment method is not particularly limited, and any of the usual means used in technical fields such as medicine, pharmaceuticals, quasi-drugs, cosmetics, food, and veterinary medicine, as well as basic technical fields such as regenerative medicine and bioengineering, can be used. Examples of detachment methods include physical treatment.

[0057] <Cryopreservation Method> As shown in FIG. 1( c), an example of the cryopreservation method of this embodiment includes a cooling step of cooling a cell sheet-attached culture carrier substrate 50, which includes a culture carrier substrate 10 inside a culture vessel 20 and a cell sheet 40 formed on the surface (culture surface 12) of the culture carrier substrate 10, in a cryopreservation solution 60. The culture vessel may be frozen with a culture lid (not shown) or a lid (not shown) that prevents liquid leakage and microbial contamination. In FIG. 1( c), the cell sheet-attached culture carrier substrate 50 is cryopreserved inside the culture vessel 20. However, the cell sheet-attached culture carrier substrate 50 may also be transferred to a cryopreservation container separate from the culture vessel 20 for cryopreservation. The cryopreservation container is not particularly limited, and may be, for example, the one exemplified for the culture vessel 20.

[0058] In the cooling step, the cell sheet-attached culture carrier substrate 50 can be configured in various ways. As shown in Figures 3(a) and 3(b), the cell sheet-attached culture carrier substrate 50 may have a structure in which a culture carrier substrate 10 is formed on one side of a cell sheet 40. Alternatively, as shown in Figure 3(c), the cell sheet 40 may be sandwiched between two culture carrier substrates 10a and 10b on both sides. In Figure 3(a), the culture carrier substrate 10 is placed facing the bottom of the culture vessel 20. In Figure 3(b), the cell sheet 40 is placed facing the bottom of the culture vessel 20. In Figure 3(c), the culture carrier substrates 10a and 10b may be made of the same material or different materials. For example, both culture carrier substrates 10a and 10b may be culture carrier substrates containing PEEK, or one may be a culture carrier substrate containing PEEK and the other may be a culture carrier substrate not containing PEEK.

[0059] (Cryopreservation Solution) The cryopreservation solution 60 is a solution for reducing cell damage due to cryopreservation. There are no particular limitations on the cryopreservation solution 60 as long as it is suitable for cryopreserving cells, but it may contain culture solution components contained in culture solution, cryoprotectants, etc. The culture solution components, such as sugars, amino acids, vitamins, inorganic salts, trace metals, and additives, may be any that satisfy the explanation in the above section (Culture Solution). Furthermore, it is preferable that the cryopreservation solution have a solidification onset temperature in the range of -15°C or higher and -5°C or lower.

[0060] Cryoprotectants are substances used to reduce damage to cells caused by freezing and thawing during cryopreservation. Examples of cryoprotectants include cell-impermeant cryoprotectants and cell-permeant cryoprotectants. Specific examples of cell-impermeant cryoprotectants include albumin, sucrose, trehalose, dextran, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and polylysine. Specific examples of cell-permeant cryoprotectants include dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, and propanediol. These cryoprotectants may be formulated alone or in combination of two or more. These cryoprotectants may be appropriately selected from known cryoprotectants depending on the type of cell, the composition of the cryopreservation solution, and the like.

[0061] Examples of commercially available cryopreservation solutions that do not contain DMSO include Stem Cell Banker (registered trademark) DMSO-free GMP grade (Nihon Zenyaku Kogyo Co., Ltd.), Bambanker (registered trademark) DMSO-free (CG Lymphotec Co., Ltd.), Cryoscarless (registered trademark) DMSO-free (BioVerde Co., Ltd.), Stem Cell Keep (BioVerde Co., Ltd.), CryoNovo (registered trademark) X12 (Akron BioProducts LCC), CryoNovo (registered trademark) P24 (Akron BioProducts LCC), DMSO-free cell cryopreservation solution for cryopreservation of human ES / iPS cells (ReproCell Co., Ltd.), Cell Reservoir One (Nacalai Tesque Co., Ltd.), ThelioKeep (registered trademark: BioVerde Co., Ltd.), and Cellvation (registered trademark: Protide Co., Ltd.). Examples include ReproCryo RM (ReproCell Pharmaceuticals), and SOFORO Cryo (Saraya). In addition, examples of commercially available cryopreservation solutions containing DMSO include Stem Cell Banker (registered trademark) GMP Grade (Nippon Zenyaku Kogyo Co., Ltd.), Stem Cell Banker (registered trademark) EX GMP Grade (Nippon Zenyaku Kogyo Co., Ltd.), Bambanker (registered trademark) hRM (CG Lymphotec Co., Ltd.), Bambanker (registered trademark) (CG Lymphotec Co., Ltd.), iStock (CG Lymphotec Co., Ltd.), CryoStor CS5 (Charles River Laboratories Cell Solutions, Inc.), and CryoStor CS10 (Charles River Laboratories Cell Solutions, Inc.).

[0062] (Cooling step) In the cooling step, the cell sheet-attached culture carrier substrate 50 is preferably cooled in a cryopreservation solution using a non-throughflow cooling device. The cell sheet 40 on an appropriate culture carrier substrate 10 can be cooled at a uniform temperature using a non-throughflow cooling device, which minimizes damage to the cells and suppresses a decrease in cell activity.

[0063] In the cooling step, the cooling rate at 0 to -5°C is, for example, 0.1°C / min to 15°C / min, preferably 0.25°C / min to 12.5°C / min, and more preferably 0.5°C / min to 10°C / min. By setting the cooling rate at or above the lower limit, unnecessary contact time between the liquid cryoprotectant and the cells can be reduced. By setting the cooling rate at or below the upper limit, intracellular ice crystal formation can be suppressed.

[0064] In the cooling step, the temperature of the freezing treatment is not particularly limited as long as it can freeze the cultured cells and the cryopreservation solution. The freezing temperature is, for example, −196°C or higher and −25°C or lower. The lower limit is more preferably −180°C or higher, even more preferably −160°C or higher, and particularly preferably −150°C or higher. On the other hand, the upper limit is more preferably −25°C or lower, even more preferably −30°C or lower, and particularly preferably −35°C or lower. The temperature for the freezing treatment is, for example, −196°C or higher and −25°C or lower, −196°C or higher and −30°C or lower, −196°C or higher and −35°C or lower, −180°C or higher and −25°C or lower, −180°C or higher and −30°C or lower, −180°C or higher and −35°C or lower, −160°C or higher and −25°C or lower, −160°C or higher and −30°C or lower, −160°C or higher and −35°C or lower, −150°C or higher and −25°C or lower, −150°C or higher and −30°C or lower, or −150°C or higher and −35°C or lower.

[0065] The cooling device used for the freezing process is not particularly limited, and examples include quick-freezing devices and cryogenic refrigeration devices. As a cooling device, a freezing device that does not come into contact with a heat transfer device and sprays cold air onto the culture vessel from multiple directions, preferably all directions, rather than one direction, to freeze the culture vessel and cells, is preferable from the viewpoint of freezing cells at a uniform temperature and increasing the survival rate of the cells after thawing. Specific examples of freezing devices that spray cold air onto the culture vessel include cooling devices that cool the object to be cooled by circulating cold air using a cooling fan, such as the non-through-flow cooling device equipped with a cooling fan disclosed in Japanese Patent Laid-Open No. 2005-127666. The term "non-through-flow system" refers to a system in which the majority of the through-flow air from the object to be cooled does not pass through (through-flow) the cooler.

