Cell sheet support, cell sheet laminate, and method for producing same

Abstract

Provided is a cell sheet support that satisfactorily adheres to cultured cells and has biodegradability. The cell sheet support comprises a first polymer that contains a structural unit derived from p-dioxanone. The cell sheet support may further contain a second polymer that contains at least one selected from the group consisting of polylactic acid, polyglycolic acid, polycaproic acid and copolymers thereof. Relative to the total amount of the first and second polymers, the content of the first polymer may be 50 mass% or more. The cell sheet support may be in the form of a sheet having an average thickness of 10-150 μm inclusive.

Description

[Technical Field]

[0001] The present invention relates to a cell sheet support, a cell sheet laminate, and a method for producing the same. [Background technology]

[0002] Transplantation methods using cell sheets have been developed for the purpose of regenerating damaged biological tissues. Many cell sheets used for transplantation are produced using animal cells, including human cells, particularly adhesion-dependent cells. To produce cell sheets, animal cells must be attached to a substrate surface, cultured in a sheet form, and then detached while maintaining their morphology. For example, International Publication No. 02 / 08387 proposes a method in which cells are cultured on a cell culture support whose substrate surface is coated with a polymer whose upper or lower critical solution temperature in water is between 0°C and 80°C. The resulting cell sheet is then adhered to a polymer membrane, and the culture medium temperature is raised above the upper critical solution temperature or below the lower critical solution temperature, thereby detaching the cell sheet together with the polymer membrane. Furthermore, Membrane, 40(3), 124-129 (2015) proposes a cultured cell sheet supported on a biodegradable polyester porous membrane. Summary of the Invention [Problem to be solved by the invention]

[0003] The polymer membrane described in Patent Document 1 sometimes had insufficient adhesiveness to cells, resulting in insufficient recovery of cultured cells. Furthermore, it was necessary to peel the polymer membrane from the cell sheet after transferring the cell sheet to the target site. The present invention aims to provide a cell sheet support that has good adhesiveness to cultured cells and is biodegradable. [Means for solving the problem]

[0004] Specific means for solving the above problems are as follows, and the present invention encompasses the following aspects. A first aspect is a cell sheet support comprising a first polymer containing a structural unit derived from p-dioxanone. The cell sheet support may further comprise a second polymer containing at least one selected from the group consisting of polylactic acid, polyglycolic acid, polycaproic acid, and copolymers thereof, and the content of the first polymer relative to the total amount of the first polymer and the second polymer may be 50 mass% or more. The cell sheet support may be in the form of a sheet with an average thickness of 10 μm or more and 150 μm or less. The cell sheet support may be used for transporting cell sheets or for stacking cell sheets.

[0005] A second aspect is a cell sheet laminate comprising the cell sheet support and a cell sheet placed on the cell sheet support.

[0006] The third aspect is a method for producing a cell sheet laminate, comprising: culturing cells on a temperature-responsive polymer layer to prepare a cell sheet; layering the cell sheet support on the cell sheet; and peeling the cell sheet from the temperature-responsive polymer layer by a temperature change to obtain a cell sheet support to which the cell sheet is adhered.

[0007] A fourth aspect is a method for transferring a cell sheet, comprising: culturing cells on a temperature-responsive polymer layer to prepare a cell sheet; layering the cell sheet support on the cell sheet; peeling the cell sheet from the temperature-responsive polymer layer by a temperature change to obtain a cell sheet support to which the cell sheet is adhered; and contacting the cell sheet on the cell sheet support with a target site.

[0008] A fifth aspect is a method for producing a cell sheet laminate, which comprises culturing cells on the cell sheet support having a hydrophilic coating layer containing a hydrophilic polymer formed on the surface thereof to form a cell sheet.

[0009] A sixth aspect is a method for transferring a cell sheet, comprising culturing cells on the cell sheet support having a hydrophilic coating layer containing a hydrophilic polymer formed on its surface to form a cell sheet, and contacting the cell sheet on the cell sheet support with a target site. [Effects of the Invention]

[0010] According to the present invention, a cell sheet support that has good adhesiveness to cultured cells and is biodegradable can be provided. [Brief explanation of the drawings]

[0011] [Figure 1A] 10 is a microscope image showing the state of the culture vessel after the cell sheet has been attached onto the cell sheet support. [Figure 1B] 10 is a microscope image after Hoechst staining showing the state of the culture vessel after the cell sheet was adhered onto the cell sheet support. [Figure 1C] This is a microscope image showing the state of the culture vessel after the cell sheet was attached to the CellShifter™. [Figure 1D] This is a microscope image after Hoechst staining showing the state of the culture vessel after the cell sheet was attached to the CellShifter™. [Figure 2A] This is an image taken immediately after the start of hydrolysis showing the hydrolysis of the cell sheet support. [Figure 2B] 10 shows images after 2 weeks showing the hydrolytic properties of the cell sheet support. [Figure 2C] 10 shows images after 4 weeks showing the hydrolytic properties of the cell sheet support. [Figure 3A] 1 is an enlarged image of the surface of a cell sheet support. [Figure 3B] 1 is an enlarged image of the surface of a cell sheet support. [Figure 4] This is an enlarged image of the CellShifter(TM) surface. [Figure 5] FIG. 1 shows the proliferation rate of cultured cells on a cell sheet support. [Figure 6]1 shows a schematic diagram and a fluorescence microscope image showing the state of the adhesive surface where different cell sheets are layered. DETAILED DESCRIPTION OF THE INVENTION

