Method for producing film substrate for cell culture, film substrate for cell culture, quality management method, package, cell sheet-bearing film substrate, method for producing cell sheet-bearing film substrate, frozen product of cell sheet-bearing film substrate, metal foil-attached film substrate, packed body, and method for producing packed body

Abstract

A method according to the present invention for producing a film substrate for cell culture comprises: a step for performing, under an inert gas atmosphere, a first plasma treatment on a culture surface of a film substrate for cell culture that has a culture surface on at least one surface; and, after the step for performing the first plasma treatment, a step for performing a second plasma treatment under an oxygen atom-containing gas atmosphere.

Description

Method for manufacturing a film substrate for cell culture, film substrate for cell culture, quality control method, package, film substrate with cell sheet, method for manufacturing a film substrate with cell sheet, frozen film substrate with cell sheet, film substrate with metal foil, package, and method for manufacturing a package

[0001] The present invention relates to a method for manufacturing a film substrate for cell culture, a film substrate for cell culture, a quality control method, a package, a film substrate with a cell sheet, a method for manufacturing a film substrate with a cell sheet, a frozen film substrate with a cell sheet, a film substrate with a metal foil, a package, and a method for manufacturing a package.

[0002] Various developments have been made on cell sheet scaffold materials. One known example of this type of technology is described in Patent Document 1. Patent Document 1 describes a culture method for producing a cell sheet on the temperature-responsive polymer layer of a film fixed to the bottom surface of a cell culture vessel, the film comprising a resin layer bonded to the bottom surface of the vessel and a temperature-responsive polymer layer provided on the resin layer (see, for example, claim 1 and paragraph 0026 of Patent Document 1).

[0003] JP 2016-013111 A

[0004] However, as a result of investigations by the present inventors, it was found that when a temperature-responsive polymer layer is not used in a film such as that described in Patent Document 1, there is room for improvement in terms of cell adhesiveness for forming a cell sheet after storage.

[0005] After further investigation, the inventors discovered that cell adhesion after storage can be improved by performing a first plasma treatment on the culture surface of the film substrate in an inert gas atmosphere and a second plasma treatment in a gas atmosphere containing oxygen atoms, and thus completed the present invention.

[0006] According to one aspect of the present invention, there are provided the following methods for manufacturing a film substrate for cell culture, a film substrate for cell culture, a quality control method, a package, a film substrate with a cell sheet, a method for manufacturing a film substrate with a cell sheet, a frozen film substrate with a cell sheet, a film substrate with a metal foil, a package, and a method for manufacturing a package.

[0007] 1. A method for producing a film substrate for cell culture having a culture surface on at least one surface, comprising: performing a first plasma treatment on the culture surface of the film substrate for cell culture in an inert gas atmosphere; and, after the first plasma treatment, performing a second plasma treatment in an oxygen atom-containing gas atmosphere. 2. A method for producing a film substrate for cell culture according to 1., wherein the first plasma treatment and the second plasma treatment use capacitively coupled plasma. 3. A method for producing a film substrate for cell culture according to 1. or 2., wherein the pressure of the first plasma treatment is 0.2 Pa or more and 100 Pa or less. 4. A method for producing a film substrate for cell culture according to any one of 1. to 3., wherein the pressure of the second plasma treatment is 0.2 Pa or more and 100 Pa or less. 5. A method for producing a film substrate for cell culture according to 1. to 4. 5. A method for producing a cell culture film substrate according to any one of 1. to 4., wherein the difference in pressure between the first plasma treatment and the second plasma treatment is 20 Pa or less. 6. A method for producing a cell culture film substrate according to any one of 1. to 5., wherein the discharge treatment intensity of the second plasma treatment is lower than the discharge treatment intensity of the first plasma treatment. 7. A cell culture film substrate having a culture surface on at least one surface, wherein the polar component of the surface free energy of the culture surface, as determined by the Owens-Wendt method, after 28 days of storage under the following storage conditions, is 12 mJ / m ２or more. (Storage conditions) The cell culture film substrate is stored at 25°C, a relative humidity of 50%, and an air atmosphere with the culture surface of the cell culture film substrate covered with a surface protection material. 8. The cell culture film substrate according to 7., wherein the polar component ratio calculated from [polar component immediately after 28 days of storage under the storage conditions] / [polar component immediately after 14 days of storage under the storage conditions] is 0.7 or more and 1.2 or less. 9. The cell culture film substrate according to 7. or 8., wherein the polar component of the surface free energy on the culture surface after 28 days of storage is 20 mJ / m ２ 10. The film substrate for cell culture according to any one of 7. to 9., wherein the polar component of the surface free energy of the culture surface is 12 mJ / m or less during the period from immediately after the plasma treatment to immediately after storage for 28 days. ２ or above. 11. The film substrate for cell culture according to any one of 7. to 10., wherein the material constituting the film substrate for cell culture comprises an organic polymer compound having an aromatic ring, or an organic polymer compound having an aromatic ring and an oxygen atom. 12. The film substrate for cell culture according to any one of 7. to 11., wherein the material constituting the film substrate for cell culture comprises one or more selected from the group consisting of polyether ether ketone, polyethylene terephthalate, and polystyrene. 13. The film substrate for cell culture according to any one of 7. to 12., wherein the thickness is 5 μm or more and 250 μm or less. 14. A quality control method for a cell culture film substrate having a culture surface on at least one surface, comprising: comparing the polar component of the surface free energy of the culture surface, determined based on the Owens-Wendt method, between the value immediately after production and the value two days after production, and determining whether the difference is 11.5 mJ / m ２15. A quality control method comprising a step of determining that a product is acceptable when the following condition is met: 15. The quality control method according to 14., wherein in the step of determining that a product is acceptable, the difference between the polar components immediately after 2 days and immediately after 20 days is 15 mJ / m ２A quality control method in which a product is judged to be acceptable when the following conditions are further satisfied: 16. A package in which the film substrate for cell culture described in any one of 7. to 13. is packaged in a packaging material. 17. A film substrate with a cell sheet, comprising: the film substrate for cell culture described in any one of 7. to 13.; and a cell sheet laminated on the culture surface of the film substrate for cell culture. 18. A method for producing a film substrate with a cell sheet, comprising a culturing step of culturing a cell sheet on the culture surface of the film substrate for cell culture produced by the method for producing a film substrate for cell culture described in any one of 1. to 6. after a predetermined period of time has passed. 19. A method for producing a film substrate with a cell sheet described in 18., in which the predetermined period of time has passed after packaging, sterilization, transportation, and storage. 20. A method for producing a film substrate with a cell sheet described in 7. to 13. 21. A frozen product of a cell sheet-attached film substrate, comprising: the film substrate for cell culture according to any one of items 1 to 2; and a cell sheet laminated on the culture surface of the film substrate for cell culture, wherein the film substrate for cell culture and the cell sheet are in a frozen state. 21. A film substrate with a metal foil, comprising: a film substrate for cell culture having a culture surface on at least one surface; and at least one metal foil, wherein the metal foil is fixed in a state of covering the culture surface on the plasma-treated surface. 22. The film substrate with a metal foil according to item 21, wherein the metal foil comprises aluminum foil. 23. The film substrate with a metal foil according to item 21 or 22, wherein, when viewed from a direction normal to the surface of the film substrate for cell culture, the metal foil has a margin that extends outside the outer edge of the film substrate for cell culture. 24. A package obtained by packaging the film substrate with a metal foil according to any one of items 21 to 23 in a packaging material. 25. 24. A package comprising a packaging box containing at least one package according to 26. 25. The package according to 26., wherein the polar component of the surface free energy of the culture surface of the film substrate for cell culture, as determined based on the Owens-Wendt method, is 12 mJ / m２ 27. A package as described in 25. or 26., which is in a state of being sterilized using radiation. 28. A method for manufacturing a package, comprising the steps of: preparing a film substrate for cell culture having a culture surface, at least one surface of which is plasma-treated; covering the culture surface on the plasma-treated surface with metal foil to obtain a film substrate with metal foil; packaging the film substrate with metal foil using a packaging material to obtain a package; placing at least one of the packages in a packaging box and sealing it to obtain a package; and sterilizing the package using radiation.

[0008] According to the present invention, there are provided a method for manufacturing a film substrate for cell culture having excellent cell adhesiveness for forming a cell sheet after storage, a film substrate for cell culture, a quality control method, a package, a film substrate with a cell sheet, a method for manufacturing a film substrate with a cell sheet, a frozen film substrate with a cell sheet, a film substrate with a metal foil, a package, and a method for manufacturing a package.

[0009] Fig. 1 is a cross-sectional view schematically showing an example of the configuration of a cell culture film substrate of this embodiment; Fig. 2 is a process cross-sectional view schematically showing an example of the process of a method for producing a cell sheet-attached film substrate of this embodiment; Fig. 3 is a process cross-sectional view schematically showing a modified example of the freezing method of this embodiment; (a) is a top view schematically showing an example of the configuration of a film substrate with a metal foil of this embodiment; (b) is a cross-sectional view of (a); (c) is a top view schematically showing an example of the configuration of a package of this embodiment;

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

[0011] <Method for manufacturing a cell culture film substrate> An outline of a method for manufacturing a cell culture film substrate of this embodiment will be described. The method for manufacturing a cell culture film substrate of this embodiment includes a step of performing a first plasma treatment on at least one culture surface of a cell culture film substrate in an inert gas atmosphere, and a step of performing a second plasma treatment in an oxygen atom-containing gas atmosphere after the first plasma treatment.

[0012] The inventors have found that performing the first plasma treatment and the second plasma treatment in this order can prevent a decrease in cell adhesiveness for forming a cell sheet in the film substrate after storage, thereby providing a film substrate that can maintain a high cell adhesion rate and is applicable to the mass production of cell sheets.

[0013] Although the detailed mechanism is unclear, it is speculated that the first plasma treatment in an inert gas atmosphere can clean and activate the culture surface of the film substrate, and the second plasma treatment in an oxygen-atom-containing gas atmosphere can hydrophilize the activated culture surface; therefore, the synergistic effect of the two plasma treatments allows the film substrate to maintain sufficient cell adhesion before and after storage.

[0014] Furthermore, commonly known plasmas used in plasma treatment include capacitively coupled plasma (CCP), inductively coupled plasma (ICP), and electron cyclotron resonance plasma (ECR). Based on the findings of the present inventors, among these, a method was found in which capacitively coupled plasma is suitable for the first and second plasma treatments described above. A plasma device using a CCP typically includes two parallel plate electrodes. Such a CCP device is more widely used than an ECR device, and its lower plasma density and simpler equipment reduce operating costs. Using a CCP with a lower plasma density than an ECR is expected to reduce surface damage and thermal degradation of the film substrate and improve the uniformity of surface modification.

[0015] The first and second plasma treatments may be carried out under atmospheric pressure or under reduced pressure, but are preferably carried out under reduced pressure in order to control the atmosphere.

[0016] When performing the plasma treatment under reduced pressure, the pressure of the first and / or second plasma treatment is preferably 0.2 to 100 Pa, and more preferably 1 to 50 Pa. Setting the pressure of the first plasma treatment to the above-mentioned lower limit or higher has the advantages of stabilizing the state of the generated plasma, shortening the time required for reducing the pressure, and enabling more efficient surface treatment.