[0066] (Freezing and preservation step) The freezing and preservation method preferably includes a freezing and preservation step of preserving the cell sheet-attached culture carrier substrate 50 after the cooling step, for example, at a temperature of −196 to −60° C., preferably −180 to −65° C., and more preferably −150 to −80° C. This allows the cell sheet to be stably maintained for a long period of time. The temperature in the cooling step may be the set temperature of the cooling device.

[0067] The cryopreservation method is not particularly limited as long as the cells can be stably cryopreserved. Examples of cryopreservation methods include contact with the liquid or gas phase of a coolant, and use of an ultra-low temperature freezer. A preferred cryopreservation method is contact with the liquid or gas phase of a coolant from the viewpoint of temperature. Examples of coolants include liquid nitrogen, liquid ethane, liquid propane, liquid helium, and dry ice.

[0068] The cryopreservation temperature is not particularly limited as long as the cells can be stably cryopreserved. The cryopreservation temperature may be, for example, within any range of −196°C or higher to −60°C or lower, −196°C or higher to −134°C or lower, or −134°C or higher to −60°C or lower. The cryopreservation temperature may be the surface temperature of the cell sheet 40 to be frozen. The surface temperature can be measured, for example, using a K thermocouple.

[0069] The frozen culture carrier substrate 50 with a cell sheet includes the culture carrier substrate 10 and the cell sheet 40 formed on the surface (culture surface 12) of the culture carrier substrate 10. In the culture carrier substrate 50 with a cell sheet, the culture carrier substrate 10 and the cell sheet 40 are in a frozen state.

[0070] In the above-mentioned frozen product, the culture carrier substrate 10 and the cell sheet 40 are frozen at the above-mentioned cryopreservation temperature, specifically, preferably at or below −60° C., more preferably at or below −135° C. Alternatively, the frozen product may be frozen at a temperature in the range of −134° C. to −60° C. Note that the frozen product is frozen at or above −196° C.

[0071] The frozen cell sheet-attached culture carrier substrate frozen inside a culture vessel or cryopreservation container can be packaged in a packaging material either while frozen in the culture vessel or cryopreservation container or after being removed from the container. The sealed packaging prevents microbial contamination of the cell sheet and allows the cell sheet to be stored and distributed without contamination. For example, a frozen product package can be obtained by packaging a cell sheet-attached culture carrier substrate and a container containing a cryopreservation solution in a film-like packaging material and freezing the container in a sealed state. Preferred packaging materials include aluminum, polyethylene terephthalate, ionomer, polyethylene, polyvinylidene chloride, polyvinyl alcohol, polypropylene, polyester, polycarbonate, polyacrylonitrile, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, ethylene-methacrylic acid copolymer, polyimide, fluororesins such as perfluoroalkoxy fluororesins and tetrafluoroethylene-hexafluoropropylene copolymer (FEP), and nylon. These materials may be used alone or in combinations of two or more.

[0072] (Thawing Step) When the transplantation method of this embodiment includes the above-mentioned cryopreservation method, the cryopreservation method further includes a thawing step of thawing the culture carrier substrate 50 with the cell sheet attached.

[0073] The thawing method is not particularly limited, and conventional means used in technical fields such as medicine, pharmaceuticals, quasi-drugs, cosmetics, food, and veterinary medicine, as well as basic technical fields such as regenerative medicine and bioengineering, can be used. Thawing methods include, for example, using a water bath, incubator, hot plate, defroster, immersion in a melting liquid at a temperature higher than the freezing temperature, or leaving in an environment at a temperature higher than the freezing temperature. The environmental temperature in contact with the frozen material during thawing is not particularly limited, as long as it is higher than the freezing temperature and lower than 50°C. The upper limit of the environmental temperature is, for example, 49°C or lower, preferably 45°C or lower, and more preferably 40°C or lower. Meanwhile, the lower limit of the environmental temperature is, for example, 0°C or higher, preferably 5°C or higher, more preferably 10°C, and even more preferably 15°C or higher. The range of environmental temperatures in contact with the frozen material during thawing is, for example, 0°C to 49°C, 0°C to 45°C, 0°C to 40°C, 5°C to 49°C, 5°C to 45°C, 5°C to 40°C, 10°C to 49°C, 10°C to 45°C, 10°C to 40°C, 15°C to 49°C, 15°C to 45°C, and 15°C to 40°C. Thawing temperatures of 50°C or higher are not preferred due to the possibility of thermal damage to cells. Furthermore, thawing may be temporarily performed in an environment at a temperature below the freezing point. For example, frozen material stored at -80°C can be exposed to an environmental temperature of -30°C and then thawed at an environmental temperature above the freezing point. The time required to thaw a frozen material is not particularly limited as long as freezing-induced damage to cells does not occur. Typically, thawing can be performed without problems within a time period of more than 10 seconds and less than 60 minutes. The lower limit of the time required to thaw a frozen product is sufficient if it is more than 10 seconds, preferably 20 seconds or more, more preferably 30 seconds or more, and even more preferably 1 minute or more, and the upper limit is sufficient if it is 60 minutes or less, preferably 50 minutes or less, more preferably 40 minutes or less, and even more preferably 30 minutes or less.For example, the time required for thawing is more than 10 seconds and less than 60 minutes, more than 10 seconds and less than 50 minutes, more than 10 seconds and less than 40 minutes, more than 10 seconds and less than 30 minutes, 20 seconds and less than 60 minutes, 20 seconds and less than 50 minutes, 20 seconds and less than 40 minutes, 20 seconds and less than 30 minutes, 30 seconds and less than 60 minutes, 30 seconds and less than 50 minutes, 30 seconds and less than 40 minutes, 30 seconds and less than 30 minutes, 1 minute and less than 60 minutes, 1 minute and less than 50 minutes, 1 minute and less than 40 minutes, or 1 minute and less than 30 minutes. If thawing occurs too quickly, the frozen material may crack due to thermal shock caused by the temperature difference, potentially damaging the cell sheet. If thawing occurs too slowly, water molecules may recrystallize under sub-zero conditions, causing ice crystals to grow larger and causing serious damage to the cells, which is undesirable. During the thawing time, the environmental temperature may be set to a constant temperature or may be set to fluctuate, such as by gradually increasing.

[0074] The thawing solution is not particularly limited as long as it does not damage the cultured cells. Examples of components contained in the thawing solution include sucrose, glucose, maltose, trehalose, and fructose. The thawing solution may also contain the components described above in the section (Culture Solution).

[0075] The temperature of the melting liquid is not particularly limited as long as it is higher than the freezing temperature. The temperature of the melting liquid is, for example, 0°C or higher and 45°C or lower. The lower limit is more preferably 4°C or higher, even more preferably 25°C or higher, and particularly preferably 28°C or higher. On the other hand, the upper limit is more preferably 40°C or lower, even more preferably 39°C or lower, and particularly preferably 38°C or lower. The temperature of the melting liquid is, for example, 0°C or higher and 45°C or lower, 0°C or higher and 40°C or lower, 0°C or higher and 39°C or lower, 0°C or higher and 38°C or lower, 4°C or higher and 45°C or lower, 4°C or higher and 40°C or lower, 4°C or higher and 39°C or lower, 4°C or higher and 38°C or lower, 25°C or higher and 45°C or lower, 25°C or higher and 40°C or lower, 25°C or higher and 39°C or lower, 25°C or higher and 38°C or lower, 28°C or higher and 45°C or lower, 28°C or higher and 40°C or lower, 28°C or higher and 39°C or lower, and 28°C or higher and 38°C or lower.