[0012] As used herein, the term "process" refers not only to an independent process, but also to processes that cannot be clearly distinguished from other processes, as long as the intended purpose of the process is achieved. Furthermore, the content of each component in a composition refers to the total amount of the multiple substances present in the composition, unless otherwise specified, when multiple substances corresponding to each component are present in the composition. Furthermore, the upper and lower limits of the numerical ranges described herein can be arbitrarily selected and combined. Hereinafter, embodiments of the present invention will be described in detail. However, the embodiments described below are merely examples of cell sheet supports for embodying the technical concept of the present invention, and the present invention is not limited to the cell sheet supports described below.

[0013] Cell sheet support The cell sheet support contains a first polymer containing a structural unit derived from p-dioxanone and is formed into a sheet shape. By including the first polymer as a constituent component, the cell sheet support exhibits excellent adhesiveness to cultured cells and good biodegradability.

[0014] The first polymer containing structural units derived from p-dioxanone may be polydioxanone (poly(p-dioxanone; PDS)), which is a homopolymer of p-dioxanone, or may be a copolymer of p-dioxanone with glycolic acid, lactic acid, caproic acid, or the like. The polydioxanone copolymer may preferably be a copolymer of p-dioxanone with at least one other monomer selected from the group consisting of glycolic acid, lactic acid, and caproic acid, or a copolymer of glycolic acid and p-dioxanone. The molar content of the structural units derived from p-dioxanone in the polydioxanone copolymer may be, for example, 75% or more, preferably 80% or more, or 90% or more, based on the total number of moles of the structural units. The polydioxanone copolymer may be a random copolymer or a block copolymer. The polydioxanone copolymer is preferably a block copolymer.

[0015] Polydioxanone (PDS) is a polymer prepared by ring-opening polymerization of p-dioxanone. Compared to other biodegradable polymers, polydioxanone has excellent in vivo absorption, flexibility, and bendability, as well as low toxicity. Therefore, it is used in a variety of biomedical applications, such as sutures, meshes, staples, clips, implantable orthopedic devices, and drug delivery systems. Furthermore, copolymerization of p-dioxanone with glycolic acid, lactic acid, etc., allows for controlled biodegradation in addition to the inherent flexibility of PDS.

[0016] The polydioxanone and polydioxanone copolymer can be appropriately selected from commercially available products, and specifically, they can be obtained from, for example, Sigma-Aldrich Corporation.

[0017] The cell sheet support may be composed of a first polymer, or may contain, in addition to the first polymer, a second polymer containing at least one selected from the group consisting of polylactic acid, polyglycolic acid, polycaproic acid, and copolymers thereof. By including the second polymer, the flexibility, biodegradability, etc. of the cell sheet support can be easily controlled.

[0018] The second polymer may comprise at least one polymer selected from the group consisting of polylactic acid, polyglycolic acid, and lactic acid-glycolic acid copolymer (PLGA), and may comprise at least PLGA. The weight-average molecular weight of the second polymer may be, for example, 1,000 to 50,000, and preferably 5,000 to 20,000. Furthermore, when the second polymer is a copolymer, the biodegradation rate of the cell sheet support can be controlled by appropriately selecting the monomer composition ratio. For example, when the second polymer is PLGA, the molar ratio of lactic acid to glycolic acid (L / G) may be, for example, 0.3 to 5, and preferably 1 to 4.

[0019] When the cell sheet support contains a first polymer and a second polymer, the content of the first polymer relative to the total amount of the first polymer and the second polymer may be, for example, 50% by mass or more, preferably 60% by mass or more or 70% by mass or more. The upper limit of the content of the first polymer relative to the total amount of the first polymer and the second polymer may be, for example, less than 100% by mass, preferably 95% by mass or less, 90% by mass or less, or 85% by mass or less.

[0020] The cell sheet support may be formed in a sheet shape. The planar shape of the cell sheet support may be appropriately selected depending on the purpose, etc., and may be, for example, rectangular, polygonal, approximately circular, approximately elliptical, etc. The size of the cell sheet laminate may be appropriately selected depending on the purpose, etc. The size of the cell sheet laminate may be, for example, 1 cm in area. 2 More than 100cm 2 It may be the following:

[0021] The average thickness of the cell sheet support may be appropriately selected from the viewpoints of, for example, biodegradability, ease of handling, etc., and may be selected according to the polymer composition. The average thickness of the cell sheet support may be, for example, 10 μm or more and 150 μm or less, and preferably 15 μm or more, 20 μm or more, or 30 μm or more. The upper limit of the average thickness may be, for example, 140 μm or less, 120 μm or less, or 110 μm or less. The average thickness of the cell sheet support is calculated as the arithmetic mean of the thicknesses at any three points.

[0022] The cell sheet support can be formed, for example, by forming a solution of a support-forming composition containing a first polymer, a liquid medium, and an optional second polymer into a film, and then removing the liquid medium. The liquid medium may be any solvent capable of dissolving the first polymer, and may be volatile. Specific examples of the liquid medium include hexafluoro-2-propanol (HFIP). The solids concentration of the support-forming composition may be, for example, 0.1% by mass or more and 5% by mass or less, and preferably 1% by mass or more and 4% by mass or less.

