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

Abstract

A transplantation method according to the present invention comprises: a culture step for culturing a cell sheet on a culture surface of a culture carrier substrate; and a transplantation step in which the cell sheet formed on the culture surface of the culture carrier substrate is attached to the site to be transplanted, and then the culture carrier substrate is peeled off from the cell sheet.

Description

Technical Field

[0001] This invention relates to transplantation methods, culture carrier substrates, packaging bodies, culture carrier substrates with cell sheets, methods for manufacturing culture carrier substrates with cell sheets, cryopreservation methods, and frozen products of culture carrier substrates with cell sheets. Background Technology

[0002] To date, various developments have been made regarding the "culture" and "delivery" of cell slices, which are technical elements constituting transplantation techniques. For example, the technique described in Patent Document 1 is known as such.

[0003] The background art of Patent Document 1 describes a series of operations in order to transplant cell sheets to the target affected area, including peeling cell sheets cultured on a temperature-responsive material by temperature change, covering the obtained cell sheets with a separately prepared sheet, removing the cell sheets from the container while they are attached to the sheet, transporting them to the affected area, and transplanting (attaching) them to the affected area.

[0004] In this regard, claim 1 and paragraph 0007 of Patent Document 1 describe the following technology: as a method for transporting cell sheets cultured in a cell culture container having a temperature-responsive polymer layer, the bottom of the cell culture container is peeled off while the cell sheets are attached, and the cell sheets attached to the bottom are transported together with the peeled bottom.

[0005] Existing technical documents Patent documents Patent Document 1: Japanese Patent Application Publication No. 2016-013111 Summary of the Invention

[0006] (a) Technical problems to be solved However, the inventors of this application conducted research and found that in the transplantation method that effectively utilizes the technology of Patent Document 1, since the cell sheet needs to be debonded by low temperature treatment when peeling off the temperature-responsive polymer layer, the tightness between the cell sheet and the substrate is released. Furthermore, since the low temperature treatment reduces the tightness between the substrate and the cell sheet, it is difficult to cryopreserve the cell sheet cultured in the cell culture container with the temperature-responsive polymer layer while it is still attached to the cell culture container with the temperature-responsive polymer layer.

[0007] (II) Technical Solution The inventors of this application conducted in-depth research and, as a result, integrated the concepts of "culture" and "transportation" of cell slices, which were studied separately, into a new integrated concept, and concretized the transplantation method and culture carrier substrate, thereby completing this invention.

[0008] According to one aspect of the present invention, the following are provided: transplantation method, culture carrier substrate, packaging body, culture carrier substrate with cell sheets, method for manufacturing culture carrier substrate with cell sheets, cryopreservation method, and frozen product of culture carrier substrate with cell sheets.

[0009] 1. A transplantation method, comprising: The culturing process involves culturing cell sheets on the culture surface of a culture carrier substrate; and In the transplantation process, the cell sheet formed on the culture surface of the culture carrier substrate is attached to the transplantation site, and then the culture carrier substrate is peeled off from the cell sheet.

[0010] 2. The transplantation method as described in item 1, wherein the thickness of the culture carrier substrate is 5 μm or more and 250 μm or less. The culture carrier substrate is a polyetheretherketone membrane or a polyethylene terephthalate membrane. The culture surface of the culture carrier substrate has been hydrophilized.

[0011] 3. The transplantation method as described in item 1 or 2, wherein the substrate thickness of the culture carrier substrate is greater than 10 μm, and in the culture process, the culture carrier substrate is not fixedly attached to the inside of the culture container to function as a self-supporting membrane.

[0012] 4. The transplantation method according to any one of items 1 to 3, wherein, in the transplantation process, the time from attachment to peeling is less than 10 minutes.

[0013] 5. The transplantation method according to any one of items 1 to 4, wherein the area of ​​the cell sheet obtained by the culture process is 0.3 cm². 2 above.

[0014] 6. A culture carrier substrate for transporting cell sheets cultured on a culture surface, wherein, The substrate thickness of the culture carrier is greater than 5 μm and less than 250 μm.

[0015] 7. The culture carrier substrate as described in item 6, wherein the substrate thickness of the culture carrier substrate is greater than 10 μm, and the culture carrier substrate does not fixally attach to the inside of the culture container to function as a self-supporting membrane during the culture of the cell sheet.

[0016] 8. The culture carrier substrate as described in item 6 or 7, wherein the culture surface is subjected to a hydrophilic treatment.

[0017] 9. The culture carrier substrate as described in item 8, wherein plasma treatment is performed as the hydrophilic treatment.

[0018] 10. The culture carrier substrate as described in any one of items 6 to 9, wherein the culture carrier substrate is a polyetheretherketone membrane or a polyethylene terephthalate membrane.

[0019] 11. The culture carrier substrate as described in any one of items 6 to 10, wherein the culture surface is free of temperature-responsive polymers.

[0020] 12. A packaging body, which is a packaging body made by packaging the culture carrier substrate described in any one of items 6 to 11 using packaging materials.

[0021] 13. A culture carrier substrate with cell sheets, comprising a laminate of the culture carrier substrate and cell sheets as described in any one of 6 to 11, wherein... The cell sheet is in a state where at least 50% of the surface of the culture carrier substrate is covered on the culture side.

[0022] 14. The culture carrier substrate with cell sheets as described in item 13, wherein the area of ​​the cell sheets is 0.3 cm². 2 above.

[0023] 15. A method for manufacturing a culture carrier substrate with cell sheets, comprising: The cultivation process involves introducing multiple cells, culture medium, and a culture carrier substrate as described in any one of 6 to 11 into the interior of a culture container, and culturing cell sheets on the culture surface of the culture carrier substrate; and The removal process involves removing the culture carrier substrate containing the cell sheet from the culture container.

[0024] 16. A method for manufacturing a culture carrier substrate with cell sheets as described in claim 15, wherein, in the culture step, the cell sheets are cultured in a manner that does not fix the culture carrier substrate to the inside of the culture container.

[0025] 17. A method for manufacturing a culture carrier substrate with cell sheets as described in item 15 or 16, wherein, in the removal step, it is not necessary to physically separate the culture carrier substrate from the culture container.

[0026] 18. A cryopreservation method comprising: a cooling step of cooling a culture carrier substrate with cell sheets in a cryopreservation solution, wherein the culture carrier substrate with cell sheets comprises a culture carrier substrate and cell sheets formed on the surface of the culture carrier substrate.

[0027] 19. The cryopreservation method as described in item 18, wherein, in the cooling step, a non-flow cooling device is used to cool the culture carrier substrate with cell sheets in a cryopreservation solution.

[0028] 20. The cryopreservation method as described in 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.

[0029] 21. The cryopreservation method according to any one of items 18 to 20, wherein, after the cooling step, a cryopreservation step is included to preserve the culture carrier substrate with cell sheets at -196 to -60°C.

[0030] 22. A cryogenic product of a culture carrier substrate with cell sheets, comprising: A culture carrier substrate and cell sheets formed on the surface of the culture carrier substrate, wherein, The culture carrier substrate and the cell sheet are in a cryopreserved state.

[0031] 23. The frozen product of the culture carrier substrate with cell sheets as described in item 22, which is cryopreserved at -196°C to -60°C.

[0032] 24. The frozen product of the culture carrier substrate with cell sheets as described in item 22, which is cryopreserved at -196°C to -135°C.

[0033] 25. The frozen product of the culture carrier substrate with cell sheets as described in item 22, which is cryopreserved at -134°C to -60°C.

[0034] 26. A packaging body, which is a packaging body made by packaging the frozen product described in any one of items 22 to 25 using packaging materials.

[0035] (III) Beneficial Effects According to the present invention, a transplantation method capable of both culturing and transporting cell sheets, a culture carrier substrate, a packaging body, a culture carrier substrate with cell sheets, a method for manufacturing the culture carrier substrate with cell sheets, a cryopreservation method, and a frozen product of the culture carrier substrate with cell sheets can be provided. Attached Figure Description

[0036] Figure 1 A process cross-sectional view is shown to illustrate an example of the process steps of the transfer method of this embodiment.

[0037] Figure 2 A cross-sectional view illustrating an example of the composition of the cell-sheet culture carrier substrate of this embodiment is shown.

[0038] Figure 3 A process cross-sectional view is shown schematically for a variation of the freezing method of this embodiment. Detailed Implementation

[0039] The embodiments of the present invention will be described below using the accompanying drawings. Furthermore, in all the drawings, the same symbols are used to denote the same constituent elements, and descriptions are omitted where appropriate. Also, the figures are schematic diagrams and do not correspond to actual dimensions.

[0040] <Transplantation Methods> The transplantation method of this embodiment includes: a culture step, in which cell sheets are cultured on the culture surface of a culture carrier substrate; and a transplantation step, in which the cell sheets formed on the culture surface of the culture carrier substrate are attached to the transplantation site, and then the culture carrier substrate is peeled off from the cell sheets.

[0041] The transplant site can be, for example, at least a part of the body of a transplant recipient, such as mammals, birds, amphibians, fish, insects, plants, or microorganisms. Specific examples of mammals and birds include humans, monkeys, chimpanzees, cattle, horses, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, and chickens. The transplant site excludes both internal and external parts of the human body. Transplant sites other than those of the transplant recipient can include other cell fragments, medical instruments, and single tissues or organs separated from the transplant recipient. Examples of single tissues or organs include skin, oral tissues, esophagus, trachea, bronchi, lungs, lung lobes, stomach, duodenum, pancreas, spleen, small intestine, large intestine, muscle tissue, and bones (excluding cases where material taken from the transplant recipient is re-implanted into the same person for therapeutic purposes).

[0042] use Figure 1 The transplantation method of this implementation plan is explained in detail.

[0043] Figure 1 A process cross-sectional view is shown to schematically illustrate an example of the process of the transplantation method. Figure 1 (a) shows cell seeding, (b) shows cell culture, (c) shows cryopreservation / thawing, (d) shows removal, and (e) shows the various attachment processes. Cryopreservation / thawing in (c) is an arbitrary process. This section describes a transplantation method without (c); details of (c) will be provided in the subsequent section on <Cryopreservation Methods>.

[0044] The cultivation process may include Figure 1 The cell seeding process shown in (a) and Figure 1 The cell culture process is shown in (b). Figure 2 A cross-sectional schematic diagram of an example of a culture carrier substrate 50 with cell sheets obtained in the culture process is shown.

[0045] exist Figure 1In the cell seeding process of (a), the culture medium 30 containing cells is brought into contact with the culture carrier substrate 10 disposed in the culture container 20. The cells may be pre-suspended in the culture medium 30, or a cell suspension containing cells may be added to the culture medium 30.

[0046] During the culture process, the culture carrier substrate 10 may be partially or entirely immersed in the culture solution 30. Specifically, in the cell seeding process, the culture solution 30 may be added dropwise to 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, such that at least a portion of the culture surface 12 and the side surfaces of the culture carrier substrate 10 are in contact with the culture solution 30. Compared to the former dropwise method, the latter immersion method can stably maintain the culture environment in subsequent cell culture processes and / or can achieve a large-area culture surface 12 of the culture carrier substrate 10.

[0047] exist Figure 1 In the cell culture process of (b), cell culture is carried out under appropriate culture conditions, and cell sheets 40 are formed on the culture surface 12 of the culture carrier substrate 10. The cells can also be cultured with a cap (not shown).

[0048] In the cell culture process, cell sheets 40 can be cultured without the culture carrier substrate 10 being fixedly attached to the inside of the culture container 20. In this case, the culture carrier substrate 10 functions as a self-supporting membrane during cell culture. Because the culture carrier substrate 10 is not fixedly attached, the culture carrier substrate 50 with cell sheets can be easily removed from the culture container 20 during the removal process. Furthermore, damage to the cell sheets 40 during removal can be suppressed. Fixed attachment refers to a tight seal that requires physical peeling during removal. Fixation methods that use a load other than their own weight to prevent movement are not included in this "fixed attachment" method.

[0049] In this specification, physical means may be mentioned, such as methods of peeling off by holding the film or methods of peeling off by damaging it with a knife or the like.

[0050] Furthermore, as one method for immersing the culture carrier substrate 10 in the culture medium 30 without fixing it in place, one or more of the following methods can be used: a method to make the specific gravity of the culture carrier substrate 10 greater than that of the culture medium 30; a method to apply a weight to a portion of the culture surface 12 of the culture carrier substrate 10 for load fixation; or a method to fix the position of the culture carrier substrate 10 using a device. That is, in the above-described cell culture process, cell culture can be performed while the culture carrier substrate 10 is immersed in the culture medium 30 by load fixation and / or position fixation.

[0051] The transplantation process may include: Figure 1 The removal process of the culture carrier substrate 50 with cell sheets shown in (d) and Figure 1 The cell sheet 40 attachment process shown in (e)

[0052] exist Figure 1 In the removal process of (d), the culture carrier substrate 50 with cell sheets is removed from the culture container 20 and transported, for example, to the desired location such as the transplantation site.

