WO2020067434A1 - Method for cryopreserving cells - Google Patents

Abstract

The purpose of the present invention is to provide: a method that is for cryopreserving cells for transplantation and that can be suitably used in treatment of various diseases, particularly heart disease; a method for culturing the cells thawed after being cryopreserved; a production method for a transplant including a sheet-like cultured product containing the cells for transplantation; a transplant produced by the method; a composition and medical product containing the transplant; a method for treating a disease using the transplant; etc. This method is for cryopreserving cells and is characterized by freezing a cell suspension containing cells for transplantation on a culture base material. This method is for culturing cells for transplantation and is characterized by incubating a cell population in a medium containing a cryoprotectant.

Description

Cell cryopreservation method

The present disclosure relates to a method for cryopreserving cells for transplantation, a method for culturing the cells for transplantation, a method for producing a graft containing the cells for transplantation, and a method for the method that can be suitably used in the treatment of various diseases, particularly heart diseases. The present invention relates to a graft containing the sheet-shaped cell culture produced in 1., a composition and a medical product containing the graft, a method for treating a disease using the graft, and the like.

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

As a part of such an attempt, a cell structure formed using a scaffold and a sheet-shaped cell culture in which cells are formed in a sheet shape have been developed (Patent Document 1, Non-Patent Document 2).
For the application of sheet-shaped cell culture to treatment, use of cultured epidermis sheet for skin damage due to burns, use of corneal epithelial sheet-shaped cell culture for corneal injury, oral mucosal sheet for endoscopic resection of esophageal cancer Studies on the use of cell cultures are underway, and some of them are in the stage of clinical application.

In order to clinically use such a sheet cell culture, it must be aseptically manufactured and transported. However, at present, in order to use the sheet-shaped cell culture, etc., the sheet-shaped cell culture, etc. is used in a large-scale facility such as a cell preparation facility (CPC) that is aseptically managed in the medical institution used. At present, it is difficult to use the sheet-shaped cell culture or the like easily in a medical institution or the like that does not have such a facility without producing it aseptically.

JP-T 2007-528755

The present disclosure relates to a cryopreservation method for transplant cells, a method for culturing the cells thawed after cryopreservation, and a sheet-like cell culture containing the transplant cells, which can be suitably used in the treatment of various diseases, particularly heart diseases. It is an object of the present invention to provide a method for producing an implant containing the same, a graft produced by the method, a composition and a medical product containing the implant, a method for treating a disease using the implant, and the like.

As described above, in order to use a transplant containing transplanted cells such as a sheet-shaped cell culture, it is necessary to have a facility such as a CPC in a medical institution or the like. The present inventors have been conducting research to enable the use of a sheet-shaped cell culture or the like without having a facility such as CPC, and have transferred from cryopreservation of cells for transplantation to culture and production of transplants. It was conceived that if it could be realized in a closed container that could be used, it would be possible to easily use a transplant such as a sheet-shaped cell culture, but in such a closed container, some operations such as cell washing were extremely difficult. Faced the challenge of becoming complicated. While further research is being carried out to solve this problem, even if a sufficient amount of medium is added to the cell suspension obtained by thawing the cryopreserved cells and cultured, sufficient cell culture as a medical grade is obtained. Have been found, and as a result of further research, the present invention has been completed.

