How to transfer cell sheets

Abstract

To provide a method for holding a cell sheet shape and simply transferring the sheet.SOLUTION: A method for transferring a cell sheet onto a subject includes the steps of: adhering a cell sheet face of a laminate, which includes a support layer constituted of polymers on a film, a cover layer constituted of temperature-responsive polymers on a film laminated on the support layer, and a cell sheet laminated on the cover layer, onto a subject; cooling the laminate to the lower critical solution temperature (LCST) of a temperature-responsive polymer to water or less; and peeling off the cell sheet by peeling the support layer and the cover layer.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to Method for targeting cell sheets Regarding. [Background technology]

[0002] In recent years, much attention has been focused on regenerative medicine, which involves transplanting cells cultured in vitro into a patient to regenerate lost cells, tissues, and organs and restore their functions. Among these, medical methods using cell sheets are said to be more effective than simply transplanting cells, and there is a demand for technology to easily obtain cell sheets.

[0003] As a method for obtaining a cell sheet, for example, Patent Document 1 discloses a method for obtaining a cardiomyocyte cell sheet from a dish coated with poly-N-isopropylacrylamide having a lower critical dissolution temperature. After culturing cardiomyocytes until they become confluent, a cardiomyocyte cell sheet can be isolated by cooling at a temperature lower than the lower critical temperature of poly-N-isopropylacrylamide, and the isolated cell sheet can be transplanted to the affected area for treatment. On the other hand, cell sheets exist in a sheet shape when a support such as a dish is present, but there is a problem that after isolation, they shrink and form cell masses that are unsuitable for transplantation. Therefore, a material and method that can maintain the sheet shape until transplantation is performed has been required. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2003-513132 Summary of the Invention [Problem to be solved by the invention]

[0005] The object of the present invention is to A method for easily transferring a cell sheet while maintaining its shape The purpose of this invention is to provide [Means for solving the problem]

[0006] In view of the above, the present inventors have conducted extensive research and have completed the present invention.

[0007] That is, one aspect of the present invention is A method for transferring a cell sheet to a target, comprising: a step of attaching a cell sheet surface of a laminate comprising a support layer composed of a polymer on a film, a cover layer composed of a temperature-responsive polymer on a film laminated on the support layer, and a cell sheet laminated on the cover layer, to a target; cooling the laminate to a temperature equal to or lower than a lower critical solution temperature (LCST) of the temperature-responsive polymer in water; peeling the support layer and the cover layer off the cell sheet, A method in which the subject is a living body (excluding humans), cultured cell tissue, or experimental gel. It is. Effect of the Invention

[0008] The present invention makes it possible to easily transfer a cell sheet onto a subject. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] One embodiment of the present invention will be described in detail below, but the present invention is not limited to the following content. The present invention can be carried out by appropriately modifying it within the scope of the spirit of the present invention.

[0010] The laminate of the present invention comprises a support layer composed of a film-like polymer, a cover layer composed of a film-like temperature-responsive polymer laminated on the support layer, and a cell sheet laminated on the cover layer.

[0011] There are no particular limitations on the material of the film that serves as the support layer, and it may be a synthetic polymer compound or a natural polymer compound, but a synthetic polymer compound is preferable because it is easy to form into a film shape. Specific examples include polyethylene, polypropylene, polystyrene, polyvinylidene chloride, polyvinyl chloride, polyester, polyolefin, and copolymers containing these.

[0012] The thickness of the support layer is not particularly limited, and from the viewpoint of ease of handling, it is preferably 1 to 400 μm, more preferably 10 to 100 μm.

[0013] The material of the film that becomes the cover layer is not particularly limited as long as it is a temperature-responsive polymer that contains a segment that exhibits a lower critical solution temperature (LCST) in water (hereinafter sometimes referred to as an LCST segment), but is preferably a polymer that contains a segment that exhibits an LCST in the range of 0 to 50° C. The LCST is the temperature below which a polymer dissolves in water to form a transparent solution, but above which it becomes insoluble and becomes cloudy or precipitates, resulting in phase separation.