[0023] The cell sheet support can be formed into a desired shape, for example, by pouring the support-forming composition into a mold having a desired shape and removing the liquid medium. Furthermore, the thickness can be controlled by adjusting the total solid content of the support-forming composition poured into the mold. Alternatively, the support-forming composition can be dropped onto a flat substrate without a mold to form a desired shape. The material of the mold and substrate may be any material as long as the cell sheet support formed can be peeled off, and examples thereof include glass and resins such as polypropylene.

[0024] The method for removing the liquid medium may be selected depending on the properties of the liquid medium. Specifically, the liquid medium may be removed by volatilizing it at room temperature or under heated conditions. The cell sheet support may be formed in a porous form or in a non-porous, dense membrane form.

[0025] Cell sheet laminate The cell sheet laminate comprises the above-mentioned cell sheet support and a cell sheet placed on the cell sheet support. The cell sheet laminate can be produced by the production method described below.

[0026] Because the cell sheet support has excellent adhesive properties with cultured cells, cell sheets can be stably adhered and held on the cell sheet support with an excellent recovery rate. Therefore, the cell sheet support can be used to transfer a cell sheet formed on a cell culture vessel from the culture vessel to a desired site. Even if the site to which the cell sheet is to be transferred is a living body, the cell sheet support is biodegradable, so there is no need to remove the cell sheet support after the cell sheet is transferred. Furthermore, the cell sheet support can be used to form a cell sheet laminate in which multiple cell sheets are stacked via the cell sheet support by stacking multiple cell sheet supports each holding a cell sheet on one side of the cell sheet support. After the cell sheet support is degraded, a cell sheet laminate is formed in which multiple cell sheets are directly stacked.

[0027] Here, the cell sheet in this specification refers to a sheet-like assembly of cultured cells cultured on one surface of a culture vessel. Specifically, it may be a sheet-like assembly of cultured cells confluently cultured on a cell culture dish.

[0028] The cells that make up the cell sheet are not particularly limited in species or tissue of origin as long as they are animal cells, and examples include cells immediately after collection from a living organism and established cell lines. Animal cells can be derived from mammals, including humans. The cells may be somatic cells or stem cells.

[0029] In one embodiment, the cell sheet laminate may further comprise a hydrophilic coating layer containing a hydrophilic polymer between the cell sheet support and the cell sheet.

[0030] Method for producing a cell sheet laminate The method for producing a cell sheet laminate includes a preparation step of culturing cells on a temperature-responsive polymer layer to prepare a cell sheet, a lamination step of laminating the above-mentioned cell sheet support on the cell sheet, a temperature adjustment step of detaching the cell sheet from the temperature-responsive polymer layer by changing the temperature, and an adhesion step of adhering the cell sheet to the cell sheet support. Because the cell sheet is cultured on the temperature-responsive polymer layer, it can be easily detached from the temperature-responsive polymer layer by applying a predetermined temperature change and easily migrate to and adhere to the cell sheet support, which has a high affinity for the cultured cells. This results in a cell sheet laminate in which the cell sheet is arranged on the cell sheet support.

[0031] In the preparation step, cells are cultured on a temperature-responsive polymer to prepare a cell sheet. Specifically, a cell sheet is formed by culturing desired cells on a cell culture support having an area coated with a temperature-responsive polymer.

[0032] Examples of materials for the substrate coated with the temperature-responsive polymer that constitutes the cell culture support include polyvinyl resins such as polystyrene, polyethylene, and polypropylene, and glass. The shape and size of the substrate are not particularly limited as long as they allow cell culture, and may be selected appropriately depending on the shape and size of the desired cell sheet. The substrate can be selected appropriately from commercially available substrates for cell culture depending on the purpose, etc.

[0033] A temperature-responsive polymer is a polymeric material whose adhesiveness to cultured cells reversibly changes depending on the environmental temperature. A polymer whose adhesiveness to cultured cells decreases in an environment lower than the cell culture temperature (hereinafter also referred to as a "first temperature-responsive polymer") can be used as the temperature-responsive polymer. For example, the polymers described in JP-A-2-211865 and the like are known as the first temperature-responsive polymer. Specifically, a polymer obtained by homopolymerization or copolymerization of at least one monomer selected from the group consisting of (meth)acrylamide compounds, alkyl-substituted (meth)acrylamide derivatives, and vinyl ether derivatives can be used. For example, poly(N-isopropylacrylamide) can be preferably used.

[0034] Furthermore, a polymer (hereinafter also referred to as a "second temperature-responsive polymer") whose adhesiveness to cultured cells decreases when placed in an environment slightly higher than the cell culture temperature can also be used as the temperature-responsive polymer. The use of a second temperature-responsive polymer can effectively prevent low-temperature damage to cultured cells when the cell sheet is detached. Examples of the second temperature-responsive polymer include the polymers described in JP 2018-102296 A. Specifically, a copolymer containing a first structural unit derived from a (meth)acrylate having an alkyl group with 14 to 22 carbon atoms and a second structural unit derived from (meth)acrylic acid can be preferably used.