[0017] Furthermore, the difference in pressure between the first plasma treatment and the second plasma treatment is preferably 20 Pa or less, and more preferably 10 Pa or less. Alternatively, the value (P1 / P2) obtained by dividing the pressure (P1) of the first plasma treatment by the pressure (P2) of the second plasma treatment is preferably 0.3 to 3, and more preferably 0.5 to 2. This increases the continuity of the manufacturing process, thereby improving productivity.

[0018] The first plasma treatment and the second plasma treatment may be performed consecutively in the same apparatus or discontinuously in different apparatuses. In the discontinuous case, one or more known treatments may be performed between the plasma treatments, or the substrate may be stored.

[0019] The plasma in the first and / or second plasma treatment is not particularly limited, but it is preferable to use high-frequency low-pressure plasma because the use of high-frequency plasma can achieve stability in the processing process and sufficient surface treatment ability. A preferred processing mode may be selected from RIE (reactive ion etching) processing, in which anisotropic etching is performed toward an ion sheath formed in the processing section, and PE (plasma etching) processing, in which isotropic etching is performed without forming an ion sheath in the processing section, but RIE processing is preferably used for the first plasma treatment.

[0020] The process gas used in the first plasma treatment is an inert gas. Examples of the inert gas include a rare gas such as argon, nitrogen gas, etc. These may be contained alone or in any combination of two or more.

[0021] The process gas used in the second plasma treatment is a gas containing oxygen atoms. Examples of the gas containing oxygen atoms include oxygen gas, carbon dioxide gas, etc. These may be contained alone or in combination of any two or more. The gas containing oxygen atoms may also be used in combination with an inert gas. The content of the gas containing oxygen atoms in the process gas used in the second plasma treatment is, for example, preferably 30% by volume or more, more preferably 50% by volume or more, of the total process gas (100% by volume).

[0022] The discharge treatment intensity in the first and / or second plasma treatment is 150 to 15,000 W·min / m ２ It is preferable that the power consumption is 1000 to 12000 W·min / m ２ It is more preferable that the discharge treatment intensity of the second plasma treatment is set to a condition lower than the discharge treatment intensity of the first plasma treatment. Specifically, the discharge treatment intensity ratio calculated from [discharge treatment intensity of the second plasma treatment] / [discharge treatment intensity of the first plasma treatment] is preferably 0.01 to 0.5, more preferably 0.02 to 0.4. This makes it possible to further improve cell adhesiveness.

[0023] <Cell Culture Film Substrate> The cell culture film substrate of this embodiment was stored under the following storage conditions for 28 days, and the polar component of the surface free energy on the culture surface, as determined by the Owens-Wendt method, was found to be 12 mJ / m ２ (Storage Conditions) The cell culture film substrate is stored at 25° C., 50% relative humidity, and in an air atmosphere with the culture surface covered with a surface protection material.

[0024] In addition, the polar component of the surface free energy on the culture surface after 28 days of storage from immediately after the completion of the plasma treatment was 12 mJ / m ２The above is preferable. In this specification, "after 28 days of storage" refers to the period thereafter, including the base day 28, when the day on which the storage treatment to cover the plasma-treated surface with the surface protective material was performed is set as day 0 and the 28th day is set as the base day. However, if the day on which the plasma treatment was completed and the day on which the storage treatment was performed are the same day, the day of the plasma treatment may be calculated as day 0. Furthermore, "immediately after X days of storage" refers to within +1 day from the base day X. For example, "immediately after 14 days of storage" refers to the 14th and 15th days, and "immediately after 28 days of storage" refers to the 28th and 29th days, where X is a natural number. Furthermore, "up to X days" refers to including the end day of the X day.

[0025] The lower limit of the polar component of the surface free energy on the culture surface after storage for 28 days is, for example, 12 mJ / m ２ or more, preferably 20 mJ / m ２ Furthermore, the polar component of the surface free energy is 12 mJ / m ２ The period for maintaining the above conditions after 28 days of storage does not necessarily need to be set as long as there are no quality problems, but from the viewpoint of inventory management, it may be set to, for example, up to 365 days, up to 270 days, up to 180 days, or up to 90 days. ２ The period for maintaining the above mentioned conditions does not necessarily need to be set as a deadline if there are no quality problems after 28 days of storage, but from the viewpoint of inventory management, it may be set to, for example, up to 365 days, up to 270 days, up to 180 days, or up to 90 days. The lower limit of the polar component of the surface free energy on the culture surface immediately after 28 days of storage is, for example, 12 mJ / m ２ or more, preferably 20 mJ / m ２ More preferably, 25 mJ / m ２ This increases the adhesiveness of the cells. On the other hand, the upper limit of the polar component of the surface free energy on the culture surface immediately after storage for 28 days is, for example, 55 mJ / m ２ Below, 50mJ / m ２ It may be less than 45 mJ / m ２This can prevent a decrease in the detachability of the cell sheet. Note that the film substrate used for cell sheet formation and the film substrate used for physical property evaluation should not be the same, and equivalent products produced in the same lot should be used as evaluation samples.

[0026] The polar component ratio calculated from [polar component immediately after 28 days of storage under the above storage conditions] / [polar component immediately after 14 days of storage under the above storage conditions] is, for example, preferably 0.7 to 1.2, more preferably 0.8 to 1.1, thereby enhancing the stability of cell adhesion.

[0027] The procedure for measuring surface free energy is as follows. First, a contact angle meter is used to measure the contact angle with water and the contact angle with diiodomethane on the culture surface of the culture film substrate. The contact angle θ with water or diiodomethane is measured by placing 2.0 μL of pure water or 2.0 μL of diiodomethane on the surface of the film substrate in a 23°C environment, measuring the angle between the droplet and the surface of the film substrate with a contact angle meter, and analyzing using the θ / 2 method. Contact angle meter: DMo-502 (Kyowa Interface Science Co., Ltd.), dispenser: ADC-311 (Kyowa Interface Science Co., Ltd.), and analysis software: Interface Measurement / Analysis Integrated System FAMAS ver. 7.2.0 (Kyowa Interface Science Co., Ltd.) are used to measure and analyze contact angles obtained using the following measurement mode (sessile drop method). Contact angle measurements and analysis are performed with n = 3, and the average value of these contact angles is used to calculate surface free energy (Owens-Wendt method). <Contact angle measurement conditions> Measurement method: Sessile drop method (static contact angle) Measurement solvent: Pure water, diiodomethane Analysis method: θ / 2 method Amount of dropped liquid: 2.0 μL Waiting time from dropping to measurement: 1000 ms Measurement temperature: 23°C <θ / 2 method> The θ / 2 method is explained below. When a liquid lands on a solid surface, it becomes round due to surface tension, forming part of a sphere. The shape at this point is captured as an image, and image processing is used to find the left and right endpoints and vertex of the droplet, and the radius (r) and height (h) of the droplet sphere are then calculated. The calculated values ​​are then substituted into the following formula to calculate the contact angle θ. tan θ = (h / r) → θ = 2arctan(h / r)

[0028] <Calculation of surface free energy (Owens-Wendt method)> The values ​​of the contact angle θ1 with water and the contact angle θ2 with diiodomethane obtained by the above method and the dispersion component γ of each liquid were calculated. Ｌ ｄ and the polar component γ Ｌ ｈ Substituting this into the Young-Dupre equation of the Owens-Wendt theory in "3" below, and solving the simultaneous equations for water and diiodomethane, the surface free energy between the solid and gas (γ ｓ ) variance component (γ ｓ ｄ ) and polar component (γ ｓ ｈ ) is calculated.

[0029]

[0030] The cell culture film substrate may be sterilized. Sterilization methods include electron beam sterilization, gamma ray sterilization, EOG sterilization, and high-pressure steam sterilization. Even after sterilization, the polar component of the surface free energy on the culture surface of the cell culture film substrate remains below 12 mJ / m ２ The polar component of the surface free energy on the culture surface during the period from immediately after plasma treatment to immediately after 28 days of storage is 12 mJ / m ２ The above-mentioned range of values ​​for the surface free energy immediately after storage for 28 days can be adopted. The sterilization treatment described above may also be carried out on a package containing the cell culture film substrate or a package containing the cell culture film substrate.

[0031] <Quality Control Method> This embodiment also provides a quality control method for a cell culture film substrate having a culture surface on at least one surface. This quality control method involves comparing the polar component of the surface free energy of the culture surface, determined based on the Owens-Wendt method, immediately after production with the value two days after production, and determining whether the difference is 11.5 mJ / m ２ It includes a process of determining that the product is acceptable when the following conditions are met:

[0032] According to the findings of the present inventors, it has been found that the degree of fluctuation in the polar component of the surface free energy immediately after production can be used as an index to predict the degree of fluctuation thereafter. In other words, it has been found that if the degree of fluctuation in the polar component in the initial period is small, the degree of fluctuation in the polar component thereafter also tends to be small.

[0033] In addition, in the above quality control method, in the process of determining whether a product is acceptable, the difference between the polar components immediately after 2 days and immediately after 20 days is 15 mJ / m ２ If the following condition is further satisfied, the sample may be judged as passing. This makes it possible to more accurately predict the fluctuation of polar components after the 20th day.

[0034] The configurations of the film substrate with metal foil and the film substrate with cell sheet using the film substrate for cell culture will be described in detail below.

[0035] Fig. 4(a) is a top view showing a schematic example of the configuration of a film substrate with a metal foil, and Fig. 4(b) is a cross-sectional view of Fig. 4(a). Here, a metal foil 80 is used as an example of a surface protection material.

[0036] The film substrate with metal foil shown in Figures 4(a) and (b) comprises a film substrate for cell culture 10 having a culture surface 12 on at least one surface thereof, and at least one metal foil 80, the metal foil 80 being fixed in a state in which it covers the culture surface 12 on the plasma-treated surface.

[0037] In order to protect the culture surface 12 on the plasma-treated surface, the cell culture film substrate 10 preferably has a surface protection material (e.g., metal foil 80) that covers at least the culture surface 12. This can prevent a decrease in cell adhesiveness after long-term storage.

[0038] The cell culture film substrate 10 in storage may have the culture surface 12 covered with a surface protective material, or may be stored in a state where the culture surface 12 is not in contact with anything. Covering the culture surface 12 with a surface protective material can prevent unexpected contamination by dust or the like on the culture surface 12 of the cell culture film substrate 10, and can maintain the surface free energy at a predetermined value.

[0039] Examples of surface protection materials include metal foil 80, resin sheets, and fiber sheets, but those that do not allow components from the surface protection material to migrate or adhere to the culture surface 12 are preferred, with metal foil 80 being more preferred. By using metal foil 80, the polar component values ​​on the culture surface 12 can be maintained high for four months and beyond. Although the detailed mechanism is unclear, it is presumed that metal foil is excellent at preventing a decrease in cell adhesiveness because it can prevent organic matter contained in fibers and components blended in resins from adhering to the culture surface 12.

[0040] Examples of materials that can be used for the metal foil 80 include metal materials such as aluminum and copper. Among these, it is preferable that the metal foil 80 includes aluminum foil. Examples of materials that can be used for the resin sheet include polyethylene terephthalate, ionomer, polyethylene, polyvinyl chloride, polyvinylidene chloride, polyvinyl alcohol, polypropylene, polyester, polycarbonate, polystyrene, polyacrylonitrile, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, ethylene-methacrylic acid copolymer, perfluoroalkoxy fluororesin, nylon, and cellophane. Examples of materials for the fiber sheet include paper. These materials may be used alone or in combination of two or more.