[0014] Examples of the repeating unit and LCST of the LCST segment include N-cyclopropylacrylamide (LCST = 46 ° C), N-isopropylacrylamide (LCST = 32 ° C), Nn-propylmethacrylamide (LCST = 22 ° C), N-tetrahydrofurfuryl acrylamide (LCST = 28 ° C), N-ethoxyethylacrylamide (LCST = 35 ° C), N,N-diethylacrylamide (LCST = 32 ° C), N-isopropylmethacrylamide (LCST = 44 ° C), Nn-propylmethacrylamide (LCST = 28 ° C), N-tetrahydrofurfurylmethacrylamide (LCST = 35 ° C), N-methyl-N-isopropylacrylamide (LCST = 23 ° C), N-methyl-Nn-propylacrylamide (LCST = 20 ° C), and N,N-dimethylaminoethyl methacrylate (LCST = 47 ° C). Among these, it is preferable to use the repeating unit of N-isopropylacrylamide because it is easy to polymerize. The LCST segment may be composed of one type of repeating unit or two or more types of repeating units.

[0015] The temperature-responsive polymer may be a homopolymer of only the LCST segment, or may be a random copolymer or a block copolymer containing a segment other than the LCST segment. In the case of a block copolymer, it is preferable that the block copolymer contains a hydrophobic segment and a hydrophilic segment as disclosed in JP-A-2018-087316, and specifically, a block copolymer composed of repeating units of 2-methoxyethyl acrylate (hydrophilic segment), n-butyl acrylate (hydrophobic segment), and N-isopropylacrylamide (LCST segment) is particularly preferable.

[0016] The thickness of the cover layer is not particularly limited, but if the cover layer is too thin, the cell sheet may be difficult to peel off even when cooled, and if the cover layer is too thick, the cell sheet may be difficult to adhere, so a thickness of 1 to 1000 nm is preferable, and 5 to 50 nm is more preferable.

[0017] The cell sheet is a sheet-like cell aggregate in which cells are connected to each other by intercellular junctions, and may have a single layer structure or a laminated structure. There is no particular limitation on the cells that can be used, and they may be animal cells or plant cells, but it is preferable that they are adhesive cells derived from animal cells. More preferable are epithelial cells and mesenchymal stem cells. As an example of epithelial cells, they may be cells derived from epithelia that are functionally classified as secretory epithelium, absorptive epithelium, ductal epithelium, covering epithelium, etc., or they may be cancer cells for research purposes. As examples of cancer epithelial cells, A549 (human alveolar basal epithelial adenocarcinoma cells) and HCT116 (human colon adenocarcinoma cells) can be used. There is also no particular limitation on the origin of the mesenchymal cells, and bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, etc. can be used. Furthermore, the mesenchymal stem cells may be autologous cells or allogeneic cells.

[0018] The laminate of the present invention may contain other components in addition to the above-mentioned film and cell sheet. For example, the surface of the laminate may contain extracellular matrix components such as fibronectin and collagen, germicides such as acrinol and benzalkonium chloride, culture medium components, and antibiotics.

[0019] The laminate of the present invention can be produced by the following production method. A step of laminating a film-shaped temperature-responsive polymer onto a film-shaped polymer (hereinafter, sometimes referred to as a lamination step); a step of fixing the laminated film-like temperature-responsive polymer to the bottom surface of a container having a smooth bottom surface with a fixing jig so that the film-like temperature-responsive polymer is on top (hereinafter, sometimes referred to as a fixing step); A step of culturing cells on the immobilized film-like temperature-responsive polymer (hereinafter, sometimes referred to as a culturing step); However, the method for producing the laminate of the present invention is not limited to the above-mentioned method.

[0020] Examples of the lamination process include a method of connecting the support layer surface and the cover layer with a covalent bond by electron beam polymerization or the like, and a method of bonding the support layer surface and the cover layer by hydrophobic interaction. In the latter case, the cover layer may contain a hydrophobic portion, or an adhesive layer made of an epoxy adhesive or the like may be included between the support layer and the cover layer. When the cover layer contains a hydrophobic portion, it is preferable that the temperature-responsive polymer contains a repeating unit having a high hydrophobic interaction.