[0076] The thawed cell sheet 40 and culture carrier substrate 10 (cell sheet-attached culture carrier substrate 50) may be washed with a cell washing solution immediately after the thawing process, if necessary. The cell washing solution is not particularly limited and may contain the components described in the above section (Culture Solution). The temperature of the cell washing solution is not particularly limited. For example, the temperature of the cell washing solution is 0°C or higher and 45°C or lower. The lower limit is more preferably 4°C or higher, even more preferably 25°C or higher, and particularly preferably 28°C or higher. On the other hand, the upper limit is more preferably 40°C or lower, even more preferably 39°C or lower, and particularly preferably 38°C or lower. The number of times the cultured cells are washed is not particularly limited and may be one or multiple times (e.g., two, three, four, five, etc.).

[0077] In the present disclosure, since the culture surface 12 does not contain a temperature-responsive polymer, it is possible to prevent the cell sheet 40 from peeling off from the culture carrier substrate 10 in a low-temperature environment such as a cryopreservation process, and it is possible to obtain a culture carrier substrate 50 with a cell sheet that maintains the adhesiveness between the cell sheet and the film even after undergoing the cryopreservation process and the thawing process.

[0078] Although the embodiments of the present invention have been described above, these are merely illustrative examples, and various other configurations may be employed. Furthermore, the present invention is not limited to the above-described embodiments, and modifications, improvements, and the like are included within the scope of achieving the object of the present invention. Examples of Reference Forms A to J are listed below. <A. Culture Carrier Substrate> Reference Form A of the culture carrier substrate includes the following 1. to 13. 1. A culture carrier substrate used for transporting a cell sheet cultured on a culture surface, wherein the culture carrier substrate has a base thickness of 5 μm to 250 μm. 2. The culture carrier substrate described in 1., wherein the culture carrier substrate has a base thickness of more than 10 μm and functions as a free-standing membrane without being adhered to the inside of a culture vessel during cell sheet culture. 3. The culture carrier substrate described in 1. or 2., wherein the culture surface has been subjected to a hydrophilization treatment. 4. The culture carrier substrate described in 3., wherein the hydrophilization treatment is UV ozone treatment or plasma treatment. 5. The culture carrier substrate according to any one of 1. to 4., wherein the culture carrier substrate is a polyether ether ketone film or a polyethylene terephthalate film. 6. The culture carrier substrate according to any one of 1. to 5., wherein the culture surface does not contain a temperature-responsive polymer. 7. The culture carrier substrate according to any one of 1. to 6., wherein the area of ​​the culture carrier substrate is 0.3 cm ２ More than 1000cm ２A culture carrier substrate that is the following: 8. A culture carrier substrate according to any one of 1. to 7., wherein the culture carrier substrate comprises one or more resin layers made of a resin material. 9. A culture carrier substrate according to any one of 1. to 8., wherein, when the culture surface is measured with a laser microscope, there are three or fewer holes with a diameter of 1 μm to 100 μm and a depth of 0.5 μm to 100 μm in at least one 100 μm square observation area. 10. A culture carrier substrate according to any one of 1. to 9., wherein the porosity is 15% or less. 11. A culture carrier substrate according to any one of 1. to 10., wherein the water contact angle on the culture surface is 70° or less. 12. A culture carrier substrate according to any one of 1. to 11., wherein the culture carrier substrate is used for cryopreserving a cell sheet formed on the culture surface. 13. 1. to 12. The culture carrier substrate according to any one of the above, which is used for attaching a cell sheet formed on the culture surface to a transplantation site. <B. Cell Sheet-Attached Culture Carrier Substrate> Reference form B of the cell sheet-attached culture carrier substrate includes the following 1. to 21.: 1. A culture carrier substrate with a cell sheet, which has a laminate of a culture carrier substrate and a cell sheet, wherein the cell sheet covers at least 50% of the surface on the culture surface side of the culture carrier substrate. 2. A culture carrier substrate with a cell sheet according to 1., wherein the culture carrier substrate has a substrate thickness of 5 μm or more and 250 μm or less. 3. A culture carrier substrate with a cell sheet according to 1. or 2., wherein the culture surface has been subjected to a hydrophilization treatment. 4. A culture carrier substrate with a cell sheet according to 3., wherein the hydrophilization treatment is a UV ozone treatment or a plasma treatment.5. A culture carrier substrate with a cell sheet according to any one of 1. to 4., wherein the culture carrier substrate is a polyether ether ketone film or a polyethylene terephthalate film. 6. A culture carrier substrate with a cell sheet according to any one of 1. to 5., wherein the culture surface does not contain a temperature-responsive polymer. 7. A culture carrier substrate with a cell sheet according to any one of 1. to 6., wherein the area of ​​the culture carrier substrate is 0.3 cm. ２ More than 1000cm ２A cell sheet-attached culture carrier substrate which is one of the following: 8. A cell sheet-attached culture carrier substrate according to any one of 1. to 7., wherein the culture carrier substrate comprises one or more resin layers made of a resin material. 9. A cell sheet-attached culture carrier substrate according to any one of 1. to 8., wherein, when the culture surface is measured with a laser microscope, there are three or fewer holes with a diameter of 1 μm to 100 μm and a depth of 0.5 μm to 100 μm in at least one 100 μm square observation area. 10. A cell sheet-attached culture carrier substrate according to any one of 1. to 9., wherein the porosity is 15% or less. 11. A cell sheet-attached culture carrier substrate according to any one of 1. to 10., wherein the water contact angle on the culture surface is 70° or less. 12. A cell sheet-attached culture carrier substrate according to any one of 1. to 11. 1. A culture carrier substrate with a cell sheet according to any one of 1. to 12., used for transporting a cell sheet cultured on a culture surface. 13. A culture carrier substrate with a cell sheet according to any one of 1. to 12., used for cryopreserving a cell sheet formed on the culture surface. 14. A culture carrier substrate with a cell sheet according to any one of 1. to 13., used for attaching a cell sheet formed on the culture surface to a transplant site. 15. A culture carrier substrate with a cell sheet according to any one of 1. to 14., used for cell transplantation therapy. 16. A culture carrier substrate with a cell sheet according to any one of 1. to 15., wherein the cell transplantation therapy includes at least one of suppressing and preventing the onset and recurrence of symptoms associated with loss, dysfunction, and dysfunction of cells, tissues, and organs.17. A cell sheet-attached culture carrier substrate according to any one of 1. to 16., used for treating at least one of spinal cord injury, knee joint cartilage injury, ischemic heart disease, age-related macular degeneration, corneal epithelial stem cell exhaustion, aplastic anemia, severe limb ischemia, intractable skin ulcers, prevention of postoperative complications, and burns. 18. A cell sheet-attached culture carrier substrate according to any one of 1. to 17., used for a transplantation method comprising at least one of a cell sheet culturing step, a cell sheet cryopreserving step, a cell sheet thawing step, and a cell sheet attachment step to a transplantation site. 19. A transplant material comprising a cell sheet-attached culture carrier substrate according to any one of 1. to 18.. 20. A transplant material according to 19., used for attaching a cell sheet to a diseased site. 21. 19. or 20. The transplant material according to 1. above is a therapeutic transplant material for treating at least one of spinal cord injury, knee joint cartilage injury, ischemic heart disease, age-related macular degeneration, corneal epithelial stem cell exhaustion, aplastic anemia, severe limb ischemia, intractable skin ulcer, prevention of postoperative complications, and burns. The above-mentioned culture carrier substrate with a cell sheet may comprise any one of the culture carrier substrates 1. to 13. of Reference Form A. <C. Frozen Culture Carrier Substrate with a Cell Sheet> Reference Form C of a frozen culture carrier substrate with a cell sheet includes the following 1. to 8.: 1. A frozen product comprising a culture carrier substrate and a cell sheet formed on the surface of the culture carrier substrate, wherein the culture carrier substrate and the cell sheet are in a frozen state. 2. The frozen product according to 1. above, which is frozen and stored at -196°C to -60°C. 3. The frozen product according to 1. above, which is frozen and stored at -196°C to -135°C. 4. 1. The frozen product according to item 1, which is frozen and preserved at -134°C to -60°C. 5. The frozen product according to any one of items 1 to 4, which comprises a cryopreservation solution. 6. The frozen product according to item 5, which comprises the cryopreservation solution comprising at least one of a culture medium component and a cryoprotectant.7. The frozen product according to 5. or 6., wherein the preservation solution does not contain DMSO. 8. The frozen product according to any one of 1. to 4., wherein the culture carrier substrate and the cell sheet are in a frozen preservation state in which they are entirely covered with a cryopreservation solution. The above-mentioned frozen product of a culture carrier substrate with a cell sheet may include any one of the culture carrier substrates 1. to 13. of Reference Form A, or any one of the culture carrier substrates 1. to 21 of Reference Form B. <D. Package> Reference Form D of the package includes the following 1. to 8. 1. A package comprising a culture carrier substrate and a packaging material for packaging the culture carrier substrate. 2. A package comprising a cell sheet-attached culture carrier substrate comprising a culture carrier substrate and a cell sheet formed on the culture surface of the carrier substrate, and a packaging material for packaging the cell sheet-attached culture carrier substrate. 3. A package comprising a frozen product of a cell sheet-attached culture carrier substrate, the cell sheet comprising a culture carrier substrate and a cell sheet formed on the culture surface of the carrier substrate, and a packaging material for packaging the frozen product. 4. The package according to any one of 1. to 3., comprising a culture container and / or a cryopreservation container inside the packaging material. 5. The package according to any one of 1. to 4., wherein the inside of the packaging material is sealed. 6. The package according to any one of 1. to 5., used for transportation and / or storage. The above package may comprise any one of the culture carrier substrates 1. to 13. of Reference Form A, any one of the cell sheet-attached culture carrier substrates 1. to 21 of Reference Form B, or any one of the cell sheet-attached culture carrier substrates 1. to 8 of Reference Form C. <E. Cryopreservation method or thawing method> Reference Form E of the cryopreservation method or thawing method includes the following 1. to 10.. 1. A cryopreservation method comprising a cooling step of cooling a cell sheet-bearing culture carrier substrate, which comprises a culture carrier substrate and a cell sheet formed on the surface of the culture carrier substrate, in a cryopreservation solution.2. The cryopreservation method according to 1., wherein in the cooling step, the culture carrier substrate with the cell sheet is cooled in a cryopreservation solution using a non-throughflow cooling device. 