[0041] The thickness of the metal foil 80 is, for example, 5 μm to 50 μm, preferably 6 μm to 30 μm, and more preferably 8 μm to 20 μm. By making the thickness equal to or greater than the lower limit, the mechanical strength can be increased, and the handleability of the metal foil 80 can be improved. By making the thickness equal to or less than the upper limit, the metal foil 80 and the cell culture film substrate 10 can be laminated with good adhesion, and the thickness of the laminate structure can be reduced.

[0042] In one embodiment, the metal foil 80 may be disposed on the culture surface 12 side of the cell culture film substrate 10, or may also be disposed on the back surface 14 side. One metal foil 80 may be folded over to cover both sides of the cell culture film substrate 10, or two metal foils 80 may be disposed on each side of the cell culture film substrate 10. In another embodiment, at least one cell culture film substrate 10 is disposed on one metal foil 80, or two or more cell culture film substrates 10 may be disposed at different intervals in the in-plane direction.

[0043] In another embodiment, when viewed from the normal direction to the surface (culture surface 12) of the cell culture film substrate 10, the metal foil 80 may have a margin that extends beyond the outer edge of the cell culture film substrate 10. This margin can be used to separate the cell culture film substrate 10 from the metal foil 80, improving operability.

[0044] In this embodiment, the cell culture film substrate 10 alone or a metal foil-attached film substrate including a cell culture film substrate 10 with a metal foil 80 provided on the culture surface 12 can be stored in a package or wrapping. FIG. 4( c) is a top view schematically illustrating an example of the configuration of the packaging body 100. One preferred form of packaging body 100 includes a metal foil-attached film substrate (a cell culture film substrate 10 having a metal foil 80) and a packaging material 90, and at least one metal foil-attached film substrate is packaged in the packaging material 90. Examples of packaging materials include aluminum, polyethylene terephthalate, ionomer, polyethylene, polyvinyl chloride, polyvinylidene chloride, polyvinyl alcohol, polypropylene, polyester, polycarbonate, polystyrene, polyacrylonitrile, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, ethylene-methacrylic acid copolymer, perfluoroalkoxy fluororesin, nylon, cellophane, and paper. These materials may be used alone or in combination of two or more. When sterilization requiring gas permeability, such as EOG sterilization or high-pressure steam sterilization, is performed, a material combining a paper material or a nonwoven fabric material with the above-mentioned material is preferably used. The structure of the packaging material in the thickness direction may be multi-packaging, or further packaging may be stacked after sterilization, and the outermost part of the package is preferably a resin film-like packaging material. The shape of the packaging material is preferably a bag, but is not limited thereto. The bag may be a bag sealed by heat sealing or a bag with a zipper, and specifically, a two-sided bag, a three-sided sealed bag, a three-sided bag with a zipper, a stand-up pouch, etc. are used. The sealing method may be selected according to the sealing performance required for the package.

[0045] Another preferred embodiment of the package includes the package and a packaging box, and at least one package is housed in the packaging box. The packaging box is, for example, a paper box such as a cardboard box.

[0046] An example of a method for manufacturing a package according to the present embodiment may include the steps of: preparing a film substrate 10 for cell culture having a culture surface 12 on at least one surface of which the culture surface 12 has been plasma-treated; coating the culture surface 12 on the plasma-treated surface with a metal foil 80 to obtain a film substrate with the metal foil; and packaging the film substrate with the metal foil using a packaging material to obtain a package. Another example of a method for manufacturing a package according to the present embodiment may include the steps of: preparing a film substrate 10 for cell culture having a culture surface 12 on at least one surface of which the culture surface 12 has been plasma-treated; coating the culture surface 12 on the plasma-treated surface with a metal foil 80 to obtain a film substrate with the metal foil; packaging the film substrate with the metal foil using a packaging material to obtain a package; and placing at least one of the packages in a packaging box and sealing it to obtain a package. In the above-described package or package manufacturing method, the plasma treatment preferably includes the first plasma treatment and the second plasma treatment described above. In the above-described package and manufacturing method of the package, a sterilization treatment is preferably performed. However, the sterilization treatment may be performed any time from after the step of obtaining the package to after the step of obtaining the package. Examples of sterilization treatment include electron beam sterilization, gamma ray sterilization, EOG sterilization, and high-pressure steam sterilization, but sterilization using radiation is preferred. In another embodiment, the manufacturing method of the package preferably includes a step of sterilizing the package using radiation after the step of obtaining the package. The cell culture film substrate 10 can also be placed in a culture vessel 20 and then packaged in a packaging material to form a package. Using such a package or packaging allows the cell culture film substrate 10 to be stored and distributed without contamination of the culture surface 12 before use in cell sheet culture.

[0047] In one embodiment, the polar component of the surface free energy of the culture surface 12 of the cell culture film substrate 10, as determined based on the Owens-Wendt method, is 12 mJ / m ２In another embodiment, in the film substrate with metal foil in the packaging or the packaging, the polar component of the surface free energy on the culture surface 12 of the film substrate for cell culture 10, as determined based on the Owens-Wendt method, is 12 mJ / m ２ The polar component here may be a value immediately after 28 days of storage, or may be a value after 28 days of storage, for example, a value from after 28 days of storage until the 90th day, the 180th day, the 270th day, or the 365th day.

[0048] The package or packaging may be in a state where it has been sterilized using radiation. It is preferable that the above-mentioned polar component values ​​are maintained even after irradiation.

[0049] 1 is a cross-sectional view schematically illustrating an example of the configuration of a cell sheet-attached film substrate 50 of this embodiment. The cell sheet-attached film substrate 50 includes a cell culture film substrate 10 and a cell sheet 40 that covers at least a portion of the culture surface 12 of the cell culture film substrate 10. The culture surface 12 of the cell culture film substrate 10 is the plasma-treated surface described above. The back surface 14 may or may not be plasma-treated.

[0050] The cell culture film substrate 10 can be used both as a cell sheet scaffold for culturing cells to form a cell sheet 40 and as a cell sheet support for transporting the cultured cell sheet 40. Such a cell culture film substrate 10 can be used as a medical substrate for transplantation, which is separated from the cell sheet 40 after the cell sheet 40 has been attached to the transplantation site.

[0051] The area of ​​the cell culture film substrate 10 is not particularly limited, but may be, for example, 0.3 cm ２ More than 1000cm ２ The lower limit of the area of ​​the cell culture film substrate 10 is more preferably 0.6 cm ２ More preferably, 1.6 cm or more ２ More than 8 cm, especially preferred ２ More than 3 cm ２More than 4cm ２ Above, 5cm ２ More than 6cm ２ More than 7cm ２ or more or 10 cm ２ On the other hand, the upper limit of the area of ​​the cell culture film substrate 10 is more preferably 900 cm ２ More preferably, 800 cm or less ２ Particularly preferably 500 cm ２ is less than or equal to 100 cm ２ Below, 50cm ２ Less than or equal to 20cm ２ If the area of ​​the cell culture film substrate 10 is larger than the culture vessel, it may be impossible to obtain a cell sheet. For example, the area of ​​the cell culture film substrate 10 may be 0.6 cm or less. ２ More than 900cm ２ Below, 1.6cm ２ More than 800cm ２ Below, 3cm ２ More than 500cm ２ Below, 6cm ２ More than 100cm ２ Below, 8cm ２ More than 50cm ２ Less than or equal to 10 cm ２ 20cm or more ２ The shape of the cell culture film substrate 10 when viewed from the surface normal direction is shown as a circle in Fig. 1, but it may be any suitable shape other than a circle, such as a regular polygon such as a square, a regular pentagon, a regular hexagon, or a regular octagon, or an oval or rectangle. When viewed from the surface normal direction, the cell culture film substrate 10 may not have an opening penetrating from the front to the back surface, and preferably does not have an opening that exposes the bottom surface of the culture vessel 20 when placed in the culture vessel 20.

[0052] The thickness of the cell culture film substrate 10 is not particularly limited, but is preferably 5 μm or more and 250 μm or less. The lower limit of the thickness is more preferably 6 μm or more, even more preferably 8 μm or more, 10 μm or more, or 12 μm or more. On the other hand, the upper limit of the thickness is more preferably 50 μm or less, 30 μm or less, 25 μm or less, and even more preferably 20 μm or less. For example, the thickness of the cell culture film substrate 10 is 6 μm or more and 50 μm or less, 8 μm or more and 30 μm or less, or 10 μm or more and 20 μm or less. By setting the thickness at or above the lower limit, the cell culture film substrate 10 can be prevented from tearing or curling during transportation, improving handleability. By setting the thickness at or below the upper limit, the conformability to the affected area during transplantation can be improved. In particular, a thickness of 20 μm or less can improve conformability to affected areas with higher curvatures, and can be applied to, for example, areas where internal organs have been sutured during surgery. The cell culture film substrate 10 may have a sheet shape with a flat surface as a whole when viewed in cross section in the thickness direction.

[0053] Furthermore, from the viewpoint of functioning as a free-standing membrane during culture, the lower limit of the thickness of the cell culture film substrate 10 is preferably greater than 10 μm, more preferably 11 μm or greater, and even more preferably 12 μm or greater. Thus, by using a cell culture film substrate 10 with a thickness greater than 10 μm, it is possible to produce a cell sheet 40 during the cell culture process without fixing or adhering the cell culture film substrate 10 to the inside of the culture vessel 20. A free-standing membrane is preferably a membrane that maintains the planar state of the culture surface 12 of the cell culture film substrate 10 even in the culture medium 30, and more preferably maintains this planar state even after the cell sheet 40 is formed on the culture surface 12. Because the planar state of the culture surface 12 is maintained, the cell culture film substrate 10 does not curl, improving cell culture characteristics in the culture medium 30 without adhering. Furthermore, this free-standing membrane may be capable of retaining its sheet shape to a certain extent when one end is pinched and lifted with tweezers.

[0054] Examples of materials that can be used to form the cell culture film substrate 10 include polyether ether ketone (PEEK), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polystyrene (PS), polycarbonate (PC), modified polyphenylene ether (mPPE), polyphenylene sulfide (PPS), polysulfone (PSU), polyarylate (PAR), liquid crystal polymer (LCP), polyethylene (PE), polypropylene (PP), nylon 66 (N66), ethylene-tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA), acrylonitrile-butadiene-styrene copolymer (ABS), polyethersulfone (PES), silicone, polyvinylidene fluoride (PVDF), polyacetal (POM), polyimide (PI), polyamide (PA), polyglycolic acid (PGA), polylactic acid (PLA), fibroin, cellulose, regenerated cellulose, cyclic olefin polymer, gelatin, and collagen. Among these, it is preferable that such a material contains an organic polymer compound having an aromatic ring, or an organic polymer compound having an aromatic ring and an oxygen atom.

