Method for producing cell sheets

Abstract

To reduce stimulation applied to a cell sheet when peeling the cell sheet from a substrate.SOLUTION: A method for producing a cell sheet according to one mode of the present invention includes the steps of: disseminating cells on a substrate at the dissemination density of 2.6×104 to 6.0×105 cells / cm2; and culturing disseminated cells in culture medium to form a cell sheet on the substrate. The substrate contains fluorinated polyimide resin.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing a cell sheet.

Background Art

[0002] Conventionally, in order to detach a cell sheet formed on a culture substrate from the substrate, stimuli such as enzymes or temperature have been used. For example, Patent Document 1 describes that a cell sheet was produced using a temperature-responsive culture dish capable of detaching cells by a temperature change.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] In the method described in Patent Document 1, since a stimulus of a certain level or more is applied to the cells due to a temperature change, the cell sheet may be damaged.

[0005] The present invention has been made in view of the above problems, and an object thereof is to reduce the stimulus applied to the cell sheet when detaching the cell sheet from the substrate.

Means for Solving the Problems

[0006] A method for producing a cell sheet according to one embodiment of the present invention includes a step of seeding cells at a seeding density of 2.6×10 4 ~6.0×10 5 cells / cm 2 on a substrate, and a step of culturing the seeded cells in a medium to form a cell sheet on the substrate, wherein the substrate contains a fluorinated polyimide resin. The seeding density is 1.6×10 5 ~6.0×10 5 cells / cm 2It may be.

[0007] The fluorinated polyimide resin may include an aromatic polyimide resin containing a structural unit represented by the following formula (I).

Chemical formula

[0008] The fluorinated polyimide resin may be a copolymer of 4,4'-hexafluoroisopropylidenediphthalic anhydride and 1,4-bis(aminophenoxy)benzene, 1,3-bis(4-aminophenoxy)benzene, 4,4'-oxydiphthalic anhydride / 1,4-bis(aminophenoxy)benzene, 2,2-bis(4-(4-aminophenoxy)phenyl)propane, 2,2'-bis(trifluoromethyl)benzidine, 4,4'-diaminodiphenyl ether, 4,4'-bis(4-aminophenoxy)biphenyl, or 2,2-bis(4-(4-aminophenoxy)phenyl)sulfone, or 4,4'-(4,4'-isopropylidenediphenoxy)diphthalic acid / 2,2-bis[4-(4-aminophenoxy)phenyl]hexafluoropropane. The cells may be human adipose-derived stem cells, human dermal fibroblasts, or human epidermal keratinocytes. The method for producing the cell sheet may further include a step of peeling the cell sheet from the substrate, and the peeling of the cell sheet may be performed by stirring the medium or using a scraper.

Advantages of the Invention

[0009] According to the method of the present invention, the cell sheet can be easily peeled from the substrate, so that the stimulus applied to the cell sheet during peeling can be reduced.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Mode for Carrying Out the Invention

[0011] The method for producing a cell sheet according to one embodiment of the present invention includes a step of seeding cells at a seeding density of 2.6×10 4 ~6.0×10 5 cells / cm 2 onto a substrate, and a step of culturing the seeded cells in a medium to form a cell sheet on the substrate.

[0012] In this specification, a cell sheet is a sheet-like cell aggregate in which cells are directly connected to each other by cell-cell junctions or cells are connected to each other via an extracellular matrix, and is distinguished from a spheroid. The size of the cell sheet is not particularly limited, and the area of the cell sheet, more specifically, the area of the main surface of the cell sheet, may be, for example, 0.1 cm 2 ~400 cm 2 for example.

[0013] The substrate contains a fluorinated polyimide resin. The substrate used in the method according to this embodiment can adhere and culture cells because it exhibits appropriate cell adhesiveness by containing a fluorinated polyimide resin. In other words, the fluorinated polyimide resin is a fluorine-containing polyimide resin. The fluorinated polyimide resin may be an aromatic polyimide resin containing a structural unit represented by the following formula (I).

[0014]

Chemical formula

[0015] When p = 0, X 0 may not be present (in other words, the two benzene rings may be directly bonded). When p = 1, the two benzene rings are bonded via X 0 .