3. The cryopreservation method according to 1. or 2., wherein in the cooling step, the cooling rate at 0 to -5°C is 0.1°C / min or more and 15°C / min or less. 4. The cryopreservation method according to any one of 1. to 3., comprising a cryopreservation step of storing the culture carrier substrate with the cell sheet at -196 to -60°C after the cooling step. 5. The cryopreservation method according to any one of 1. to 4., wherein a frozen product comprising the culture carrier substrate and the cell sheet is obtained by the cooling step. 6. A thawing method comprising a culture carrier substrate and a cell sheet formed on the surface of the culture carrier substrate, comprising a thawing step of thawing a frozen product in which the culture carrier substrate and the cell sheet are in a frozen state. 7. 6. The thawing method according to any one of items 1. to 4., comprising the thawing step after the cooling step of the cryopreservation method according to any one of items 1. to 4. 8. The thawing method according to item 6. or 7., comprising a thawing step of thawing a frozen material obtained by the cryopreservation method according to item 5. 9. The thawing method according to any one of items 6. to 8., wherein in the thawing step, the environmental temperature during thawing is 0°C or higher and lower than 50°C. 10. The thawing method according to any one of items 6. to 9., wherein in the thawing step, the thawing time is longer than 10 seconds and shorter than 60 minutes. The above cryopreservation method or freezing method may comprise a culture carrier substrate according to any one of items 1. to 13. of Reference Form A, or a cell sheet-attached culture carrier substrate according to any one of items 1. to 21 of Reference Form B. The frozen material obtained by the cryopreservation method may comprise a frozen material of a cell sheet-attached culture carrier substrate according to any one of items 1. to 8. of Reference Form C. Furthermore, the frozen material obtained by the cryopreservation method and / or the thawed material obtained by the thawing method can be used in the transplantation method described below. <F. Method for producing a culture carrier substrate with a cell sheet> Reference form F of the method for producing a culture carrier substrate with a cell sheet includes the following 1. to 3.1. A method for producing a cell sheet-attached culture carrier substrate, comprising: a culturing step of introducing a plurality of cells, a medium, and a culture carrier substrate into a culture vessel and culturing a cell sheet on the culture surface of the culture carrier substrate; and a removal step of removing the culture carrier substrate with the cell sheet attached from the culture vessel. 2. A method for producing a cell sheet-attached culture carrier substrate according to 1., wherein in the culturing step, the cell sheet is cultured without adhering the culture carrier substrate to the inside of the culture vessel. 3. A method for producing a cell sheet-attached culture carrier substrate according to 1. or 2., wherein in the removal step, an operation of physically separating the culture carrier substrate from the culture vessel is not required. The method for producing a cell sheet-attached culture carrier substrate may include any one of the culture carrier substrates 1. to 13. of Reference Form A. <G. Transplantation Method> Reference Form G of the transplantation method includes the following 1. to 25.. 1. A transplantation method comprising: a cell sheet-attached culture carrier substrate comprising a culture carrier substrate and a cell sheet formed on the culture surface of the culture carrier substrate, attaching the cell sheet to a transplantation site, and then peeling off the cell sheet from the culture carrier substrate. 2. The transplantation method according to 1., wherein the transplantation step uses a cell sheet-attached culture carrier substrate having a laminate of the culture carrier substrate and the cell sheet, with the cell sheet covering at least 50% of the surface on the culture surface side of the culture carrier substrate. 3. The transplantation method according to 1. or 2., wherein the transplantation step involves removing the cell sheet-attached culture carrier substrate from a package and using it, or removing a frozen product of the cell sheet-attached culture carrier substrate from a package and thawing the frozen product before using it. 4. The transplantation method according to any one of 1. to 3., comprising a culture step of culturing the cell sheet on the culture surface of the culture carrier substrate to obtain the cell sheet-attached culture carrier substrate.5. The transplantation method according to 4., wherein in the culturing step, the cell sheet is cultured in a state in which at least a portion of each of the culture surface and side surface of the culture carrier substrate is in contact with a culture solution. 6. The transplantation method according to 4. or 5., wherein in the culturing step, the cell sheet is cultured in a state in which the culture carrier substrate is loaded and fixed by a weight and / or the culture carrier substrate is fixed in position by an instrument. 7. The transplantation method according to any one of 4. to 6., wherein in the culturing step, the density of the cells cultured is 5 x 10 ２ cells / cm ２ 1x10 or more ９ cells / cm ２ A transplantation method as set forth below. 8. The transplantation method as set forth in any one of 4. to 7., wherein the substrate thickness of the culture carrier substrate exceeds 10 μm, and in the culturing step, the culture carrier substrate functions as a free-standing membrane without being fixed to the inside of the culture vessel. 9. The transplantation method as set forth in any one of 1. to 8., comprising a cooling and preservation step of cooling the cell sheet-attached culture carrier substrate in a cryopreservation solution to obtain a frozen product of the cell sheet-attached culture carrier substrate. 10. The transplantation method as set forth in any one of 1. to 9., comprising a thawing step of thawing the frozen product of the cell sheet-attached culture carrier substrate. 11. The transplantation method as set forth in any one of 1. to 10., wherein the substrate thickness of the culture carrier substrate is 5 μm or more and 250 μm or less, the culture carrier substrate is a polyether ether ketone film or a polyethylene terephthalate film, and the culture surface of the culture carrier substrate has been subjected to a hydrophilization treatment. 12. The transplantation method as set forth in any one of 1. to 11. 13. The transplantation method according to any one of 1. to 12., wherein in the transplantation step, the time from attachment to detachment is 10 minutes or less. ２14. The transplantation method according to any one of 1. to 13., wherein after the transplantation step, the survival rate of the cell sheet on the culture carrier substrate after the detachment is 50% or less in terms of area ratio. 15. The transplantation method according to any one of 1. to 14., wherein the thickness of the cell sheet is 0.001 mm or more and 2.0 mm or less. 16. The transplantation method according to any one of 1. to 15., wherein the area of ​​the culture carrier substrate is 0.3 cm or less. ２ More than 1000cm ２A transplantation method as set forth in any one of the following: 17. The transplantation method according to any one of 1. to 16., wherein the culture carrier substrate comprises one or more resin layers made of a resin material. 18. The transplantation method according to any one of 1. to 17., wherein the culture carrier substrate has three or fewer holes with a diameter of 1 μm to 100 μm and a depth of 0.5 μm to 100 μm in at least one 100 μm square observation area when the culture surface is measured with a laser microscope. 19. The transplantation method according to any one of 1. to 18., wherein the porosity of the culture carrier substrate is 15% or less. 20. The transplantation method according to any one of 1. to 19., wherein the water contact angle on the culture surface of the culture carrier substrate is 70° or less. 21. The transplantation method according to any one of 1. to 20. 22. The transplantation method according to any one of 1. to 21., wherein a culture carrier substrate with a cell sheet is obtained, in which the cell sheet covers 50% to 100% of the cell deposition area on the culture surface of the culture carrier substrate. 22. The transplantation method according to any one of 1. to 21., wherein the cell sheet is used in cell transplantation therapy. 23. The transplantation method according to 22., wherein the cell transplantation therapy includes at least one of suppressing and preventing the onset and recurrence of symptoms associated with loss, dysfunction, and dysfunction of cells, tissues, and organs. 24. The transplantation method according to any one of 1. to 23., wherein the cell sheet is attached to the diseased site in the transplantation step. 25. 1. to 24. The transplantation method according to any one of the above, wherein the disease includes at least one of spinal cord injury, knee joint cartilage injury, ischemic heart disease, age-related macular degeneration, corneal epithelial stem cell exhaustion, aplastic anemia, severe limb ischemia, intractable skin ulcer, prevention of postoperative complications, and burns. The transplantation method may include any one of the culture carrier substrates 1. to 13. of Reference Form A, any one of the culture carrier substrates 1. to 21. of Reference Form B, or any one of the frozen culture carrier substrates 1. to 8. of Reference Form C.The transplantation method may include a method for producing a cell sheet-attached culture carrier substrate or a culturing step thereof according to any one of 1. to 3. of Reference Form F, or a cryopreservation method or thawing method according to any one of 1. to 10. of Reference Form E. Reference Form H also includes a treatment method using at least one of a culture carrier substrate according to any one of 1. to 13. of Reference Form A, a cell sheet-attached culture carrier substrate according to any one of 1. to 21. of Reference Form B, and a frozen product of a cell sheet-attached culture carrier substrate according to any one of 1. to 8. of Reference Form C, the treatment method including at least one of suppressing and preventing the onset and recurrence of symptoms associated with cell, tissue, or organ loss, dysfunction, or dysfunction. The associated symptoms of this treatment method may include at least one of the diseases described above. Reference Form I also includes a treatment method using at least one of a culture carrier substrate according to any one of 1. to 13. of Reference Form A and a frozen product of a cell sheet-attached culture carrier substrate according to any one of 1. to 21. of Reference Form B for cell, tissue, or organ loss, dysfunction, or dysfunction. Examples of the use include the use of at least one of a cell sheet-attached culture carrier substrate of any one of 1. to 21. and a frozen product of a cell sheet-attached culture carrier substrate of any one of 1. to 8. of Reference Form C in Reference Form A. The associated symptoms of this use may include at least one of the diseases described above. Reference Form J also includes the use of at least one of a cell sheet-attached culture carrier substrate of any one of 1. to 13. of Reference Form A, a cell sheet-attached culture carrier substrate of any one of 1. to 21. of Reference Form B, and a frozen product of a cell sheet-attached culture carrier substrate of any one of 1. to 8. of Reference Form C in Reference Form A, in the manufacture of a therapeutic agent for treating, including at least one of suppressing and preventing the onset and recurrence of symptoms associated with loss, dysfunction, and dysfunction of cells, tissues, and organs. The associated symptoms of this use may include at least one of the diseases described above.