[0053] In the removal process of this embodiment, the removal method is not particularly limited, but for example, the method of pinching and removing the culture carrier substrate 10 that constitutes the culture carrier substrate 50 with cell sheets, the method of contacting the culture carrier substrate 10 with the suction nozzle and lifting it up while suction, and the method of hanging the culture carrier substrate 10 with a string and pulling it up, etc.

[0054] exist Figure 1 In the attachment process of (e), after attaching the cell sheet 40 of the culture carrier substrate 50 with cell sheet to the transplantation site 70, the culture carrier substrate 10 is peeled off from the cell sheet 40.

[0055] In this embodiment, the time from attachment to peeling can be shortened during the application process. By shortening the time, the cell sheets are prevented from remaining as foreign matter for extended periods, thus reducing the risk of immune rejection of the substrate membrane, decreasing the likelihood of cell death, and simplifying the operation. It can also be used in surgeries such as open-chest surgery to attach cell sheets in vivo.

[0056] The self-attachment to peel-off time is preferably less than 10 minutes, more preferably less than 5 minutes, further preferably less than 3 minutes, and even more preferably less than 1 minute.

[0057] The detailed mechanism is not yet clear, but it can be inferred that because the culture surface 12 of the culture carrier substrate 10 and the surface of the cell sheet 40 are in moderate strength of adhesion, the culture carrier substrate 10 can be detached from the cell sheet 40 even before the cell sheet 40 bio-binds to the transplanted site 70.

[0058] Furthermore, in this embodiment, the residual cell sheets 40 on the culture carrier substrate 10 after peeling can be suppressed. Calculated by area ratio, the residual rate of cell sheets 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.

[0059] Cell transplantation therapy aims to inhibit and prevent the onset and recurrence of symptoms related to defects, dysfunctions, and insufficiency of cells, tissues, and organs. Examples of diseases suitable for cell transplantation therapy include spinal cord injury, knee cartilage damage, ischemic heart disease, age-related macular degeneration, limbal stem cell deficiency, aplastic anemia, severe limb ischemia, refractory skin ulcers, prevention of postoperative complications (such as incomplete suturing of various organs, bronchial stump fistula, pancreatic juice fistula, bile fistula), and burns.

[0060] Furthermore, the cells used in cell transplantation therapy can be autologous cells, allogeneic non-autologous cells, or xenogeneic cells. From the perspective of clinical application and safety, autologous cells are preferred, while from the perspective of clinical application and productivity, non-autologous cells are preferred.

[0061] The following describes the key elements of each step in the transplantation process.

[0062] (Cultivation container) The culture container 20 is a container for culturing cells in a culture medium, and there are no particular restrictions as long as it is suitable for the type of cells being cultured and the intended use.

[0063] Examples of culture containers 20 include, for example, petri dishes, Petri dishes, tissue culture dishes, multi-well dishes, flasks, tissue culture flasks, microplates, microwell plates, multiplates, multiwell plates, chamber slides, shallow culture dish trays, test tubes, culture trays, culture bags, and roller flasks.

[0064] Furthermore, the culture container 20 can be a surface-treated culture container that imparts cell adhesion (for cell adhesion) or an untreated culture container (for suspension cells). Whether or not the culture container 20 has undergone surface treatment does not affect the culture carrier substrate 10. Unless otherwise specified, either type may be used.

[0065] There are no particular restrictions on the material of the culture container 20 as long as it does not permeate the culture medium 30. However, examples that can be listed include polystyrene, polyethylene, polypropylene, polyvinyl alcohol, polyethylene terephthalate, polyacetal, polyvinyl chloride, acrylic resin, polycarbonate, polyetheretherketone, polyethersulfone, polytetrafluoroethylene, polyimide, polyamide, cellulose, silicone, nylon 6,6, glass, stainless steel, or aluminum and other metals.

[0066] There are no particular restrictions on the area of ​​the culture container 20; any commercially available culture container can be used without any problems. For example, the area of ​​culture container 20 is 0.3 cm². 2Above and 1000cm 2 The following is a lower limit value, more preferably 0.35cm. 2 The above is further preferred to be 1.0cm. 2 The above, especially preferred, is 1.9cm. 2 That's all. On the other hand, as an upper limit, 900cm is more preferable. 2 The following is a further preferred length of 800cm. 2 The following is particularly preferred: 500cm 2 the following.

[0067] (cell) Cells are clinically useful cells used to treat or prevent symptoms related to defects, dysfunctions, or insufficiency of cells, tissues, or organs, or culturable cells used in non-clinical trials, as long as they are isolated from an organism, without particular restrictions.

[0068] 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 differentiation-induced stem cells or precursor cells. Furthermore, cells can be adherent cells or suspension cells.

[0069] Specific examples of biological tissue cells include fibroblasts, myofibroblasts, corneal epithelial cells, retinal cells, nerve cells, muscle cells, cardiomyocytes, myoblasts, osteoblasts, chondrocytes, adipocytes, hepatocytes, pancreatic cells, kidney cells, gingival cells, periosteal cells, skin cells, endothelial cells, etc.

[0070] Specific examples of mesenchymal stem cells include mesenchymal stem cells derived from adipose tissue, mesenchymal stem cells derived from bone marrow, mesenchymal stem cells derived from umbilical cord blood, and mesenchymal stem cells derived from the umbilical cord.

[0071] 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.

[0072] Furthermore, these cells can be cultured individually or in combination of two or more types. Appropriate settings can be made for these cells based on their intended use and other known cell types.

[0073] Furthermore, there are no particular restrictions on the source of cells; examples include mammals, birds, amphibians, fish, insects, plants, and microorganisms. 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.

[0074] (Cultivation conditions) Cell culture has no particular limitations and can be used in common techniques used in the fields of medicine, pharmaceuticals, quasi-pharmaceuticals, cosmetics, food, veterinary drugs, and basic technologies such as regenerative medicine and bioengineering.

[0075] There are no particular restrictions on culture conditions as long as they allow the cultured cells to reach the target state. Typical culture conditions include, for example, 37°C and 5% CO2 using the prepared basal culture medium.

[0076] There are no particular restrictions on the duration of cell culture, as long as the cells reach the target state. Suitable culture times include, for example, within 28 days, 21 days, 14 days, 7 days, 5 days, and 3 days. For long-term culture, the culture medium can be changed once. There are no particular restrictions on the frequency and method of medium exchange. Generally, it is preferred to change the medium every 1 to 7 days. Particularly preferred is every 1 to 5 days. At this time, all the culture medium can be exchanged, or a portion can remain and new medium can be added.

[0077] There are no particular restrictions on the density of the cultured cells, as long as it is suitable for the cells, the culture container, and the intended use of the cells; for example, 5 × 10⁶ cells / year. 2 cells / cm 2 Above and 1×10 9 cells / cm 2 The following is a preferred lower limit for cell density: 1 × 10⁻⁶. 3 cells / cm 2 The above is further optimized to be 5×10 3 cells / cm 2 The above is particularly preferred, with 5×10 4 cells / cm 2 That's all. On the other hand, as an upper limit for cell density, 1×10⁻⁶ is more preferable. 8 cells / cm 2 The following is a further preferred option: 5×10 7 cells / cm 2 The following is particularly preferred: 1×10 7 cells / cm 2 the following.

[0078] The density of the cultured cells, for example, is 5 × 10⁻⁶. 2 cells / cm 2 Above and 1×10 9 cells / cm 2 Below, 5×10 2 cells / cm 2 Above and 1×108 cells / cm 2 Below, 5×10 2 cells / cm 2 Above and 5×10 7 cells / cm 2 Below, 5×10 2 cells / cm 2 Above and 1×10 7 cells / cm 2 Below, 1×10 3 cells / cm 2 Above and 1×10 9 cells / cm 2 Below, 1×10 3 cells / cm 2 Above and 1×10 8 cells / cm 2 Below, 1×10 3 cells / cm 2 Above and 5×10 7 cells / cm 2 Below, 1×10 3 cells / cm 2 Above and 1×10 7 cells / cm 2 Below, 5×10 3 cells / cm 2 Above and 1×10 9 cells / cm 2 Below, 5×10 3 cells / cm 2 Above and 1×10 8 cells / cm 2 Below, 5×10 3 cells / cm 2 Above and 5×10 7 cells / cm 2 Below, 5×10 3 cells / cm 2 Above and 1×10 7 cells / cm 2 Below, 5×10 4 cells / cm 2 Above and 1×10 9 cells / cm 2 Below, 5×10 4 cells / cm 2 Above and 1×10 8 cells / cm 2 Below, 5×10 4 cells / cm 2 Above and 5×10 7cells / cm 2 Below, 5×10 4 cells / cm 2 Above and 1×10 7 cells / cm 2 the following.

[0079] (culture medium) There are no particular restrictions on the culture medium as long as it is suitable for the cells being cultured. Examples of components of the culture medium include, for instance, carbohydrates, amino acids, vitamins, inorganic salts, trace metals, and additives.

[0080] Furthermore, these culture medium components can be blended individually or in combination of two or more. The appropriate formulation of these components can be determined from known culture media, depending on the type of cells being cultured.

[0081] Specific examples of sugars include monosaccharides such as glucose or fructose, mannose, and galactose; disaccharides such as sucrose or sucralose, trehalose, maltose, and lactose; trisaccharides such as glucosyl sucrose or lactulose and raffinose; tetrasaccharides such as acarbose or maltotetrasaccharide; and cyclodextrin and oligosaccharides.

[0082] Specific examples of amino acids include L-glutamic acid, L-glutamine, L-arginine, L-cysteine, 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.

[0083] Specific examples of vitamins include sodium L-ascorbate, L-ascorbic acid pyrophosphate, choline, folic acid, niacin, biotin, pantothenic acid, pyridoxine, riboflavin, thiamine, thymidine, and vitamin B12.

[0084] 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, and magnesium carbonate.

[0085] Specific examples of trace metals include ferric sulfate, ferric nitrate, copper sulfate, copper nitrate, and zinc sulfate.

[0086] Specific examples of additives include bovine serum, horse serum, human serum, etc.; growth factors such as FGF2, EGF, HGF, VEGF, PDGF, etc.; proteins such as albumin; antioxidants such as glutathione, ascorbic acid, and ascorbic acid derivatives; antibiotics such as penicillin or streptomycin; pH adjusters such as HEPES; organic acids such as lactic acid or propionic acid; lipids such as cholesterol; fatty acids such as linolenic acid; amines such as ethanolamine or putrescine; reducing agents such as mercaptoethanol or 3-mercapto-1,2-propanediol; thickeners such as sodium alginate, polyvinylpyrrolidone, carboxymethyl cellulose, pullulan, etc.; and pH indicators such as phenol red.

[0087] Culture medium 30 containing the above-mentioned culture medium components may include, for example, AIM V medium, HFDM-1 medium, Dublin phosphate buffer (D-PBS) or Hanks balanced salt solution (HBSS), etc.; DMEM (Dulbecco's Modified Eagle Medium) or EMEM (Eagle's Minimum Essential Medium), α-MEM (Minimum Essential Medium alpha Modification), IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), Ham's F-10 medium, Ham's F-12 medium, Ham's F-12K medium, RPMI 1640 medium, M-199 medium, L-15 medium, McCoy's Basic culture media include 5A medium, MCDB105 medium, MCDB107 medium, MCDB131 medium, MCDB153 medium, MCDB201 medium, NCTC109 medium, NCTC135 medium, Waymouth's MB752 / 1 medium, CMRL-1066 medium, Williams' E medium, Brinster's BMOC-3 medium, E8 medium, etc.

[0088] Furthermore, these basal culture media can be used alone or in combination of two or more. Moreover, the components of the basal culture media can be added, removed, increased, or decreased according to the type or state of the cells. These basal culture media can be appropriately formulated based on known culture media, such as those used for culturing the cells.

[0089] (Cell slice) Cell sheet 40 is a cell sheet with a sheet structure in which cells are physically and functionally connected to each other through adhesion molecules, extracellular matrix, etc.

[0090] Cell sheet 40 can be a single-layer structure consisting of one cell layer, or a stacked structure consisting of two or more cell layers. There are no particular restrictions on the stacked structure; multi-layer structures with two, three, four, or five layers can be listed.

[0091] When the cell sheet 40 has a multilayer structure, it is sometimes obtained during culture using the culture carrier substrate 10, or it can be obtained by stacking cell sheets with a single-layer structure. In particular, by preparing multiple culture carrier substrates 50 of the present invention with cell sheets, overlapping one cell sheet on one cell sheet, and peeling the culture carrier substrate from the other cell sheet, a cell sheet with a multilayer structure can be obtained.