[0021] When the cover layer is adhered to the surface of the support layer by hydrophobic interaction, for example, any temperature-responsive polymer solution can be used, and various commonly known methods such as brush coating, dip coating, spin coating, bar coating, flow coating, spray coating, roll coating, air knife coating, and blade coating can be used. In addition, since there is no problem as long as the cover layer is laminated on the surface of the support layer, there is no problem even if the support layer is covered with the cover layer. When the cover layer is to be covered on the support layer, in addition to the above-mentioned method, the support layer can be immersed in a polymer solution to cover it. There is no particular limitation on the solvent used in the polymer solution as long as it does not corrode the support. When polyethylene, polypropylene, or polyester is used as the support layer, 2-methoxyethanol can be used as an example. There is no particular limitation on the concentration of the polymer solution, and for example, it is a value of 0.01 to 20.00 wt%.

[0022] In the fixing step, the laminate of the support layer and cover layer obtained in the lamination step is fixed to the bottom of a vessel having a smooth bottom surface with a fixing jig so that the cover layer is on top (so that the support layer is in contact with the bottom of the vessel). If the laminate is one in which the support layer is covered by the cover layer, there is no need to worry about the surface that contacts the bottom of the vessel, and this is simple. A cell culture dish can be used as the vessel, and the fixing jig is preferably made of a material that has a higher specific gravity than the culture medium, is not cytotoxic, and has a shape that does not interfere with the cell culture described below. One example is a ring-shaped PLA resin.

[0023] In the culture step, cells are cultured on the laminate fixed in the fixation step. The method of culturing cells may be a commonly used method, but since cells are cultured on a temperature-responsive polymer, it is necessary to perform the culture at a temperature equal to or higher than the LCST of the segment exhibiting LCST. Since the area of ​​the cell sheet is preferably 50% or more of the area of ​​the cover layer, cells may be seeded in a number that is 50% or more, and even if the number of seeded cells is small, there is no problem as long as the cells are sufficiently increased. There is no particular limitation on the medium used for culturing cells, and examples include a medium in which fetal bovine serum is added to a basal medium such as D-MEM, a medium further containing an antibiotic, and a serum-free medium. In addition, in the case of cells having differentiation ability such as mesenchymal stem cells, a differentiation process may be performed to make them useful cells. As an example, after seeding and culturing bone marrow-derived mesenchymal stem cells, a differentiation process may be performed using a chondrocyte differentiation medium to differentiate into chondrocytes.

[0024] The laminate of the present invention can transfer the cell sheet to an object by attaching the cell sheet surface of the laminate to an object and cooling it. The object to be attached is not particularly limited, and may be a living organism or cultured cell tissue, or may be substituted with gel in an experimental case. When gel is substituted, a cell sheet training kit from CellSeed Co., Ltd. can be used as an example. The cooling is not particularly limited as long as it is at a temperature below the LCST of the segment showing the LCST of the laminate, and the laminate or the object to be attached may be cooled. EXAMPLES

[0025] Examples of the present invention are described below, but the present invention is not limited to these examples. Unless otherwise specified, commercially available reagents were used.

[0026] <Compositional analysis of block copolymers> The amount was determined by proton nuclear magnetic resonance spectroscopy (1H-NMR) spectrum analysis using a nuclear magnetic resonance measuring device (manufactured by JEOL Ltd., trade name JNM-ECZ400S / L1).

[0027] <Analysis of molecular weight and molecular weight distribution of block copolymer> Weight average molecular weight (Mw), number average molecular weight (Mn) and molecular weight distribution (Mw / Mn) were measured by gel permeation chromatography (GPC). The GPC apparatus used was a Tosoh Corporation HLC-8320GPC, and two Tosoh Corporation TSKgel SuperAWM-H columns were used. The column temperature was set to 40°C, and the eluent was 2,2,2-trifluoroethanol containing 10 mM sodium trifluoroacetate. The measurement sample was prepared at 1.0 mg / mL and measured. The molecular weight calibration curve was made using polymethylmethacrylate (Sigma-Aldrich) with known molecular weight.

[0028] <Film thickness analysis> The thickness of the film was measured using a microspectrophotometric film thickness meter (manufactured by Otsuka Electronics Co., Ltd., product name OPTM-F1).