[0055] An example of the cell culture film substrate 10 may be a film containing at least one of these materials as a primary component. Preferably, the material is transparent, has a specific gravity greater than 1.0, and exhibits excellent durability, mechanical strength, and processability. Polyether ether ketone film, polyethylene terephthalate film, or polystyrene is preferred, and polyether ether ketone film is even more preferred. The cell culture film substrate 10 is preferably a resin film containing one or more resin layers made from the above-mentioned resin materials. The two or more resin layers may contain different materials. The cell culture film substrate 10 may be composed of a resin substrate containing a resin layer made from the above-mentioned material, or a laminate substrate containing a resin layer made from the above-mentioned material and a resin layer and / or inorganic layer made from another material. However, the resin layer does not need to include a layer formed by chemical vapor deposition such as parylene or a wet coating layer. Furthermore, the inorganic layer in the laminate substrate preferably does not contain glass and may contain metal foil. Furthermore, the cell culture film substrate 10 preferably does not contain a nonwoven fabric or a fiber substrate on the culture surface 12 side. The cell culture film substrate 10 containing PEEK is preferable compared to one containing PET, considering overall the low linear expansion coefficient, solvent resistance, heat resistance, impact resistance, and sterilization resistance. Furthermore, the cell culture film substrate 10 is preferably made of a material with a higher specific gravity than the culture medium 30.

[0056] The surface roughness Ra of the culture surface 12 of the cell culture film substrate 10 has a lower limit of 0.3 nm or more, preferably 0.5 nm or more, and an upper limit of 100 nm or less, preferably 10 nm or less, more preferably 2 nm or less. Here, the surface roughness Ra refers to the arithmetic mean roughness in a square area with sides of 100 nm, measured using surface profile data measured with an atomic force microscope (AFM). When the culture surface 12 of the cell culture film substrate 10 is measured with a laser microscope, the cell culture film substrate has three or fewer holes with a diameter of 1 μm to 100 μm and a depth of 0.5 μm to 100 μm within a 100 μm square area. The diameter of the holes is, for example, 1 μm to 100 μm, preferably 2 μm to 50 μm, more preferably 3 μm to 30 μm. The depth of the hole is, for example, 0.5 μm or more and 100 μm or less, preferably 1 μm or more and 50 μm or less, and more preferably 2 μm or more and 20 μm or less. The combination of the range of the hole diameter and the hole depth is, for example, 1 μm or more and 100 μm or less and the hole depth is 0.5 μm or more and 100 μm or less, 1 μm or more and 100 μm or less and the hole diameter is 1 μm or more and 100 μm or less and the hole depth is 1 μm or more and 50 μm or less, 1 μm or more and 100 μm or less and the hole depth is 2 μm or more and 20 μm or less, 2 μm or more and 50 μm or less and the hole depth is 0.5 μm or more and 100 μm or less, The hole diameter is 2 μm to 50 μm and the hole depth is 1 μm to 50 μm, the hole diameter is 2 μm to 50 μm and the hole depth is 2 μm to 20 μm, the hole diameter is 3 μm to 30 μm and the hole depth is 0.5 μm to 100 μm, the hole diameter is 3 μm to 30 μm and the hole depth is 1 μm to 50 μm, or the hole diameter is 3 μm to 30 μm and the hole depth is 2 μm to 20 μm. The upper limit of the porosity of the cell culture film substrate 10 is, for example, 15% or less, preferably 10% or less, and more preferably 5% or less. The lower limit of the porosity of the cell culture film substrate 10 is not particularly limited, but may be 0% or more. By having three or less holes on the culture surface 12 and / or by making the porosity of the cell culture film substrate 10 below the upper limit value, the adhesion to the cell sheet 40 can be made appropriate.The presence or absence of holes may be measured on the surface of the resin layer formed on the culture surface 12 side of the cell culture film substrate 10. The porosity is calculated from the theoretical density and the actually measured density. Specifically, it is calculated using the formula: porosity = {1 - (actual density / theoretical density)} x 100.

[0057] Furthermore, the culture surface 12 of the cell culture film substrate 10 may be configured to not contain a temperature-responsive polymer. This prevents a decrease in adhesion between the cell sheet 40 and the cell culture film substrate 10 in low-temperature environments, such as those during cryopreservation. A temperature-responsive polymer is a material that exhibits cell adhesive properties at the temperature used for cell culture and exhibits cell non-adhesive properties upon temperature change, allowing for easy detachment of the cell sheet. A temperature range in which the temperature-responsive polymer exhibits cell adhesive properties of 10°C to 45°C, particularly 33°C to 40°C, is preferred because it allows stable cell culture. Furthermore, a temperature range in which the temperature-responsive polymer exhibits cell non-adhesive properties of 1°C to 36°C, particularly 4°C to 32°C, is preferred because it reduces damage to the detachment of the cell sheet. Specific examples of materials constituting the temperature-responsive polymer include temperature-responsive polymers such as poly-N-isopropylacrylamide (PNIPAAm), poly-N-n-propylacrylamide, poly-N-n-propylmethacrylamide, poly-N-ethoxyethylacrylamide, poly-N-tetrahydrofurfurylacrylamide, poly-N-tetrahydrofurfurylmethacrylamide, and poly-N,N-diethylacrylamide, with PNIPAAm, poly-N-n-propylmethacrylamide, and poly-N,N-diethylacrylamide being particularly preferred. The bottom surface of the culture vessel 20 may also be configured so as not to contain a temperature-responsive polymer.

[0058] <Method for manufacturing cell sheet-attached film substrate> By culturing a cell sheet 40 on the cell culture film substrate 10 that has been subjected to the first and second plasma treatments, a cell sheet-attached film substrate 50 including a laminate of the cell sheet 40 and the cell culture film substrate 10 is obtained, as shown in Figure 1. Specifically, an example of a method for manufacturing the cell sheet-attached film substrate 50 of this embodiment may include, for example, a culture step of introducing a plurality of cells, a culture medium 30, and the first and second plasma-treated cell culture film substrate 10 into a culture vessel 20, and culturing the cell sheet 40 on the culture surface 12 of the cell culture film substrate 10.

[0059] In the above-described culturing process, it is also possible to culture a cell sheet 40 on the culture surface 12 of the cell culture film substrate 10 after a predetermined period of time has elapsed. That is, the first and second plasma-treated cell culture film substrates 10 can be used to culture a cell sheet 40 at least after storage. The predetermined period of time may be, for example, after 14 days or after 28 days. Alternatively, the predetermined period of time may be after the steps of packaging, sterilization, storage, and opening have been performed.

[0060] The method for producing a cell sheet-attached film substrate of this embodiment will be specifically described using FIG. 2. FIG. 2 is a cross-sectional view showing a schematic example of the steps of the production method. In FIG. 2, (a) shows the processes of cell seeding, (b) shows cell culture, (c) shows cryopreservation and thawing, and (d) shows removal. The cryopreservation and thawing process in (c) is an optional process. These steps may be performed manually by a person or may be performed automatically using an appropriate device, and can be selected appropriately based on quality and cost. Here, a production method that does not perform (c) will be described, and details of (c) will be explained in the <Cryopreservation Method> section below.

[0061] The culture process may include a cell seeding process shown in FIG. 2( a) and a cell culture process shown in FIG. 2( b). In the cell seeding process shown in FIG. 2( a), a cell culture film substrate 10 with a plasma-treated culture surface 12 is placed in a culture vessel 20, and a cell-containing culture medium 30 is brought into contact with the culture surface 12 of the cell culture film substrate 10. The cells may be suspended in the culture medium 30 in advance, or a cell suspension containing cells may be added to the culture medium 30. Before placing the cell culture film substrate 10 in the culture vessel 20, a cleaning process may be added in which the cell culture film substrate 10 is washed with an ethanol aqueous solution or the like, or a destaticizing process may be added in which static electricity or other charges on the cell culture film substrate 10 are removed. The cell culture film substrate 10 may be partially or entirely immersed in the culture medium 30 during the culture process. Specifically, in the cell seeding step, the culture medium 30 may be dripped onto a portion of the surface of the cell culture film substrate 10, or the cell culture film substrate 10 may be immersed in the culture medium 30 so that at least a portion of each of the culture surface 12 and side surfaces of the cell culture film substrate 10 is in contact with the culture medium 30. Compared to the former dripping method, the latter immersion method makes it possible to maintain a stable culture environment during the subsequent cell culture step and / or to increase the area of ​​the culture surface 12 of the cell culture film substrate 10.

[0062] 2(b), the cells are cultured under appropriate culture conditions to form a cell sheet 40 on the culture surface 12 of the cell culture film substrate 10. A lid (not shown) may be placed over the cells during the culture.

[0063] In the cell culture process, the cell sheet 40 can be cultured without adhering the cell culture film substrate 10 to the inside of the culture vessel 20. In this case, the cell culture film substrate 10 functions as a free-standing membrane during cell culture. Because the cell culture film substrate 10 is not adhered, the cell sheet-attached film substrate 50 can be easily removed from the culture vessel 20 during the removal process. Furthermore, damage to the cell sheet 40 during removal can be suppressed. Here, "adhesion" refers to a state in which the film is in close contact with the cell substrate 10 to the extent that physical means are required for removal. Fixation means that hold the film down with a load other than its own weight to prevent movement are not included in the "adhesion" means used here. In this specification, physical means include, for example, a method of gripping the film and peeling it off, or a method of scratching it with a knife or the like to remove it. As a method for immersing the cell culture film substrate 10 in the culture medium 30 without adhering it to the culture medium 30, one or more of the following may be employed: a method for making the specific gravity of the cell culture film substrate 10 greater than that of the culture medium 30; a method for immobilizing a load on a part of the culture surface 12 of the cell culture film substrate 10 using a weight; or a method for fixing the position of the cell culture film substrate 10 using an instrument. That is, during the cell culture step, cells can be cultured in a state where the cell culture film substrate 10 is immersed in the culture medium 30 by immobilizing the load and / or fixing the position.

[0064] The manufacturing method may include a removal step of the cell sheet-attached film substrate 50 shown in Figure 2(d). In the removal step of Figure 2(d), the cell sheet-attached film substrate 50 is removed from the culture vessel 20. Thereafter, for example, the cell sheet-attached film substrate 50 may be transported to a desired location, such as a transplantation site. In the removal step of this embodiment, the removal method is not particularly limited, but examples include a method of pinching and removing the cell culture film substrate 10 constituting the cell sheet-attached film substrate 50, a method of bringing the cell culture film substrate 10 into contact with a suction nozzle and lifting it while suctioning, and a method of threading the cell culture film substrate 10 and lifting it up.

[0065] (Culture Vessel) The culture vessel 20 is a vessel for culturing cells in a medium. It is not particularly limited as long as it is suitable for the type of cells to be cultured and the intended use. Examples of the culture vessel 20 include dishes, Petri dishes, tissue culture dishes, multi-dishes, flasks, tissue culture flasks, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, Petri dishes, tubes, trays, culture bags, and roller bottles. The culture vessel 20 may be a surface-treated culture vessel with cell adhesive properties (for adherent cells) or an untreated culture vessel (for suspension cells). The presence or absence of a surface treatment on the culture vessel 20 does not affect the cell culture film substrate 10. Hereinafter, unless otherwise specified, either type may be used.

[0066] The material of the culture vessel 20 is not particularly limited as long as it is impermeable to the culture medium 30, and examples thereof include polystyrene, polyethylene, polypropylene, polyvinyl alcohol, polyethylene terephthalate, polyacetal, polyvinyl chloride, acrylic resin, polycarbonate, polyether ether ketone, polyether sulfone, polytetrafluoroethylene, polyimide, polyamide, polycycloolefin, cellulose, silicone, nylon 6,6, glass, and metals such as stainless steel and aluminum.

