WO2021065970A1

WO2021065970A1 - Method for producing sheet-like material containing somatic cells

Info

Publication number: WO2021065970A1
Application number: PCT/JP2020/037057
Authority: WO
Inventors: 賢二大山; 文哉大橋
Original assignee: テルモ株式会社
Priority date: 2019-09-30
Filing date: 2020-09-30
Publication date: 2021-04-08
Also published as: JPWO2021065970A1

Abstract

The present invention addresses the problem of providing: a method for producing a sheet-like material containing somatic cells; a sheet-like material produced by the method; a kit for producing the sheet-like material; and a method for applying the sheet-like material. The problem can be solved by a method for producing a sheet-like material containing somatic cells, the method comprising the steps of: arranging cells on a substrate with gaps interposed between the cells; and filling gaps (a plurality of pores) between the cells with a gel to form a sheet-like material.

Description

Method for producing a sheet containing somatic cells

The present invention relates to a method for producing a sheet-like material containing somatic cells, a sheet-like material produced by the method, a kit for producing the sheet-like material, and a method for applying the sheet-like material.

In recent years, attempts have been made to transplant various cells for repairing damaged tissues. For example, use of fetal cardiomyocytes, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, iPS cells, etc. for repair of myocardial tissue damaged by ischemic heart disease such as angina and myocardial infarction. Has been attempted (Non-Patent Document 1).

As an example of such an attempt, the purpose is to transplant a cell structure formed by using a scaffold (Patent Document 1) or a sheet-shaped cell culture in which cells are formed into a sheet shape (Non-Patent Document 2). Cell preparations have been developed, some of which are in the stage of clinical application.
Gels have been used for a variety of purposes in the preparation of such cell preparations. For example, a gel is laminated on a sheet-shaped cell culture to improve operability (Patent Document 2), or a cardiomyocyte, smooth muscle, and fibroblast are embedded in a hydrogel and then cultured. Cardiopatch (Non-Patent Document 3) and the like.

Japanese Patent No. 4943844 Japanese Unexamined Patent Publication No. 2016-52272

The present invention provides a method for producing a sheet-like substance containing somatic cells, a sheet-like substance produced by the method, a kit for producing the sheet-like substance, and a method for applying the sheet-like substance. The purpose was to do.

When preparing a sheet-shaped cell culture, an operation of freezing or thawing the cells is performed, and these operations may reduce the cell adhesion ability. Moreover, even if these operations are not performed, the cell adhesion ability may decrease without knowing the cause. When cells in such a low adhesive state are used in the conventional method for producing a sheet-like cell culture, the cells form aggregates, and the formed aggregates are exfoliated from the substrate. As a result, uniform cell-cell adhesion between cells is not formed, and a sheet-like cell culture cannot be obtained.
The present inventors have found that a sheet containing cells can be produced even with cells having such a low adhesive ability, and based on such findings, further research has been carried out to complete the present invention. I came to let you.

That is, the present invention relates to the following.
[1] A method for producing a sheet-like substance containing somatic cells, which includes the following:
The step of placing the cell population on the substrate through the gap,
A step of filling the gaps between the cells with gel to form a sheet,
The method described above.
[2] The method according to [1], further comprising the step of adhering the cells to the substrate.
[3] The method according to [1] or [2], wherein the cell is in the form of a cell mass.
[4] Any one of [1] to [3], wherein the cell contains any one or more of hepatocytes, fibroblasts, myoblasts, pancreatic cells, renal cells, vascular endothelial cells, and corneal epithelial cells. The method described in one.

[5] The method according to any one of [1] to [4], wherein the cell is a myoblast.
[6] The method according to any one of [1] to [5], wherein the gel contains fibrin gel, gelatin or collagen which is in a solidified state at room temperature.
[7] The method according to any one of [1] to [6], wherein the gel is gelled by mixing the two liquids.
[8] The method according to any one of [1] to [7], wherein the base material is a base material having a stimulus-responsive material.

[9] A sheet-like substance containing somatic cells produced by the method according to any one of [1] to [8].
[10] A kit for producing a sheet-like substance containing the somatic cells according to [9].
The kit comprising cells, a substrate for arranging the cells, and a gel for forming the cells into a sheet.
[11] A method for treating a disease improved by application of a sheet-like substance containing somatic cells, wherein the sheet-like substance produced by the method according to any one of [1] to [8] is used. The method described above, comprising applying it to an object that requires it.