That is, the present invention relates to the following:
[1] A method for cryopreserving cells, comprising freezing a cell suspension containing cells for transplantation on a culture substrate.
[2] The method of [1], wherein the transplant cell is an adherent cell.
[3] The method of [1] or [2], wherein the transplant cell is a myoblast or a cardiomyocyte.
[4] The method of [1] to [3], wherein the culture substrate has a cell-adhesive culture surface.
[5] The method of [1] to [4], wherein the culture substrate is coated with a temperature-responsive material.
[6] The method of [1] to [3], wherein the culture substrate has a culture surface for spheroid formation.
[7] The method of [1] to [6], wherein the cell suspension contains a cryoprotectant.
[8] The method of [1] to [7], wherein the cell suspension contains 5 to 20% of a cryoprotectant.
[9] the cell suspension, the area of the culture substrate, present on the culture substrate at a density of ^{5μL / cm 2 ~ 1000μL / cm} 2, [1] any one of to [8] The method described in.
[10] The cells for transplantation are present in the cell suspension at a density of 7.5 × 10 ⁵ cells / cm ² to 3.0 × 10 ⁶ cells / cm ² with respect to the area of the culture substrate. The method of [1] to [9].
[11] A cryopreserved cell frozen on a cell culture substrate.
[12] A method for culturing cells for transplantation, which comprises incubating a cell population with a medium containing a cryoprotectant.
[13] The following steps;
(A) thawing a cell population cryopreserved on a culture substrate to obtain a cell suspension containing a cryoprotectant;
(B) adding a culture medium to the cell suspension obtained in (a) to dilute the cryoprotectant;
(C) incubating the cell suspension obtained in (b) on the culture substrate;
The method according to [12], comprising:
[14] The method of [12] or [13], wherein the cell population contains adherent cells.
[15] The method of [12] to [14], wherein the cell population contains myoblasts or cardiomyocytes.
[16] The method of [12] to [15], wherein the cryoprotectant is DMSO.
[17] The method of [13] to [16], wherein the culture substrate has a cell-adhesive culture surface.
[18] The method of [13] to [17], wherein the culture substrate is coated with a temperature-responsive material.
[19] The method of [13] to [16], wherein the culture substrate has a culture surface for spheroid formation.
[20] The method of [13] to [19], wherein all the steps are performed in a closed vessel.
[21] The following steps;
(A) thawing a cell population containing cells for transplantation cryopreserved on a culture substrate to obtain a cell suspension containing a cryoprotectant;
(B) adding a culture medium to the cell suspension obtained in (A) to dilute the cryoprotectant;
(C) culturing the cell suspension obtained in (B) on an explant forming culture on the culture substrate;
A method for producing an implant, comprising:
[22] The method of [21], wherein the transplant is a sheet-shaped cell culture.
[23] The method of [21] or [22], wherein the transplant cell is a myoblast or a cardiomyocyte.
[24] The cells for transplantation are present in the cell suspension at a density of 7.5 × 10 ⁵ cells / cm ² to 3.0 × 10 ⁶ cells / cm ² with respect to the area of the culture substrate. [21] The method according to [23].
[25] The method of [21] to [24], wherein all the steps are performed in a closed vessel.
[26] A storage container 1 enclosing a cell culture substrate and cryopreserved cells frozen on the cell culture substrate, and a storage container enclosing a cell culture medium that can be closedly connected to the storage container 1 2. An implant manufacturing kit comprising:

According to the present invention, cells for transplantation contained in a sheet-shaped cell culture or the like can be easily stored and used. For this reason, it is possible to aseptically produce and transport the graft in a closed container without requiring complicated procedures and equipment for aseptically producing and transporting the graft. It is expected to contribute to the spread of medical treatments used. In addition, a large facility or the like is not required in the production of such an implant, so that it is possible to significantly reduce the cost.

FIG. 1 is a graph showing the results of thawing and recovering cryopreserved cells. No significant difference was observed in the cell recovery rate when the cells were suspended in 200 µL or more of a cryopreservation solution (at a density of 1.0 × 10 ⁷ cells / mL or more). Viability did not differ significantly between suspensions of any density. FIG. 2 is a photograph of a sheet-shaped cell culture prepared by collecting and seeding cryopreserved cells of each density. In each case, sheets could be formed without any problem.

Hereinafter, the present disclosure will be described in detail.
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referred to herein are hereby incorporated by reference in their entirety. In the case of inconsistencies between the publication referred to in this specification and the description of this specification, the description of this specification shall control.

に お い て In the present disclosure, “transplant cell” means a cell to be transplanted into a living body. In the present disclosure, “graft” refers to a structure for transplantation into a living body, and particularly refers to a structure for transplantation containing the cell for transplantation as a component. The so-called suspension state, in which at least one state in which cells are adhered to each other in a transplant to form a certain shape as a whole, and each and every cell is present separately, is referred to as the present disclosure. Are not included in the “grafts” In a preferred embodiment, the implant is an implantable structure that does not include structures other than cells and cell-derived substances (eg, a scaffold). Examples of the graft in the present disclosure include, but are not limited to, a sheet-shaped cell culture, a spheroid, a cell aggregate, a cell suspension, a cell suspension containing fibrin gel, and a cell using a nanofiber. Cultures and the like are preferable, and a sheet cell culture or a spheroid is preferable, and a sheet cell culture is more preferable.

に お い て In the present disclosure, the “sheet-shaped cell culture” refers to a cell in which cells are connected to each other to form a sheet. In the present disclosure, “spheroid” refers to a cell in which cells are connected to each other to form a substantially spherical shape. The cells may be connected to each other directly (including via a cellular element such as an adhesion molecule) and / or via an intermediary substance. The intervening substance is not particularly limited as long as it is a substance capable of at least physically (mechanically) connecting cells, and examples thereof include an extracellular matrix. The intervening substance is preferably derived from cells, particularly from cells constituting a sheet-shaped cell culture or spheroid. Cells are at least physically (mechanically) linked, but may be further functionally linked, eg, chemically or electrically. The sheet-shaped cell culture may be composed of one cell layer (single layer) or composed of two or more cell layers (laminate (multilayer), for example, two or three layers, Four layers, five layers, six layers, etc.). Further, the sheet-shaped cell culture may have a three-dimensional structure having a thickness exceeding the thickness of one cell without the cells showing a clear layer structure. For example, in a vertical cross section of a sheet-shaped cell culture, a plurality of cells are arranged in a non-uniform (eg, mosaic) vertical direction without being uniformly arranged in a horizontal direction. Is also good.