[0079] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the descriptions of these examples.

[0080] Example 1: Production of a culture carrier substrate Plasma treatment was performed on the culture surface (surface) of a PEEK film (Shin-Etsu Polymer Co., Ltd., polyether ether ketone film, Shin-Etsu Sepla Film® low crystallinity type, 12 μm thick). The surface roughness after plasma treatment was rougher than before plasma treatment, with an Ra of 0.6 nm. Ra was measured as follows: An AFM (Shimadzu Corporation, SPM-9700) was used, and a silicon single crystal probe with a small tip diameter (radius of curvature approximately 10 nm) was used. The surface was observed over a 1 μm × 1 μm area. Ra was calculated using the analysis software provided with the device. After smoothing in the X and Y directions, surface roughness analysis was performed over a 100 nm × 100 nm area, avoiding foreign matter and surface scratches. The arithmetic mean roughness Ra is the average value of the distance from a reference line within the reference length. The PEEK film had a porosity of 5% or less, and when the culture surface was measured with a laser microscope, it was found to be a solid film with no holes with a diameter of 1 μm to 100 μm and a depth of 0.5 μm to 100 μm within a 100 μm square area. The porosity was calculated using the formula "{1 - (measured density / theoretical density)} x 100". The surface-treated film was then cut into a Φ33.5 mm size to obtain a disc-shaped culture carrier substrate.

[0081] <Production of cell sheets> The culture carrier substrates produced in the above <Production of culture carrier substrates> were washed with 70% ethanol, phosphate buffer, and culture medium in that order, and then placed on the flat bottoms of each well of a cell culture multi-well plate (6 wells). At this time, the culture surface of the culture carrier substrate was placed facing the opening of the well plate, i.e., the back surface of the culture carrier substrate was placed in contact with the flat bottom of the well plate hole. However, the culture carrier substrate was not adhered to the inside of the well plate (culture vessel). Cryopreserved human fibroblasts (derived from human oral tissue) were thawed at 37°C and washed with culture medium. 5 x 10 ５ The cells were suspended in a 5% serum-containing medium and placed in two dishes (60.1 cm ２ ) to 2.5 x 10 ５The cultured cells were collected, suspended in a 5% serum-containing medium, and then placed in four flasks (225 cm ２ ) to 2.5 x 10 ５ The cultured cells were collected, suspended in a 2% serum-containing medium, and placed in a cell culture multi-well plate with a culture carrier substrate in a density of 55.8 × 10 cells per well. ４ pieces / cm ２ and incubated at 37°C and 5% CO ２ The culture medium was incubated for 1 day under the above conditions to prepare a cell sheet on the culture surface of the culture carrier substrate.