According to the method of the present invention, a sheet-like substance can be obtained even from cells in a state of low adhesiveness. In the Cardio patch described in Non-Patent Document 2, cells have entered the inside of the patch, whereas in the sheet-like material according to the present invention, the cells are arranged on a substrate and then a sheet is formed by gel. Therefore, the cells are localized on one side of the sheet-like material and are exposed on that side. Therefore, in the sheet-like substance according to the present invention, the cells can ingest oxygen, nutrients, etc. from the exposed surface, and the cytokines produced by those cells are sheet-like without being hindered by the gel. It can act directly and / or indirectly on the cells around the site of application.
In addition, unlike the conventional sheet-like cell culture, the sheet-like material according to the present invention does not shrink after exfoliation, so that a sheet-like material having the same size as the base material used for preparation can be obtained and has the same size. A sheet larger than the sheet-like cell culture prepared from the substrate can be obtained.

FIG. 1 is a photograph taken in Example 1 before treatment with fibrin gel. (A) is a photograph of the appearance, (B) is a photomicrograph having a magnification of 4 times, and (C) is a photomicrograph having a magnification of 40 times. FIG. 2 is a photograph taken after treatment with fibrin gel in Example 1. (A) is a photograph of the appearance, (B) is a photomicrograph having a magnification of 4 times, and (C) is a photomicrograph having a magnification of 40 times. FIG. 3 shows a photograph of what was obtained in Comparative Example 1. (A) is a photograph of the appearance, (B) is a photomicrograph having a magnification of 4 times, and (C) is a photomicrograph having a magnification of 40 times. FIG. 4 shows a photograph of what was obtained in Comparative Example 2. (A) is a photograph of the appearance, (B) is a photomicrograph having a magnification of 4 times, and (C) is a photomicrograph having a magnification of 40 times.

Hereinafter, the present invention will be described in detail.
One aspect of the present invention is a method for producing a sheet-like substance containing somatic cells, which is a step of arranging cells on a substrate through gaps, and filling the gaps between the cells with gel to form a sheet. It relates to a method including a step of shaping.

In the present invention, the "sheet-like substance" refers to a sheet-like cell. In one embodiment of the present invention, the sheet-like material contains cells and a gel, preferably does not contain a scaffold (support) other than the gel. Scaffolds may be used in the art to attach cells to and / or inside the scaffold to maintain the physical integrity of the sheet, eg, polyvinylidene fluoride (PVDF). Made of film and the like are known. The sheet-like material of the present invention can maintain its physical integrity without such a scaffold.

The cells contained in the sheet-like substance are not particularly limited as long as they can form the sheet-like substance, and are, for example, somatic cells. In a preferred embodiment, the cell contained in the sheet is a cell into which no gene has been introduced. In a more preferred embodiment, the cells contained in the sheet are somatic cells into which no gene has been introduced. The cells contained in the sheet-like substance are preferably adherent cells (adhesive cells). Adhesive cells include, for example, adherent somatic cells (eg, myocardial cells, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, renal cells, adrenal cells, root membrane cells, gingival cells, bone membrane cells, skin. Includes cells, synovial cells, chondrocytes, etc.) and stem cells (eg, tissue stem cells such as myoblasts, cardiac stem cells, embryonic stem cells, pluripotent stem cells, mesenchymal stem cells, etc.) and the like. Somatic cells may be differentiated from stem cells. Non-limiting examples of cells contained in the sheet form include, for example, myoblasts (eg, skeletal myoblasts), mesenchymal stem cells (eg, bone marrow, adipose tissue, peripheral blood, skin, hair roots, muscle tissue). , Endometrial, placenta, umbilical cord blood, etc.), myocardial cells, fibroblasts, heart stem cells, embryonic stem cells, synovial cells, cartilage cells, epithelial cells (eg, oral mucosal epithelial cells, retinal pigment epithelial cells) , Nasal mucosal epithelial cells, etc.), endothelial cells (eg, vascular endothelial cells, etc.), hepatocytes (eg, hepatic parenchymal cells, etc.), pancreatic cells (eg, pancreatic islet cells, etc.), renal cells, adrenal cells, root membrane cells, Examples include gingival cells, epithelial cells, skin cells and the like.

In the present disclosure, "myoblast" is a progenitor cell of striated muscle cell, and includes skeletal myoblast and myocardial blast (myocardial progenitor cell).
In the present disclosure, "skeletal myoblast" means a myoblast present in skeletal muscle. Skeletal myoblasts are well known in the art and can be prepared from skeletal muscle by any known method (eg, the method described in JP-A-2007-89442) or commercially. It is also available (eg Lonza, Cat # CC-2580). Skeletal myoblasts are not limited to markers such as, for example, CD56, α7 integrin, myosin heavy chain IIa, myosin heavy chain IIb, myosin heavy chain IId (IIx), MyoD, Myf5, Myf6, myogenin, desmin, PAX3. Can be identified by. In certain embodiments, skeletal myoblasts are CD56 positive. In a further specific embodiment, the skeletal myoblasts are CD56 positive and desmin positive. Skeletal myoblasts are any organism with skeletal muscle, including, but not limited to, humans, non-human primates, rodents (mouses, rats, hamsters, guinea pigs, etc.), rabbits, dogs, cats, pigs, etc. It may be derived from mammals such as horses, cows, goats, and sheep. In one embodiment, the skeletal myoblasts are mammalian skeletal myoblasts. In certain embodiments, the skeletal myoblasts are human skeletal myoblasts.
In the present invention, the cells are preferably hepatocytes, fibroblasts, myoblasts, pancreatic cells, renal cells, vascular endothelial cells, or corneal epithelial cells, more preferably myoblasts, and most preferably myoblasts. Skeletal myoblasts.