[0092] The thickness of the cell sheet 40 is not particularly limited, for example, it is 0.001 mm or more and 2.0 mm or less. As a lower limit for the thickness of the cell sheet 40, it is more preferably 0.01 mm or more, further preferably 0.03 mm or more, and particularly preferably 0.05 mm or more. On the other hand, as an upper limit for the thickness of the cell sheet 40, it is more preferably 1.5 mm or less, further preferably 1.2 mm or less, and particularly preferably 1.0 mm or less. The thickness of the cell sheet 40 can be, for example, 0.001 mm or more and 2.0 mm or less, 0.001 mm or more and 1.5 mm or less, 0.001 mm or more and 1.2 mm or less, 0.001 mm or more and 1.0 mm or less, 0.01 mm or more and 2.0 mm or less, 0.01 mm or more and 1.5 mm or less, 0.01 mm or more and 1.2 mm or less, 0.01 mm or more and 1.0 mm or less, 0.03 mm or more and 2.0 mm or less, 0.03 mm or more and 1.2 mm or less, 0.03 mm or more and 1.0 mm or less, 0.05 mm or more and 2.0 mm or less, 0.05 mm or more and 1.5 mm or less, 0.05 mm or more and 1.2 mm or less, and 0.05 mm or more and 1.0 mm or less. By setting the thickness of the cell sheet 40 within the above ranges, high cell viability and excellent shape maintenance capabilities beneficial for cell transplantation can be achieved within the cell sheet 40.

[0093] There is no particular limitation on the area of ​​cell sheet 40, for example, it can be 0.3 cm. 2 Above and 1000cm 2 The following is a preferred lower limit for the area of ​​cell sheet 40: 0.6 cm². 2 The above is further preferred to be 1.6cm. 2 The above is particularly preferred, with 8cm being the ideal size. 2 The above can also be 3cm. 2 Above, 6cm 2 Above or 10cm 2That's all. On the other hand, as an upper limit for the area of ​​the cell sheet 40, 900 cm² is more preferable. 2 The following is a further preferred length of 800cm. 2 The following is particularly preferred: 500cm 2 The following can also be 100cm 2 Below, 50cm 2 Below or 20cm 2 For example, the area of ​​cell slice 40 is 0.6 cm². 2 Above and 900cm 2 Below, 1.6cm 2 Above and 800cm 2 Below, 3cm 2 Above and 500cm 2 Below, 6cm 2 Above and 100cm 2 Below, 8cm 2 Above and 50cm 2 Below or 10cm 2 Above and 20cm 2 The following is a summary of previous methods. Previously, when transplanting individual cell sheets, the low strength of the cell sheets led to a high risk of breakage during large-area transport. However, in this invention, the cell sheet 40 is supported by a culture carrier substrate, thus preventing breakage during transplantation. Therefore, the size of the cell sheet 40 can be increased to 8 cm. 2 That's all. In addition, large cell sheets can be prepared and appropriately adjusted to fit the affected area for use.

[0094] In addition, the shape of the culture carrier substrate is Figure 1 The shape shown is a circle, but it can also be a suitable shape other than a circle, such as a square, a regular pentagon, a regular hexagon, a regular octagon, or a regular polygon, or an ellipse, a rectangle, etc.

[0095] <Cultivation Carrier Substrate> The culture carrier substrate 10 of this embodiment is a substrate used for both the cell sheet scaffold material for culturing cells to form cell sheets 40 and the cell sheet support for transporting the cultured cell sheets 40, and is peeled off from the cell sheets after the cell sheets are attached to the transplantation site.

[0096] There are no particular limitations on the area of ​​the culture carrier substrate 10, but it is desirable to be smaller than the culture container and the same as or larger than the cell sheet. For example, 0.3 cm². 2 Above and 1000cm 2 The following is a lower limit for the area of ​​the culture carrier substrate 10, which is more preferably 0.6 cm². 2 The above is further preferred to be 1.6cm. 2The above is particularly preferred, with 8cm being the ideal size. 2 The above can also be 3cm. 2 Above, 6cm 2 Above or 10cm 2 That's all. On the other hand, the upper limit of the area of ​​the culture carrier substrate 10 is more preferably 900 cm². 2 The following is a further preferred length of 800cm. 2 The following is particularly preferred: 500cm 2 The following can also be 100cm 2 Below, 50cm 2 Below or 20cm 2 The following applies. If the culture container is larger than the specified size, cell sheets may not be obtainable. For example, the culture carrier substrate 10 has an area of ​​0.6 cm². 2 Above and 900cm 2 Below, 1.6cm 2 Above and 800cm 2 Below, 3cm 2 Above and 500cm 2 Below, 6cm 2 Above and 100cm 2 Below, 8cm 2 Above and 50cm 2 Below or 10cm 2 Above and 20cm 2 the following.

[0097] The thickness of the culture carrier substrate 10 is not particularly limited, but is preferably 5 μm or more and 250 μm or less. As a lower limit for the substrate thickness, 6 μm or more is more preferred, and 8 μm or more, 10 μm or more, or 12 μm or more are even more preferred. On the other hand, as an upper limit for the substrate thickness, 50 μm or less, 30 μm or less, or 25 μm or less are more preferred, and 20 μm or less are even more preferred. 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.

[0098] By setting the thickness to the lower limit or above, the culture carrier substrate 10 can be prevented from cracking or curling during transport, thus improving operability. By setting it to the upper limit or below, the ability to follow the affected area during transplantation can be improved. In particular, if the substrate thickness is set to 20 μm or less, the ability to follow the affected area with high curvature can be improved, for example, it can be applied to sites where organs are sutured in the body during surgery.

[0099] Furthermore, from the perspective of maximizing the function of the self-supporting membrane during culture, the lower limit of the thickness of the culture carrier substrate 10 is preferably greater than 10 μm, more preferably 11 μm or more, and even more preferably 12 μm or more. Thus, by using a culture carrier substrate 10 with a substrate thickness greater than 10 μm, it becomes possible to manufacture cell sheets 40 during the cell culture process without fixing or attaching the culture carrier substrate 10 to the inside of the culture container 20.

[0100] A self-supporting 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 medium 30, and more preferably a membrane that maintains its planar state even after cell sheets 40 are formed on the culture surface 12. Because the planar state of the culture surface 12 is maintained, the cell culture characteristics in the culture medium 30 can be improved even without fixed attachment of the uncurled culture carrier substrate 10.

[0101] Furthermore, this self-supporting membrane can maintain its sheet shape to some extent even when one end is pinched and lifted with tweezers. In this case, the area of ​​the culture carrier substrate 10 is preferably 1.6 cm². 2 above.

[0102] The culture surface 12 of the culture carrier substrate 10 is preferably subjected to a hydrophilic treatment. This improves cell adhesion. Examples of such hydrophilic treatments include UV ozone treatment and plasma treatment.

[0103] Preferably, the culture carrier substrate 10 with the culture surface 12 having undergone plasma treatment is used.

[0104] In this specification, a hydrophilized surface refers to a surface where the upper limit of the water droplet contact angle θ, measured using the θ / 2 method, is 70° or less. On the other hand, the lower limit of the water droplet contact angle θ of a hydrophilized surface is preferably 3° or more, but is not limited to this. The water droplet contact angle θ is determined by placing 2 μL of pure water on a substrate at 25°C and measuring the angle between the water droplet and the substrate using a contact angle meter according to the θ / 2 method of ISO 19403-2:2017.

[0105] Furthermore, the lower limit of the surface roughness Ra of the culture surface that has undergone hydrophilic treatment 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.

[0106] Furthermore, the surface roughness Ra refers to the arithmetic mean roughness in a square region with sides of 100 nm, measured using surface shape data obtained by atomic force microscopy (AFM).

[0107] Materials constituting the culture carrier substrate 10 include polyetheretherketone (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), polypropylene (PP), and other resin materials.

[0108] An example of the culture carrier substrate 10 may be composed of a membrane containing at least one of these resin materials as a main component, preferably a polyetheretherketone membrane or a polyethylene terephthalate membrane, more preferably a polyetheretherketone membrane.

[0109] The culture carrier substrate 10 preferably comprises one or more resin layers made of the aforementioned resin material. The two or more resin layers may contain different types of materials.

[0110] The culture carrier substrate 10 may be a resin substrate comprising a resin layer containing the aforementioned materials, or a laminated substrate comprising an inorganic layer comprising the aforementioned materials and other materials. The resin layer may not contain a layer formed by chemical vapor deposition such as parylene or a wet-coated layer. Furthermore, the inorganic layer in the laminated substrate preferably does not contain a glass layer and may contain metal foil. Additionally, the culture carrier substrate 10 preferably does not contain nonwoven fabric or fiber substrate on the culture surface 12 side.

[0111] Considering factors such as low coefficient of linear expansion, solvent resistance, and heat resistance, the culture carrier substrate 10 containing PEEK is preferred over the case containing PET.

[0112] Furthermore, the culture carrier substrate 10 is preferably made of a material with a specific gravity higher than that of the culture medium 30.

[0113] The culture carrier substrate 10, when measured using a laser microscope on its culture surface 12, contains no more than three pores with a diameter of 1 μm or more and 100 μm or less, and a depth of 0.5 μm or more and 100 μm, within a 100 μm square area. The diameter of these pores is, for example, 1 μm or more and 100 μm or less, preferably 2 μm or more and 50 μm or less, more preferably 3 μm or more and 30 μm or less. The depth of these pores is, for example, 0.5 μm or more and 100 μm or less, preferably 1 μm or more and 50 μm or less, more preferably 2 μm or more and 20 μm or less.

[0114] Combinations of the range of hole diameter and hole depth, such as: hole diameter greater than or equal to 1 μm and less than 100 μm and hole depth greater than or equal to 0.5 μm and less than 100 μm; hole diameter greater than or equal to 1 μm and less than 100 μm and hole depth greater than or equal to 1 μm and less than 50 μm; hole diameter greater than or equal to 1 μm and less than 100 μm and hole depth greater than or equal to 2 μm and less than 20 μm; hole diameter greater than or equal to 2 μm and less than 50 μm and hole depth greater than or equal to 0.5 μm and less than 100 μm; hole diameter of... Holes with a diameter of 2μm or more and less than 50μm and a depth of 1μm or more and less than 50μm; holes with a diameter of 2μm or more and less than 50μm and a depth of 2μm or more and less than 20μm; holes with a diameter of 3μm or more and less than 30μm and a depth of 0.5μm or more and less than 100μm; holes with a diameter of 3μm or more and less than 30μm and a depth of 1μm or more and less than 50μm; holes with a diameter of 3μm or more and less than 30μm and a depth of 2μm or more and less than 20μm.

[0115] Furthermore, 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. On the other hand, the lower limit of the porosity of the culture carrier substrate 10 is not particularly limited and can be 0% or more.

[0116] By making the number of pores on the culture surface 12 three or less and / or setting the porosity of the culture carrier substrate 10 to below the aforementioned upper limit value, a suitable adhesion force with the cell sheet 40 can be achieved.

[0117] In addition, the presence or absence of pores can be determined by measuring the surface of the resin layer formed on the culture surface 12 side of the culture carrier substrate 10.

[0118] Porosity is calculated based on theoretical density and measured density. Specifically, it is calculated using the formula: porosity = {1 - (measured density / theoretical density)} × 100.

[0119] Furthermore, the culture surface 12 of the culture carrier substrate 10 can be constructed without temperature-responsive polymers. This helps to prevent a decrease in the adhesion between the cell sheet 40 and the culture carrier substrate 10 under low-temperature conditions such as cryopreservation.

[0120] Temperature-responsive polymers are materials that exhibit cell adhesion at the temperature during cell culture and become non-adhesive to facilitate easy detachment of cell sheets by changing the temperature thereafter. It is preferable if the temperature range in which the temperature-responsive polymer exhibits cell adhesion is 10°C to 45°C, particularly 33°C to 40°C, allowing for stable cell culture. Furthermore, it is preferable if the temperature range in which the temperature-responsive polymer exhibits cell non-adhesion is 1°C to 36°C, particularly 4°C to 32°C, as this reduces damage to the cell sheets during detachment. Materials constituting temperature-responsive polymers include poly-N-isopropylacrylamide (PNIPAAm), poly-N-n-propylacrylamide, poly-N-n-propylmethylacrylamide, poly-N-ethoxyethylacrylamide, poly-N-tetrahydrofurfurylacrylamide, poly-N-tetrahydrofurfurylmethylacrylamide, and poly-N,N-diethylacrylamide, among which PNIPAMAm, poly-N-n-propylmethylacrylamide, and poly-N,N-diethylacrylamide are preferred.

[0121] The culture carrier substrate 10 used for pre-culturing cell slices can be packaged in packaging materials. By creating a sealed package, it can be stored and distributed without contaminating the culture surface. Furthermore, the package can be sterilized. Sterilization methods include electron beam sterilization, gamma ray sterilization, EOG sterilization, and autoclaving.