[0035] Specific examples of the monomer that forms the first structural unit include (meth)acrylates having a linear alkyl group, such as tetradecyl (meth)acrylate, hexadecyl (meth)acrylate, stearyl (meth)acrylate, eicosanyl (meth)acrylate, and behenyl (meth)acrylate.

[0036] The molar ratio of the first constitutional unit to the second constitutional unit contained in the second temperature-responsive polymer (first constitutional unit:second constitutional unit) is, for example, from 2:8 to 5:5, and preferably from 2:8 to 4:6. The second temperature-responsive polymer may be a random copolymer or a block copolymer, but is preferably a block copolymer.

[0037] The second temperature-responsive polymer may further contain other structural units in addition to the first structural unit and the second structural unit to the extent that the effects of the present invention are not impaired. The monomers constituting the other structural units are not particularly limited as long as they are copolymerizable with (meth)acrylates having an alkyl group with 14 to 22 carbon atoms and (meth)acrylic acid, and examples thereof include (meth)acrylates having an alkyl group with 12 or less carbon atoms, such as styrene and butyl acrylate.

[0038] The molecular weight of the second temperature-responsive polymer may be, for example, a weight-average molecular weight (Mw) of 3,500 to 200,000, preferably 6,000 to 70,000. Furthermore, the polydispersity (Mw / Mn) may be, for example, 1.05 to 15, preferably 1.2 to 2. The weight-average molecular weight and polydispersity can be determined using GPC in terms of polystyrene.

[0039] When the second temperature-responsive polymer is a block copolymer, the weight-average molecular weight of the first constitutional unit portion may be, for example, 500 or more, 1,000 or more, 2,000 or more, 3,000 or more, 4,000 or more, or 5,000 or more, and 100,000 or less, 50,000 or less, 10,000 or less, 8,000 or less, or 7,000 or less. The weight-average molecular weight of the second constitutional unit portion may be, for example, 500 or more, 1,000 or more, or 2,000 or more, and 100,000 or less, 20,000 or less, or 15,000 or less. The degree of polymerization of the first constitutional unit portion may be, for example, 2 or more, 5 or more, or 10 or more, and 800 or less, 500 or less, 100 or less, or 50 or less. The degree of polymerization of the second constitutional unit portion may be, for example, 5 or more, 10 or more, or 30 or more, and may be 800 or less, 500 or less, or 100 or less.

[0040] The amount of the temperature-responsive polymer disposed on the surface of the substrate is, for example, 30 μg / cm 2 More than 160μg / cm 2 may be less than or equal to 50 μg / cm 2 More than 80μg / cm 2 The following are preferred: The polymer may be used alone, or two or more polymers having different structures may be used in combination.

[0041] The cells cultured on the cell culture support are not particularly limited in species or tissue of origin as long as they are animal cells, and examples thereof include cells immediately after collection from a living organism and established cell lines. Animal cells can be derived from mammals, including humans. The cells may be somatic cells or stem cells.

[0042] A commonly used medium can be used for cell culture. The medium used for culture may be any medium commonly used for culturing animal cells, including various serum-free basal culture media (standard culture media) such as RPMI medium, Dulbecco's Modified Eagle Medium (DMEM), MEM medium, and F12 medium. Serum may be added to this medium to promote cell proliferation, or alternatives to serum may include cell growth factors such as FGF, EGF, and PDGF, and known serum components such as transferrin. When serum is added, the concentration can be adjusted appropriately depending on the culture conditions at the time, for example, to 5% to 10% by volume. The medium may also contain various vitamins, antibiotics such as streptomycin, differentiation inducers, and the like.

[0043] The density of cells seeded on the cell culture support may be selected appropriately depending on the cell type, etc., as long as a cell sheet can be formed. For example, 3 × 10 cells per culture area may be seeded. 4 From 6 x 10 4 cells / cm 2 It can be said that:

[0044] Cell culture conditions can be selected appropriately depending on the cells. The culture temperature can be, for example, 35°C to 37°C. Cells may be cultured in an incubator with a 5% CO2 concentration. Cell sheets derived from various cells can be formed by culturing them under normal culture conditions for approximately 3 to 5 days until they reach a confluent state. Cell sheet formation can be confirmed by microscopic observation.

[0045] In the layering step, the above-mentioned cell sheet support is layered on the prepared cell sheet. The size of the cell sheet support layered on the cell sheet may be approximately the same as the size of the prepared cell sheet, or may be larger than the cell sheet. When using a cell sheet support larger than the cell sheet, a cell sheet support having an area approximately 1% to 10% larger than the area of ​​the cell sheet may be used.

[0046] In the layering step, at least a portion of the culture medium covering the cell sheet may be removed before layering the cell sheet support on the cell sheet. By removing the culture medium and exposing the cell sheet, the cell sheet and the cell sheet support can be easily brought into contact with each other. In the layering step, a holding time may be set during which the cell sheet support is layered on the cell sheet and the cell sheet and the cell sheet support are maintained in contact with each other. The holding time may be, for example, 5 to 30 minutes.