The cells constituting the cells contained in the sheet-like substance can be derived from any organism that can be treated with the sheet-like substance. Such organisms include, but are not limited to, for example, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents (eg, mice, rats, hamsters, guinea pigs, etc.), rabbits, etc. Is included. Further, the number of types of cells constituting the sheet-like substance is not particularly limited, and may be composed of only one type of cells, or may be composed of two or more types of cells. When there are two or more types of cells forming a sheet-like substance, the content ratio (purity) of the most abundant cells is 50% or more, preferably 60% or more, more preferably 70% or more at the end of the formation of the sheet-like substance. , More preferably 75% or more.

The cell may be a heterologous cell or an allogeneic cell. Here, "heterologous cell" means a cell derived from an organism of a species different from the recipient when the sheet-like substance is used for transplantation. For example, when the recipient is a human, cells derived from monkeys and pigs correspond to heterologous cells. In addition, "homogeneous cell" means a cell derived from an organism of the same species as the recipient. For example, when the recipient is a human, the human cell corresponds to an allogeneic cell. Allogeneic cells include autologous cells (also referred to as autologous cells or autologous cells), ie, recipient-derived cells and allogeneic non-autologous cells (also referred to as allogeneic cells). Autologous cells are preferred in the present disclosure because they do not cause rejection when transplanted. However, it is also possible to utilize heterologous cells and allogeneic non-autologous cells. When using heterologous cells or allogeneic non-autologous cells, immunosuppressive treatment may be required to suppress rejection. In the present specification, cells other than autologous cells, that is, allogeneic non-self-derived cells and allogeneic non-self-derived cells may be collectively referred to as non-autologous cells. In one aspect of the disclosure, the cell is an autologous cell or an allogeneic cell. In one aspect of the disclosure, the cell is an autologous cell. In another aspect of the disclosure, the cell is an allogeneic cell.

In the present invention, "arranging cells on a substrate through a gap" means that the cells are in contact with or adhered to the bottom surface on the substrate while the cells have gaps. Refers to doing. The cells need only have a gap in a part thereof, and does not mean that the cells are not in contact with each other. In the present invention, in the state in which the cells are arranged on the base material through the gap, for example, a plurality of island-shaped cell populations exist on the base material, and the island-shaped cell populations have gaps between the island-shaped cell populations. The state of being in is also included. In the present invention, the state in which one surface of the base material is continuously covered with cells is not included in the state in which the cells are arranged on the base material with a gap.
In one embodiment, the placement of the cells on the substrate is done by spontaneously precipitating the cells after seeding the cells. Spontaneous sedimentation is carried out, for example, by allowing to stand for 1 minute to 24 hours, preferably 5 minutes to 6 hours, and more preferably 5 minutes to 1 hour.
In one embodiment, the placement of the cells on the substrate is done by seeding the cells and then centrifuging. Centrifugation is performed by appropriately setting the centrifugal force, the time to centrifuge, and the temperature inside the centrifuge using a centrifuge. The centrifugal force is set to, for example, 10 G to 1000 G, preferably 50 G to 1000 G, and more preferably 50 G to 400 G. The time to centrifuge is set to, for example, 10 seconds to 2 hours, preferably 1 minute to 60 minutes, more preferably 1 to 15 minutes, and even more preferably 1 minute to 10 minutes. The in-flight temperature is set to, for example, 0 ° C. to 42 ° C., preferably 4 ° C. to 37 ° C.

In one embodiment, the placed cells are in contact with the substrate. In the present invention, the state in which the cell is in contact with the base material is the state in which the cell is in contact with the base material, but the cell and the base material are joined via the extracellular matrix produced by the cell. Refers to the state where it is not.
In the present invention, the contact between the cell and the substrate depends on various conditions, but for example, 5 minutes to 24 hours after seeding of the cell, for example, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes. About 40 minutes, about 50 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 It is produced by culturing for about 12 hours, about 18 hours, and about 24 hours.

In another embodiment, the placed cells are attached to the substrate. In the present invention, the state in which the cell is adhered to the substrate refers to the state in which the cell and the substrate are bonded via the extracellular matrix produced by the cell.
In one aspect, the method of the present invention further comprises the step of adhering cells to a substrate.
In the present invention, the adhesion between the cell and the substrate depends on various conditions, but for example, after seeding the cell, 24 hours or more, for example, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, It is produced by culturing for 60 hours, 66 hours, and 72 hours.