[0122] Packaging materials may include, for example, aluminum, polyethylene terephthalate, ionomers, 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 fluoropolymers, nylon, cellophane, and paper. These materials can be used alone or in combination of two or more. In cases requiring breathable sterilization, such as EOG sterilization or autoclaving, packaging materials combining paper with the aforementioned materials are preferred. Furthermore, packaging materials can be made into multiple bundles or further stacked after sterilization; preferably, the outermost part of the packaging is a resin film-like material.

[0123] By culturing cell sheets 40 on culture carrier substrate 10, a culture carrier substrate 50 containing cell sheets 40 and culture carrier substrate 10 can be obtained.

[0124] In the culture carrier substrate 50 with cell sheets, the cell sheets 40 are in a state where cells can accumulate on the surface of the culture surface 12, covering, for example, 50% to 100% of it. When using a method that fixes a portion of the culture surface 12 of the culture carrier substrate 10 with a weight or a method that fixes the culture carrier substrate 10 in position using a device, the area where cells can accumulate refers to the area where cells can accumulate without contact with the weight or device. Furthermore, when the culture surface 12 of the culture carrier substrate 10 has undergone a hydrophilic treatment, the area where cells can accumulate refers to the area where the hydrophilic treatment has been performed. The lower limit of the coverage area of ​​the cell sheets 40 is preferably 70% or more, more preferably 90% or more. The upper limit of the coverage area of ​​the cell sheets 40 can be 100% or less, 99% or less, or 98% or less. The coverage area of ​​the cell sheet 40 is, for example, 50% or more and less than 100%, 50% or more and less than 99%, 50% or more and less than 98%, 70% or more and less than 100%, 70% or more and less than 99%, 70% or more and less than 98%, 90% or more and less than 100%, 90% or more and less than 99%, or 90% or more and less than 98%. Furthermore, the area of ​​the culture carrier substrate 10 is preferably the same as or larger than the area of ​​the cell sheet 40 where cells can be stacked. By setting it to the lower limit or above, the curative effect can be improved.

[0125] Furthermore, by forming cell sheets on the culture carrier substrate, the possibility of cell sheet breakage when removing them from the culture container or cryopreservation container is reduced, allowing for easy removal. Additionally, the cell sheets can be easily peeled off from the culture carrier substrate after being attached to the transplantation site. Because the cell sheets are easier to handle, large-sized cell sheets can be produced.

[0126] <Manufacturing Method of Culture Carrier Substrate with Cell Sheets> An example of the method for manufacturing the cell-sheet culture carrier substrate 50 of this embodiment may include, for example: a culture step, introducing multiple cells, culture medium (culture solution 30) and culture carrier substrate 10 into the interior of a culture container 20, and culturing cell sheets 40 on the culture surface 12 of the culture carrier substrate 10; and a removal step, removing the cell-sheet culture carrier substrate 50 from the culture solution 30.

[0127] In this embodiment, the cell sheet 40 is a cell sheet peeled off from the culture carrier substrate 50, which has its own cell sheet, and can be used for treatment, prevention, etc., such as cell transplantation therapy.

[0128] There are no particular limitations on the stripping method; common techniques used in the fields of medical, pharmaceutical, quasi-pharmaceutical, cosmetic, food, and veterinary medicine, as well as in fundamental fields such as regenerative medicine and bioengineering, can be employed. Examples of stripping methods include physical treatments.

[0129] <Freezing preservation method> An example of the cryopreservation method in this implementation scheme, such as Figure 1 As shown in (c), the process includes a cooling step in which the culture carrier substrate 10 containing the interior of the culture container 20 and the cell sheet 40 formed on the surface (culture surface 12) of the culture carrier substrate 10 are cooled in a cryopreservation solution 60. The culture container may be frozen by applying a cap for culture (not shown) or a cap to prevent leakage and microbial contamination. Figure 1 In (c), the culture carrier substrate 50 with cell sheets is cryopreserved inside the culture container 20, but the culture carrier substrate 50 with cell sheets can also be transferred to a cryopreservation container different from the culture container 20 for cryopreservation. There are no particular limitations on the cryopreservation container, and the container exemplified in the culture container 20 can be used, for example.

[0130] During the cooling process, the culture carrier substrate 50 with cell sheets can take various forms. For example... Figure 3 As shown in (a) and (b), the culture carrier substrate 50 with cell sheets can have a structure in which the culture carrier substrate 10 is formed on one side of the cell sheet 40, as shown in (a) and (b). Figure 3 As shown in (c), it can also have a structure in which two culture carrier substrates 10a and 10b are added to both sides of the cell sheet 40. Figure 3 In (a), the culture carrier substrate 10 is arranged with its bottom surface facing the culture container 20. Figure 3 In (b), the cell sheet 40 is positioned with the bottom surface of the culture container 20 facing towards it. Figure 3 In (c), the culture carrier substrates 10a and 10b may be made of the same material or may be made of 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 without PEEK.

[0131] (Cryopreservation solution) Cryopreservation solution 60 is a solution used to reduce cell damage caused by cryopreservation.

[0132] There are no particular restrictions on cryopreservation solution 60 as long as it is suitable for cell cryopreservation; it may contain the same components as the culture medium, cryoprotectants, etc.

[0133] For sugars, amino acids, vitamins, inorganic salts, trace metals, and additives used as components of the culture medium, as long as the descriptions in the (culture medium) section above are met, they are acceptable. Furthermore, the cryopreservation solution preferably contains substances with a freezing point in the range of -15°C to -5°C.

[0134] Cryoprotectants are substances used to reduce cell damage caused by freezing or thawing during cryopreservation. Examples of cryoprotectants include cell-non-permeable cryoprotectants and cell-permeable cryoprotectants.

[0135] Specific examples of cell-nonpermeable cryoprotectants include albumin, sucrose, trehalose, dextran, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and polylysine.

[0136] Specific examples of cell-permeable cryoprotectants include dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, and propanediol.

[0137] Furthermore, these cryoprotectants can be blended individually or in combination of two or more. The appropriate settings for these cryoprotectants can be made from known cryoprotectants, depending on the cell type, the composition of the cryopreservation solution, and other relevant factors.

[0138] Examples of commercially available cryopreservation solutions that do not contain DMSO include: STEM-CELLBANKER (registered trademark) DMSO-free GMP grade (NIPPON ZENYAKU KOGYO CO.,LTD.), BAMBANKER (registered trademark) DMSO-free (GCLymphotec, Inc.), Cryo Scarless (registered trademark) DMSO-free (Bioverde Co., Ltd.), StemCellKeep (Bioverde Co., Ltd.), CryoNovo (registered trademark) X12 (Akron BioProducts LLC), CryoNovo (registered trademark) P24 (Akron BioProducts LLC), DMSO-free cell cryopreservation solutions for human ES / iPS cells (REPROCELL, Inc.), Cell Reservoir One (NACALAI TESQUE, Inc.), ThelioKeep (registered trademark: Bioverde Co., Ltd.), Cellvation (registered trademark: Protide Pharmaceuticals Inc.), and ReproCryo. RM (REPROCELL Inc.), SOFORO Cryo (SARAYA Co., Ltd.), etc. In addition, commercially available cryopreservation solutions containing DMSO include, for example, STEM-CELLBANKER (registered trademark) GMP grade (NIPPON ZENYAKU KOGYO CO.,LTD.), STEM-CELLBANKER (registered trademark) EX GMP grade (NIPPON ZENYAKU KOGYO CO.,LTD.), BAMBANKER (registered trademark) hRM (GC Lymphotec, Inc.), BAMBANKER (registered trademark) (GC Lymphotec, Inc.), iStock (GC Lymphotec, Inc.), CryoStor CS5 (Charles River Laboratories Cell Solutions, Inc.), CryoStor CS10 (Charles River Laboratories Cell Solutions, Inc.), etc.

[0139] (Cooling process) In the cooling process, it is preferable to use a non-flow cooling device in the cryopreservation solution to cool the culture carrier substrate 50 containing cell sheets. Since the cell sheets 40 can be cooled at a uniform temperature on a suitable culture carrier substrate 10 by a non-flow cooling device, the damage to the cells is minimal and the decrease in cell viability can be suppressed.

[0140] In the cooling process, the cooling rate at 0 to -5°C is, for example, 0.1°C / min or more and 15°C / min or less, preferably 0.25°C / min or more and 12.5°C / min or less, and more preferably 0.5°C / min or more and 10°C / min or less.

[0141] Setting the value above the lower limit reduces unnecessary contact time between the liquid cryoprotectant and the cells. Setting it below the upper limit inhibits the formation of intracellular ice crystals.

[0142] In the cooling process, the freezing temperature is not particularly limited as long as it can freeze the cultured cells and the cryopreservation solution. For example, the freezing temperature is -196°C or higher and -25°C or lower. As a lower limit, -180°C or higher is more preferred, further preferred is -160°C or higher, and particularly preferred is -150°C or higher. On the other hand, as an upper limit, -25°C or lower is more preferred, further preferred is -30°C or lower, and particularly preferred is -35°C or lower. The freezing temperatures are, for example, above -196°C and below -25°C, above -196°C and below -30°C, above -196°C and below -35°C, above -180°C and below -25°C, above -180°C and below -30°C, above -180°C and below -35°C, above -160°C and below -25°C, above -160°C and below -30°C, above -160°C and below -35°C, above -150°C and below -25°C, above -150°C and below -30°C, and above -150°C and below -35°C.

[0143] There are no particular limitations on the cooling device used in the freezing process; examples include rapid freezing devices and ultra-low temperature refrigeration devices. From the perspective of freezing cells at a uniform temperature to improve the survival rate of thawed cells, a freezing device that does not contact the heat transfer tool and freezes the culture container and cells by blowing cold air from multiple directions rather than one direction, preferably from all directions, is more preferable than freezing the culture container and cells by contacting the heat transfer tool with the culture container. Specifically, a freezing device that uses the circulation of cold air from a cooling fan to cool the object being cooled can be cited as an example, such as the non-cross-flow cooling device with a cooling fan disclosed in Japanese Patent Application Publication No. 2005-127666. Furthermore, "non-cross-flow" means that most of the cross-flow air from the object being cooled does not pass through a (cross-flow) cooler.

[0144] (Cryopreservation process) The cryopreservation method preferably includes a cryopreservation step after the cooling step, wherein the cryopreservation step preserves the culture carrier substrate 50 containing cell sheets at, for example, -196 to -60°C, preferably -180 to -65°C, more preferably -150 to -80°C. This allows for the stable maintenance of the cell sheets over a long period.

[0145] The temperature of the cooling process can also be the set temperature of the cooling device.

[0146] There are no particular restrictions on cryopreservation as long as the cells can be stably cryopreserved.

[0147] Methods of cryopreservation include, for example, contact with a liquid or gaseous coolant, and the use of an ultra-low temperature freezer. From a temperature perspective, the preferred cryopreservation method is contact with the liquid or gaseous phases of a coolant.

[0148] Examples of coolants include liquid nitrogen, liquid ethane, liquid propane, liquid helium, and dry ice.

[0149] There are no particular restrictions on the cryopreservation temperature, as long as the cells can be stably cryopreserved. For example, the cryopreservation temperature can be any of the following ranges: above -196°C and below -60°C, above -196°C and below -134°C, or above -134°C and below -60°C.

[0150] The cryopreservation temperature can also be determined using the surface temperature of the cell slice 40 to be cryopreserved. The surface temperature can be measured, for example, using a K thermocouple.

[0151] The frozen culture carrier substrate 50 with cell sheets includes a culture carrier substrate 10 and cell sheets 40 formed on the surface (culture surface 12) of the culture carrier substrate 10. In the culture carrier substrate 50 with cell sheets, both the culture carrier substrate 10 and the cell sheets 40 are in a cryopreserved state.

[0152] In the aforementioned frozen products, the culture carrier substrate 10 and cell sheets 40 are cryopreserved, for example, at the aforementioned cryopreservation temperature, specifically preferably below -60°C, and more preferably below -135°C. Furthermore, the frozen products may also be cryopreserved in a range of -134°C to -60°C. Additionally, the frozen products are cryopreserved at temperatures above -196°C.

[0153] Frozen products containing cell-bearing culture carrier substrates, frozen inside culture or cryopreservation containers, can be packaged using packaging materials while frozen in the culture or cryopreservation container or removed from the container. By creating a sealed package, microorganisms can be prevented from contaminating the cell slices, and the cells can be stored and circulated without being damaged. As an example, a packaged frozen product can be obtained by freezing a culture carrier substrate containing cell slices and a container of cryopreservation solution in a sealed state using a membrane-like packaging material. Preferred packaging materials include aluminum, polyethylene terephthalate, ionomers, polyethylene, polyvinylidene chloride, polyvinyl alcohol, polypropylene, polyester, polycarbonate, polyacrylonitrile, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, ethylene-methacrylic acid copolymer, polyimide, perfluoroalkoxy fluoropolymers, or tetrafluoroethylene-hexafluoropropylene copolymer (FEP), and nylon. These materials can be used alone or in combination of two or more.