[0047] Temperature adjustment process In the temperature adjustment step, the cell sheet is detached from the temperature-responsive polymer layer by temperature change. When the temperature-responsive polymer is a first temperature-responsive polymer, the temperature change is made by changing the temperature from the cell sheet culture temperature to a temperature lower than the culture temperature. Specifically, the cell sheet can be detached from the temperature-responsive polymer layer by changing the temperature, for example, from 20°C to 25°C. When the temperature-responsive polymer is a second temperature-responsive polymer, the temperature change is made by changing the cell sheet culture temperature to a temperature higher than the culture temperature. Specifically, the cell sheet can be detached from the temperature-responsive polymer layer by changing the temperature, for example, from 38°C to 45°C. In the temperature adjustment step, the changed temperature is maintained for, for example, 30 to 60 minutes.

[0048] The temperature adjustment step may be performed after the lamination step or before the lamination step. By adjusting the temperature after the lamination step, the cell sheets adhered to the cell sheet support in the lamination step are detached from the temperature-responsive polymer, thereby obtaining a cell sheet laminate in a free state. Alternatively, by adjusting the temperature before the lamination step, the cell sheet support is laminated on the cell sheets detached from the temperature-responsive polymer, thereby obtaining a cell sheet laminate in a free state.

[0049] Adhesion process In the adhesion step, the cell sheet is adhered to a cell sheet support to obtain a cell sheet laminate. Since the cell sheet support has a high affinity for cultured cells, the cell sheet adheres to the cell sheet support by being layered on the cell sheet and brought into contact with it. The adhesion step may be performed as a layering step before the temperature adjustment step or as a layering step after the temperature adjustment step.

[0050] Another embodiment of the method for producing a cell sheet laminate is a method for producing a cell sheet laminate, which comprises culturing cells on the above-mentioned cell sheet support having a hydrophilic coating layer containing a hydrophilic polymer formed on its surface. Another embodiment of the method for producing a cell sheet laminate may be a method for producing a cell sheet laminate, which comprises forming a hydrophilic coating layer containing a hydrophilic polymer on the above-mentioned cell sheet support, and culturing cells on the hydrophilic coating layer to form a cell sheet.

[0051] Examples of hydrophilic polymers contained in the hydrophilic coating layer include synthetic polymers such as the above-mentioned temperature-responsive polymers; and biopolymers such as type I collagen, type III collagen, type IV collagen, type V collagen, laminin, polylysine, and extracellular matrix (ECM) hydrogels. The hydrophilic coating layer may contain one hydrophilic polymer alone or two or more hydrophilic polymers in combination. The hydrophilic polymer preferably contains at least one selected from the group consisting of copolymers containing structural units derived from (meth)acrylates having an alkyl group with 14 to 22 carbon atoms and structural units derived from (meth)acrylic acid, type I collagen, type III collagen, type IV collagen, type V collagen, laminin, polylysine, and ECM hydrogels. The hydrophilic polymer may contain at least a copolymer containing structural units derived from (meth)acrylates having an alkyl group with 14 to 22 carbon atoms and structural units derived from (meth)acrylic acid.

[0052] The thickness of the hydrophilic coating layer may be, for example, 10 nm to 2000 μm, or 20 nm to 1000 μm. When the hydrophilic polymer contained in the hydrophilic coating layer is a synthetic polymer such as the above-mentioned temperature-responsive polymer, or a biopolymer such as collagen, laminin, or polylysine, the thickness of the hydrophilic coating layer may be preferably 20 nm or more, or 50 nm or more, and preferably 500 nm or less, or 200 nm or less. Furthermore, when the hydrophilic polymer is a hydrogel having an ECM composition, the thickness of the hydrophilic coating layer may be preferably 100 μm or more, or 300 μm or more, and preferably 1000 μm or less, or 500 μm or less. The hydrophilic coating layer may be disposed on only one surface of the cell sheet support, or on both surfaces.

[0053] The hydrophilic coating layer can be formed on the cell sheet support, for example, by applying a solution of a hydrophilic polymer to the surface of the cell sheet support and then removing at least a portion of the solvent.

[0054] Regarding the method for culturing cells on the above-mentioned cell sheet support on which a hydrophilic coating layer has been formed to form a cell sheet, a method similar to the method for culturing cells on the above-mentioned temperature-responsive polymer to form a cell sheet can be applied.

[0055] Cell sheet transfer method The cell sheet transfer method includes a preparation step of culturing cells on a temperature-responsive polymer layer to prepare a cell sheet, a lamination step of laminating the above-mentioned cell sheet support on the cell sheet, a temperature adjustment step of detaching the cell sheet from the temperature-responsive polymer layer by temperature change, an adhesion step of adhering the cell sheet on the cell sheet support, and a transfer step of contacting the cell sheet on the cell sheet support with a target site.

[0056] By transporting the cell sheet together with the cell sheet support to the target site and bringing them into contact with each other, damage to the cell sheet can be suppressed and the handling of the cell sheet can be improved.

[0057] The preparation step, layering step, temperature adjustment step, and adhesion step in the cell sheet transfer method are the same as those in the above-described cell sheet laminate production method.

[0058] In the transfer step, the cell sheet on the cell sheet support is transferred to a target site and brought into contact with the target site. That is, the cell sheet laminate obtained in the adhesion step is transferred to a target site and the cell sheet on the cell sheet support is brought into contact with the target site. The target site may be in vivo or ex vivo. Examples of ex vivo target sites include another cell sheet, a laminate of multiple cell sheets, another cell sheet laminate, tissues and organs collected from a living organism, etc. The target site may also be a tissue or organ in vivo.