The medium used for culturing may be any known medium used for culturing cells. Such media is not limited to these, but is, for example, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM. , RITC80-7, DMEM / F12 and the like.

In one aspect of the invention, the cell is in the form of a cell mass. In the present invention, the "cell mass" refers to a cell mass in which two or more cells are closely adhered to each other to form a mass. In the present invention, the size of the cell mass is, for example, 10 μm to 1000 μm in diameter. It is preferably 50 μm to 500 μm in diameter, and more preferably 100 μm to 300 μm in diameter. In one aspect, the size of the cell mass does not have to be uniform. In the present invention, uniform cell mass means that, for example, the relative standard deviation with respect to the individual size of the cell mass measured by a cell analyzer or the like is less than 40%, preferably less than 30%, more preferably. Means less than 20%, and non-uniform cell mass size means that the relative standard deviation is 60% or more, preferably 50% or more, more preferably 45% or more. The cell mass is also called a spheroid.

Another aspect of the present invention is a method for producing a sheet-like substance containing somatic cells, in which a cell mass is placed on a substrate through a gap, and the gap between the cell masses is gelled. It relates to a method including a step of filling and forming a sheet.
In one aspect, the method according to the invention further comprises culturing the cells to form a cell mass prior to the step of placing the cell mass on the substrate through a gap.

Any known method can be used to form the cell mass, and the method is not limited to, for example, the inner surface is coated with the cell adhesion inhibitor, and a plurality of depressions are formed on the surface of the substrate. There is a method (Japanese Patent Application No. 2012-533946) of culturing using a base material which has and has a non-flat space between the recesses.
When the cell mass is seeded, there is a difference in the thickness of the cell portion seen from the side from a minimum of 10 μm to a maximum of 100 μm, and the difference is filled with a gel to form a sheet. Therefore, the thickness of the sheet itself is uniform regardless of the thickness of the cell portion, and even when formed from a cell mass, the sheet-like material can be easily handled.

In one embodiment, seeding to obtain the cells used in the present invention is about 1.0 × 10 ⁵ cells / cm ² to about 5.0 × 10 ⁷ cells / cm ² , about 3.0 × 10 ⁵ cells. / Cm ² to about 5.0 x 10 ⁷ pieces / cm ² , about 3.5 x 10 ⁵ pieces / cm ² to about 5.0 x 10 ⁷ pieces / cm ² , about 1.0 x 10 ⁶ pieces / cm ² to about 5.0 x 10 ⁷ pieces / cm ² , about 1.0 x 10 ⁵ pieces / cm ² to about 3.4 x 10 ⁶ pieces / cm ² , about 3.0 x 10 ⁵ pieces / cm ² to Approximately 3.4 x 10 ⁶ pcs / cm ² , Approximately 3.5 x 10 ⁵ pcs / cm ² to Approximately 3.4 x 10 ⁶ pcs / cm ² , Approximately 1.0 x 10 ⁶ pcs / cm ² to Approximately 3 .4 x 10 ⁶ pieces / cm ² , about 3.0 x 10 ⁵ pieces / cm ² to about 1.7 x 10 ⁶ pieces / cm ² , about 3.5 x 10 ⁵ pieces / cm ² to about 1.7 The density can be such as × 10 ⁶ pieces / cm ² , about 1.0 × 10 ⁶ pieces / cm ² to about 1.7 × 10 ⁶ pieces / cm ^2.

In the present invention, any gel used for forming a sheet-like substance can be used as long as it does not adversely affect the living body. Gels that do not adversely affect the living body include, but are not limited to, fibrin gel, gelatin, collagen that is solidified at room temperature, and sodium alginate. Room temperature refers to, for example, 15 ° C to 25 ° C. In the present invention, being in a solidified state at room temperature means having no fluidity at room temperature, for example, 15 ° C. to 25 ° C., and having a recoverable strength, and preferably the temperature-responsive substrate is used. It means that it has no fluidity and has recoverable strength at a reaction temperature, for example, a temperature of 20 to 25 ° C. In one embodiment, the gel is an in vivo degradable gel, which is degraded in vivo, absorbed into the body, metabolized and excreted.
In one embodiment, the gel is formed by mixing the two liquids. In one aspect, the gel is in a solidified state near room temperature. In one aspect, the gel does not show temperature reversibility. Such gels include, but are not limited to, fibrin gel obtained by mixing fibrinogen solution and thrombin solution, anti-adhesion agent obtained by mixing NHS-modified CM dextrin and trehalose aqueous solution with sodium carbonate and sodium hydrogen carbonate aqueous solution. Can be mentioned. These are commercially available, for example, for Bolheel (registered trademark) tissue bonding (Teijin Pharma Limited), Veriplast (registered trademark) tissue bonding (CSL Bering), and Adspray (registered trademark) (Termo). It is possible and can be used in the present invention. In the present invention, the gel is preferably fibrin gel, gelatin or collagen in a solidified state at room temperature, more preferably fibrin gel, gelatin or collagen in a solidified state at 20 to 25 ° C., and even more preferably fibrin. It is a gel.
In the present invention, the gel may serve as a support for preventing damage to the sheet-like material when the sheet-like material is peeled from the substrate and when the sheet-like material is moved for application.