[0082] <Transplantation> After the above <Cell Sheet Production>, the culture carrier substrate with the cell sheet was removed from the multi-plate. The removed culture carrier substrate with the cell sheet was transported onto a piece of pork (transplantation site) prepared in a separate location, simulating cell tissue. After the surface of the cell sheet was attached to the surface of the pork piece, the culture carrier substrate was peeled off from the cell sheet. Note that the substrate was not specifically cooled between attachment and removal, so the temperature of the substrate was maintained at 30°C or higher.

[0083] [Example 2] In the above <Production of culture carrier substrate>, <Production of cell sheet> and <Transplantation> were carried out in the same manner as in Example 1, except that a 25 μm thick PET film (polyethylene terephthalate film, Lumirror (registered trademark), manufactured by Toray Industries, Inc.) was used instead of the polyether ether ketone film.

[0084] [Example 3] In the above <Production of culture carrier substrate>, the cutting size of the surface-treated film was changed from Φ33.5 mm to Φ14.0 mm, and in the above <Production of cell sheet>, a 24-hole flat-bottom cell culture multiwell plate was used for seeding, and the seeding density was changed to 27.9 × 10 ４ The <Cell sheet production> and <Transplantation> were carried out in the same manner as in Example 1, except that the cell density was changed to cells / cm.

[0085] Comparative Example 1 The culture carrier substrate produced in the above-mentioned <Culture carrier substrate production> was not used. Instead, in <Cell sheet production>, a cell sheet was produced on the flat bottom inside the well of a multi-well plate for adherent cells. In <Transplantation>, the cell sheet was peeled off from the flat bottom and attached to the surface of a pork piece. Except for this, <Cell sheet production> and <Transplantation> were carried out in the same manner as in Example 1.

[0086] [Comparative Example 2] In the above <Production of culture carrier substrate>, <Production of cell sheet> and <Transplantation> were carried out in the same manner as in Example 1, except that plasma treatment was not performed on the culture surface of the substrate.

[0087] [Comparative Example 3] In the above <Production of culture carrier substrate>, <Production of cell sheet> and <Transplantation> were carried out in the same manner as in Example 2, except that plasma treatment was not performed on the culture surface of the substrate.

[0088] The culture and transplantation of cell sheets were evaluated for each example and comparative example. The results are shown in Table 1.

[0089]

[0090] In Examples 1 to 3, a cell sheet-attached culture carrier substrate was obtained in the <Cell Sheet Production> step, in which the cell sheet covered 100% or more of the substrate culture surface. In the <Transplantation> step, the cell sheet-attached culture carrier substrate was easily removed because it was not adhered to the multi-plate. While the PET film in Example 2 required slightly pressing the cell sheet with tweezers to peel the substrate, the PEEK films in Examples 1 and 3 were more easily peeled by sliding the substrate without pressing the cell sheet, demonstrating superior ease of handling. Furthermore, in the <Transplantation> step, the culture carrier substrate was easily removed from the cell sheet. Specifically, the cell sheet and pork meat quickly adhered to each other, allowing for application and removal within one minute. This simplified the transplantation process, shortening the handling time. Furthermore, there was no need to culture the cell sheet on the pork meat after the <Transplantation> step. The results of Examples 1 to 3 confirmed that both cell sheet cultivation and transplantation can be achieved by using plasma-treated PEEK film or plasma-treated PET film as a culture carrier substrate. Furthermore, after the detachment of the cell sheet in the <Transplantation> step in Examples 1 to 3, the area ratio of the cell sheet remaining on the culture surface of the culture carrier substrate was 1% or less. This makes it possible to reduce the amount of cell sheet remaining during the transplantation process. Furthermore, when detaching the culture carrier substrate from the cell sheet, it was not necessary to treat the culture surface with dispase before culturing.

[0091] In Comparative Example 1, in the <Cell Sheet Production> step, a cell sheet was successfully produced in the multiwell plate for adherent cells. However, in the <Transplantation> step, when removing the cell sheet from the multiwell plate for adherent cells, it was difficult to peel the cell sheet from the flat bottom of the multiwell plate for adherent cells so as not to damage the cell sheet with tweezers or the like. Even if the cell sheet could be peeled off, it was difficult to grasp the cell sheet, making it difficult to attach it. In Comparative Examples 2 and 3, a cell sheet could not be produced, so the transplantation procedure was not performed.

[0092] Example 4 Production of Cell Sheets Using a culture carrier substrate obtained by changing the cutting size of the surface-treated film in the production of a culture carrier substrate in Example 1 to 14.0 mm instead of 33.5 mm, a 24-well flat-bottom cell culture multiwell plate was used for seeding, and the seeding density was 27.9 × 10 ４ pieces / cm ２ A cell sheet (cell sheet with culture carrier substrate) was produced on the culture surface of the culture carrier substrate in the same manner as in <Production of cell sheet> in Example 1, except for changing the procedure to: The cell activity rate before freezing was measured according to the following procedure.

[0093] <Cryopreservation of Cell Sheets> After the above <Cell Sheet Production>, the cell culture multiwell plate was placed on ice, and the cell sheet with the culture carrier substrate was washed with phosphate buffer. Then, 0.3 mL / well of cryoprotectant (STEMCELL-BANKER GMP grade, manufactured by Nippon Zenyaku Kogyo Co., Ltd.) was added. The cell culture multiwell plate was then packaged in a zippered plastic bag, and the resulting package was placed in a non-throughflow cooling device (3D Freezer (registered trademark), KSS-40BLW-2400V, manufactured by Kogasan Co., Ltd.) pre-cooled to -35°C and maintained for 30 minutes. The cooling rate from 0 to -5°C was 3.4°C / min. The cooled package was then promptly placed in a -80°C freezer (RDE50086FD ultra-low temperature freezer, manufactured by Thermoscientific Co., Ltd.) and maintained for 1 hour. The temperature of the cell sheet (cell sheet with culture carrier substrate) stored in a freezer at 80°C was measured using a K thermocouple and found to be -70°C.

[0094] <Thawing of cell sheet> After the above-mentioned freezing period, the package was removed from the -80°C freezer, and the cell culture multiwell plate contained therein was thawed at room temperature in a safety cabinet. The thawing time was 16 minutes. After thawing, the appearance of the cell sheet was good. After thawing, the cryoprotectant was aspirated, and 2 mL of 2% serum-containing medium was added. Then, the cells were stored at 37°C and 5% CO ２The cells were incubated for 24 hours under the 50°C environment. After incubation, cell activity was measured. As a result, when the cell activity before freezing was defined as C0 and the cell activity after freezing as C1, the "percent change in cell activity before and after freezing," calculated as (C1 / C0) x 100, was found to be 89%. Furthermore, the culture supernatant was collected, and vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were measured the day after freezing at -80°C. VEGF and HGF in the supernatant were found to be 1451 pg and 4608 pg, respectively. (Method for Measuring Thawing Time) The thawing time was measured as the time required to thaw a frozen product that had been cryoprotectant-added and cryopreserved. The thawing time was measured from the time of exposure to the thawing environment until the frozen product became completely liquid.