[0059] Another aspect of the method for transferring a cell sheet may include culturing cells on the above-mentioned cell sheet support having a hydrophilic coating layer containing a hydrophilic polymer formed on its surface to form a cell sheet, and contacting the cell sheet on the cell sheet support with a target site. [Example]

[0060] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0061] Example 1 Polydioxanone (PDS; Sigma-Aldrich) was used as the first polymer, and lactic-co-glycolic acid copolymer (PLGA5005; Fujifilm Wako Pure Chemical Industries, Ltd.; L / G 50%, Mw 5000) was used as the second polymer. As shown in Table 1, PDS and PLGA5005 were mixed, and 1 mL of hexafluoro-2-propanol (Sigma-Aldrich) was added and mixed by shaking to prepare a polymer solution. The polymer solution was spread onto a glass plate using a pipette, left to dry at room temperature for approximately 1 hour, and then peeled off from the glass plate with tweezers to obtain cell sheet supports No. 1-1 to 1-3.

[0062] Average thickness and peelability The thickness of the cell sheet support obtained in Example 1 was measured at three locations using a digital caliper (DC-10, TOPMIGHTY), and the average thickness was calculated as the arithmetic mean of the measured values. Peelability from the glass plate was also evaluated visually. The results are shown in Table 1.

[0063] [Table 1]

[0064] Cell recovery rate The adhesiveness of the cell sheet substrate obtained in Example 1 to a cell sheet was evaluated as follows. The results are shown in FIG.

[0065] A temperature-responsive polymer composed of tetradecyl acrylate (TDA) and acrylic acid (AA) with a weight-average molecular weight of TDA:AA = 10,000:10,000 was synthesized with reference to the description in JP 2018-102296 A. The synthesized temperature-responsive polymer was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 0.05% (w / v) to prepare a composition for coating cell culture dishes.

[0066] 1.5 mL of the obtained composition was dropped onto a 35 mm untreated polystyrene (PS) dish (IWAKI #1000-035), left to stand at room temperature for 2 hours, and then 2 mL of PBS was added to rinse, producing a cell culture support with a temperature-responsive polymer attached to its surface. Next, Myocyte Basal Medium (supplemented with 5% FCS, hEGF (0.5 ng / mL), hbFGF (2 ng / mL), and insulin (5 μg / mL)) was added to the cell culture support, and 1 × 10 human cardiomyocyte cell line (HCM-c) was cultured. 5 Cells were seeded at 1000 cells / dish. Cell culture was carried out for 28 days in an incubator at 37°C and 5% CO2 concentration to form a cell sheet. The cells were then shaken at 40°C for 30 to 60 minutes, the medium was aspirated, and the cell sheet support obtained in Example 1 was layered on the cell sheet and left to stand for 5 minutes. The cell sheet was then removed from the cell culture support to recover the cell sheet. 1 μL of Hoechst 33342 reagent was diluted with 500 μL of PBS and added to each culture dish after cell sheet recovery. The dish was then left to stand in an incubator at 37°C and 5% CO2 concentration for 30 minutes. After recovery, the cells remaining in the culture dish were visualized by fluorescence using Hoechst staining and observed under a microscope.

[0067] Figure 1(A) is a microscopic image showing the state of the incubator after cell sheet recovery using the cell sheet support No. 1-2, and Figure 1(B) is a microscopic image (405 nm) after Hoechst staining. Figure 1(C) shows the state of the incubator after cell sheet recovery using the CellShifter (TM)Figure 1(D) is a microscopic image (405 nm) showing the state of the incubator after cell sheet recovery using a cell sheet support (manufactured by CellSeed), and Figure 1(E) is a microscopic image (405 nm) after Hoechst staining. It can be seen that by using a cell sheet support, no cultured cells remain in the incubator, and cell sheets can be recovered with a good recovery rate.

[0068] Example 2 Cell sheet supports No. 2-1 to 2-8 were obtained in the same manner as in Example 1, except that in addition to PLGA5005, PLGA5010 (L / G 50%, Mw 10000), PLGA5020 (L / G 50%, Mw 20000), PLGA0010 (L / G 100%, Mw 10000), and PLGA7510 (L / G 75%, Mw 10000) (all manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were prepared as the second polymer, and the compositions were as shown in Table 2.

[0069] [Table 2]

[0070] Hydrolyzable The cell sheet support was trimmed to fit the bottom of a 35 mm dish and placed on the bottom of the dish. 1.5 mL of PBS was added dropwise, and the plate was left to stand at 37°C for 4 weeks and observed over time. The results are shown in Figure 2.

[0071] Figure 2(A) shows images taken immediately after application (Day 0), Figure 2(B) shows images taken two weeks later (Day 14), and Figure 2(C) shows images taken four weeks later (Day 28). It was found that the time required for degradation and flexibility can be controlled by adjusting the lactic acid to glycolic acid ratio (L / G) and molecular weight of the PLGA contained in the cell sheet support. Specifically, the larger the molecular weight, the longer the time required for degradation, and the higher the molecular weight, the harder the support tended to be.

[0072] Example 3 A depression with a diameter of 31 mm was formed on a glass plate to serve as a mold for preparing a cell sheet support. Cell sheet supports No. 3-1 to 3-6 were obtained in the same manner as in Example 1, except that the compositions shown in Table 3 were used and the polymer solution was spread inside the mold.