[0067] The area of ​​the culture vessel 20 is not particularly limited, but any commercially available culture vessel can be used for culture without any problems. For example, a size of 0.3 cm ２ More than 1000cm ２ The lower limit is more preferably 0.35 cm. ２ More preferably, 1.0 cm or more ２ More than 1.9 cm, particularly preferably ２ On the other hand, the upper limit is more preferably 900 cm ２ More preferably, 800 cm or less ２ Particularly preferably 500 cm ２ The following is the result.

[0068] (Cells) The cells are not particularly limited as long as they are clinically useful cells for treating or preventing symptoms associated with cell, tissue, or organ deficiency, dysfunction, or dysfunction, or culturable cells for use in non-clinical trials, and are cells isolated from a living organism. Examples of cells include biological tissue cells, mesenchymal stem cells capable of differentiating into cells belonging to mesenchymal tissue, pluripotent stem cells capable of differentiating into various biological tissues, and stem cells and progenitor cells that can be induced to differentiate. The cells may be adherent cells or suspension cells.

[0069] Specific examples of biological tissue cells include fibroblasts, myofibroblasts, corneal epithelial cells, retinal cells, nerve cells, muscle cells, cardiac muscle cells, myoblasts, bone cells, osteoblasts, chondrocytes, adipocytes, hepatocytes, pancreatic cells, kidney cells, gingival cells, periosteal cells, skin cells, and endothelial cells. Specific examples of mesenchymal stem cells include adipose tissue-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, and umbilical cord-derived mesenchymal stem cells. Specific examples of pluripotent stem cells include induced pluripotent stem cells, embryonic stem cells, nuclear transfer embryonic stem cells, embryonic tumor cells, and embryonic germ cells. These cells may be cultured alone or in combination of two or more types. These cells may be appropriately selected from known types depending on the intended use of the cells.

[0070] The origin of the cells is not particularly limited, and examples thereof include mammals, birds, amphibians, fish, insects, plants, microorganisms, etc. Specific examples of mammals and birds include humans, monkeys, chimpanzees, cows, horses, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, chickens, etc.

[0071] (Culture conditions) The cell culture is not particularly limited, and conventional means used in technical fields such as medical care, pharmaceuticals, quasi-drugs, cosmetics, food, and veterinary medicine, as well as basic technical fields such as regenerative medicine and bioengineering, can be used.

[0072] The culture conditions are not particularly limited as long as they can bring the cultured cells into the desired state. Typical culture conditions include, for example, culture at 37°C, 5% CO using a prepared basal medium. ２ The cell culture period is not particularly limited as long as the cultured cells reach the desired state. The cell culture period is, for example, within 28 days, within 21 days, within 14 days, within 7 days, within 5 days, or within 3 days. When culturing for a long period of time, the medium may be replaced. The frequency and method of medium replacement are not particularly limited. In general, it is preferable to replace the medium every 1 to 7 days. It is particularly preferable to replace the medium every 1 to 5 days. At this time, the entire medium may be replaced, or a portion of the medium may be left and new medium may be added.

[0073] The density of the cells to be cultured is not particularly limited as long as it is suitable for the cells to be cultured, the culture vessel, and the intended use of the cultured cells. For example, ２ cells / cm ２ 1x10 or more ９ cells / cm ２ The lower limit of the cell density is more preferably 1 × 10 ３ cells / cm ２ More preferably, 5×10 ３ cells / cm ２ More preferably, 5 × 10 ４ cells / cm ２ On the other hand, the upper limit of the cell density is more preferably 1 × 10 ８ cells / cm ２ More preferably, 5 × 10 ７ cells / cm ２ Particularly preferably, 1 × 10 ７ cells / cm ２ The density of the cells to be cultured is, for example, 5 × 10 ２ cells / cm ２ 1x10 or more ９ cells / cm ２ Below, 5 x 10 ２ cells / cm ２ 1x10 or more ８ cells / cm ２ Below, 5 x 10 ２ cells / cm ２Above 5×10 ７ Cells / cm ２ Below, 5×10 ２ Cells / cm ２ Above 1×10 ７ Cells / cm ２ Below, 1×10 ３ Cells / cm ２ Above 1×10 ９ Cells / cm ２ Below, 1×10 ３ Cells / cm ２ Above 1×10 ８ Cells / cm ２ Below, 1×10 ３ Cells / cm ２ Above 5×10 ７ Cells / cm ２ Below, 1×10 ３ Cells / cm ２ Above 1×10 ７ Cells / cm ２ Below, 5×10 ３ Cells / cm ２ Above 1×10 ９ Cells / cm ２ Below, 5×10 ３ Cells / cm ２ Above 1×10 ８ Cells / cm ２ Below, 5×10 ３ Cells / cm ２ Above 5×10 ７ Cells / cm ２ Below, 5×10 ３ Cells / cm ２ Above 1×10 ７ Cells / cm ２ Below, 5×10 ４ Cells / cm ２ Above 1×10 ９ Cells / cm ２ Below, 5×10 ４ Cells / cm ２ Above 1×10 ８ Cells / cm ２ Below, 5×10 ４ Cells / cm ２ Above 5×10 ７ Cells / cm ２ Below, 5×10 ４ Cells / cm ２ Above 1×10 ７ Cells / cm２ The following is the result.

[0074] (Culture Medium) The culture medium 30 is a liquid containing culture medium components, and is not particularly limited as long as it is suitable for the cells to be cultured. Examples of culture medium components include sugars, amino acids, vitamins, inorganic salts, trace metals, and additives. These culture medium components may be blended alone or in combination of two or more. These culture medium components may be appropriately selected from known components depending on the cells to be cultured, or may be uniquely blended components.

[0075] Specific examples of sugars include monosaccharides such as glucose, fructose, mannose, and galactose; disaccharides such as sucrose, sucralose, trehalose, maltose, and lactose; trisaccharides such as glucosylsucrose, lactosucrose, and raffinose; tetrasaccharides such as acarbose and maltotetraose; cyclodextrins; and oligosaccharides.

[0076] Specific examples of amino acids include L-glutamic acid, L-glutamine, L-arginine, L-cystine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-alanine, L-asparagine, L-aspartic acid, L-cysteine, and L-hydroxyproline.

[0077] Specific examples of vitamins include sodium L-ascorbate, L-ascorbic acid-diphosphate, choline, folic acid, niacin, biotin, pantothenic acid, pyridoxine, riboflavin, thiamine, thymidine, and vitamin B12.

[0078] Specific examples of inorganic salts include sodium chloride, sodium hydroxide, sodium sulfate, sodium phosphate, disodium hydrogen phosphate, sodium carbonate, sodium bicarbonate, potassium chloride, potassium hydroxide, potassium sulfate, potassium phosphate, dipotassium hydrogen phosphate, potassium carbonate, potassium bicarbonate, calcium chloride, calcium sulfate, calcium nitrate, calcium phosphate, calcium carbonate, magnesium chloride, magnesium sulfate, magnesium nitrate, magnesium phosphate, magnesium carbonate, etc. Specific examples of trace metals include iron sulfate, iron nitrate, copper sulfate, copper nitrate, zinc sulfate, etc.

[0079] Specific examples of additives include serum such as bovine serum, horse serum, and human serum, or artificial serum; growth factors such as FGF2, EGF, HGF, VEGF, and PDGF; proteins such as albumin; antioxidants such as glutathione, ascorbic acid, and ascorbic acid derivatives; antibiotics such as penicillin and streptomycin; pH adjusters such as HEPES; organic acids such as lactic acid and propionic acid; lipids such as cholesterol; fatty acids such as linolenic acid; amines such as ethanolamine and putrescine; reducing agents such as mercaptoethanol and 3-mercapto-1,2-propanediol; thickeners such as sodium alginate, polyvinylpyrrolidone, carboxymethylcellulose, and pullulan; and pH indicators such as phenol red.

[0080] Examples of the medium 30 containing the above-mentioned medium components include AIM V medium, HFDM-1 medium, equilibrated buffer solutions such as Dulbecco's phosphate buffered saline (D-PBS) and Hank's balanced salt solution (HBSS), DMEM (Dulbecco's Modified Eagle Medium), EMEM (Eagle's Minimum Essential Medium), α-MEM (Minimum Essential Medium alpha Modification), IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), and Ham's F-10. medium, Ham's F-12 medium, Ham's F-12K medium, RPMI medium 1640, M-199 medium, L-15 medium, McCoy's 5A Medium, MCDB105 medium, MCDB107 medium, MCDB131 medium, MCDB153 medium, MCDB201 medium, NCTC109 medium, NCTC135 medium, Waymouth's MB752 / 1 medium, CMRL-1066 Examples of suitable basal media include basal media such as basal medium E, Williams' medium E, Brinster's BMOC-3 medium, and E8 medium. These basal media may be used alone or in combination of two or more. Furthermore, medium components may be added, removed, increased, or decreased in amount depending on the type and condition of the cells. These basal media may be appropriately selected from known media depending on the cells to be cultured, or a uniquely formulated composition may be used.

[0081] (Cell Sheet) The cell sheet 40 has a sheet structure in which cells are physically and functionally connected to one another via adhesion molecules, extracellular matrix, etc. The cell sheet 40 may have a single-layer structure consisting of one cell layer, or a laminated structure consisting of two or more cell layers. The laminated structure is not particularly limited, but examples include multi-layer structures such as two-layer, three-layer, four-layer, and five-layer structures.

[0082] When the cell sheet 40 has a multilayer structure, the multilayer structure may be obtained when the cell sheet is cultured on the cell culture film substrate 10, or it may be obtained by stacking cell sheets with a single layer structure. In particular, a cell sheet with a multilayer structure can be obtained by preparing multiple cell sheet-attached film substrates 50 of the present invention, overlaying one cell sheet on another, and peeling off the cell culture film substrate from the other cell sheet.

[0083] The thickness of the cell sheet 40 is not particularly limited, but is, for example, 0.001 mm or more and 2.0 mm or less. The lower limit of the thickness of the cell sheet 40 is more preferably 0.01 mm or more, even more preferably 0.03 mm or more, and particularly preferably 0.05 mm or more. On the other hand, the upper limit of the thickness of the cell sheet 40 is more preferably 1.5 mm or less, even more preferably 1.2 mm or less, and particularly preferably 1.0 mm or less. The thickness of the cell sheet 40 may be, for example, 0.001 mm to 2.0 mm, 0.001 mm to 1.5 mm, 0.001 mm to 1.2 mm, 0.001 mm to 1.0 mm, 0.01 mm to 2.0 mm, 0.01 mm to 1.5 mm, 0.01 mm to 1.2 mm, 0.01 mm to 1.0 mm, 0.03 mm to 2.0 mm, 0.03 mm to 1.2 mm, 0.03 mm to 1.0 mm, 0.05 mm to 2.0 mm, 0.05 mm to 1.5 mm, 0.05 mm to 1.2 mm, or 0.05 mm to 1.0 mm. By keeping the thickness of the cell sheet 40 within the above ranges, high cell activity within the cell sheet 40 and excellent shape retention ability, which are advantageous for cell transplantation, can be achieved.