[0029] <Counting the number of seeded cells> The number of cells to be seeded was counted using a hemocytometer. 2 The total cells in the compartment were counted, and the total cell number was calculated by (average number of all live cells in the compartment) × dilution factor × medium volume (mL) × 10,000.

[0030] <Synthesis of block copolymer MBI> 0.650 g (5 mmol) of 2-methoxyethyl acrylate (MEA) was added to a 100 mL two-neck flask, followed by 31.8 mg (100 μmol) of cyanomethyl dodecyl carbonate, 1.6 mg (10 μmol) of azobisisobutyronitrile, and 10 mL of 1,4-dioxane. After replacing with argon gas, the mixture was heated and stirred at 62°C for 24 hours.

[0031] After the first heating and stirring, 3.845 g (30 mmol) of n-butyl acrylate (BA) was added to the above, and then 1.6 mg (10 μmol) of azobisisobutyronitrile and 5 mL of 1,4-dioxane were added. After replacing with argon gas, the mixture was heated and stirred at 62° C. for 48 hours.

[0032] After the second heating and stirring, 7.355 g (65 mmol) of N-isopropylacrylamide (IPAAm, LCST = 32 °C) was added to the above, and 1.6 mg (10 μmol) of azobisisobutyronitrile and 35 mL of 1,4-dioxane were further added. After replacing with argon gas, the mixture was heated and stirred at 62 °C for 48 hours.

[0033] After the third heating and stirring, the reaction solution was purified by reprecipitation with water and dried under reduced pressure to obtain a yellow solid. The obtained yellow solid was dissolved in chloroform, and the chloroform phase was collected using a separatory funnel. The collected chloroform phase was concentrated with an evaporator and purified by reprecipitation with hexane. The precipitate was collected by filtration and dried under reduced pressure to obtain 5.805 g of block copolymer MBI. The composition ratio of the obtained block copolymer MBI was MEA / BA / IPAAm = 5 / 26 / 69 (mol%), the number average molecular weight Mn was 85,000, and the molecular weight distribution Mw / Mn was 1.78.

[0034] <Preparation of Fixture> Using a 3D printer (manufactured by FLASHFOEGE), a PLA resin fixture was created with a ring having an inner diameter of 47.7 mm, an outer diameter of 50.00 mm, and a height of 3.00 mm, and two handles on the top of the ring.

[0035] Example 1 A circular polyethylene sheet with a diameter of 51 mm was cut from a 0.04 mm thick polyethylene sheet using a laser cutter. The circular polyethylene sheet was immersed in a 0.02 wt% solution of block copolymer MBI / 2-methoxyethanol. After air drying, a film PE2 coated with block copolymer MBI was prepared. The film thickness of the block copolymer MBI, which was the cover layer, was 5.9 nm.

[0036] Film PE2 was placed on the bottom of an IWAKI tissue culture dish (diameter 60 mm) and fixed to the bottom of the dish with a fixing jig. 5 × 10 A549 cells were 5A 4 mL cell suspension containing 10% fetal bovine serum and antibiotics was used as the medium, and the cells were cultured at 37°C and 5% CO2 for 6 days. A laminate PE2A549 was obtained, in which A549 cells were confluently grown on the film PE2.

[0037] The laminate PE2A549 was removed from the dish, and the cell surface of the laminate was attached to the surface of a gel (Cell Sheet Practice Kit - Practice Gel (CellSeed Co., Ltd.)) packed in a tray with a surface temperature of 24°C, and left to stand for 2 minutes. When film PE2 was turned over, the wrinkled cell sheet had been transferred from film PE2 to the gel surface. When PBS was dropped onto the cell sheet after transfer, the wrinkles in the cell sheet were smoothed out, and a cell sheet with a diameter of 46 mm (90% of the area of ​​the cover layer) was formed on the gel surface.

[0038] Example 2 A film PE5 coated with block copolymer MBI was prepared in the same manner as in Example 1, except that a 0.05 wt % solution of block copolymer MBI / 2-methoxyethanol was used. The thickness of the block copolymer MBI cover layer was 14.3 nm.

[0039] A laminate PE5A549 in which A549 cells were confluently grown on the film PE5 was obtained in the same manner as in Example 1, except that the film PE5 was used.