[0095] (Measurement of Cell Viability) Cell activity was measured using a viable cell count reagent (Cell Count Reagent SF, manufactured by Nacalai Tesque) and a plate reader (iMark, manufactured by BIO-RAD). The viable cell count reagent was diluted 20-fold with culture medium (reagent / culture medium = 1 / 19 volume ratio) to prepare a test solution. The supernatant of each well of the cell culture multi-well plate containing the cultured cell sheet was aspirated, and 0.5 mL of the test solution was added to each well. The cells were then incubated at 37°C and 5% CO ２ The cells were incubated for 1 hour in an environment of 0.1 mL / well. After incubation, 0.1 mL of the supernatant was added to a cell culture multi-well plate (96 wells, manufactured by Corning) and the supernatant was measured using a plate reader. The calculation of cell activity is as follows: Cell activity [Abs.] = {absorbance of measurement sample [absorbance (450 nm) - absorbance of measurement sample (630 nm)]} - {absorbance of blank [absorbance (450 nm) - absorbance of blank (630 nm)]} The test solution was used as the blank.

[0096] (Measurement of Vascular Endothelial Growth Factor (VEGF) and Hepatocyte Growth Factor (HGF)) VEGF and HGF were measured as cytokines involved in angiogenesis. These cytokines were measured using a VEGF, Human, ELISA Kit, Quantikine (96-well) (R&D Systems, DVE00) and an HGF, Human, ELISA Kit, Quantikine (96-well) (R&D Systems, DHG00B), respectively, and a plate reader (iMark, BIO-RAD). Each kit was used according to its instruction manual. After thawing, the test samples were cultured for 24 hours, and the supernatant was collected and frozen for storage. The test samples were thawed for measurement the day after freezing. The test samples were diluted 2- to 4-fold with the diluent provided with the kit and then measured. The calibration curve was prepared using the standard provided with each kit. The absorbance of each sample and the standard for the calibration curve was calculated using the following formula, and the obtained absorbance was applied to the calibration curve to calculate the concentrations of VEGF and HGF. Absorbance of sample and standard for the calibration curve [Abs.] = absorbance (450 nm) - absorbance (570 nm)

[0097] <Transplantation after thawing> The thawed cell sheet with the culture carrier substrate was removed from the cell culture multi-plate, and the cell sheet surface was attached to pork, and the substrate was peeled off. Note that the substrate was not heated or cooled during the period from attachment to peeling, so the temperature of the substrate was maintained at room temperature (23°C).

[0098] [Examples 5 to 11] <Production of cell sheets>, <Cryopreservation of cell sheets>, <Thawing of cell sheets>, and <Transplantation after thawing> were carried out in the same manner as in Example 4, except that the freezing period in the above <Cryopreservation of cell sheets> was changed to the values ​​shown in Table 2.

[0099] [Comparative Example 4] In Comparative Example 4, the <Production of cell sheets> was carried out in the same manner as in Example 4, except that no culture carrier substrate was used and the cell culture multi-well plate was changed to a Petri dish (UpCell (registered trademark), manufactured by CellSeed Co., Ltd.).

[0100] [Comparative Example 5] In Comparative Example 5, the <Production of cell sheets>, <Cryopreservation of cell sheets>, and <Thawing of cell sheets> were carried out in the same manner as in Example 4, except that no culture carrier substrate was used and the cell culture multi-well plate was changed to a multi-well plate for adherent cells (24 wells).

[0101] For each example and comparative example, evaluation was performed on cryopreservation and transplantation after thawing. The results are shown in Table 2.

[0102]

[0103] In Comparative Example 4, the cell sheet detached from the dish during cooling, making it impossible to cryopreserve the cell sheet. Therefore, measurement of the cell activity rate after freezing and transplantation after thawing were not performed. In Comparative Example 5, the cell sheet was cryopreserved while still in close contact with the multiwell plate, but the results showed a decrease in the rate of change in the cell activity rate before and after freezing.

[0104] In contrast, in Examples 4 to 11, cell sheets could be frozen while still adhered to the culture carrier substrate, and the percentage change in cell activity before and after freezing, as well as at least one of VEGF and HGF, were higher than in Comparative Example 5. Furthermore, even after thawing and transplantation, the cell sheet could be easily detached from the culture carrier substrate. Because the cell sheet quickly adhered to the pork, the process from attachment to detachment was completed within one minute, eliminating the need to culture the cell sheet on the pork. The results of Examples 4 to 11 confirmed that using plasma-treated PEEK film as a culture carrier substrate enabled cell sheets to be frozen and then thawed and transplanted, and that the thawed cell sheet maintained a high cell activity even after being frozen for a long period of time.

[0105] Examples 12 to 23: <Cell sheet production>, <Cryopreservation of cell sheets>, <Thawing of cell sheets>, and <Transplantation after thawing> were carried out in the same manner as in Example 4, except that the freezing period in the above <Cryopreservation of cell sheets> was changed to the values ​​shown in Table 3, Bambanker (registered trademark) hRM (CG Lymphotec) was used as the cryoprotectant, and a -150°C freezer was used. The thawing time in this case was 18 minutes. After thawing, the appearance of the cell sheets was good. Furthermore, the temperature of the cell sheets (cell sheets with culture carrier substrates) stored in a -150°C freezer was measured using a K thermocouple and found to be -140°C. For each example, cryopreservation and transplantation after thawing were evaluated. The results are shown in Table 3.

[0106]

[0107] In Examples 12 to 23, the cell sheets could be frozen while still adhered to the culture carrier substrate, and the percentage change in cell activity before and after freezing, as well as VEGF and HGF, were all higher than in Comparative Example 5. Furthermore, even after thawing and transplantation, the cell sheets could be easily detached from the culture carrier substrate. Because the cell sheet quickly adhered to the pork, the process from attachment to detachment was completed within one minute, eliminating the need to culture the cell sheet on the pork.

[0108] Examples 24-26: Cell sheet production, cell sheet cryopreservation, cell sheet thawing, and post-thaw transplantation were performed in the same manner as in Example 12, except that the freezing period in the above-mentioned cell sheet cryopreservation was changed to the value shown in Table 4. However, the frozen state in each example was configured as shown in Figures 3(a) to 3(c). In Figure 3(a), the culture carrier substrate 10 was positioned facing the bottom of the culture vessel 20. In Figure 3(b), the cell sheet 40 was positioned facing the bottom of the culture vessel 20. In Figure 3(c), the cell sheet 40 was sandwiched between two culture carrier substrates 10. Cryopreservation and post-thaw transplantation were evaluated for each example. The results are shown in Table 4.

[0109]

[0110] In Examples 24 to 26, the cell sheets could be frozen while still adhered to the culture carrier substrate, and the change in cell activity rate before and after freezing was higher than in Comparative Example 5. Furthermore, even after thawing, the cell sheets could be easily detached from the culture carrier substrate during transplantation. Because the cell sheet quickly adhered to the pork, the process from attachment to detachment was completed within one minute, eliminating the need to culture the cell sheet on the pork. Furthermore, because PEEK film has low linear expansion, it was found to be useful as a culture carrier substrate that is also useful for freezing treatment.