細胞 The cells (transplantation cells) constituting a graft such as a sheet-shaped cell culture are not particularly limited as long as they can form a graft, and include, for example, adherent cells (adherent cells). Adherent cells include, for example, adherent somatic cells (eg, cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin Cells, synovial cells, chondrocytes, etc.) and stem cells (eg, tissue stem cells such as myoblasts and cardiac stem cells, embryonic stem cells, pluripotent stem cells such as iPS (induced pluripotent stem) cells, mesenchymal stem cells, etc.) Including. The somatic cells may be stem cells, particularly those differentiated from iPS cells (iPS cell-derived adherent cells). Non-limiting examples of cells constituting the graft include, for example, myoblasts (eg, skeletal myoblasts), mesenchymal stem cells (eg, bone marrow, adipose tissue, peripheral blood, skin, hair root, muscle tissue, Endometrium, placenta, cord blood, etc.), cardiomyocytes, fibroblasts, cardiac stem cells, embryonic stem cells, iPS cells, synovial cells, chondrocytes, 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.), kidney cells, adrenal cells, periodontal ligament Cells, gingival cells, periosteal cells, skin cells and the like. Non-limiting examples of iPS cell-derived adherent cells include iPS cell-derived cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, renal cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells , Skin cells, synovial cells, chondrocytes and the like.

In the present disclosure, “myoblasts” are precursor cells of striated muscle cells, and include skeletal myoblasts and myoblasts.
In the present disclosure, “skeletal myoblasts” means myoblasts 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 available. It is also available (eg, Lonza, Cat # CC-2580). Skeletal myoblasts include, but are not limited to, markers such as CD56, α7 integrin, myosin heavy chain IIa, myosin heavy chain IIb, myosin heavy chain IId (IIx), MyoD, Myf5, Myf6, myogenin, desmin, PAX3, and the like. Can be identified by In certain embodiments, the skeletal myoblasts are CD56 positive. In a more particular embodiment, the skeletal myoblasts are CD56 positive and desmin positive. Skeletal myoblasts include any organism having skeletal muscle, including, but not limited to, humans, non-human primates, rodents (such as mice, rats, hamsters, guinea pigs), rabbits, dogs, cats, pigs, It may be derived from mammals such as horses, cows, goats and sheep. In one aspect, the skeletal myoblast is a mammalian skeletal myoblast. In certain embodiments, the skeletal myoblast is a human skeletal myoblast.

In the present disclosure, “cardioblast” refers to myoblasts present in the heart muscle. Cardioblasts are well known in the art and can be identified by a marker such as Isll. Myocardial blasts are any organisms with myocardium, including, but not limited to, humans, non-human primates, rodents (mouse, rat, hamster, guinea pig, etc.), rabbits, dogs, cats, pigs, horses, It may be derived from mammals such as cows, goats and sheep. In one embodiment, the cardiac myoblast is a mammalian cardiac myoblast. In certain embodiments, the cardiac myoblast is a human cardiac myoblast.
In the present disclosure, “cardiomyocyte” means a cell having the characteristics of a cardiomyocyte, and the characteristics of the cardiomyocyte include, but are not limited to, for example, the expression of a cardiomyocyte marker, the presence of an autonomous beat, and the like . Non-limiting examples of cardiomyocyte markers include, for example, c-TNT (cardiac troponin T), CD172a (alias SIRPA or SHPS-1), KDR (alias CD309, FLK1 or VEGFR2), PDGFRA, EMILIN2, VCAM and the like. . Preferred examples of the cardiomyocytes include cardiomyocytes derived from iPS cells.

細胞 The cells constituting the graft can be derived from any organism that can be treated with the graft. Such organisms include, without limitation, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents (eg, mice, rats, hamsters, guinea pigs, etc.), rabbits, etc. Is included. The number of types of cells constituting the graft is not particularly limited, and may be composed of only one type of cells, or may be a type using two or more types of cells. When there are two or more types of cells forming the graft, 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 graft formation. It is preferably at least 75%.