In the present invention, the thickness of the sheet-like material is, for example, 10 μm to 2000 μm. It is preferably 10 μm to 500 μm, more preferably 50 μm to 200 μm.

In the present invention, the step of filling the gaps between the cells with gel to form a sheet is performed, for example, by gently adding the gel onto a substrate on which the cells are adhered to the surface. The amount added may be any amount, but is preferably an amount at which the sheet-like material has the above-mentioned thickness. In one aspect, the gel is gelled by mixing the two liquids, and any known method can be used as the method for gelling using such a gel. As such a method, for example, a method of adding a fibrinogen solution and a thrombin solution at the same time, or a method of dropping the fibrinogen solution onto cells and then spraying the thrombin solution to form a fibrin gel (Patent No. 6495603) is used. be able to.
In this step, the gaps (pores) that were present at the time of placement are filled with gel, and the cells are present through the gel on one side where the cells of the sheet are localized, and are continuously present over one side. Not.

In the present invention, the "base material" is not particularly limited as long as the cells can form a cell culture on the container, for example, a container of various materials, a solid or semi-solid surface in the container, and the like. Including. The container preferably has a structure / material that does not allow a liquid such as a culture solution to permeate. Such materials include, without limitation, for example, polyethylene, polypropylene, Teflon®, polyethylene terephthalate, polymethylmethacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, polydimethyl. Acrylamide, metals (eg, iron, stainless steel, aluminum, copper, brass) and the like can be mentioned. Also, the container preferably has at least one flat surface. Examples of such containers include, without limitation, cell culture dishes, cell culture bottles, and the like. Further, the container may have a solid or semi-solid surface inside the container. Examples of the solid surface include plates and containers of various materials as described above, and examples of the semi-solid surface include gels and soft polymer matrices. The base material may be produced using the above materials, or a commercially available material may be used. Preferred base materials include, without limitation, for example, base materials having an adhesive surface suitable for forming a sheet-like material. Specifically, a base material having a hydrophilic surface, for example, a base material coated with a hydrophilic compound such as corona discharge-treated polystyrene, collagen gel or hydrophilic polymer, and further, collagen, vitronectin, laminin. , Vitronectin, proteoglycan, glycosaminoglycan and other extracellular matrices, and base materials coated with cell adhesion factors such as cadoherin family, selectin family and integrin family on the surface. In addition, such a base material is commercially available (for example, Corning ™ TC-Treated Culture Dish, Corning, etc.).

The surface of the base material may be coated with a material whose physical properties change in response to a stimulus, for example, temperature or light. Such materials include, but are not limited to, for example, (meth) acrylamide compounds, N-alkyl substituted (meth) acrylamide derivatives (eg, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, etc. N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacryl (Amid, etc.), N, N-dialkyl-substituted (meth) acrylamide derivatives (eg, N, N-dimethyl (meth) acrylamide, N, N-ethylmethylacrylamide, N, N-diethylacrylamide, etc.), and having cyclic groups (eg, Meta) Acrylamide derivatives (eg 1- (1-oxo-2-propenyl) -pyrrolidine, 1- (1-oxo-2-propenyl) -piperidin, 4- (1-oxo-2-propenyl) -morpholin, 1 -(1-oxo-2-methyl-2-propenyl) -pyrrolidine, 1- (1-oxo-2-methyl-2-propenyl) -piperidin, 4- (1-oxo-2-methyl-2-propenyl) -A temperature-responsive material consisting of a homopolymer or copolymer of a vinyl ether derivative (eg, methylvinyl ether) or a homopolymer or copolymer of a vinyl ether derivative (eg, methylvinyl ether), a photoabsorbable polymer having an azobenzene group, a vinyl derivative of triphenylmethane leucohydrooxide and an acrylamide-based single amount. Known materials such as a copolymer with a body and a photoresponsive material such as N-isopropylacrylamide gel containing spirobenzopyran can be used (see, for example, JP-A-2-21186 and JP-A-2003-33177). ). By giving a predetermined stimulus to these materials, their physical characteristics, for example, hydrophilicity and hydrophobicity can be changed, and the exfoliation of the cell culture adhering on the material can be promoted. Culture dishes coated with a temperature-responsive material are commercially available (for example, UpCell® from CellSeed Corporation and Cepallet® from DIC Corporation), and these can be used in the present invention.