[0016] X 0The divalent organic group which is an example may be, for example, an alkylene group, an arylene group, an aryleneoxy group, or an arylenthio group. Among these, an alkylene group, an aryleneoxy group, or an arylenthio group is preferable, an alkylene group or an aryleneoxy group is more preferable, and these may be substituted with a fluorine atom. The number of carbon atoms of the alkylene group is, for example, 1 to 12, preferably 1 to 6.

[0017] X 0 The alkylene group substituted with a fluorine atom which is an example may be, for example, -C(CF 3 ) 2 -, or -C(CF 3 ) 2 -C(CF 3 ) 2 -, and -C(CF 3 ) 2 - is preferable.

[0018] Examples of the arylene group include the following.

Chemical formula

[0019] Examples of the aryleneoxy group include the following.

Chemical formula

[0020] Examples of the arylenthio group include the following.

Chemical formula

[0021] X 0 The divalent organic group which is an example is preferably a group selected from the group consisting of b-2 to b-10 and c-2 to c-10, more preferably a group selected from the group consisting of b-7 to b-9 and c-7 to c-9, and even more preferably the group of b-8.

[0022] The above-mentioned arylene group, aryleneoxy group, and arylenedithio group may each independently be substituted with at least one atom or group selected from the group consisting of a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, preferably a fluorine atom or a chlorine atom, more preferably a fluorine atom.), a methyl group, and a trifluoromethyl group. These substituents may be plural, and in that case, the types of substituents may be the same or different from each other. The arylene group, aryleneoxy group, or arylenedithio group is preferably substituted with a fluorine atom and / or a trifluoromethyl group, and more preferably substituted with a fluorine atom. When Y does not contain a fluorine atom, the arylene group, aryleneoxy group, or arylenedithio group is preferably substituted with at least one or more fluorine atoms.

[0023] In formula (I), the divalent organic group represented by Y is not particularly limited, and may be, for example, a divalent organic group having an aromatic ring. Specifically, the divalent organic group represented by Y may be a group consisting of one benzene ring, or a group having a structure in which two or more benzene rings are bonded via a carbon atom (that is, a single bond or an alkylene group), an oxygen atom, or a sulfur atom or directly bonded. Specifically, the following groups can be exemplified.

[0024]

Chemical formula

[0025]

Chemical formula

[0026]

Chemical formula

[0027]

Chemical formula

[0028] The divalent organic group having an aromatic ring, which is an example of Y, may be substituted with at least one atom or group selected from the group consisting of a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, preferably a fluorine atom or a chlorine atom, more preferably a fluorine atom), a methyl group, and a trifluoromethyl group. These substituents may be plural, and in that case, the types of the substituents may be the same or different from each other. X 0 When the divalent organic group having an aromatic ring does not contain a fluorine atom, it is preferably substituted with a fluorine atom and / or a trifluoromethyl group, more preferably a fluorine atom.

[0029] In the above formula (I), Y is preferably a group selected from the group consisting of d-3, d-9, e-1 to e-4, f-6, and f-7, and more preferably a group of e-1, e-3, or e-4.

[0030] In a preferred embodiment, in the above formula (I), X 0 The divalent organic group which is an example of is selected from the group consisting of -C(CF 3 ) 2 -, the above b-2 to b-10, and c-2 to c-10, and Y is selected from the group consisting of d-3, d-9, e-1 to e-4, f-6, and f-7. In a more preferred embodiment, in the above formula (I), X 0 The divalent organic group which is an example of is selected from the group consisting of -C(CF 3 ) 2 -, b-7 to b-9 and c-7 to c-9, and Y is selected from the group consisting of e-1, e-3, and e-4.

[0031] The fluorinated polyimide resin containing the structural unit represented by the above formula (I) may be, for example, a 4,4'-hexafluoroisopropylidenediphthalic anhydride (6FDA) / 1,4-bis(aminophenoxy)benzene (TPEQ) copolymer, a 6FDA / 1,3-bis(4-aminophenoxy)benzene (TPER) copolymer, a 6FDA / 4,4'-oxydiphthalic anhydride (ODPA) / TPEQ copolymer, a 4,4'-(4,4'-isopropylidenediphenoxy)diphthalic acid (BPADA) / 2,2-bis[4-(4-aminophenoxy)phenyl]hexafluoropropane (HFBAPP) copolymer, a 6FDA / 2,2-bis(4-(4-aminophenoxy)phenyl)propane (BAPP) copolymer, a 6FDA / 2,2'-bis(trifluoromethyl)benzidine (TFMB) copolymer, a 6FDA / 4,4'-diaminodiphenyl ether (ODA) copolymer, a 6FDA / 4,4'-bis(4-aminophenoxy)biphenyl (BAPB) copolymer, or a 6FDA / 2,2-bis(4-(4-aminophenoxy)phenyl)sulfone (BAPS) copolymer.