[0073] [Table 3]

[0074] Cell recovery rate A temperature-responsive polymer composed of tetradecyl acrylate (TDA) and acrylic acid (AA) with a weight-average molecular weight of 10,000:10,000 was synthesized based on the description in JP 2018-102296 A. The resulting temperature-responsive polymer was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 0.05% (w / v) to prepare a composition for coating cell culture dishes. 1.5 mL of the resulting composition was dropped onto a 35 mm untreated polystyrene (PS) dish (IWAKI #1000-035), left to stand at room temperature for 2 hours, and then rinsed with 2 mL of PBS to produce a cell culture support with a temperature-responsive polymer attached to its surface.

[0075] DMEM medium (with 10% FCS) was added to the cell culture support as the medium, and 3 × 10 human oral cancer cell line NA was added. 5 The cells were seeded at 100 cells / dish and cultured at 37°C and 5% CO2 for 7 days until they became confluent, forming a cell sheet on the temperature-responsive polymer layer.

[0076] The cell culture support on which the cell sheet was formed was heated at 40°C for 30 minutes, and the cell sheet was detached from the temperature-responsive polymer layer. Next, after removing the medium, the cell sheet support was layered on the cell sheet and left to stand for 5 minutes, and then the cell sheet support with the cell sheet attached was removed from the cell culture support. The cells remaining on the cell culture support were stained with Hoechst, and the cell survival rate was calculated based on the fluorescence intensity, with the fluorescence intensity before the cell sheet detachment being used as the reference (100%). The results are shown in Table 4. Note that the reference example was performed using CellShifter(TM) is.

[0077] flexibility The flexibility of the cell sheet support was evaluated by calculating the ratio of the area of ​​the cell sheet support adhered to the bottom surface of the cell culture support when the cell sheet support was layered on the cell sheet to the bottom surface of the cell culture support, and then evaluating the ratio according to the following scoring criteria. The results are shown in Table 4.

[0078] Scoring Criteria A Bottom area of ​​90% or more B: Base area 60% or more but less than 90% C. Bottom area: 50% or more and less than 60% D. Bottom area: 30% or more and less than 50% E. Less than 30% of base area F Not tightly attached

[0079] [Table 4]

[0080] Surface observation For cell sheet substrates No. 3-2 and No. 3-5 obtained in Example 3, the surface of the cell sheet substrate was observed using a tabletop microscope (Miniscope® TM4000Plus; manufactured by HITACHI Corporation) to obtain a magnified image (1000x). The results are shown in Figure 3. (TM) A magnified image of the surface is shown in Figure 4.

[0081] Figure 3(A) is an enlarged image of the center of the surface of No. 3-2, and (B) is an enlarged image of No. 3-5. As shown in Figures 3 and 4, Cellshifter (TM) It can be seen that the PDS film and PDS / PLGA film, which are cell sheet supports, have a solid structure that is neither fibrous nor porous, while the PDS film has a structure in which polymers are arranged like paving stones, while the PDS / PLGA film has a pleated structure.

[0082] Hygroscopic The moisture absorption rate of the cell sheet support was calculated using the following method. The cell sheet support was dried in a drying device (SPH-10N, manufactured by IKEDARIKA) for at least 3 hours, and then the dry weight A (g) was measured. The dried cell sheet support was left to stand for 1 hour in an environment of 40°C and 90% RH (SCA-30D, manufactured by ASTEC), and then the weight after moisture absorption B (g) was measured. The moisture absorption rate (%) was calculated using the following formula. The results are shown in Table 5. Moisture absorption rate (%)=(BA) / A×100

[0083] [Table 5]

[0084] The contact angles of the cell sheet supports No. 3-2 and No. 3-5 were measured using the following method. Using a contact angle meter (DropMaster DM-301, manufactured by Kyowa Interface Science Co., Ltd.), the contact angle was measured by the drop method, in which 1 μL of purified water was dropped onto the cell sheet support. The results are shown in Table 6.

[0085] [Table 6]

[0086] CellShifter (TM) The moisture absorption rate of the cell sheet scaffold was lower than that of the CellShifter. (TM) Compared to the surface of the cell sheet, the cell sheet support had a larger contact angle, which was found to be a suitable contact angle for cell culture. These findings suggest that the large contact angle (surface tension) of the cell sheet support may have increased the cell sheet adhesion efficiency.

[0087] Example 4 A polymer solution was prepared by adding 1 mL of hexafluoro-2-propanol (Sigma-Aldrich) to 40 mg of dioxanone-co-glycolide copolymer (Poly(dioxanone-co-glycolide), (90:10); Sigma-Aldrich) and dissolving. The polymer solution was spread onto a glass plate using a pipette, left to dry at room temperature for about 1 hour, and then peeled off from the glass plate with tweezers to obtain the cell sheet support of Example 4.

[0088] The cell recovery rate, flexibility, and average thickness were evaluated in the same manner as above for the obtained cell sheet support of Example 4. The results were that the cell recovery rate was 78.5%, flexibility was a score of B, and the average thickness was 30 μm.