[0154] (Thawing process) In the case where the transplantation method of this embodiment includes the above-described cryopreservation method, the cryopreservation method further includes a thawing step of thawing the culture carrier substrate 50 with cell sheets.

[0155] There are no particular restrictions on the thawing method; common methods used in the fields of medical, pharmaceutical, quasi-pharmaceutical, cosmetic, food, and veterinary medicine, as well as in basic fields such as regenerative medicine and bioengineering, can be employed.

[0156] Methods for thawing include, for example, using a water bath, incubator, heating plate, thawing device, or immersing in a thawing solution at a temperature higher than the freezing temperature, or placing it in an environment at a temperature higher than the freezing temperature.

[0157] There are no particular restrictions on the ambient temperature that the frozen food comes into contact with during thawing, as long as it is above the freezing temperature and below 50°C. The upper limit of the aforementioned ambient temperature is, for example, below 49°C, preferably below 45°C, and more preferably below 40°C. On the other hand, the lower limit of the ambient temperature is, for example, above 0°C, preferably above 5°C, more preferably above 10°C, and even more preferably above 15°C.

[0158] The range of ambient temperatures that the frozen food comes into contact with during thawing, for example, above 0°C and below 49°C, above 0°C and below 45°C, above 0°C and below 40°C, above 5°C and below 49°C, above 5°C and below 45°C, above 5°C and below 40°C, above 10°C and below 49°C, above 10°C and below 45°C, above 10°C and below 40°C, above 15°C and below 49°C, above 15°C and below 45°C, and above 15°C and below 40°C.

[0159] Furthermore, thawing temperatures above 50°C are not preferred as they may cause thermal damage to cells. Alternatively, the food can be temporarily stored at temperatures below its freezing point during thawing. For example, frozen food stored at -80°C can be exposed to an ambient temperature of -30°C and then thawed at an ambient temperature above its freezing point.

[0160] There are no particular restrictions on the thawing time of frozen food, as long as it does not cause damage to the cells due to freezing. Generally, thawing is acceptable as long as it is greater than 10 seconds and less than 60 minutes. As a lower limit for the thawing time of frozen food, it is acceptable as long as it is greater than 10 seconds, preferably more than 20 seconds, more preferably more than 30 seconds, and even more preferably more than 1 minute. As an upper limit, it is acceptable as long as it is less than 60 minutes, preferably less than 50 minutes, more preferably less than 40 minutes, and even more preferably less than 30 minutes. For example, the time required for thawing is greater than 10 seconds and less than 60 minutes, greater than 10 seconds and less than 50 minutes, greater than 10 seconds and less than 40 minutes, greater than 10 seconds and less than 30 minutes, more than 20 seconds and less than 60 minutes, more than 20 seconds and less than 50 minutes, more than 20 seconds and less than 40 minutes, more than 20 seconds and less than 30 minutes, more than 30 seconds and less than 60 minutes, more than 30 seconds and less than 50 minutes, more than 30 seconds and less than 40 minutes, more than 30 seconds and less than 30 minutes, more than 1 minute and less than 60 minutes, more than 1 minute and less than 50 minutes, more than 1 minute and less than 40 minutes, and more than 1 minute and less than 30 minutes.

[0161] If thawing is too rapid, thermal shock from the temperature difference may cause cracks in the frozen material, resulting in cell breakage. If thawing is too slow, water molecules recrystallize at freezing points, causing ice crystals to grow larger and severely damaging cells; therefore, this is not the preferred method.

[0162] During the aforementioned thawing time, the ambient temperature can be set to a constant value or to a variable value such as a phased increase.

[0163] There are no particular restrictions on the thawing medium as long as it does not damage the cultured cells. Examples of components that can be included in the thawing medium include sucrose, glucose, maltose, trehalose, and fructose. Furthermore, the thawing medium may also contain any of the components listed in the section on (culture medium).

[0164] There are no particular limitations on the temperature of the melting solution as long as it is above the freezing temperature. For example, the temperature of the melting solution is 0°C or higher and 45°C or lower. As a lower limit, 4°C or higher is more preferred, 25°C or higher is even more preferred, and 28°C or higher is particularly preferred. On the other hand, as an upper limit, 40°C or lower is more preferred, 39°C or lower is even more preferred, and 38°C or lower is particularly preferred. The temperature of the melting solution, for example, is above 0°C and below 45°C, above 0°C and below 40°C, above 0°C and below 39°C, above 0°C and below 38°C, above 4°C and below 45°C, above 4°C and below 40°C, above 4°C and below 39°C, above 4°C and below 38°C, above 25°C and below 45°C, above 25°C and below 40°C, above 25°C and below 39°C, above 25°C and below 38°C, above 28°C and below 45°C, above 28°C and below 40°C, above 28°C and below 39°C, above 28°C and below 38°C.

[0165] The thawed cell sheets 40 and culture carrier substrate 10 (culture carrier substrate 50 with cell sheets) can be washed immediately with cell washing solution after thawing, as needed. There are no particular restrictions on the cell washing solution; it may contain the components described in the (culture medium) section above.

[0166] There is no particular limitation on the temperature of the cell washing solution. For example, the temperature of the cell washing solution is 0°C or higher and 45°C or lower. As a lower limit, it is more preferably 4°C or higher, further preferably 25°C or higher, and particularly preferably 28°C or higher. On the other hand, as an upper limit, it is more preferably 40°C or lower, further preferably 39°C or lower, and particularly preferably 38°C or lower.

[0167] There is no particular limit to the number of times cultured cells can be washed, either once or multiple times (e.g., 2, 3, 4, 5, etc.).

[0168] In this invention, since the culture surface 12 does not contain temperature-responsive polymers, the cell sheet 40 can be prevented from peeling off from the culture carrier substrate 10 under low-temperature conditions such as cryopreservation. Even after cryopreservation and thawing processes, a culture carrier substrate 50 with cell sheets can be obtained that maintains the adhesion between the cell sheet and the membrane.

[0169] The embodiments of the present invention have been described above, but these are examples of the present invention, and various configurations other than the above can be adopted. In addition, the present invention is not limited to the above embodiments, and modifications, improvements, etc. carried out within the scope of achieving the object of the present invention are all included in the present invention.

[0170] Examples of Reference Forms A to J are attached below.

[0171] <A. Culture Carrier Substrate> As Reference Form A of the culture carrier substrate, the following items 1 to 13 are listed.

[0172] 1. A culture carrier substrate for transporting a cell sheet cultured on a culture surface, wherein the substrate thickness of the culture carrier substrate is 5 μm or more and 250 μm or less.

[0173] 2. The culture carrier substrate according to item 1, wherein the substrate thickness of the culture carrier substrate is greater than 10 μm, and in the culture of the cell sheet, the culture carrier substrate does not fixedly adhere to the inside of the culture container and functions as a self-supporting film.

[0174] 3. The culture carrier substrate according to item 1 or 2, wherein the culture surface is subjected to a hydrophilic treatment.

[0175] 4. The culture carrier substrate according to item 3, wherein the hydrophilic treatment is a UV ozone treatment or a plasma treatment.

[0176] 5. The culture carrier substrate according to any one of items 1 to 4, wherein the culture carrier substrate is a polyether ether ketone film or a polyethylene terephthalate film.

[0177] 6. The culture carrier substrate according to any one of items 1 to 5, wherein the culture surface does not contain a temperature-responsive polymer.

[0178] 7. The culture carrier substrate according to any one of items 1 to 6, wherein the area of the culture carrier substrate is 0.3 cm 2 or more and 1000 cm 2 or less.

[0179] 8. The culture carrier substrate according to any one of items 1 to 7, wherein the culture carrier substrate includes one layer or two or more resin layers composed of a resin material.

[0180] 9. The culture carrier substrate according to any one of Items 1 to 8, wherein when measuring the culture surface using a laser microscope, in at least one observation range of 100 μm square, the number of holes with a diameter of 1 μm or more and 100 μm or less and a depth of 0.5 μm or more and 100 μm is 3 or less.

[0181] 10. The culture carrier substrate according to any one of Items 1 to 9, having a porosity of 15% or less.

[0182] 11. The culture carrier substrate according to any one of Items 1 to 10, having a water contact angle of the culture surface of 70° or less.

[0183] 12. The culture carrier substrate according to any one of Items 1 to 11, which is used for cryopreserving a cell sheet formed on the culture surface.

[0184] 13. The culture carrier substrate according to Items 1 to 12, which is used for attaching a cell sheet formed on the culture surface to a transplantation site.

[0185] <B. Culture carrier substrate with a cell sheet> As the reference form B of the culture carrier substrate with a cell sheet, the following Items 1 to 21 are listed.

[0186] 1. A culture carrier substrate with a cell sheet, having a laminate of a culture carrier substrate and a cell sheet, wherein the cell sheet is in a state of covering at least 50% of the surface on the culture surface side of the culture carrier substrate.

[0187] 2. The culture carrier substrate with a cell sheet according to Item 1, wherein the substrate thickness of the culture carrier substrate is 5 μm or more and 250 μm or less.

[0188] 3. The culture carrier substrate with a cell sheet according to Item 1 or 2, wherein the culture surface is subjected to a hydrophilic treatment.

[0189] 4. The culture carrier substrate with a cell sheet according to Item 3, wherein the hydrophilic treatment is a UV ozone treatment or a plasma treatment.

[0190] 5. The culture carrier substrate with a cell sheet according to any one of Items 1 to 4, wherein the culture carrier substrate is a polyether ether ketone film or a polyethylene terephthalate film.

[0191] 6. The culture carrier substrate with a cell sheet according to any one of Items 1 to 5, wherein the culture surface does not contain a temperature-responsive polymer.

[0192] 7. The culture carrier substrate with cell sheets as described in any one of items 1 to 6, wherein the area of ​​the culture carrier substrate is 0.3 cm². 2 Above and 1000cm 2 the following.

[0193] 8. The culture carrier substrate with cell sheets as described in any one of items 1 to 7, wherein the culture carrier substrate comprises one or more resin layers composed of resin material.

[0194] 9. The culture carrier substrate with cell sheets as described in any one of items 1 to 8, wherein, when the culture surface is measured using a laser microscope, there are 3 or fewer pores with a diameter of 1 μm or more and 100 μm or less and a depth of 0.5 μm or more and 100 μm in at least one 100 μm square observation area.

[0195] 10. The culture carrier substrate with cell sheets as described in any one of items 1 to 9, wherein the porosity is less than 15%.

[0196] 11. The culture carrier substrate with cell sheets as described in any one of items 1 to 10, wherein the water contact angle of the culture surface is 70° or less.

[0197] 12. The culture carrier substrate with cell sheets as described in any one of items 1 to 11, for transporting cell sheets cultured on a culture surface.

[0198] 13. The culture carrier substrate with cell sheets as described in any one of items 1 to 12, used for cryopreservation of cell sheets formed on the culture surface.

[0199] 14. The culture carrier substrate with cell sheets as described in any one of items 1 to 13, for attaching cell sheets formed on the culture surface to the transplantation site.

[0200] 15. The culture carrier substrate with cell sheets as described in any one of items 1 to 14, for use in cell transplantation therapy.

[0201] 16. The cell-bearing culture carrier substrate as described in any one of items 1 to 15, wherein the cell transplantation therapy includes at least one of inhibiting or preventing the onset and recurrence of symptoms related to defects and dysfunctions of cells, tissues, and organs.

[0202] 17. The cell-bearing culture carrier substrate as described in any one of items 1 to 16, used for the treatment of at least one of spinal cord injury, knee cartilage injury, ischemic heart disease, age-related macular degeneration, limbal stem cell deficiency, aplastic anemia, severe limb ischemia, refractory skin ulcers, prevention of postoperative complications, and burns.

[0203] 18. The culture carrier substrate with a cell sheet according to any one of Items 1 to 17, which is used for a transplantation method, and the transplantation method includes at least one of the following processes: a process of culturing the cell sheet, a process of cryopreserving the cell sheet, a process of thawing the cryopreserved product of the cell sheet, and a process of attaching the cell sheet to the transplantation site.

[0204] 19. A transplantation material, which includes the culture carrier substrate with a cell sheet according to any one of Items 1 to 18.

[0205] 20. The transplantation material according to Item 19, which is used for attaching the cell sheet to a diseased site.

[0206] 21. The transplantation material according to Item 19 or 20, which is a therapeutic transplantation material for treating at least one of spinal cord injury, knee joint cartilage injury, ischemic heart disease, age-related macular degeneration, limbal stem cell deficiency, aplastic anemia, severe limb ischemia, refractory skin ulcer, prevention of postoperative complications, and scald.