[0032] The fluorinated polyimide resin containing the structural unit represented by the above formula (I) can be obtained by a method of thermally imidizing or chemically imidizing a polyamic acid obtained by polymerizing an acid dianhydride and a diamine. The imidization rate of the fluorinated polyimide resin does not have to be 100%. That is, the fluorinated polyimide resin containing the structural unit represented by the above formula (I) may consist only of the structural unit represented by the above formula (I), or may partially contain a structural unit in which the cyclic imide structure remains as an amic acid without dehydrating and closing the ring as long as the effects of the invention are not impaired.

[0033] The weight average molecular weight of the fluorinated polyimide resin is, for example, from 5,000 to 2,000,000, preferably from 8,000 to 1,000,000, and more preferably from 20,000 to 500,000. In this specification, the weight average molecular weight is measured by the following method.

[0034] (Measurement of weight average molecular weight) Apparatus: HCL-8220GPC (manufactured by Tosoh Corporation) Column: TSKgel Super AWM-H Eluent (N-methylpyrrolidone containing LiBr·H 2 O and phosphoric acid): 0.01 mol / L Measurement method: A 0.5 mass% solution is prepared with the eluent, and the molecular weight is calculated based on the calibration curve prepared with polystyrene.

[0035] The thickness of the substrate is not particularly limited and may be, for example, 10 to 5000 μm or 100 to 2000 μm. From the viewpoint of culturing cells in a sheet form, the area of the culture surface of the substrate is, for example, 0.1 cm 2 ~400 cm 2 、0.1 cm 2 ~200 cm 2 、or 0.1 cm 2 ~100 cm 2 and may be. Here, the culture surface means a surface on which cells can be adhered and cultured, and means a continuous surface (preferably a flat surface).

[0036] The cells are not particularly limited, and for example, primary cells, established cell lines, or cells obtained by genetically modifying these, derived from any organ or tissue (brain, liver, pancreas, spleen, heart, lung, intestine, cartilage, bone, fat, kidney, nerve, skin, bone marrow, embryo, etc.) of humans or animals other than humans (such as monkeys, pigs, dogs, rats, mice, etc.) can be used.

[0037] The cells are not particularly limited as long as they can form a cell sheet by adherent culture, and may be, for example, stem cells, progenitor cells, or differentiated cells. Stem cells may be, for example, ES cells, iPS cells, neural stem cells, mesenchymal stem cells (such as adipose-derived stem cells such as human adipose-derived stem cells (AdSC)), tissue stem cells (somatic stem cells), hematopoietic stem cells, or cancer stem cells. Differentiated cells may be, for example, cells derived from digestive organs (such as liver cells or pancreatic cells), cells derived from cardiovascular organs (such as kidney cells, nerve cells, or cardiomyocytes), adipocytes, fibroblasts (such as skin fibroblasts such as human dermal fibroblasts (NHDF)), epithelial cells (such as keratinocytes such as human skin keratinocytes), bone cells, cartilage cells, cells derived from eye tissues, vascular cells, blood cell lineage cells, or germ cells.

[0038] From the perspective of forming a cell sheet, the seeding density of the cells is 2.6×10 4 、5.2×10 4 、1.1×10 5 、1.6×10 5 、1.7×10 5 、1.8×10 5 、1.9×10 5 、2.0×10 5 、or 2.5×10 5 cells / cm 2 or more, and may be 6.0×10 5 、5.5×10 5 、5.0×10 5 、4.5×10 5 、4.1×10 5 、4.0×10 5 、3.5×10 5 、or 2.1×10 5 cells / cm 2 or less. When the seeding density of the cells is less than 2.6×10 4 cells / cm 2 cultured cells do not form a sheet, and a plurality of small spheroids tend to be formed. On the other hand, when the seeding density of the cells is 6.0×10 5 cells / cm 2Even if it is excessive, a cell sheet is not formed, and instead, a spheroid-like mass tends to be formed. In the conventional method using a culture substrate having strong cell adhesiveness, a higher seeding density has tended to be required to form a cell sheet. On the other hand, since the substrate used in the method according to this embodiment has appropriate cell adhesiveness, the interaction in the lateral direction between cells (the direction horizontal to the main surface of the cell sheet) works strongly, and it is presumed that a cell sheet can be formed at a seeding density lower than the conventionally common seeding density.