[0111] [Example 27] <Cell sheet production>, <Cell sheet cryopreservation>, <Cell sheet thawing>, and <Post-thawed transplantation> were carried out in the same manner as in Example 12, except that, in the method described in <Cell sheet thawing> in Example 12, thawing at room temperature in a safety cabinet was performed by placing the cell culture multiwell plate on the heat sink of a defroster (manufactured by DEC). The defroster's set temperature, which indicates the heat sink temperature, was set to 37°C for thawing. The thawing time was 3 minutes. The temperature of the cell sheet (cell sheet with culture carrier substrate) stored in a -150°C freezer was measured using a K thermocouple and found to be -140°C. After thawing, the cell sheet had a good appearance. [Example 28] <Cell sheet production>, <Cell sheet cryopreservation>, <Cell sheet thawing>, and <Post-thawed transplantation> were carried out in the same manner as in Example 26, except that the defroster's set temperature in Example 27 was set to 20°C. The thawing time was 5 minutes. After thawing, the cell sheet had a good appearance. [Example 29] <Cell sheet production>, <Cell sheet cryopreservation>, <Cell sheet thawing>, and <Transplantation after thawing> were performed in the same manner as in Example 26, except that the defroster temperature setting in Example 27 was changed to 4°C. The thawing time was 8 minutes. The appearance of the cell sheet was good after thawing. [Example 30] <Cell sheet production>, <Cell sheet cryopreservation>, <Cell sheet thawing>, and <Transplantation after thawing> were performed in the same manner as in Example 26, except that in the method described in <Cell sheet thawing> in Example 12, thawing at room temperature in a safety cabinet was changed to thawing in a refrigerator set to 4°C. The thawing time was 37 minutes. The appearance of the cell sheet was good after thawing. For Examples 27 to 30, the appearance of the cell sheets, measurement of cell activity rate, and transplantation after thawing were evaluated. The results are shown in Table 5. For reference, Table 5 also shows the results of Examples 4 and 12.

[0112]

[0113] In Examples 4, 12, and 27 to 30, the appearance of the cell sheets was not affected. Furthermore, Examples 4, 12, and 27 to 30 showed a higher "percent change in cell activity before and after freezing" than Comparative Example 5. In other words, it can be said that a high cell viability was maintained after freezing. Among these, Examples 4, 12, and 27 to 29 were found to have a higher "percent change in cell activity before and after freezing" than Example 30, which had a relatively long freezing time. Furthermore, the thawed products of all Examples were successfully transplanted after thawing.

[0114] In the above Examples 1 to 30, it was confirmed that similar results were obtained when mouse fibroblasts were used instead of human fibroblasts.

[0115] This application claims priority based on Japanese Patent Application No. 2023-115623 filed on July 14, 2023, and Japanese Patent Application No. 2023-220506 filed on December 27, 2023, the disclosures of which are incorporated herein in their entireties.

[0116] REFERENCE SIGNS LIST 10 culture carrier substrate 12 culture surface (front surface) 14 non-culture surface (rear surface) 20 culture vessel 30 culture solution 40 cell sheet 50 culture carrier substrate with cell sheet 60 cryopreservation solution 70 transplantation site

Claims

1. A transplantation method comprising: a culture step of culturing a cell sheet on the culture surface of a culture carrier substrate; and a transplantation step of attaching the cell sheet formed on the culture surface of the culture carrier substrate to a site to be transplanted, and then peeling off the culture carrier substrate from the cell sheet.

2. A transplantation method as described in claim 1, wherein the substrate thickness of the culture carrier substrate is 5 μm or more and 250 μm or less, the culture carrier substrate is a polyether ether ketone film or a polyethylene terephthalate film, and the culture surface of the culture carrier substrate has been subjected to a hydrophilic treatment.

3. A transplantation method according to claim 1 or 2, wherein the substrate thickness of the culture carrier substrate exceeds 10 μm, and in the culture step, the culture carrier substrate functions as a self-supporting membrane without being fixed to the inside of the culture vessel.

4. A transplantation method according to any one of claims 1 to 3, wherein in the transplantation step, the time from application to removal is 10 minutes or less.

5. The transplantation method according to any one of claims 1 to 4, wherein the area of ​​the cell sheet obtained by the culture step is 0.3 cm ２ That's it, the transplantation method.

6. A culture carrier base material used for transporting a cell sheet cultured on a culture surface, the culture carrier base material having a base material thickness of 5 μm or more and 250 μm or less.

7. A culture carrier substrate according to claim 6, wherein the substrate thickness of the culture carrier substrate is greater than 10 μm, and the culture carrier substrate functions as a self-supporting membrane without being fixed to the inside of the culture vessel during the culture of the cell sheet.

8. A culture carrier base according to claim 6 or 7, wherein the culture surface has been subjected to a hydrophilic treatment.

9. A culture carrier substrate according to claim 8, wherein the hydrophilization treatment is performed by plasma treatment.

10. A culture carrier substrate according to any one of claims 6 to 9, wherein the culture carrier substrate is a polyether ether ketone film or a polyethylene terephthalate film.

11. The culture carrier substrate according to any one of claims 6 to 10, wherein the culture surface does not contain a temperature responsive polymer.

12. A package comprising the culture carrier substrate according to any one of claims 6 to 11 packaged in a packaging material.

13. A culture carrier substrate with a cell sheet, comprising a laminate of the culture carrier substrate according to any one of claims 6 to 11 and a cell sheet, wherein the cell sheet covers at least 50% of the surface on the culture side of the culture carrier substrate.

14. The cell sheet-attached culture carrier substrate according to claim 13, wherein the area of ​​the cell sheet is 0.3 cm ２ The above is the cell sheet-attached culture carrier substrate.

15. A method for producing a culture carrier substrate with a cell sheet, comprising: a culture step of introducing a plurality of cells, a culture medium, and the culture carrier substrate according to any one of claims 6 to 11 into a culture vessel, and culturing a cell sheet on the culture surface of the culture carrier substrate; and a removal step of removing the culture carrier substrate with the cell sheet from the culture vessel.

16. A method for producing a culture carrier substrate with a cell sheet as described in claim 15, wherein in the culture step, the cell sheet is cultured without fixing the culture carrier substrate to the inside of a culture vessel.

17. A method for producing a culture carrier substrate with a cell sheet as described in claim 15 or 16, wherein in the removal step, an operation of separating the culture carrier substrate from the culture vessel by physical means is not required.

18. A cryopreservation method comprising a cooling step of cooling a culture carrier substrate with a cell sheet, the culture carrier substrate comprising a culture carrier substrate and a cell sheet formed on the surface of the culture carrier substrate, in a cryopreservation liquid.

19. A cryopreservation method according to claim 18, wherein in the cooling step, the culture carrier substrate with the cell sheet is cooled in a cryopreservation liquid using a non-flow-through cooling device.

20. A cryopreservation method according to claim 18 or 19, wherein in the cooling step, the cooling rate at 0 to -5°C is 0.1°C / min or more and 15°C / min or less.

21. A method for freezing and preserving a culture carrier substrate according to any one of claims 18 to 20, comprising a freezing and preserving step of preserving the culture carrier substrate with the cell sheet at -196 to -60°C after the cooling step.

22. A frozen culture carrier base with a cell sheet, comprising: a culture carrier base; and a cell sheet formed on the surface of the culture carrier base, wherein the culture carrier base and the cell sheet are in a frozen state.

23. A frozen product of the culture carrier substrate with a cell sheet according to claim 22, which is stored frozen at -196°C to -60°C.

24. A frozen product of the culture carrier substrate with a cell sheet according to claim 22, which is stored frozen at -196°C to -135°C.

25. A frozen product of the culture carrier substrate with a cell sheet according to claim 22, which is stored frozen at -134°C to -60°C.

26. A package comprising a frozen product according to any one of claims 22 to 25 packaged in a packaging material.

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