[0040] A cell sheet was transferred onto the gel surface in the same manner as in Example 1, except that laminate PE5A549 was used, and a cell sheet with a diameter of 46 mm (91% of the area of ​​the cover layer) was successfully formed on the gel surface.

[0041] Comparative Example 1 A circular polyethylene sheet with a diameter of 51 mm was formed from a polyethylene sheet with a thickness of 0.04 mm using a laser cutter, and this was designated PE0.

[0042] A laminate PE0A549 in which A549 cells were confluently grown on the film PE0 was obtained in the same manner as in Example 1, except that the film PE0 was used.

[0043] An attempt was made to transfer a cell sheet onto the gel surface in the same manner as in Example 1 except that laminate PE0A549 was used, but the transfer was unsuccessful and a cell sheet could not be formed on the gel surface.

[0044] Example 3 A film PP2 coated with block copolymer MBI was prepared in the same manner as in Example 1, except that a polypropylene film with a thickness of 0.08 mm was used. The thickness of the block copolymer MBI, which was the cover layer, was 6.3 nm.

[0045] A laminate PP2A549 in which A549 cells were confluently grown on the film PP2 was obtained in the same manner as in Example 1, except that the film PP2 was used.

[0046] A cell sheet was transferred onto the gel surface in the same manner as in Example 1, except that PP2A549 was used, and a cell sheet with a diameter of 45 mm (88% of the area of ​​the cover layer) was successfully formed on the gel surface.

[0047] Example 4 A film PP5 coated with block copolymer MBI was prepared in the same manner as in Example 3, except that a 0.05 wt % solution of block copolymer MBI / 2-methoxyethanol was used. The thickness of the block copolymer MBI cover layer was 14.8 nm.

[0048] A laminate PP5A549 in which A549 cells were confluently grown on the film PP5 was obtained in the same manner as in Example 1, except that the film PP5 was used.

[0049] A cell sheet was transferred onto the gel surface in the same manner as in Example 1, except that laminate PP5A549 was used, and a cell sheet with a diameter of 45 mm (88% of the area of ​​the cover layer) was successfully formed on the gel surface.

[0050] Comparative Example 2 A circular polypropylene sheet with a diameter of 51 mm was formed from a 0.08 mm thick polypropylene sheet using a laser cutter, and this was designated PP0.

[0051] A laminate PP0A549 in which A549 cells were confluently grown on film PP0 was obtained in the same manner as in Example 1, except that film PP0 was used.

[0052] An attempt was made to transfer a cell sheet onto the gel surface in the same manner as in Example 1 except that the laminate PP0A549 was used, but the transfer was unsuccessful and a cell sheet could not be formed on the gel surface.

[0053] Example 5 A film PET2 coated with block copolymer MBI was prepared in the same manner as in Example 1, except that a polyester film with a thickness of 0.01 mm was used. The thickness of the block copolymer MBI, which was the cover layer, was 5.9 nm.

[0054] A laminate PET2A549 in which A549 cells were confluently grown on the PET2 film was obtained in the same manner as in Example 1, except that the PET2 film was used.

[0055] A cell sheet was transferred onto the gel surface in the same manner as in Example 1, except that PET2A549 was used, and a cell sheet with a diameter of 46 mm (90% of the area of ​​the cover layer) was formed on the gel surface.

[0056] Example 6 A film PET5 coated with block copolymer MBI was prepared in the same manner as in Example 5, except that a 0.05 wt% solution of block copolymer MBI / 2-methoxyethanol was used. The thickness of the block copolymer MBI cover layer was 15.0 nm.

[0057] A laminate PET5A549 in which A549 cells were confluently grown on the PET5 film was obtained in the same manner as in Example 1, except that the PET5 film was used.

[0058] A cell sheet was transferred onto the gel surface in the same manner as in Example 1, except that the laminate PET5A549 was used, and a cell sheet with a diameter of 45 mm (88% of the area of ​​the cover layer) was formed on the gel surface.

[0059] Comparative Example 3 A polyester circular sheet with a diameter of 51 mm was formed from a polyester sheet with a thickness of 0.01 mm using a laser cutter, and this was designated PET0.