[0039] The medium used for culture is not particularly limited, and depending on the type of cells, for example, any basal medium, differentiation medium, or primary culture dedicated medium can be used. The medium can be, for example, Eagle's Minimum Essential Medium (EMEM), Dulbecco's Modified Eagle Medium (DMEM), α-MEM, Glasgow MEM (GMEM), IMDM, RPMI1640, Ham's F-12, MCDB medium, Williams Medium E, Hepatocyte thaw medium, mesenchymal stem cell dedicated medium, AdSC dedicated medium, keratinocyte dedicated medium, or a mixed medium thereof. The medium may contain additive components such as serum, various growth factors, differentiation-inducing factors, antibiotics, hormones, amino acids, sugars, salts, etc. The medium is preferably a xenofree medium that does not contain components derived from other species. In the method according to this embodiment, since cells can be adherently cultured by using a substrate containing a fluorinated polyimide resin, a cell sheet can be produced even when a xenofree medium is used as the medium.

[0040] The culture temperature is not particularly limited, but is usually about 25 to 40°C. The relative humidity during culture is not particularly limited and may be, for example, 40 to 50%RH.

[0041] The culture time is not particularly limited and can be appropriately determined according to the type of cells, the cell growth rate, and the size of the cell sheet to be produced. The culture time can be, for example, 4 hours to 48 days, 4 hours to 28 days, 4 hours to 7 days, or 4 hours to 3 days.

[0042] By culturing the cells seeded on the substrate in a medium, a cell sheet is formed on the substrate. The method according to this embodiment may further include a step of peeling the cell sheet from the substrate. The peeling of the cell sheet may be performed by stirring the medium. Alternatively, as long as the cell sheet is not broken, the cell sheet can be peeled off by a general method. For example, the cell sheet may be peeled off from the substrate using a scraper, or the cell sheet may be peeled off by bringing a transfer sheet such as a non-woven fabric that can adhere to the cell sheet into close contact with the cell sheet and then peeling it off.

[0043] The method of stirring the medium is not particularly limited as long as the cell sheet is not broken. For example, the medium can be stirred by pipetting the medium. Pipetting the medium means repeating the inhalation and exhalation of the medium using a pipette. Alternatively, the medium can also be stirred by shaking the substrate.

[0044] Since the substrate used in the method according to this embodiment has an appropriate cell adhesiveness derived from a fluorinated polyimide resin, the cell sheet can be peeled off from the substrate without using the techniques that have been conventionally used to peel the cell sheet from the substrate, such as enzyme treatment and temperature change. Enzyme treatment and temperature change are not preferable because they may damage the cell sheet. Therefore, it is preferable that the method according to this embodiment does not include a step of treating the cell sheet with an enzyme or changing the temperature to peel the cell sheet from the substrate.

Example

[0045] <Test Example 1> Cultivation of Human Adipose-Derived Stem Cells In a 384-well plate made of polystyrene (the shape of the well: a 3.5 mm square) with a 6FDA / TPEQ copolymer film attached to the entire bottom surface of the well, 2.0×10 5 、1.0×10 5 、5.0×10 4 、2.5×10 4 、1.3×10 4 、6.3×10 3 、3.1×10 3, 1.6×10 3 , or 8.0×10 2 Human adipose-derived stem cells (AdSC) were seeded at a seeding density of cells / well. As AdSC, PT-5006 purchased from Lonza was used. The well plate was placed in an incubator at 37°C with 5% (v / v) CO 2 and cultured for 3 days. As the medium, KBM ADSC-2 medium (manufactured by Cosmo Bio Co., Ltd.) containing 1% antibiotic was used.