The base material may have various shapes, but is preferably flat. The area thereof is not particularly limited, but is typically about 1 cm ² to about 200 cm ² , preferably about 2 cm ² to about 100 cm ² , and more preferably about 3 cm ² to about 50 cm ² .

The base material may be coated (coated or coated) with serum. "Coated with serum" means a state in which serum components are attached to the surface of the base material. Such a state can be obtained without limitation, for example, by treating the substrate with serum. Treatment with serum involves contacting the serum with the substrate and, if necessary, incubating for a predetermined period of time.

As the serum, heterologous serum and allogeneic serum can be used. Heterologous serum means serum derived from an organism of a different species from its recipient when the sheet is used for transplantation. For example, when the recipient is human, serum derived from bovine or horse, for example, fetal bovine serum (FBS, FCS), calf serum (CS), horse serum (HS), etc. corresponds to heterologous serum. In addition, "homogeneous serum" means serum derived from an organism of the same species as the recipient. For example, if the recipient is human, human serum corresponds to allogeneic serum. Allogeneic sera include autologous sera (also referred to as autologous sera), that is, sera derived from the recipient and allogeneic sera derived from allogeneic individuals other than the recipient. In the present specification, sera other than autologous serum, that is, allogeneic serum and allogeneic serum may be collectively referred to as non-autologous serum.

Serum for coating the substrate can be commercially available or can be prepared by a conventional method from blood collected from a desired organism. Specific examples thereof include a method in which the collected blood is left at room temperature for about 20 minutes to about 60 minutes to coagulate, and the collected blood is centrifuged at about 1000 G to about 1200 G to collect a supernatant. ..

When incubating on a substrate, serum may be used as a stock solution or diluted. Dilution can be made in any medium, such as, but not limited to, water, saline, various buffers (eg, PBS, HBSS, etc.), various liquid media (eg, DMEM, MEM, F12, DME, RPMI1640, etc.). It can be performed in MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12, etc.). The dilution concentration is not particularly limited as long as the serum component can adhere to the substrate, and is, for example, about 0.5% to about 100% (v / v), preferably about 1% to about 60% (v / v). v), more preferably about 5% to about 40% (v / v).

The incubation time is also not particularly limited as long as the serum component can adhere to the substrate, for example, about 1 hour to about 72 hours, preferably about 4 hours to about 48 hours, more preferably about 5 hours to about 24 hours. The time, more preferably about 6 hours to about 12 hours. The incubation temperature is also not particularly limited as long as the serum component can adhere to the substrate, for example, at about 0 ° C. to about 60 ° C., preferably about 4 ° C. to about 45 ° C., more preferably room temperature to about 40 ° C. is there.

Serum may be discarded after incubation. As a serum disposal method, suction with a pipette or the like or a conventional liquid disposal method such as decantation can be used. In a preferred embodiment of the present invention, the substrate may be washed with a serum-free washing solution after serum disposal. The serum-free cleaning solution is not particularly limited as long as it is a liquid medium that does not contain serum and does not adversely affect the serum components adhering to the substrate. (Eg, PBS, HBSS, etc.), various liquid media (eg, DMEM, MEM, F12, DMEM / F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, etc.) can be used. Examples of the cleaning method include a conventional substrate cleaning method, for example, a method in which a serum-free cleaning solution is added onto the substrate, stirred for a predetermined time (for example, about 5 seconds to about 60 seconds), and then discarded without limitation. Can be used.

In the present invention, the base material may be coated with a growth factor. Here, "growth factor" means any substance that promotes cell growth as compared to its absence, eg, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblasts. Includes cell growth factor (FGF) and the like. In the substrate coating method, disposal method and washing method using growth factors, the dilution concentration during incubation is, for example, about 0.0001 μg / mL to about 1 μg / mL, preferably about 0.0005 μg / mL to about 0.05 μg. It is basically the same as serum except that it is / mL, more preferably about 0.001 μg / mL to about 0.01 μg / mL.

In the present invention, the base material may be coated with a steroid agent. Here, the "steroid agent" refers to a compound having a steroid nucleus that can adversely affect the living body such as adrenocortical insufficiency and Cushing's syndrome. Such compounds include, but are not limited to, for example, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone and the like. In the substrate coating method, disposal method and washing method using a steroid agent, the dilution concentration at the time of incubation is, for example, about 0.1 μg / mL to about 100 μg / mL, preferably about 0.4 μg / mL to about as dexamethasone. It is basically the same as serum except that it is 40 μg / mL, more preferably about 1 μg / mL to about 10 μg / mL.