[0089] Example 5 The cell sheet support obtained in No. 1-2 of Example 1 was cut to the same size as the bottom of each well of a non-coated 96-well plate and placed on the bottom of each well. A 0.05% (w / v) solution of the temperature-responsive polymer (hereinafter also referred to as SCCBC) obtained above was added, left at room temperature for 2 hours, and washed with PBS solution. The cell sheet support with the temperature-responsive polymer attached was removed and transferred to a new well. Next, 1 x 10 mouse melanoma cell line B16 / BL6GFP was placed on the cell sheet support. 4 Cells were seeded at 1000 cells / well and cultured for 72 hours. 24, 48, and 72 hours after the start of culture, a WST-8 assay (cell counting kit-8 (CCK-8): Dojindo Laboratories) was performed to evaluate the cell proliferation rate. The results are shown in Figure 5 as SCCBC+. The results of cell culture without the cell sheet support served as a control.

[0090] Comparative Example Cell culture was carried out in the same manner as above, except that a cell sheet support to which no temperature-responsive polymer had been attached was placed in the well. The results are shown in Figure 5 as SCCBC-.

[0091] From Figure 5, it can be seen that cells grow well when cultured on a cell sheet support with a thermoresponsive polymer, which is a hydrophilic polymer, attached to the surface.

[0092] Example 6 On the cell culture supports with the temperature-responsive polymer attached to the surface prepared in the same manner as above, DMEM medium (supplemented with 10% FCS) was added as the medium, and mouse melanoma cell line B16 / BL6 GFP (green fluorescence) was added at 3 × 10 5 The cells were seeded at 2000 cells / dish and cultured at 37°C and 5% CO2 for 5 days until confluent, forming a cell sheet on the temperature-responsive polymer layer. The cell culture support on which the cell sheet was formed was heated at 40°C for 30 minutes, and the cell sheet was detached from the temperature-responsive polymer layer. After removing the medium, the cell sheet support was layered on the cell sheet and left to stand for 5 minutes, yielding cell sheet laminate A in which a cell sheet of a mouse melanoma cell line was adhered to the cell sheet support.

[0093] Cell sheet laminate B, in which a cell sheet of a human cervical cancer cell line was adhered to a cell sheet support, was obtained in the same manner as above, except that the human cervical cancer cell line HeLa tdTomato (red fluorescence) was used instead of the mouse melanoma cell line.

[0094] Cell sheet support A and cell sheet support B were layered with the cell sheet sides facing each other and cultured for three days to obtain a cell sheet laminate in which different cell sheets were layered. The adhesive surfaces of the two types of cell sheets in the obtained laminate of different cell sheets were observed using a confocal laser fluorescence microscope (LSM710, ZEIZZ). The results are shown in Figure 6, along with a schematic diagram of the laminate.

[0095] As shown in the schematic diagram of Figure 6, the laminate is composed of a cell sheet support 10, a cell sheet 20 of a mouse melanoma cell line, a cell sheet 30 of a human cervical cancer cell line, and another cell sheet support 10 stacked in this order. Figure 6 also shows that the two different types of cell sheets are adhered together as a laminated sheet, with various types of cells present at the contact surface between the sheets in a mixed state.

[0096] The disclosure of Japanese Patent Application No. 2021-023164 (filing date: February 17, 2021) is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

Claims

1. A cell sheet support comprising a first polymer containing a structural unit derived from p-dioxanone, which can be directly layered on a cell sheet formed by culture to adhere to the cell sheet and hold the cell sheet on the cell sheet support.

2. The cell sheet support according to claim 1 , further comprising a second polymer comprising at least one selected from the group consisting of polylactic acid, polyglycolic acid, polycaproic acid, and copolymers thereof.

3. The cell sheet support according to claim 2 , wherein the content of the first polymer relative to the total amount of the first polymer and the second polymer is 50% by mass or more.

4. The cell sheet support according to any one of claims 1 to 3, which is in the form of a sheet having an average thickness of 10 µm or more and 150 µm or less.

5. The cell sheet support according to any one of claims 1 to 4, which is used for transporting a cell sheet.

6. The cell sheet support according to any one of claims 1 to 4, which is used for stacking cell sheets.

7. A cell sheet laminate comprising the cell sheet support according to claim 1 and a cell sheet placed on the cell sheet support.

8. preparing a cell sheet by culturing cells on a temperature-responsive polymer layer; directly layering the cell sheet support according to any one of claims 1 to 4 on the prepared cell sheet to bring the cell sheet and the cell sheet support into contact with each other, thereby adhering the cell sheet to the cell sheet support; and peeling the cell sheet from the temperature-responsive polymer layer by a temperature change.

9. preparing a cell sheet by culturing cells on a temperature-responsive polymer layer; directly layering the cell sheet support according to any one of claims 1 to 4 on the prepared cell sheet to bring the cell sheet and the cell sheet support into contact with each other, thereby adhering the cell sheet to the cell sheet support; Peeling the cell sheet from the temperature-responsive polymer layer by changing the temperature; A method for transferring a cell sheet, comprising: contacting a cell sheet on a cell sheet support with a target site.

10. The method according to claim 8 or 9, wherein the temperature-responsive polymer is a copolymer containing a structural unit derived from a (meth)acrylate having an alkyl group having 14 to 22 carbon atoms and a structural unit derived from (meth)acrylic acid.

11. 11. The method of claim 8, wherein the temperature change comprises maintaining the cells at a temperature higher than the temperature at which they are cultured.

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