[0046] After culturing for 3 days, using a microscope, it was confirmed whether a cell sheet was formed in the well. An example of the microscope image is shown in Fig. 1. As shown in Fig. 1, in the wells seeded with AdSC at a seeding density of 5.0×10 4 cells / well (4.1×10 5 cells / cm 2 ) or 2.5×10 4 cells / well (2.0×10 5 cells / cm 2 ), a cell sheet was formed. The formed sheet could be detached from the substrate by pipetting the medium. In the wells seeded with AdSC at a seeding density of 1.0×10 5 cells / well (8.2×10 5 cells / cm 2 ) or higher, no cell sheet was formed, and spheroid-like masses were formed. In the wells seeded with AdSC at a seeding density of 1.3×10 4 cells / well (1.1×10 5 cells / cm 2 ) or lower, the cell culture did not form a sheet and multiple spheroids were formed.

[0047] <Test Example 2> Cultivation of Human Dermal Fibroblasts Human dermal fibroblasts (NHDF) were cultured in a T225 culture flask. CC-2509 purchased from Lonza was used as the NHDF. As the medium, DMEM high glucose medium (manufactured by Nacalai Tesque, Inc.) containing 1% antibiotic and 5% serum was used. After removing the medium from the T225 culture flask and adding 5 mL of the cell detachment agent Accutase® (manufactured by PromoCell), the T225 culture flask was left standing in a 5% (v / v) CO 2 incubator at 37 °C for about 5 minutes to detach the cells. Subsequently, the detached solution was collected, 20 mL of the medium was added to wash the cells, and the cells were transferred to a tube. The tube was centrifuged at 210 × g for 5 minutes, and the cells were suspended in 1 mL of the medium to count the number of cells. Then, a cell suspension with a concentration of 2.0 × 10 5 cells / mL was prepared.

[0048] NHDF was seeded at a seeding density of 2.0 × 10 5 cells / well (1.1 × 10 5 cells / cm 2 ) or 4.0 × 10 5 cells / well (2.1 × 10 5 cells / cm 2 ) on a 24-well polystyrene plate (well shape: circular with a diameter of 15.6 mm) with a fluorinated polyimide resin film attached to the entire bottom surface of the well, and cultured in a 5% (v / v) CO 2 incubator at 37 °C for 4 days. As the fluorinated polyimide resin film, a 6FDA / TFMB copolymer film, a 6FDA / BAPP copolymer film, a 6FDA / ODA copolymer film, a 6FDA / BAPB copolymer film, or a 6FDA / BAPS copolymer film was used. After 4 days of culture, it was confirmed by microscopy that cell sheets were formed in all the well plates. Examples of the microscopic images are shown in Figures 2 and 3. Also, all the formed cell sheets could be easily detached from the substrate using a scraper.

[0049] The seeding density of the cells was 1 × 10 5 cells / well (5.2 × 10 4 cells / cm 2) was changed, and NHDF was cultured in the same manner as above except that a 6FDA / TPEQ copolymer film was used as the fluorinated polyimide resin film. After culturing for 4 days, it was confirmed by microscopy that a cell sheet was formed in the well. An example of the microscopic image is shown in Fig. 4.

[0050] <Test Example 3> Cultivation of Human Skin Keratinocytes Human skin keratinocytes (KC) were cultured in a T225 culture flask. NHEK-Neo (00192907) purchased from Lonza was used as the human skin KC. As the medium, a dedicated medium KGM-Gold (trademark) BulletKit (trademark) (manufactured by Lonza) was used. The medium was removed from the T225 culture flask, and 20 mL of HEPES-buffered saline (CC-5034, manufactured by Lonza) was added for washing. Subsequently, 6 mL of the cell detachment agent trypsin (CC-5034, manufactured by Lonza) was added, and then the T225 culture flask was placed in a 5% (v / v) CO 2 incubator at 37 °C and left standing for about 5 minutes to detach the cells. Subsequently, the detached solution was collected, 12 mL of trypsin neutralizing solution (CC-5034, manufactured by Lonza) was added to wash the cells, and the cells were transferred to a tube. The tube was centrifuged at 210×g for 5 minutes, and the cells were suspended in 2 mL of the medium to count the cell number. Then, 2.0×10 5 a cell suspension with a cell concentration of / mL was prepared.