[0084] The area of ​​the cell sheet 40 is not particularly limited, but may be, for example, 0.3 cm ２ More than 1000cm ２ The lower limit of the area of ​​the cell sheet 40 is more preferably 0.6 cm ２ More preferably, 1.6 cm or more ２ More than 8 cm, especially preferred ２ More than 3 cm ２ More than 4cm ２ Above, 5cm ２More than 6cm ２ More than 7cm ２ or more or 10 cm ２ On the other hand, the upper limit of the area of ​​the cell sheet 40 is more preferably 900 cm ２ More preferably, 800 cm or less ２ Particularly preferably 500 cm ２ is less than or equal to 100 cm ２ Below, 50cm ２ Less than or equal to 20cm ２ For example, the area of ​​the cell sheet 40 may be 0.6 cm ２ More than 900cm ２ Below, 1.6cm ２ More than 800cm ２ Below, 3cm ２ More than 500cm ２ Below, 6cm ２ More than 100cm ２ Below, 8cm ２ More than 50cm ２ Less than or equal to 10 cm ２ 20cm or more ２ Conventionally, when a cell sheet is transplanted alone, the strength of the cell sheet is low, and therefore there is a high possibility that the cell sheet will break during transportation if it is large in area. However, in the present disclosure, the cell sheet 40 is supported by a cell culture film substrate, which prevents the cell sheet from breaking during transplantation. Therefore, the size of the cell sheet 40 can be reduced to 8 cm. ２ It is also possible to produce a larger cell sheet and adjust it to the size of the affected area as needed. In the cell sheet-attached film substrate 50, it is preferable that the entire underside of the cell sheet 40 overlaps the surface (culture surface 12) of the cell culture film substrate 10.

[0085] <Cryopreservation Method> As shown in FIG. 2(c), an example of the cryopreservation method of this embodiment includes a cooling step of cooling a cell sheet-attached film substrate 50, which includes a cell culture film substrate 10 inside a culture vessel 20 and a cell sheet 40 formed on the surface (culture surface 12) of the cell culture film substrate 10, in a cryopreservation solution 60. The culture vessel may be frozen with a culture lid (not shown) or a lid (not shown) that prevents liquid leakage and microbial contamination. In FIG. 2(c), the cell sheet-attached film substrate 50 is cryopreserved inside the culture vessel 20. However, the cell sheet-attached film substrate 50 may also be transferred to a cryopreservation container separate from the culture vessel 20 for cryopreservation. The cryopreservation container is not particularly limited, and may be, for example, the one exemplified for the culture vessel 20.

[0086] In the cooling process, the cell sheet-attached film substrate 50 can be configured in various ways. As shown in Figures 3(a) and 3(b), the cell sheet-attached film substrate 50 may have a structure in which a cell culture film substrate 10 is formed on one side of a cell sheet 40. Alternatively, as shown in Figure 3(c), the cell sheet 40 may be sandwiched between two cell culture film substrates 10a and 10b on both sides. In Figure 3(a), the cell culture film substrate 10 is placed facing the bottom of the culture vessel 20. In Figure 3(b), the cell sheet 40 is placed facing the bottom of the culture vessel 20. In Figure 3(c), the cell culture film substrates 10a and 10b may be made of the same material or different materials. For example, both cell culture film substrates 10a and 10b may be cell culture film substrates containing PEEK, or one may be a cell culture film substrate containing PEEK and the other may be a cell culture film substrate not containing PEEK.

[0087] (Cryopreservation Solution) The cryopreservation solution 60 is a solution for reducing cell damage due to cryopreservation. There are no particular limitations on the cryopreservation solution 60 as long as it is suitable for cryopreserving cells, but it may also contain medium components contained in the culture medium, cryoprotectants, etc. The medium components, such as sugars, amino acids, vitamins, inorganic salts, trace metals, and additives, may be any that satisfy the explanation in the above (Culture Medium) section. Furthermore, it is preferable that the cryopreservation solution have a solidification initiation temperature in the range of -15°C or higher and -5°C or lower.

[0088] Cryoprotectants are substances used to reduce damage to cells caused by freezing and thawing during cryopreservation. Examples of cryoprotectants include cell-impermeant cryoprotectants and cell-permeant cryoprotectants. Specific examples of cell-impermeant cryoprotectants include albumin, sucrose, trehalose, dextran, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and polylysine. Specific examples of cell-permeant cryoprotectants include dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, and propanediol. These cryoprotectants may be formulated alone or in combination of two or more. These cryoprotectants may be appropriately selected from known cryoprotectants depending on the type of cell, the composition of the cryopreservation solution, and the like, or a uniquely formulated composition may be used.

[0089] Examples of commercially available cryopreservation solutions that do not contain DMSO include Stem Cell Banker (registered trademark) DMSO-free GMP grade (Nihon Zenyaku Kogyo Co., Ltd.), Bambanker (registered trademark) DMSO-free (CG Lymphotec Co., Ltd.), Cryoscarless (registered trademark) DMSO-free (BioVerde Co., Ltd.), Stem Cell Keep (BioVerde Co., Ltd.), CryoNovo (registered trademark) X12 (Akron BioProducts LCC), CryoNovo (registered trademark) P24 (Akron BioProducts LCC), DMSO-free cell cryopreservation solution for cryopreservation of human ES / iPS cells (ReproCell Co., Ltd.), Cell Reservoir One (Nacalai Tesque Co., Ltd.), ThelioKeep (registered trademark: BioVerde Co., Ltd.), and Cellvation (registered trademark: Protide Co., Ltd.). Examples include ReproCryo RM (ReproCell Pharmaceuticals), and SOFORO Cryo (Saraya). In addition, examples of commercially available cryopreservation solutions containing DMSO include Stem Cell Banker (registered trademark) GMP Grade (Nippon Zenyaku Kogyo Co., Ltd.), Stem Cell Banker (registered trademark) EX GMP Grade (Nippon Zenyaku Kogyo Co., Ltd.), Bambanker (registered trademark) hRM (CG Lymphotec Co., Ltd.), Bambanker (registered trademark) (CG Lymphotec Co., Ltd.), iStock (CG Lymphotec Co., Ltd.), CryoStor CS5 (Charles River Laboratories Cell Solutions, Inc.), and CryoStor CS10 (Charles River Laboratories Cell Solutions, Inc.).

[0090] (Cooling step) In the cooling step, the cell sheet-attached film substrate 50 is preferably cooled in a cryopreservation solution using a non-throughflow cooling device. The cell sheet 40 on an appropriate cell culture film substrate 10 can be cooled at a uniform temperature using a non-throughflow cooling device, which minimizes damage to the cells and suppresses a decrease in cell activity.

[0091] In the cooling step, the cooling rate at 0 to -5°C is, for example, 0.1°C / min to 15°C / min, preferably 0.25°C / min to 12.5°C / min, and more preferably 0.5°C / min to 10°C / min. By setting the cooling rate at or above the lower limit, unnecessary contact time between the liquid cryoprotectant and the cells can be reduced. By setting the cooling rate at or below the upper limit, intracellular ice crystal formation can be suppressed.

[0092] In the cooling step, the temperature of the freezing treatment is not particularly limited as long as it can freeze the cultured cells and the cryopreservation solution. The freezing temperature is, for example, −196°C or higher and −25°C or lower. The lower limit is more preferably −180°C or higher, even more preferably −160°C or higher, and particularly preferably −150°C or higher. On the other hand, the upper limit is more preferably −25°C or lower, even more preferably −30°C or lower, and particularly preferably −35°C or lower. The temperature for the freezing treatment is, for example, −196°C or higher and −25°C or lower, −196°C or higher and −30°C or lower, −196°C or higher and −35°C or lower, −180°C or higher and −25°C or lower, −180°C or higher and −30°C or lower, −180°C or higher and −35°C or lower, −160°C or higher and −25°C or lower, −160°C or higher and −30°C or lower, −160°C or higher and −35°C or lower, −150°C or higher and −25°C or lower, −150°C or higher and −30°C or lower, or −150°C or higher and −35°C or lower.

[0093] The cooling device used for the freezing process is not particularly limited, and examples include quick-freezing devices and cryogenic refrigeration devices. As a cooling device, a freezing device that does not come into contact with a heat transfer device and sprays cold air onto the culture vessel from multiple directions, preferably all directions, rather than one direction, to freeze the culture vessel and cells, is preferable from the viewpoint of freezing cells at a uniform temperature and increasing the survival rate of the cells after thawing. Specific examples of freezing devices that spray cold air onto the culture vessel include cooling devices that cool the object to be cooled by circulating cold air using a cooling fan, such as the non-through-flow cooling device equipped with a cooling fan disclosed in Japanese Patent Laid-Open No. 2005-127666. The term "non-through-flow system" refers to a system in which the majority of the through-flow air from the object to be cooled does not pass through (through-flow) the cooler.

[0094] (Freezing and preservation step) The freezing and preservation method preferably includes a freezing and preservation step in which the cell sheet-attached film substrate 50 is stored after the cooling step, for example, at −196 to −60°C, preferably −180 to −65°C, and more preferably −150 to −80°C. This allows the cell sheet to be stably maintained for a long period of time. The temperature in the cooling step may be the set temperature of the cooling device.

[0095] The cryopreservation method is not particularly limited as long as the cells can be stably cryopreserved. Examples of cryopreservation methods include contact with the liquid or gas phase of a coolant, and use of an ultra-low temperature freezer. A preferred cryopreservation method is contact with the liquid or gas phase of a coolant from the viewpoint of temperature. Examples of coolants include liquid nitrogen, liquid ethane, liquid propane, liquid helium, and dry ice.

[0096] The cryopreservation temperature is not particularly limited as long as the cells can be stably cryopreserved. The cryopreservation temperature may be, for example, within any range of −196°C or higher to −60°C or lower, −196°C or higher to −134°C or lower, or −134°C or higher to −60°C or lower. The cryopreservation temperature may be the surface temperature of the cell sheet 40 to be frozen. The surface temperature can be measured, for example, using a K thermocouple.

[0097] The frozen cell sheet-attached film substrate 50 includes the cell culture film substrate 10 and the cell sheet 40 formed on the surface (culture surface 12) of the cell culture film substrate 10. In the cell sheet-attached film substrate 50, the cell culture film substrate 10 and the cell sheet 40 are in a frozen state.

[0098] In the above-mentioned frozen product, the cell culture film substrate 10 and the cell sheet 40 are frozen at the above-mentioned cryopreservation temperature, specifically, preferably at or below −60° C., more preferably at or below −135° C. Alternatively, the frozen product may be frozen at a temperature between −134° C. and −60° C. The frozen product is frozen at a temperature above −196° C.

[0099] The frozen cell sheet-attached film substrate frozen inside a culture vessel or cryopreservation container can be packaged in a packaging material either while frozen in the culture vessel or cryopreservation container or after being removed from the container. The sealed packaging prevents microbial contamination of the cell sheet and allows the cell sheet to be stored and distributed without contamination. As an example, a frozen product package can be obtained by packaging a container containing the cell sheet-attached film substrate and a cryopreservation solution in a film-like packaging material and freezing it in a sealed state. Preferred packaging materials include aluminum, polyethylene terephthalate, ionomer, polyethylene, polyvinylidene chloride, polyvinyl alcohol, polypropylene, polyester, polycarbonate, polyacrylonitrile, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, ethylene-methacrylic acid copolymer, polyimide, fluororesins such as perfluoroalkoxy fluororesin and tetrafluoroethylene-hexafluoropropylene copolymer (FEP), and nylon. These materials may be used alone, or two or more of these materials may be combined to form a multilayer laminate, or a multi-layer package may be used.