[0207] The above-mentioned culture carrier substrate with a cell sheet may include the culture carrier substrate according to any one of Items 1 to 13 in Reference Form A.

[0208] <C. Cryopreserved Product of the Culture Carrier Substrate with a Cell Sheet> As Reference Form C of the cryopreserved product of the culture carrier substrate with a cell sheet, the following Items 1 to 8 are listed.

[0209] 1. A cryopreserved product, which includes 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 cryopreserved state.

[0210] 2. The cryopreserved product according to Item 1, which is in a cryopreserved state at -196°C to -60°C.

[0211] 3. The cryopreserved product according to Item 1, which is in a cryopreserved state at -196°C to -135°C.

[0212] 4. The cryopreserved product according to Item 1, which is in a cryopreserved state at -134°C to -60°C.

[0213] 5. The cryopreserved product according to any one of Items 1 to 4, which includes a cryopreservation solution.

[0214] 6. The cryopreserved product according to Item 5, wherein the cryopreservation solution includes at least one of a culture solution component and a cryoprotectant.

[0215] 7. The cryopreserved product according to Item 5 or 6, wherein the cryopreservation solution does not contain DMSO.

[0216] 8. The cryopreserved product according to any one of items 1 to 4, wherein the entire culture carrier substrate and the cell sheet are in a cryopreserved state covered with a cryopreservation solution.

[0217] In the cryopreserved product of the culture carrier substrate with a cell sheet, it may include the culture carrier substrate of any one of items 1 to 13 in Reference Form A, or may include the culture carrier substrate with a cell sheet of any one of items 1 to 21 in Reference Form B.

[0218] <D. Package> As Reference Form D of the package, the following items 1 to 8 are listed.

[0219] 1. A package comprising a culture carrier substrate and a packaging material for packaging the culture carrier substrate.

[0220] 2. A package comprising: a culture carrier substrate with a cell sheet, and a packaging material for packaging the culture carrier substrate with a cell sheet, The culture carrier substrate with a cell sheet includes a culture carrier substrate and a cell sheet formed on the culture surface of the culture carrier substrate.

[0221] 3. A package comprising: a cryopreserved product of a culture carrier substrate with a cell sheet, and a packaging material for packaging the cryopreserved product, The cryopreserved product of the culture carrier substrate with a cell sheet includes a culture carrier substrate and a cell sheet formed on the culture surface of the culture carrier substrate.

[0222] 4. The package according to any one of items 1 to 3, wherein a culture container and / or a cryopreservation container are included inside the packaging material.

[0223] 5. The package according to any one of items 1 to 4, wherein the inside of the packaging material is in a sealed state.

[0224] 6. The package according to any one of items 1 to 5, which is used for transportation and / or preservation.

[0225] In the above package, it may include the culture carrier substrate of any one of items 1 to 13 in Reference Form A, or may include the culture carrier substrate with a cell sheet of any one of items 1 to 21 in Reference Form B, or may further include the cryopreserved product of the culture carrier substrate with a cell sheet of any one of items 1 to 8 in Reference Form C.

[0226] <E. Cryopreservation Method or Thawing Method> As Reference Form E of the cryopreservation method or thawing method, the following items 1 to 10 are listed.

[0227] 1. A cryopreservation method comprising: a cooling step of cooling a culture carrier substrate with cell sheets in a cryopreservation solution, wherein the culture carrier substrate with cell sheets comprises a culture carrier substrate and cell sheets formed on the surface of the culture carrier substrate.

[0228] 2. The cryopreservation method as described in item 1, wherein, in the cooling step, a non-flow cooling device is used to cool the culture carrier substrate with cell sheets in the cryopreservation solution.

[0229] 3. The cryopreservation method as described in item 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.

[0230] 4. The cryopreservation method according to any one of items 1 to 3, wherein, after the cooling step, a cryopreservation step is included to preserve the culture carrier substrate with cell sheets at -196 to -60°C.

[0231] 5. The cryopreservation method according to any one of items 1 to 4, wherein the cooling process yields a frozen product comprising the culture carrier substrate and the cell sheet.

[0232] 6. A thawing method comprising a thawing step of thawing frozen items. The frozen material comprises a culture carrier substrate and cell sheets formed on the surface of the culture carrier substrate. The culture carrier substrate and the cell sheet are in a cryopreserved state.

[0233] 7. The thawing method as described in item 6, wherein the thawing step is included after the cooling step of the cryopreservation method as described in any one of items 1 to 4.

[0234] 8. The thawing method as described in item 6 or 7, comprising: a thawing step of thawing the frozen material obtained in the cryopreservation method as described in item 5.

[0235] 9. The thawing method according to any one of items 6 to 8, wherein, in the thawing process, the ambient temperature during thawing is above 0°C and below 50°C.

[0236] 10. The thawing method according to any one of items 6 to 9, wherein, in the thawing step, the thawing time is greater than 10 seconds and less than 60 minutes.

[0237] The above cryopreservation method or freezing method may include a culture carrier substrate of any one of items 1 to 13 of reference form A, or a culture carrier substrate with cell sheets of any one of items 1 to 21 of reference form B.

[0238] The cryopreserved product obtained by the cryopreservation method may also include a cryopreserved product of a culture carrier substrate with a cell sheet of any one of items 1 to 8 of Reference Form C.

[0239] In addition, the cryopreserved product obtained by the cryopreservation method and / or the thawed product obtained by the thawing method can be used in the transplantation method described below.

[0240] <F. Manufacturing Method of Culture Carrier Substrate with Cell Sheet> As Reference Form F of the manufacturing method of a culture carrier substrate with a cell sheet, the following items 1 to 3 are listed.

[0241] 1. A manufacturing method of a culture carrier substrate with a cell sheet, comprising: a culturing step of introducing a plurality of cells, a culture medium, and a culture carrier substrate into the interior of a culture container, and culturing a cell sheet on the culture surface of the culture carrier substrate; and a removing step of removing the culture carrier substrate with the cell sheet from the culture container.

[0242] 2. The manufacturing method of a culture carrier substrate with a cell sheet according to item 1, wherein in the culturing step, the culture carrier substrate is not fixedly attached to the interior of the culture container to culture the cell sheet.

[0243] 3. The manufacturing method of a culture carrier substrate with a cell sheet according to item 1 or 2, wherein in the removing step, there is no need for an operation of separating the culture carrier substrate from the culture container by physical means.

[0244] In the above manufacturing method of a culture carrier substrate with a cell sheet, it may include a culture carrier substrate of any one of items 1 to 13 of Reference Form A.

[0245] <G. Transplantation Method> As Reference Form G of the transplantation method, the following items 1 to 25 are listed.

[0246] 1. A transplantation method, comprising: a transplantation step of using a culture carrier substrate with a cell sheet including a culture carrier substrate and a cell sheet formed on the culture surface of the culture carrier substrate, attaching the cell sheet to the transplantation site, and then peeling the culture carrier substrate from the cell sheet.

[0247] 2. The transplantation method according to item 1, wherein in the transplantation step, the culture carrier substrate with a cell sheet is used, and the culture carrier substrate with a cell sheet has a laminate of the culture carrier substrate and the cell sheet, and the cell sheet is in a state of covering at least 50% of the surface on the culture surface side of the culture carrier substrate.

[0248] 3. The transplantation method as described in item 1 or 2, wherein, in the transplantation process, the culture carrier substrate with cell sheets is removed from the packaging for use, or a frozen portion of the culture carrier substrate with cell sheets is removed from the packaging and thawed for use.

[0249] 4. The transplantation method as described in any one of items 1 to 3, comprising: a culture step of culturing the cell sheet on the culture surface of the culture carrier substrate to obtain the culture carrier substrate with the cell sheet.

[0250] 5. The transplantation method as described in item 4, wherein, in the culture step, the cell sheet is cultured with at least a portion of each of the culture surface and side surface of the culture carrier substrate in contact with the culture medium.

[0251] 6. The transplantation method as described in item 4 or 5, wherein, in the culture step, the cell sheet is cultured in a state in which the culture carrier substrate is fixed by means of load-bearing and / or the position of the culture carrier substrate is fixed by means of instruments.

[0252] 7. The transplantation method according to any one of items 4 to 6, wherein, in the culture step, the density of the cultured cells is 5 × 10⁻⁶. 2 cells / cm 2 Above and 1×10 9 cells / cm 2 the following.

[0253] 8. The transplantation method according to any one of items 4 to 7, wherein the substrate thickness of the culture carrier substrate is greater than 10 μm, and in the culture process, the culture carrier substrate is not fixedly attached to the inside of the culture container to function as a self-supporting membrane.

[0254] 9. The transplantation method according to any one of items 1 to 8, comprising: a cooling and preservation step, wherein the culture carrier substrate with cell sheets is cooled in a cryopreservation solution to obtain a frozen product of the culture carrier substrate with cell sheets.

[0255] 10. The transplantation method according to any one of items 1 to 9, comprising: a thawing step of thawing the frozen contents of the culture carrier substrate with cell sheets.

[0256] 11. The transplantation method according to any one of items 1 to 10, wherein the thickness of the culture carrier substrate is 5 μm or more and 250 μm or less. The culture carrier substrate is a polyetheretherketone membrane or a polyethylene terephthalate membrane. The culture surface of the culture carrier substrate has been hydrophilized.

[0257] 12. The transplantation method according to any one of items 1 to 11, wherein, in the transplantation process, the time from attachment to peeling is less than 10 minutes.

[0258] 13. The transplantation method according to any one of items 1 to 12, wherein the area of ​​the cell sheet is 0.3 cm². 2 above.

[0259] 14. The transplantation method according to any one of items 1 to 13, wherein, after the transplantation process, the residual rate of the cell sheet on the peeled culture carrier substrate is 50% or less in terms of area ratio.

[0260] 15. The transplantation method according to any one of items 1 to 14, wherein the thickness of the cell sheet is 0.001 mm or more and 2.0 mm or less.

[0261] 16. The transplantation method according to any one of items 1 to 15, wherein the area of ​​the culture carrier substrate is 0.3 cm². 2 Above and 1000cm 2 the following.

[0262] 17. The transplantation method according to any one of items 1 to 16, wherein the culture carrier substrate comprises one or more resin layers composed of resin material.

[0263] 18. The transplantation method according to any one of items 1 to 17, wherein the culture carrier substrate is used, and when the culture surface is measured using a laser microscope, the culture carrier substrate has 3 or fewer pores with a diameter of 1 μm or more and 100 μm or less and a depth of 0.5 μm or more and 100 μm in at least one 100 μm square observation area.

[0264] 19. The transplantation method according to any one of items 1 to 18, wherein the porosity of the culture carrier substrate is less than 15%.

[0265] 20. The transplantation method according to any one of items 1 to 19, wherein the water contact angle of the culture surface of the culture carrier substrate is less than 70°.

[0266] 21. The transplantation method according to any one of items 1 to 20, wherein the culture carrier substrate with cell sheets is obtained in a state in which the cell sheets cover 50% to 100% of the stackable cell area of ​​the culture surface of the culture carrier substrate.

[0267] 22. The transplantation method as described in any one of items 1 to 21, used in cell transplantation therapy.

[0268] 23. The transplantation method as described in any one of items 1 to 22, wherein the cell transplantation therapy includes at least one of inhibiting or preventing the onset and recurrence of symptoms related to defects and functional impairments or insufficiency of cells, tissues, or organs.

[0269] 24. The transplantation method according to any one of items 1 to 23, wherein, in the transplantation process, the cell sheet is attached to the disease site.

[0270] 25. The transplantation method according to any one of items 1 to 24, wherein the disease includes at least one of spinal cord injury, knee cartilage injury, ischemic heart disease, age-related macular degeneration, limbal stem cell deficiency, aplastic anemia, severe limb ischemia, refractory skin ulcer, prevention of postoperative complications, and burns.

[0271] The above transplantation method may include a culture carrier substrate of any one of items 1 to 13 of reference morphology A, a culture carrier substrate with cell sheets of any one of items 1 to 21 of reference morphology B, or a cryogenic culture carrier substrate with cell sheets of any one of items 1 to 8 of reference morphology C.

[0272] Furthermore, the above-mentioned transplantation method may include a method for manufacturing a culture carrier substrate with cell sheets or a culture process thereof, which is one of the items 1 to 3 of reference morphology F, and may also include a cryopreservation method or a thawing method, which is one of the items 1 to 10 of reference morphology E.

[0273] Furthermore, as a reference form H, a manufacturing method can be provided, which uses at least one of the following: a culture carrier substrate of any one of items 1 to 13 of reference form A, a culture carrier substrate with cell sheets of any one of items 1 to 21 of reference form B, and a frozen product of a culture carrier substrate with cell sheets of any one of items 1 to 8 of reference form C, and includes at least one of the following: inhibiting or preventing the onset and recurrence of symptoms related to defects and functional disorders of cells, tissues, and organs. For this treatment method, the relevant symptoms it targets may include at least one of the aforementioned diseases.