[0051] Human skin KC was seeded at a seeding density of 2.0×10 5 cells / well (1.1×10 5 cells / cm 2 ) or 4.0×10 5 cells / well (2.1×10 5 cells / cm 2 ) on a polystyrene 24-well plate with a fluorinated polyimide resin bonded to the entire bottom surface of the well (well shape: circular with a diameter of 15.6 mm), and 5% (v / v) CO at 37 °C 2It was cultured in an incubator for 4 days. As the fluorinated polyimide resin film, a 6FDA / TPEQ copolymer film, a 6FDA / TFMB copolymer film, a 6FDA / BAPP copolymer film, a 6FDA / ODA copolymer film, or a 6FDA / BAPB copolymer film was used. After 4 days of culture, it was confirmed by microscopy that cell sheets were formed in all well plates. Examples of microscope images are shown in FIGS. 5 and 6. Also, all of the formed cell sheets could be easily peeled off from the substrate using a scraper.

[0052] The cell sheet of human skin KC obtained as described above (seeding density: 1.1×10 5 cells / cm 2 ) was fixed with a 4% paraformaldehyde phosphate buffer (manufactured by Nacalai Tesque) for 15 minutes. ZO-1, a tight junction-forming protein, was stained with a fluorescently labeled ZO-1 antibody (manufactured by Thermo Fisher Scientific). This was observed with a confocal laser microscope. An example of a microscope image is shown in FIG. 7. In the examples using a 6FDA / ODA copolymer film or a 6FDA / TFMB copolymer film, since ZO-1 expression was observed in the cells, the formation of tight junctions between cells was confirmed.

[0053] Human skin KC was cultured in the same manner as above except that the seeding density of the cells was changed to 1×10 5 cells / well (5.2×10 4 cells / cm 2 ) or 5×10 4 cells / well (2.6×10 4 cells / cm 2 ). As the fluorinated polyimide resin film, a 6FDA / TPEQ copolymer film, a 6FDA / TFMB copolymer film, or a 6FDA / ODA copolymer film was used. After 4 days of culture, it was confirmed by microscopy that cell sheets were formed in all well plates. Examples of microscope images are shown in FIG. 8. Also, all of the formed cell sheets could be easily peeled off from the substrate using a scraper.

Claims

1. 2.6 x 10 on the substrate ４ ~6.0 x 10 ５ cells / cm ２ seeding the cells at a seeding density of Culturing the seeded cells in a medium to form a cell sheet on the substrate; A method for producing a cell sheet, wherein the substrate comprises a fluorinated polyimide resin.

2. The seeding density is 1.6 x 10 ５ ~6.0 x 10 ５ cells / cm ２ The method of claim 1, wherein

3. The method according to claim 1 or 2, wherein the fluorinated polyimide resin comprises an aromatic polyimide resin containing a structural unit represented by the following formula (I): 【Chemistry 1】 [In formula (I), X ０ represents an oxygen atom, a sulfur atom, or a divalent organic group, Y represents a divalent organic group; Z １ , Z ２ , Z ３ , Z ４ , Z ５ , and Z ６ each independently represents a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, p is 0 or 1; X ０ , Y, Z １ , Z ２ , Z ３ , Z ４ , Z ５ , and Z ６ At least one of the groups is a fluorine atom or contains one or more fluorine atoms.

4. The method according to claim 3, wherein the fluorinated polyimide resin is a copolymer of 4,4'-hexafluoroisopropylidenediphthalic anhydride with 1,4-bis(aminophenoxy)benzene, 1,3-bis(4-aminophenoxy)benzene, 4,4'-oxydiphthalic anhydride / 1,4-bis(aminophenoxy)benzene, 2,2-bis(4-(4-aminophenoxy)phenyl)propane, 2,2'-bis(trifluoromethyl)benzidine, 4,4'-diaminodiphenyl ether, 4,4'-bis(4-aminophenoxy)biphenyl, or 2,2-bis(4-(4-aminophenoxy)phenyl)sulfone, or a 4,4'-(4,4'-isopropylidenediphenoxy)diphthalic acid / 2,2-bis[4-(4-aminophenoxy)phenyl]hexafluoropropane copolymer.

5. The method according to any one of claims 1 to 4, wherein the cells are human adipose-derived stem cells, human skin fibroblasts, or human skin keratinocytes.

6. The method according to any one of claims 1 to 5, further comprising a step of detaching the cell sheet from the substrate, wherein the detachment of the cell sheet is carried out by stirring the medium or by using a scraper.

Images