[0100] (Thawing Step) When the transplantation method of this embodiment includes the above-mentioned cryopreservation method, the cryopreservation method further includes a thawing step of thawing the cell sheet-attached film substrate 50 .

[0101] The thawing method is not particularly limited, and conventional means used in technical fields such as medicine, pharmaceuticals, quasi-drugs, cosmetics, food, and veterinary medicine, as well as basic technical fields such as regenerative medicine and bioengineering, can be used. Thawing methods include, for example, using a water bath, bead bath, incubator, hot plate, defroster, etc., immersing in a melting liquid at a temperature higher than the freezing temperature, or leaving in an environment at a temperature higher than the freezing temperature. The environmental temperature in contact with the frozen material during thawing is not particularly limited, as long as it is higher than the freezing temperature and lower than 50°C. The upper limit of the environmental temperature is, for example, 49°C or lower, preferably 45°C or lower, and more preferably 40°C or lower. Meanwhile, the lower limit of the environmental temperature is, for example, 0°C or higher, preferably 5°C or higher, more preferably 10°C, and even more preferably 15°C or higher. The range of environmental temperatures in contact with the frozen material during thawing is, for example, 0°C to 49°C, 0°C to 45°C, 0°C to 40°C, 5°C to 49°C, 5°C to 45°C, 5°C to 40°C, 10°C to 49°C, 10°C to 45°C, 10°C to 40°C, 15°C to 49°C, 15°C to 45°C, and 15°C to 40°C. Thawing temperatures of 50°C or higher are not preferred due to the possibility of thermal damage to cells. Furthermore, thawing may be temporarily performed in an environment at a temperature below the freezing point. For example, frozen material stored at -80°C can be exposed to an environmental temperature of -30°C and then thawed at an environmental temperature above the freezing point. The time required to thaw a frozen material is not particularly limited as long as freezing-induced damage to cells does not occur. Typically, thawing can be performed without problems within a time period of more than 10 seconds and less than 60 minutes. The lower limit of the time required to thaw a frozen product is sufficient if it is more than 10 seconds, preferably 20 seconds or more, more preferably 30 seconds or more, and even more preferably 1 minute or more, and the upper limit is sufficient if it is 60 minutes or less, preferably 50 minutes or less, more preferably 40 minutes or less, and even more preferably 30 minutes or less.For example, the time required for thawing is more than 10 seconds and less than 60 minutes, more than 10 seconds and less than 50 minutes, more than 10 seconds and less than 40 minutes, more than 10 seconds and less than 30 minutes, 20 seconds and less than 60 minutes, 20 seconds and less than 50 minutes, 20 seconds and less than 40 minutes, 20 seconds and less than 30 minutes, 30 seconds and less than 60 minutes, 30 seconds and less than 50 minutes, 30 seconds and less than 40 minutes, 30 seconds and less than 30 minutes, 1 minute and less than 60 minutes, 1 minute and less than 50 minutes, 1 minute and less than 40 minutes, or 1 minute and less than 30 minutes. If thawing occurs too quickly, the frozen material may crack due to thermal shock caused by the temperature difference, potentially damaging the cell sheet. If thawing occurs too slowly, water molecules may recrystallize under sub-zero conditions, causing ice crystals to grow larger and causing serious damage to the cells, which is undesirable. During the thawing time, the environmental temperature may be set to a constant temperature or may be set to fluctuate, such as by gradually increasing.

[0102] The thawing solution is not particularly limited as long as it does not damage the cultured cells. Examples of components contained in the thawing solution include sucrose, glucose, maltose, trehalose, and fructose. The thawing solution may also contain the components described above in the section (Culture Medium).

[0103] The temperature of the melting liquid is not particularly limited as long as it is higher than the freezing temperature. The temperature of the melting liquid is, for example, 0°C or higher and 45°C or lower. The lower limit is more preferably 4°C or higher, even more preferably 25°C or higher, and particularly preferably 28°C or higher. On the other hand, the upper limit is more preferably 40°C or lower, even more preferably 39°C or lower, and particularly preferably 38°C or lower.

[0104] The thawed cell sheet 40 and cell culture film substrate 10 (cell sheet-attached film substrate 50) may be washed with a cell washing solution immediately after the thawing process, if necessary. The cell washing solution is not particularly limited and may contain the components described in the above section (Culture Medium). The temperature of the cell washing solution is not particularly limited. For example, the temperature of the cell washing solution is 0°C or higher and 45°C or lower. The lower limit is more preferably 4°C or higher, even more preferably 25°C or higher, and particularly preferably 28°C or higher. On the other hand, the upper limit is more preferably 40°C or lower, even more preferably 39°C or lower, and particularly preferably 38°C or lower. The number of times the cultured cells are washed is not particularly limited and may be one or multiple times (e.g., two, three, four, five times, etc.).

[0105] In the present disclosure, since the culture surface 12 does not contain a temperature-responsive polymer, it is possible to prevent the cell sheet 40 from peeling off from the cell culture film substrate 10 in a low-temperature environment such as a cryopreservation process, and it is possible to obtain a cell sheet-attached film substrate 50 that maintains the adhesiveness between the cell sheet and the film even after undergoing the cryopreservation process and the thawing process.

[0106] The transplantation method may include a step of attaching a cell sheet 40 as shown in Fig. 2(e) . In the attachment step shown in Fig. 2(e) , the cell sheet 40 of the cell sheet-attached film substrate 50 is attached to the transplantation site 70, and then the cell culture film substrate 10 is peeled off from the cell sheet 40.

[0107] In this embodiment, the time from attachment to detachment during the attachment process can be shortened. By shortening the time, the cell sheet is not exposed to the substrate film as a foreign body for a long period of time, reducing the risk of immune rejection of the substrate film, reducing the possibility of cell death, and simplifying the procedure. This method can also be used in surgeries such as thoracotomy to attach a cell sheet to the body. The time from attachment to detachment is preferably 10 minutes or less, more preferably 5 minutes or less, even more preferably 3 minutes or less, and even more preferably 1 minute or less. Although the detailed mechanism is unclear, it is presumed that the adhesion between the culture surface 12 of the cell culture film substrate 10 and the surface of the cell sheet 40 is moderately strong, allowing the cell culture film substrate 10 to be detached from the cell sheet 40 even before the cell sheet 40 is biologically bound to the transplantation site 70.

[0108] Furthermore, in this embodiment, it is possible to prevent the cell sheet 40 from remaining on the cell culture film substrate 10 after peeling. The remaining rate of the cell sheet 40 on the cell culture film substrate 10 after peeling, in terms of area ratio, is preferably 50% or less, more preferably 30% or less, and even more preferably 10%.

[0109] Cell transplantation therapy suppresses or prevents the onset and recurrence of symptoms associated with cell, tissue, or organ loss, dysfunction, or dysfunction. Examples of diseases that can be treated with cell transplantation therapy include spinal cord injury, knee articular cartilage injury, ischemic heart disease, age-related macular degeneration, corneal epithelial stem cell deficiency, aplastic anemia, severe limb ischemia, refractory skin ulcers, and the prevention of postoperative complications (e.g., various organ suture failures, bronchial stump fistulas, pancreatic fistulas, and bile leaks), burns, and the like. Furthermore, cells used in cell transplantation therapy may be autologous, allogeneic non-autologous, or xenogeneic. Autologous cells are preferred from the standpoints of clinical application and safety, while non-autologous cells are preferred from the standpoints of clinical application and productivity.

[0110] The transplantation site may be at least a portion of the body of a recipient, such as a mammal, bird, amphibian, fish, insect, plant, or microorganism. Specific examples of mammals and birds include humans, monkeys, chimpanzees, cows, horses, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, and chickens. However, the transplantation site may exclude the interior and exterior of the human body. Examples of transplantation sites other than the recipient include other cell sheets, medical devices, and tissues or organs isolated from the recipient. Examples of tissues or organs isolated from the recipient include skin, oral tissue, esophagus, trachea, bronchi, lungs, lung lobes, stomach, duodenum, pancreas, spleen, small intestine, large intestine, muscle tissue, and bone (excluding tissues that are to be returned to the same recipient for treatment).

[0111] Although the embodiments of the present invention have been described above, these are merely examples of the present invention, and various other configurations may be adopted. Furthermore, the present invention is not limited to the above-described embodiments, and modifications and improvements within the scope of achieving the object of the present invention are included in the present invention.

[0112] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the descriptions of these examples.

[0113] <Production of Cell Culture Film Substrate> A cell culture film substrate was produced by surface treating a film substrate having the materials and thickness shown in Table 1 under the plasma treatment conditions shown in Table 1. Note that RIE stands for reactive ion etching. Specifically, the procedure was as follows: The film substrate was placed on a cathode electrode in a chamber. After reducing the pressure inside the chamber, a predetermined gas was flowed at a predetermined flow rate. After the pressure inside the chamber stabilized, high-frequency power (13.56 MHz) was applied at a predetermined power to generate plasma. After a predetermined treatment time had elapsed, the application of power was terminated, the gas was stopped, the chamber was opened to the atmosphere, and the film substrate was removed. The plasma-treated surface became the culture surface. Note that in the case of PE (plasma etching) treatment, the film was placed on a ground electrode. After setting, the surface treatment was carried out using the same procedure as for RIE. In Table 1, the discharge treatment intensity was calculated from RF (radio frequency) power x treatment time / electrode area. Note that the electrode area of ​​the electrode used was 736 cm. ２ The PEEK films used in Examples 1 to 3 and Examples 5 and 6 had a porosity of 5% or less, and when the culture surface was measured with a laser microscope, the films were solid, with no holes with a diameter of 1 μm to 100 μm and a depth of 0.5 μm to 100 μm within a 100 μm square area. The porosity was calculated from "{1 - (measured density / theoretical density)} x 100." Furthermore, for the films used in each Example, the surface roughness after plasma treatment was rougher than before plasma treatment. The Ra after the second plasma treatment was 0.6 nm for Example 1. The Ra was measured as follows: An AFM (Shimadzu SPM-9700) was used, and a silicon single crystal probe with a small tip diameter (radius of curvature approximately 10 nm) was used as the probe. The surface was observed within a 1 μm x 1 μm area. To calculate Ra, the analysis software attached to the device was used. After smoothing in the X and Y directions, surface roughness analysis was performed in a 100 nm x 100 nm area, avoiding foreign matter and surface scratches. The arithmetic mean roughness Ra is the average value of the distance from the reference line within the reference length.

[0114]

[0115]

[0116] <Cell Adhesion> The cell culture film substrates produced in the above <Production of Cell Culture Film Substrate> were stored for 28 days at 25°C, 50% relative humidity, and air atmosphere with the culture surface covered with a surface protection material (sterilized paper). After storage, the same number of substrates as the number of times used for culture were cut from the stored cell culture film substrate and cut into Φ14 mm sizes to obtain disc-shaped film substrates. 20 to 30 cell sheets were produced under the following (culture conditions). Cell adhesion to the entire surface of the film substrate was counted as a success. However, partial detachment was not counted. A success rate of 90% or higher, calculated as the number of successful cell sheet adhesions divided by the number of cell sheets produced, was considered a pass, and a rate of less than 90% was considered a fail. Examples 1 to 4 showed relatively higher success rates than Examples 5 and 6.