[0060] A laminate PET0A549 in which A549 cells were confluently grown on a PET0 film was obtained in the same manner as in Example 1, except that a PET0 film was used.

[0061] An attempt was made to transfer a cell sheet onto the gel surface in the same manner as in Example 1, except that the laminate PET0A549 was used, but the transfer was unsuccessful, and a cell sheet could not be formed on the gel surface.

[0062] Example 7 A film PE2 coated with block copolymer MBI was prepared in the same manner as in Example 1. The thickness of the block copolymer MBI, which was the cover layer, was 6.1 nm.

[0063] A laminate PE2HCT116 in which HCT116 cells were confluently grown on the film PE2 was obtained in the same manner as in Example 1, except that HCT116 cells were used as the cells.

[0064] A cell sheet was transferred onto the gel surface in the same manner as in Example 1, except that laminate PE2HCT116 was used, and a cell sheet with a diameter of 46 mm (88% of the area of ​​the cover layer) was successfully formed on the gel surface.

[0065] Comparative Example 4 A circular polyethylene sheet with a diameter of 51 mm was formed from a polyethylene sheet with a thickness of 0.04 mm using a laser cutter, and this was designated PE0.

[0066] A laminate PE0HCT116 in which HCT116 cells were confluently grown on the film PE0 was obtained in the same manner as in Example 7, except that the film PE0 was used.

[0067] An attempt was made to transfer a cell sheet onto the gel surface in the same manner as in Example 7, except that the laminate PE0HCT116 was used, but the transfer was unsuccessful and a cell sheet could not be formed on the gel surface.

[0068] Example 8 A film PE2 coated with block copolymer MBI was prepared in the same manner as in Example 1. The thickness of the block copolymer MBI, which was the cover layer, was 6.1 nm.

[0069] A laminate PE2bMSC in which bMSC cells were confluent on the film PE2 was obtained in the same manner as in Example 1, except that bone marrow-derived mesenchymal stem cells (bMSC) were used as the cells and cultured for 10 days.

[0070] A cell sheet was transferred onto the gel surface in the same manner as in Example 1, except that the laminated PE2bMSC was used, and a cell sheet with a diameter of 46 mm (91% of the area of ​​the cover layer) was formed on the gel surface.

[0071] Example 9 A film PE2 coated with block copolymer MBI was prepared in the same manner as in Example 1. The thickness of the block copolymer MBI, which was the cover layer, was 6.1 nm.

[0072] A laminate PE2ACS in which ACS cells were confluent on the film PE2 was obtained in the same manner as in Example 1, except that adipose-derived mesenchymal stem cells (ACS) were used as the cells and cultured for 8 days.

[0073] A cell sheet was transferred onto the gel surface in the same manner as in Example 1, except that the laminate PE2ACS was used, and a cell sheet with a diameter of 46 mm (90% of the area of ​​the cover layer) was formed on the gel surface.

[0074] [Table 1]

Claims

1. A method for transferring a cell sheet to a target, comprising: a step of attaching a cell sheet surface of a laminate comprising a support layer made of a film-like polymer, a cover layer made of a film-like temperature-responsive polymer laminated on the support layer, and a cell sheet laminated on the cover layer to a target; cooling the laminate to a temperature equal to or lower than a lower critical solution temperature (LCST) of the temperature-responsive polymer in water; peeling the support layer and the cover layer off the cell sheet, The subject is a living body (excluding humans), cultured cell tissue, or experimental gel, The temperature-responsive polymer is a block copolymer composed of repeating units of 2-methoxyethyl acrylate, n-butyl acrylate, and N-isopropylacrylamide, The method, wherein the material of the film is polyethylene, polypropylene, polystyrene, polyvinylidene chloride, polyvinyl chloride, polyester, polyolefin, or a copolymer containing these.

2. 2. The method according to claim 1, wherein the thickness of the support layer is from 1 to 400 μm.

3. 3. The method according to claim 1, wherein the cover layer has a thickness of 1 to 1000 nm.

4. The method according to any one of claims 1 to 3, wherein the cell sheet is made of epithelial cells or mesenchymal stem cells.

5. 5. The method according to claim 1, wherein the area of ​​the cell sheet is 50% or more of the area of ​​the cover layer.

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