The substrate may be coated with any one of serum, growth factors and steroids and any combination thereof, namely serum and growth factors, serum and steroids, serum and growth factors and steroids, or It may be coated with a combination of growth factors and steroids. When coating with a plurality of components, these components may be mixed and coated at the same time, or may be coated in separate steps.

The base material may be seeded with cells immediately after being coated with serum or the like, or may be stored after being coated and then seeded with cells. The coated substrate can be stored for a long period of time, for example, by keeping it at about 4 ° C. or lower, preferably about −20 ° C. or lower, more preferably about −80 ° C. or lower.

The seeding of cells on the substrate can be performed by any known method and conditions. The seeding of cells on a base material may be carried out, for example, by injecting a cell suspension in which cells are suspended in a culture solution into a base material (culture container). For injection of the cell suspension, an instrument suitable for the injection operation of the cell suspension, such as a dropper or a pipette, can be used.

In one embodiment, the medium may be removed prior to adding the gel to the cells attached to the substrate. In a further embodiment, after removing the medium, the cells adhered to the substrate may be washed with a washing solution. Any cleaning solution can be used as long as it does not destroy the adhesion between the cells and the substrate. Cleaning solutions that do not destroy the adhesion between cells and the substrate include, but are not limited to, Hanks equilibrium salt buffer, HEPES, DMEM, RPMI, Ham's F-12, and the like.
In one aspect, the method for making a sheet according to the invention can further include any known step for culturing cells. Examples of such steps include, but are not limited to, thawing frozen cells, adding lavage fluid to thawed cells, centrifuging cells to which lavage fluid has been added, and the like. Be done.

Another aspect of the invention contains somatic cells made by a method comprising arranging cells on a substrate through gaps, filling the gaps of the cells with gel to form a sheet. Regarding sheet-like objects. In the present invention, the somatic cells contained in the sheet-like material are preferably selected from the group including hepatocytes, fibroblasts, myoblasts, pancreatic cells, renal cells, vascular endothelial cells, and corneal epithelial cells. One or more types of cells. In the present invention, the somatic cells contained in the sheet-like material are more preferably myoblasts, and even more preferably skeletal myoblasts. In the present invention, the gel for forming the sheet-like material into a sheet-like material is preferably fibrin gel, gelatin or collagen in a solidified state at room temperature, and more preferably fibrin gel. In the present invention, the base material for arranging cells is preferably a base material having a stimulus-responsive material, and more preferably a base material having a temperature-responsive material. In one aspect of the invention, the cell is in the form of a cell mass.

Another aspect of the present invention relates to a kit for producing a sheet containing somatic cells, which comprises a cell, a substrate for arranging the cell, and a gel for forming the cell into a sheet. ..
The kit in the present invention is not limited to these, and may further include, for example, a medium, a washing solution, a buffer solution, a tube, an instrument used for cell culture, a shipping container, an instruction manual, and the like.

Examples of instruments used for cell culture include pipettes, cell strainers, and cell scrapers.
Examples of the instruction regarding the usage method include a usage manual, a medium on which information on the manufacturing method and the usage method is recorded, for example, a flexible disc, a CD, a DVD, a Blu-ray disc, a memory card, a USB memory, and the like.

Yet another aspect of the present invention is a method of treating a disease improved by application of a sheet-like substance containing somatic cells, which requires a sheet-like substance prepared by the method according to the present invention. Regarding methods including application to the subject.