[0117] (Culture Conditions) Cryopreserved human fibroblasts (derived from human oral tissue) were thawed at 37°C and washed with medium. ５ The cells were suspended in a 5% serum-containing medium and placed in two dishes (60.1 cm ２ ) to 2.5 x 10 ５ The cultured cells were collected, suspended in a 5% serum-containing medium, and then placed in four flasks (225 cm ２ ) to 2.5 x 10 ５ The cells were seeded and subcultured at a time and then cultured for 4 days. The above film substrates were washed with 70% ethanol aqueous solution, phosphate buffer, and culture medium in that order, and then placed on the flat bottom of each well of a cell culture multi-well plate (6 wells). At this time, the culture surface of the film substrate was placed facing the opening of the well plate, i.e., the back surface of the film substrate was placed in contact with the flat bottom of the well plate hole. However, the film substrate was not adhered to the inside of the well plate (culture vessel). The cells cultured as described above were collected, suspended in 2% serum-containing medium, and 27.9 x 10 cells were placed in each well of the cell culture multi-well plate with the film substrate already placed therein. ４ pieces / cm ２ and incubated at 37°C and 5% CO ２The film substrate was incubated in this environment for 1 day to prepare a cell sheet on the culture surface of the film substrate.

[0118] <Releasability> Products that could be peeled off from the film base without using degrading enzymes (the cell sheet and film could be peeled off simply by lightly pressing the cell sheet surface against a piece of pork (the transplant site) and then sliding the film) were judged to have good releasability, and products that could not be peeled off without using a separating enzyme were judged to have poor releasability. Note that products with poor cell adhesion were not tested for releasability.

[0119] <Surface Free Energy (SFE) Analysis> A film substrate was cut out from the cell culture film substrate produced in the above <Production of Cell Culture Film Substrate>, and SFE analysis was performed according to the procedure for measuring surface free energy described in paragraphs 0027 to 0029 of the specification. A contact angle meter DMo-502 manufactured by Kyowa Interface Science was used. Contact angle measurements and analysis were performed with n=3, and the average value of these contact angles was used to calculate the surface free energy (Owens-Wendt method). Specifically, the values ​​of the contact angle θ1 with water and the contact angle θ2 with diiodomethane obtained by the above method, and the dispersion component γ of each liquid were used. Ｌ ｄ and the polar component γ Ｌ ｈ Substituting this into the "Young-Dupre equation" of the Owens-Wendt theory, and solving the simultaneous equations for water and diiodomethane, the surface free energy between the solid and gas (γ ｓ ) variance component (γ ｓ ｄ ) and polar component (γ ｓ ｈ After measurement immediately after plasma treatment (day 0), the culture surface of the film substrate was covered with a surface protection material (sterilized paper) and stored in an air atmosphere at 25°C and 50% relative humidity, and the surface free energy was similarly measured on any day up to 28 days and on any day after 28 days.

[0120] The results in Table 2 above show that the cell culture film substrates produced by the plasma treatment of Examples 1 to 6 have superior cell adhesiveness after storage compared to Comparative Examples 1 to 4.

[0121] <Long-term storage stability> (Example A) A cell culture film substrate was produced in the same manner as in Example 3. On the day of the plasma treatment, the plasma-treated culture surface of the obtained cell culture film substrate was not covered with a surface protective material, and the film was sealed in a resealable polyethylene bag and stored for 4 months (112 days). (Example B) A cell culture film substrate was produced in the same manner as in Example 3, except that the second plasma treatment time was changed to 1 minute. On the day of the plasma treatment, the plasma-treated culture surface of the obtained cell culture film substrate was covered with sterilized paper, and the film was sealed in a resealable polyethylene bag and stored for 4 months (112 days).

[0122] (Example C1) A cell culture film substrate was produced in the same manner as in Example 3. On the day of plasma treatment, the plasma-treated culture surface of the obtained cell culture film substrate was covered with aluminum foil, sealed in a polyethylene bag with a zipper, and stored for 4 months (112 days). (Example C2) The cell culture film substrate was stored in the same manner as in Example C1, except that the second plasma treatment time was changed to 1 minute. In Examples A, B, C1, and C2, electron beam sterilization was performed immediately after plasma treatment.

[0123] In Examples A, B, C1, and C2, the polar component was determined based on the above-mentioned <SFE (surface free energy) analysis>. As a result, the polar component immediately after the plasma treatment was about 40 mJ / m ２ However, the polar components immediately after 4 months of storage were about 16 mJ / m in Examples A and B. ２ , and about 27 mJ / m for Examples C1 and C2. ２In addition, in Examples A, B, C1, and C2, the success rate was calculated based on the above-described <Cell Adhesion>, calculated from the number of successful cell sheet adhesions / number of cell sheet productions. However, the storage conditions were replaced with those of Examples A, B, C1, and C2. As a result, it was shown that the success rate of cell adhesion was higher in Examples C1 and C2 than in Example B, and Example B was higher than in Example A. From the above, it was shown that storing the film substrate with the culture surface protected by a metal foil such as aluminum foil resulted in high cell adhesion even after long-term storage, and therefore excellent long-term storage properties.

[0124] This application claims priority based on Japanese Patent Application No. 2024-052061 filed on March 27, 2024, and Japanese Patent Application No. 2024-209496 filed on December 2, 2024, the disclosures of which are incorporated herein in their entireties.

[0125] REFERENCE SIGNS LIST 10 Film substrate for cell culture 12 Culture surface (surface) 14 Back surface 20 Culture vessel 30 Culture medium 40 Cell sheet 50 Film substrate with cell sheet 60 Cryopreservation solution 70 Transplantation site 80 Metal foil 90 Packaging material 100 Package

Claims

1. A method for producing a film substrate for cell culture having a culture surface on at least one surface, the method comprising: a step of performing a first plasma treatment on the culture surface of the film substrate for cell culture in an inert gas atmosphere; and a step of performing a second plasma treatment in an oxygen atom-containing gas atmosphere after the first plasma treatment.

2. A method for producing a film substrate for cell culture according to claim 1, wherein the first plasma treatment and the second plasma treatment use capacitively coupled plasma.

3. A method for producing a film substrate for cell culture according to claim 1 or 2, wherein the pressure of the first plasma treatment is 0.2 Pa or more and 100 Pa or less.

4. A method for producing a film substrate for cell culture according to claim 1 or 2, wherein the pressure of the second plasma treatment is 0.2 Pa or more and 100 Pa or less.

5. A method for producing a film substrate for cell culture according to claim 1 or 2, wherein the difference in pressure between the first plasma treatment and the second plasma treatment is 20 Pa or less.

6. A method for producing a cell culture film substrate according to claim 1 or 2, wherein the discharge treatment intensity of the second plasma treatment is lower than the discharge treatment intensity of the first plasma treatment.

7. A film substrate for cell culture having a culture surface on at least one surface, wherein the polar component of the surface free energy on the culture surface, as determined by the Owens-Wendt method, is 12 mJ / m after storage for 28 days under the following storage conditions: ２ The cell culture film substrate is as described above. (Storage Conditions) The cell culture film substrate is stored at 25° C., 50% relative humidity, and in an air atmosphere with the culture surface covered with a surface protection material.

8. A film substrate for cell culture according to claim 7, wherein the polar component ratio calculated from [polar component immediately after 28 days of storage under the storage conditions] / [polar component immediately after 14 days of storage under the storage conditions] is 0.7 or more and 1.2 or less.

9. The cell culture film substrate according to claim 7 or 8, wherein the polar component of the surface free energy on the culture surface after 28 days of storage is 20 mJ / m ２ This is the above-mentioned film substrate for cell culture.

10. The cell culture film substrate according to claim 7 or 8, wherein the polar component of the surface free energy on the culture surface is 12 mJ / m during the period from immediately after plasma treatment to immediately after 28 days of storage. ２ This is the above-mentioned film substrate for cell culture.

11. A film substrate for cell culture according to claim 7 or 8, wherein the material constituting the film substrate for cell culture comprises an organic polymer compound having an aromatic ring, or an organic polymer compound having an aromatic ring and an oxygen atom.

12. A film substrate for cell culture according to claim 7 or 8, wherein the material constituting the film substrate for cell culture comprises one or more selected from the group consisting of polyether ether ketone, polyethylene terephthalate, and polystyrene.

13. The film substrate for cell culture according to claim 7 or 8, having a thickness of 5 μm or more and 250 μm or less.

14. A quality control method for a cell culture film substrate having a culture surface on at least one surface, comprising: comparing the polar component of the surface free energy of the culture surface, determined based on the Owens-Wendt method, between the value immediately after production and the value two days after production, and determining whether the difference is 11.5 mJ / m ２ A quality control method that includes a process of determining that a product is acceptable when it meets the following conditions:

15. The quality control method according to claim 14, wherein in the step of determining that the product is acceptable, the difference between the polar components immediately after 2 days and immediately after 20 days is 15 mJ / m ２ A quality control method in which a product is deemed to have passed if the following conditions are further met:

16. A packaged product in which the cell culture film substrate according to claim 7 or 8 is packaged in a packaging material.

17. A film substrate with a cell sheet, comprising: the film substrate for cell culture according to claim 7 or 8; and a cell sheet laminated on the culture surface of the film substrate for cell culture.

18. A method for producing a film substrate with a cell sheet, comprising a culture step of culturing a cell sheet on the culture surface of a film substrate for cell culture produced by the method for producing a film substrate for cell culture according to claim 1 or 2 after a predetermined period of time has elapsed.

19. A method for producing a film substrate with a cell sheet according to claim 18, wherein the predetermined period of time after the film substrate has been packaged, sterilized, transported, and stored.

20. A frozen product of a film substrate with a cell sheet, comprising: the film substrate for cell culture according to claim 7 or 8; and a cell sheet laminated on the culture surface of the film substrate for cell culture, wherein the film substrate for cell culture and the cell sheet are stored in a frozen state.

21. A film substrate with metal foil, comprising: a film substrate for cell culture, at least one surface of which has a culture surface; and at least one metal foil, wherein the metal foil is fixed in a state in which it covers the culture surface on the plasma-treated surface.

22. The metal foil-attached film substrate according to claim 21, wherein the metal foil comprises aluminum foil.

23. A film substrate with metal foil according to claim 21 or 22, wherein, when viewed from the normal direction to the surface of the film substrate for cell culture, the metal foil has a margin that extends beyond the outer edge of the film substrate for cell culture.

24. A package obtained by packaging the metal foil-coated film substrate according to claim 21 or 22 in a packaging material.

25. A package comprising a packaging box containing at least one package according to claim 24.

26. A packaging body according to claim 25, wherein the polar component of the surface free energy of the culture surface of the film substrate for cell culture, as determined by the Owens-Wendt method, is 12 mJ / m ２ That's it, the package.

27. The package of claim 25, which is in a state where it has been sterilized using radiation.

28. A method for manufacturing a package, comprising the steps of: preparing a film substrate for cell culture, at least one of whose surfaces has a culture surface that has been plasma-treated; covering the culture surface on the plasma-treated surface with metal foil to obtain a film substrate with metal foil; packaging the film substrate with metal foil using packaging material to obtain a package; placing at least one of the packages in a packaging box and sealing the box to obtain a package; and sterilizing the package using radiation.

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