[0274] Furthermore, as a reference form I, examples include the use of at least one of the following: a culture carrier substrate from any of items 1 to 13 of reference form A; a culture carrier substrate with cell sheets from any of items 1 to 21 of reference form B; and a frozen culture carrier substrate with cell sheets from any of items 1 to 8 of reference form C, for treating at least one of the following: a culture carrier substrate with cell sheets from any of items 1 to 21 of reference form B; and a culture carrier substrate with cell sheets from any of items 1 to 8 of reference form C. For this use, the relevant symptoms may include at least one of the aforementioned diseases.

[0275] Furthermore, as a reference form J, the use of at least one of the following in the manufacture of a therapeutic agent for treating at least one of the following: a culture carrier substrate of any one of items 1 to 13 of reference form A; a culture carrier substrate with cell sheets of any one of items 1 to 21 of reference form B; and a frozen product of a culture carrier substrate with cell sheets of any one of items 1 to 8 of reference form C. For this use, the relevant symptoms may include at least one of the aforementioned diseases.

[0276] Example The present invention will now be described in detail with reference to embodiments, but the present invention is not limited in any way by these embodiments.

[0277] [Example 1] <Manufacturing of Culture Carrier Substrates> The culture surface of the PEEK film (manufactured by Shin-Etsu Polymer Co., Ltd., polyetheretherketone film, Shin-Etsu SeplaFilm (registered trademark), low crystallinity, 12 μm thick) was subjected to plasma treatment. The surface roughness after plasma treatment was rougher than before plasma treatment, with an Ra of 0.6 nm. Furthermore, Ra was measured as follows: An AFM (Shimadzu SPM-9700) was used, with a small-tip-diameter silicon single-crystal probe (radius of curvature approximately 10 nm). The surface was observed within a 1 μm × 1 μm range. Ra was calculated using the analysis software included with the instrument. After smoothing in the X and Y directions, surface roughness analysis was performed within a 100 nm × 100 nm range, avoiding foreign objects or surface damage. The arithmetic mean roughness Ra is the average distance from the reference line over a reference length.

[0278] Furthermore, the porosity of the PEEK membrane is less than 5%. When measured using a laser microscope on the culture surface, there are no pores with a diameter greater than 1 μm and less than 100 μm and a depth greater than 0.5 μm and greater than 100 μm within a 100 μm square area, indicating that it is a solid membrane. The porosity is calculated using the formula: {1 - (measured density / theoretical density)} × 100.

[0279] Then, the surface-treated film is cut into... A 33.5mm diameter is used to obtain a disc-shaped culture carrier substrate.

[0280] <Cell Slice Manufacturing> After cleaning the culture carrier substrate manufactured in the above-mentioned "Manufacturing of Culture Carrier Substrate" step by step with 70% ethanol, phosphate buffer, and culture medium, it is placed on the flat bottom of each well in a cell culture multi-well plate (6 wells). At this time, it is arranged with the culture surface of the culture carrier substrate facing the opening of the well plate, that is, with the back of the culture carrier substrate in contact with the flat bottom of the well portion of the well plate. It is important to ensure that the culture carrier substrate is not fixedly attached to the inside of the well plate (culture container).

[0281] Cryopreserved human fibroblasts (derived from human oral cavity tissue) were thawed at 37°C and washed with culture medium. 5 × 10⁻⁶ cells were then... 5 Cells were suspended in a culture medium containing 5% serum and cultured on two 60.1 cm² plates. 2 Sow 2.5 × 10⁶ seeds on each of the above-ground areas. 5 After culturing for 3 days, the cultured cells were recovered and suspended in medium containing 5% serum, and then transferred to four 225 cm⁻¹ flasks. 2 Sow 2.5 × 10⁻⁶ seeds in each of the plants. 5 After each generation, the cells are cultured for 4 days.

[0282] The cultured cells were recovered and suspended in a medium containing 2% serum at a temperature of 55.8 × 10⁻⁶. 4 pcs / cm 2 The cells were seeded at the specified density in each well of a cell culture plate containing a culture carrier substrate, and cultured for one day at 37°C and 5% CO2. Cell sheets were then prepared on the culture surface of the culture carrier substrate.

[0283] <Transplantation> Following the aforementioned <Cell Sheet Fabrication>, the culture carrier substrate with the cell sheets is removed from the porous plate. The removed culture carrier substrate with the cell sheets is then transported to a pork slice (the transplantation site) prepared elsewhere to simulated cell tissue. After attaching the surface of the cell sheet to the surface of the pork slice, the culture carrier substrate is peeled off from the cell sheet.

[0284] Furthermore, since no special cooling of the substrate was performed from the time of application to the time of peeling, the temperature of the substrate remained above 30°C.

[0285] [Example 2] In the above-mentioned <manufacturing of culture carrier substrate>, a 25μm thick PET film (manufactured by Toray Industries, Ltd., polyethylene terephthalate film, Lumirror (registered trademark)) was used instead of a polyetheretherketone film. Otherwise, the <manufacturing of culture carrier substrate>, <manufacturing of cell sheets> and <transplantation> were carried out in the same manner as in Example 1.

[0286] [Example 3] In the aforementioned <manufacturing of culture carrier substrate>, the cut size of the surface-treated membrane is made into... 14.0mm to replace 33.5 mm. In the above-mentioned <Cell Sheet Manufacturing>, the seeding was changed to use a 24-well / flat-bottomed cell culture multi-well plate, and the seeding density was changed to 27.9 × 10⁻⁶. 4 Cells / cm, except that the <cell sheet fabrication> and <transplantation> were carried out in the same manner as in Example 1.

[0287] [Comparative Example 1] Instead of using the culture carrier substrate manufactured in the above-mentioned <manufacturing of culture carrier substrate>, cell sheets are manufactured on the flat bottom inside the pores of a multi-well plate for adherent cells in <manufacturing of cell sheets>, and the material obtained by peeling the cell sheets from the flat bottom is attached to the surface of pork slices in <transplantation>. Otherwise, <manufacturing of cell sheets> and <transplantation> are carried out in the same manner as in Example 1.

[0288] [Comparative Example 2] In the above-described <manufacturing of culture carrier substrate>, the culture surface of the substrate was not subjected to plasma treatment. Otherwise, the <manufacturing of culture carrier substrate>, <manufacturing of cell sheets>, and <transplantation> were performed in the same manner as in Example 1.

[0289] [Comparative Example 3] In the above-described <manufacturing of culture carrier substrate>, the culture surface of the substrate was not subjected to plasma treatment. Otherwise, the <manufacturing of culture carrier substrate>, <manufacturing of cell sheets>, and <transplantation> were performed in the same manner as in Example 2.

[0290] For each embodiment and comparative example, the culture and transplantation of cell slices were evaluated. The results are shown in Table 1.

[0291] [Table 1] Examples 1-3 show that in the <Cell Sheet Manufacturing>, a culture carrier substrate with cell sheets covering more than 100% of the substrate culture surface can be obtained.

[0292] In the transplantation process, the culture carrier substrate with cell sheets was not fixedly attached to the porous plate, thus allowing for easy removal. Furthermore, in the PET membrane of Example 2, the substrate was peeled off by gently pressing the cell sheets with tweezers, while in the PEEK membranes of Examples 1 and 3, the substrate could be peeled off even more easily by sliding without pressing the cell sheets, demonstrating excellent ease of operation.

[0293] Furthermore, during transplantation, the culture medium substrate can be easily removed from the cell sheet. Specifically, since the cell sheet adheres immediately to the pork, the entire process from attachment to detachment takes less than one minute. That is, the transplantation procedure is simplified, thus enabling a shorter operation time. Moreover, after transplantation, it is not necessary to culture the cell sheet on the pork.

[0294] The results of Examples 1-3 confirm that cell sheet culture and transplantation can be carried out by using plasma-treated PEEK membranes or plasma-treated PET membranes as culture carrier substrates.

[0295] Furthermore, after the <transplantation> in Examples 1-3, the amount of cell sheets remaining on the culture surface of the culture carrier substrate, calculated by area ratio, is less than 1%. Therefore, it becomes possible to reduce cell sheet residue during the transplantation process. Additionally, when peeling the cell sheets from the culture carrier substrate, it is not necessary to perform dispersing enzyme treatment on the culture surface before culture.

[0296] In Comparative Example 1, in the <Preparation of Cell Sheets>, cell sheets can be prepared within a multi-well plate for adherent cells. However, in the <Transplantation> process, it is difficult to peel the cell sheet from the flat bottom of the multi-well plate without damaging it with forceps or similar means when removing the cell sheet. Furthermore, even if the cell sheet can be peeled off, it is difficult to hold it in place, making cell sheet attachment difficult.

[0297] In Comparative Examples 2 and 3, the transplantation procedure was not performed because cell slices could not be prepared.

[0298] [Example 4] <Cell Slice Manufacturing> The cut size of the membrane after surface treatment in Example 1, <Manufacturing of Culture Carrier Substrate>, is made into... 14.0mm to replace 33.5 mm, using the culture carrier substrate obtained therefrom, the seeding was changed to a 24-well / flat-bottomed cell culture multi-well plate, and the seeding density was changed to 27.9 × 10⁻⁶. 4 pcs / cm2 In addition, cell sheets (cell sheets with culture carrier substrate) are manufactured on the culture surface of the culture carrier substrate in the same manner as in <Cell sheet manufacturing> of Example 1.

[0299] In addition, the cell viability rate before freezing was determined according to the following steps.

[0300] <Cryopreservation of Cell Slices> After the above <Cell Slice Fabrication>, the cell culture multi-well plate was placed on ice. After washing the cell slices with culture carrier substrate with phosphate buffer, cryoprotectant (STEMCELL-BANKER GMPgrade, manufactured by NIPPON ZENYAKU KOGYO CO.,LTD.) was added at a rate of 0.3 mL / well.

[0301] Then, the cell culture multiwell plates, packaged in zip-lock plastic bags, were placed in a non-flow cooling device (3DFreezer, KSS-40BLW-2400V, manufactured by KOGASUN Co., Ltd.) cooled to -35°C and held for 30 minutes. Furthermore, the cooling rate was 3.4°C / minute between 0 and -5°C.

[0302] Then, the cooled packaging is quickly placed into a -80°C freezer (RDE50086FD type, cryogenic refrigerator, manufactured by Thermoscientific) and kept there for 1 hour.

[0303] Using a K thermocouple, the temperature of cell slices (cell slices with culture carrier substrate) stored in an 80°C freezer was measured, and the result was -70°C.

[0304] Thawing of Cell Slices After the freezing period described above, the package was removed from the -80°C freezer, and the cell culture plates inside were thawed at room temperature in a biosafety cabinet. The thawing time was 16 minutes. After thawing, the cell slices appeared in good condition.

[0305] After thawing, aspirate the cryoprotectant and add 2 mL of culture medium containing 2% serum. Then, incubate at 37°C with 5% CO2. 2Cells were cultured for 24 hours under controlled conditions. After culture, cell viability was measured. The results showed that when the cell viability before freezing was set as C0 and the cell viability after freezing was set as C1, the change in cell viability before and after freezing, calculated as (C1 / C0)×100, was 89%. Furthermore, the culture supernatant was recovered and frozen at -80℃. The following day, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were measured. The results showed that the supernatant contained 1451 pg of VEGF and 4608 pg of HGF.

[0306] (Method for determining thawing time) Thawing time is the time required to thaw frozen food after adding cryoprotectant. It is measured from the point when the food is exposed to the thawing environment until the point when the frozen food becomes completely liquid.

[0307] (Determination of cell viability) Cell viability was measured using a live cell count assay reagent (Cell Count Reagent SF, manufactured by NACALAI TESQUE, Inc.) and a reading instrument (iMark, manufactured by BIO-RAD). The live cell count assay reagent was diluted 20-fold with culture medium (reagent / culture medium = 1 / 19 volume ratio) to prepare the reagent solution. The supernatant containing cultured cell sheets was aspirated from each well of a multi-well cell culture plate, and the test solution was added at 0.5 mL / well. The plate was incubated at 37°C and 5% CO2 for 1 hour. After incubation, 0.1 mL / well of supernatant was added to each well of a 96-well cell culture plate (manufactured by CORNING), and the supernatant was measured using the reading instrument. Cell viability was calculated as follows.

[0308] • Cell viability [Abs.] = {Absorbance of the measured sample [Absorbance (450nm) - Absorbance of the measured sample (630nm)]} - {Absorbance of the blank [Absorbance (450nm) - Absorbance of the blank (630nm)]} The blank sample used the test solution.