Example 1. Preparation of sheet-like material containing somatic cells (1) Formation of cell mass Skeletal myoblasts (CD56 positive rate 85%) cryopreserved in a cell preservation solution (MCDB medium containing 10% DMSO) at 37 ° C. Thaw, dilute with 10% FBS / DMEM, centrifuge, remove supernatant, then ^{suspend 1 × 10 6} cells in 2 mL DMEM / F12 medium (manufactured by Gibco) containing 20% human serum. Then, the cells were seeded on a substrate having a diameter of 35 mm (EZSPERE (registered trademark), manufactured by AGC Technoglass Co., Ltd.). After seeding, the cells were cultured for 1 day in an incubator (BNA-121D, manufactured by ESPEC) set at 37 ° C. and 5% CO _2. After culturing, the substrate was removed from the incubator and the cell mass was collected.
^{(2) Arrangement of cell masses 1 × 10 6} cells / well of the cell masses prepared in (1) on a 48-well temperature-responsive substrate (UpCell®, 48-well multi-well, CS3001, manufactured by Cellseed). The cell mass was inoculated in an incubator set at 37 ° C. and 5% CO ₂ for 24 hours, and the cell mass was placed. The culture supernatant was then discarded and washed with 2 mL of Hanks balanced salt solution (HBSS (+), Cat No. 14025, manufactured by Life Technologies).
A photograph taken after the above processing is shown in FIG. (A) is a photograph of the appearance, (B) is a photomicrograph having a magnification of 4 times, and (C) is a photomicrograph having a magnification of 40 times.
(3) Treatment with gel The contents of vial 1 of 10 μl of fibrinogen solution (Veriplast (registered trademark) for tissue adhesion (manufactured by CSL Bering Co., Ltd.)) (fibrinogen lyophilized powder is added to the contents of vial 2) on the substrate after washing. (Fibrinogen solution) dissolved, fibrinogen concentration 80 mg / mL)) and 10 μl thrombin solution (Thrombin lyophilized powder) of vial 3 for tissue adhesion (CSL Bering Co., Ltd.) The contents of the vial 4 (thrombin solution) dissolved, thrombin concentration 300 units / mL)) were added dropwise, and the mixture was allowed to stand for about 5 minutes to form a fibrin gel. After fibrin gel formation, 2 mL of Hanks balanced salt solution (HBSS (+), Cat No. 14025, manufactured by Life Technologies) was added and washed 3 times to remove unreacted fibrinogen and thrombin. Then, the temperature-responsive material was allowed to stand at room temperature (20 to 25 ° C.) for 5 to 30 minutes for temperature treatment.
(4) Evaluation of sheet-like material FIG. 2 shows a photograph taken after the above processing. (A) is a photograph of the appearance, (B) is a photomicrograph having a magnification of 4 times, and (C) is a photomicrograph having a magnification of 40 times. It was confirmed that the sheet-like material was formed because it could be peeled off from the base material by pipetting.

Comparative Example 1
Instead of fibrin gel, a collagen solution cell matrix type IP (Nitta Gelatin) in a solidified state at 37 ° C. was used to prepare a sheet.
A cell mass was formed in the same manner as in (1) of Example 1 above, the cell mass was arranged in the same manner as in (2), 2 ml of the collagen solution was added, and the mixture was allowed to stand for 30 minutes. Then, the temperature-responsive material was allowed to stand at room temperature (20 to 25 ° C.) for 5 to 30 minutes for temperature treatment.
A photograph of what was obtained by the above processing is shown in FIG. (A) is a photograph of the appearance, (B) is a photomicrograph having a magnification of 4 times, and (C) is a photomicrograph having a magnification of 40 times. When the temperature was returned to room temperature, the collagen was dissolved and the gel state was not maintained. Further, when an attempt was made to peel the cell mass from the substrate by pipetting, the cell mass was peeled apart, and a sheet-like product in which the cell mass was formed into a sheet was not obtained.

Comparative Example 2
An attempt was made to prepare a sheet-like product without using a gel.
After forming a cell mass in the same manner as in (1) of Example 1 above and arranging the cell mass in the same manner as in (2), an additional 24 cells were placed in an incubator set at _{37 ° C. and 5% CO 2 without treatment with a gel.} Incubated for hours. Then, the temperature-responsive material was allowed to stand at room temperature (20 to 25 ° C.) for 5 to 30 minutes for temperature treatment.
A photograph of what was obtained by the above processing is shown in FIG. (A) is a photograph of the appearance, (B) is a photomicrograph having a magnification of 4 times, and (C) is a photomicrograph having a magnification of 40 times. When an attempt was made to peel the cell mass from the substrate by pipetting, the cell mass was separated into pieces, and a sheet-like product in which the cell mass was formed into a sheet was not obtained.

Claims

A method for producing a sheet containing somatic cells, which is described below.
The step of placing the cell population on the substrate through the gap,
A step of filling the gaps between the cells with gel to form a sheet,
The method described above.
The method of claim 1, further comprising the step of adhering the cells to the substrate.
The method according to claim 1 or 2, wherein the cell is in the form of a cell mass.
The method according to any one of claims 1 to 3, wherein the cell comprises any one or more of hepatocytes, fibroblasts, myoblasts, pancreatic cells, renal cells, vascular endothelial cells, and corneal epithelial cells. ..
The method according to any one of claims 1 to 4, wherein the cell is a myoblast.
The method according to any one of claims 1 to 5, wherein the gel contains fibrin gel, gelatin or collagen which is in a solidified state at room temperature.
The method according to any one of claims 1 to 6, wherein the gel is gelled by mixing the two liquids.
The method according to any one of claims 1 to 7, wherein the base material is a base material having a stimulus-responsive material.
A sheet-like substance containing somatic cells produced by the method according to any one of claims 1 to 8.
A kit for producing a sheet-like substance containing the somatic cells according to claim 9.
The kit comprising cells, a substrate for arranging the cells, and a gel for forming the cells into a sheet.
A method for treating a disease improved by application of a sheet-like substance containing somatic cells, which requires a sheet-like substance prepared by the method according to any one of claims 1 to 8. The method, which comprises applying to an object to be subjected to.