[0309] (Measurement of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF)) VEGF and HGF, cytokines associated with angiogenesis, were measured. These cytokines were measured using a human VEGF ELISA kit (96well) (R&D Systems, Inc., DVE00) and a human HGF ELISA kit (96well) (R&D Systems, Inc., DHG00B), respectively, and a reader (iMark, BIO-RAD). The usage methods were performed according to the instructions for each kit. For test samples, the supernatant was recovered after thawing and cultured for 24 hours and then frozen. The test samples were thawed the following day before measurement. Furthermore, the test samples were diluted 2–4 times with the diluent provided with the kit before measurement. The standard curve was generated using the standards provided with the kit. The absorbance of each sample and the standard curve was calculated using the following formula; the concentrations of VEGF and HGF were then calculated by substituting the obtained absorbance into the standard curve.

[0310] • The absorbance of the sample and the standard used in the standard curve [Abs.] = absorbance (450nm) - absorbance (570nm) <Transplantation after thawing> The thawed cell slices with the culture carrier substrate were removed from the cell culture perforated plate, the cell slices were attached to pork, and the substrate was peeled off. Furthermore, since no special heating or cooling was applied to the substrate from attachment to peeling, the substrate temperature was maintained at room temperature (23°C).

[0311] [Examples 5-11] The freezing time for the above-mentioned <cryopreservation of cell sheets> was changed to the values ​​shown in Table 2. Otherwise, the <manufacturing 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.

[0312] [Comparative Example 4] In Comparative Example 4, the cell culture substrate was not used and the cell culture multi-well plate was replaced with a shallow culture dish (UpCell (registered trademark), manufactured by CellSeed Inc.). Otherwise, the <preparation of cell sheets> was carried out in the same manner as in Example 4.

[0313] [Comparative Example 5] In Comparative Example 5, the culture carrier substrate was not used and the cell culture multi-well plate was changed to an adherent cell multi-well plate (24 wells). Otherwise, the <preparation of cell sheets>, <cryopreservation of cell sheets> and <thawing of cell sheets> were carried out in the same manner as in Example 4.

[0314] For each embodiment and comparative example, the cryopreservation and transplantation after thawing were evaluated. The results are shown in Table 2.

[0315] [Table 2] In Comparative Example 4, the cell sheets could not be cryopreserved because they detached from the shallow bottom of the culture dish during cooling. Therefore, the determination of cell viability after freezing and the transplantation procedure after thawing were not performed.

[0316] Comparative Example 5 showed that cell sheets could be cryopreserved in a sealed state within a multi-well plate, but the change in cell viability before and after freezing was reduced.

[0317] In contrast, Examples 4-11 showed that cell sheets could be cryopreserved while tightly bound to the culture carrier substrate, and the changes in cell viability before and after freezing, as well as at least one of VEGF and HGF, were higher than those in Comparative Example 5. Furthermore, during transplantation after thawing, the culture carrier substrate could be easily detached from the cell sheets. Moreover, since the cell sheets immediately adhered to the pork, the attachment and detachment process took less than one minute, eliminating the need to culture the cell sheets on the pork.

[0318] The results of Examples 4-11 confirm that by using plasma-treated PEEK membranes as culture carrier substrates, cell sheets can be frozen and transplanted after thawing. Furthermore, even cell sheets that have undergone long-term freezing and thawing can maintain a high cell viability rate.

[0319] [Examples 12-23] The freezing time for the <Cryopreservation of Cell Slices> was changed to the values ​​shown in Table 3. BAMBANKER (registered trademark) hRM (GC Lymphotec, Inc.) was used as the cryoprotectant, and a -150°C freezer was used. Otherwise, the <Cell Slice Preparation>, <Cell Slice Cryopreservation>, <Cell Slice Thawing>, and <Post-Thaw Transplantation> were performed in the same manner as in Example 4. The thawing time was 18 minutes. After thawing, the cell slices had a good appearance.

[0320] In addition, using a K thermocouple, the temperature of cell slices (cell slices with culture carrier substrate) stored in a -150°C freezer was measured to be -140°C.

[0321] For each embodiment, the cryopreservation and transplantation after thawing were evaluated. The results are shown in Table 3.

[0322] [Table 3] Examples 12-23 demonstrated that cell sheets could be cryopreserved while tightly bound to the culture carrier substrate, and the changes in cell viability before and after freezing, as well as the results for either VEGF or HGF, were higher than those in Comparative Example 5. Furthermore, during transplantation after thawing, the culture carrier substrate could be easily detached from the cell sheets. Moreover, since the cell sheets immediately adhered to the pork, the attachment and detachment process took less than one minute, eliminating the need to culture the cell sheets on the pork.

[0323] [Examples 24-26] The freezing time for the above-mentioned <cryopreservation of cell slices> was changed to the values ​​shown in Table 4. Otherwise, the <manufacturing of cell slices>, <cryopreservation of cell slices>, <thawing of cell slices> and <transplantation after thawing> were carried out in the same manner as in Example 12.

[0324] Among them, such as Figure 3 (a)~ Figure 3 (c) shows the state during freezing of each embodiment.

[0325] exist Figure 3 In (a), the <cryopreservation of cell sheets> and <thawing of cell sheets> are performed with the culture carrier substrate 10 side facing the bottom surface of the culture container 20. Figure 3 In (b), the <cryopreservation of cell sheets> and <thawing of cell sheets> are performed with the cell sheet 40 side facing the bottom of the culture container 20. Figure 3 In (c), the <cryopreservation of cell sheets> and <thawing of cell sheets> are performed with the cell sheet 40 held between two culture carrier substrates 10.

[0326] For each embodiment, the cryopreservation and transplantation after thawing were evaluated. The results are shown in Table 4.

[0327] [Table 4] In Examples 24-26, it was demonstrated that cell sheets could be cryopreserved while tightly bound to the culture carrier substrate, and the change in cell viability before and after freezing was greater than that in Comparative Example 5. Furthermore, even during transplantation after thawing, the culture carrier substrate could be easily detached from the cell sheets. Moreover, since the cell sheets immediately adhered to the pork, the process from attachment to detachment took less than one minute, eliminating the need to culture the cell sheets on the pork.

[0328] In addition, it is known that PEEK membranes have low linear expansion characteristics, so they can be used as a culture carrier substrate that is beneficial for cryogenic processing.

[0329] [Example 27] In the method described in Example 12, "Thawing of Cell Slices," the thawing at room temperature in a biosafety cabinet is replaced with thawing of cell culture multiwell plates placed on the heat dissipation device of a thawing apparatus (manufactured by DEC). Otherwise, the processes of "Cell Slice Preparation," "Cell Slice Cryopreservation," "Cell Slice Thawing," and "Post-Thawing Transplantation" are performed in the same manner as in Example 12. The thawing apparatus is set to a temperature of 37°C, representing the temperature of the heat dissipation device. The thawing time is 3 minutes.

[0330] In addition, using a K thermocouple, the temperature of cell slices (cell slices with culture carrier substrate) stored in a freezer at -150°C was measured to be -140°C. After thawing, the cell slices had a good appearance.

[0331] [Example 28] The thawing apparatus of Example 27 was set to a temperature of 20°C. Otherwise, the processes of <cell slide fabrication>, <cell slide cryopreservation>, <cell slide thawing>, and <transplantation after thawing> were performed in the same manner as in Example 26. The thawing time was 5 minutes. After thawing, the cell slides had a good appearance.

[0332] [Example 29] The thawing apparatus of Example 27 was set to a temperature of 4°C. Otherwise, the processes of <cell slide fabrication>, <cell slide cryopreservation>, <cell slide thawing>, and <transplantation after thawing> were performed in the same manner as in Example 26. The thawing time was 8 minutes. After thawing, the cell slides had a good appearance.

[0333] [Example 30] In the method shown in Example 12, "Thawing of Cell Slices," the thawing at room temperature in a biosafety cabinet was changed to thawing in a refrigerator set to 4°C. Otherwise, the processes of "Cell Slice Preparation," "Cell Slice Cryopreservation," "Cell Slice Thawing," and "Transplantation After Thawing" were performed in the same manner as in Example 26. The thawing time was 37 minutes. After thawing, the cell slices had a good appearance.

[0334] For Examples 27-30, the appearance of the cell slices, the determination of cell viability, and the transplantation after thawing were evaluated. The results are shown in Table 5. In addition, for reference, the results of Examples 4 and 12 are shown again in Table 5.

[0335] [Table 5] In Examples 4, 12, and 27-30, the appearance of the cell sheets was unaffected. Furthermore, Examples 4, 12, and 27-30, compared to Comparative Example 5, showed a higher percentage change in cell viability before and after freezing. In other words, it can be said that the viability of frozen cells was maintained at a relatively high level. Specifically, Examples 4, 12, and 27-29, compared to Example 30 with a relatively longer freezing time, showed a higher percentage change in cell viability before and after freezing. Moreover, the thawed samples from all examples were successfully transplanted after thawing.

[0336] In Examples 1 to 30 above, it was confirmed that the same results were observed even when mouse fibroblasts were used instead of human fibroblasts.

[0337] This application asserts priority based on Japanese Patent No. 2023-115623 filed in Japan on July 14, 2023 and Japanese Patent No. 2023-220506 filed in Japan on December 27, 2023, all of the disclosures of which are incorporated herein by reference.

[0338] Explanation of reference numerals in the attached figures 10: Culture carrier substrate; 12: Culture side (surface); 14: Non-culture side (back); 20: Culture container; 30: Culture medium; 40: Cell sheet; 50: Culture carrier substrate with cell sheet; 60: Cryopreservation solution; 70: Transplantation site.

Claims

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

2. The transplantation method according to claim 1, wherein the culture carrier substrate has a substrate thickness of 5 μm or more and 250 μm or less, the culture carrier substrate is a polyether ether ketone film or a polyethylene terephthalate film, the culture surface of the culture carrier substrate is subjected to a hydrophilization treatment.

3. The transplantation method according to claim 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 does not fixedly adhere to the inside of a culture vessel and functions as a self-supporting film.

4. The transplantation method according to any one of claims 1 to 3, wherein in the transplantation step, the time from attachment to peeling 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 The above.

6. A culture carrier substrate 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 claim 6, wherein, the culture carrier substrate has a substrate thickness of more than 10 μm, and in the culturing of the cell sheet, the culture carrier substrate does not fixedly adhere to the inside of a culture vessel and functions as a self-supporting film.

8. The culture carrier substrate according to claim 6 or 7, wherein the culture surface is subjected to a hydrophilization treatment.

9. The culture carrier substrate according to claim 8, wherein as the hydrophilization treatment, a plasma treatment is performed.

10. The 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 that is a package of the culture carrier substrate according to any one of claims 6 to 11 with a packaging material.

13. A culture carrier substrate with a cell sheet having a laminate of the culture carrier substrate according to any one of claims 6 to 11 and a cell sheet, wherein the cell sheet is in a state of covering at least 50% of the surface of the culture surface side of the culture carrier substrate.

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

15. A method for manufacturing a culture carrier substrate with a cell sheet, comprising: a culturing 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 the inside of a culture vessel to culture a cell sheet on a culture surface of the culture carrier substrate; and a taking-out step of taking out the culture carrier substrate with the cell sheet from the culture vessel.

16. The method for manufacturing a culture carrier substrate with a cell sheet according to claim 15, wherein In the culturing step, the cell sheet is cultured in a manner that the culturing carrier substrate is not fixedly attached to the inside of the culturing vessel.

17. The method for producing a cell sheet-bearing culturing carrier substrate according to claim 15 or 16, wherein In the taking-out step, the culturing carrier substrate is not separated from the culturing vessel by physical means.

18. A method for cryopreservation, comprising: a cooling step of cooling a cell sheet-bearing culturing carrier substrate in a cryopreservation solution, the cell sheet-bearing culturing carrier substrate comprising a culturing carrier substrate and a cell sheet formed on the surface of the culturing carrier substrate.

19. The method for cryopreservation according to claim 18, wherein In the cooling step, the cell sheet-bearing culturing carrier substrate is cooled in the cryopreservation solution using a cooling device that is not of the through-flow type.

20. The method for cryopreservation 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. The method for cryopreservation according to any one of claims 18 to 20, wherein After the cooling step, a cryopreservation step of storing the cell sheet-bearing culturing carrier substrate at -196 to -60°C is included.

22. A cryogenic product of a cell sheet-bearing culturing carrier substrate, comprising a culturing carrier substrate and a cell sheet formed on the surface of the culturing carrier substrate, wherein the culturing carrier substrate and the cell sheet are in a cryopreserved state.

23. The cryogenic product of a cell sheet-bearing culturing carrier substrate according to claim 22, which is in a cryopreserved state at -196 to -60°C.

24. The cryogenic product of a cell sheet-bearing culturing carrier substrate according to claim 22, which is in a cryopreserved state at -196 to -135°C.

25. The cryogenic product of a cell sheet-bearing culturing carrier substrate according to claim 22, which is in a cryopreserved state at -134 to -60°C.

26. A package that is a package obtained by packaging the cryogenic product according to any one of claims 22 to 25 with a packaging material.

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