シ ー ト The sheet-shaped cell culture of the present disclosure preferably does not include a scaffold (support). Scaffolds are sometimes used in the art to attach cells on and / or to their surfaces and maintain the physical integrity of sheet cell cultures, such as polyvinylidene difluoride ( Although PVDF) membranes and the like are known, the sheet-shaped cell culture of the present disclosure can maintain its physical integrity without such a scaffold. Further, the sheet-shaped cell culture of the present disclosure preferably includes only a substance derived from cells (such as an extracellular matrix) constituting the sheet-shaped cell culture, and does not include other substances.

The cell may be a cell derived from a different species or a cell derived from the same species. As used herein, “heterologous cell” means a cell derived from an organism of a different species from the recipient when a sheet-shaped cell culture is used for transplantation. For example, when the recipient is a human, cells derived from monkeys and pigs correspond to xenogeneic cells. "Allogeneic cell" means a cell derived from an organism of the same species as the recipient. For example, when the recipient is human, human cells correspond to cells derived from the same species. Allogeneic cells include autologous cells (also called autologous cells or autologous cells), that is, cells derived from the recipient and allogeneic non-autologous cells (also called allogeneic cells). Autologous cells are preferred in the present disclosure because rejection does not occur even when transplanted. However, it is also possible to use xenogeneic cells or allogeneic non-autologous cells. When xenogeneic cells or allogeneic non-autologous cells are used, immunosuppressive treatment may be necessary to suppress rejection. In this specification, cells other than autologous cells, that is, non-autologous cells of the same species as cells of xenogeneic origin may be collectively referred to as non-autologous cells. In one embodiment of the present disclosure, the cells are autologous cells or allogeneic cells. In one embodiment of the present disclosure, the cell is an autologous cell (including an autologous cell derived from an autologous iPS cell and an autologous differentiation-inducing cell obtained by inducing differentiation of an autologous iPS cell). In another aspect of the present disclosure, the cells are allogeneic cells (including allogeneic cells derived from allogeneic iPS cells, and allogeneic differentiation-inducing cells obtained by inducing differentiation of allogeneic iPS cells).

A sheet-shaped cell culture can be produced by any known method (for example, refer to Patent Document 1, Patent Document 2, JP-A-2010-081829, JP-A-2011-110368, etc.). A method for producing a sheet-shaped cell culture typically involves seeding cells on a culture substrate, forming the seeded cells into a sheet, and peeling the formed sheet-shaped cell culture from the culture substrate. Includes but is not limited to steps. Prior to the step of seeding the cells on the culture substrate, a step of freezing the cells and a step of thawing the cells may be performed. Further, a step of washing the cells may be performed after the step of thawing the cells. Each of these steps can be performed by any known technique suitable for producing a sheet-shaped cell culture. The production method of the present disclosure may include a step of producing a sheet-shaped cell culture, in which case, the step of producing the sheet-shaped cell culture is a step according to the method of producing the sheet-shaped cell culture as a sub-step. Or one or more of the following. In one embodiment, the method does not include the step of growing the cells after thawing the cells and before seeding the cells on a culture substrate.

Spheroids can be produced by any known method (for example, see Japanese Patent No. 5523830). The method for producing a spheroid typically includes seeding cells on a culture substrate having a concave portion, spheroidizing the seeded cells, and obtaining the formed spheroids from the culture substrate. However, the present invention is not limited to this. For example, examples of the spheroid include a graft in which 1,000 cardiomyocytes derived from pluripotent stem cells are formed in a spherical shape having a diameter of 0.2 mm each.

The culture substrate is not particularly limited as long as cells can form a cell culture thereon, and includes, for example, containers of various materials and / or shapes, a solid or semi-solid surface in the container, and the like. . The container is preferably made of a structure / material that does not allow the passage of a liquid such as a culture solution. Examples of such a material include, but are not limited to, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, and polydimethyl. Acrylamide, metal (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 a container include, without limitation, a culture container having a bottom surface formed of a culture substrate capable of forming a cell culture and a liquid impermeable side surface. Specific examples of such culture vessels include, but are not limited to, cell culture dishes, cell culture bottles, and the like. The bottom of the container may be transparent or opaque. If the bottom surface of the container is transparent, observation and counting of cells can be performed from the back side of the container. Further, the container may have a solid or semi-solid surface inside. Examples of the solid surface include plates and containers made of various materials as described above, and examples of the semi-solid surface include a gel and a soft polymer matrix. The culture substrate may be prepared using the above materials, or a commercially available substrate may be used.
Preferred culture substrates include, but are not limited to, for example, a substrate having an adhesive surface suitable for forming a sheet-shaped cell culture, and a substrate having a low adhesive surface suitable for forming a spheroid. And / or a substrate having a uniform well-like structure. Specifically, in the case of the formation of a sheet-shaped cell culture, for example, polystyrene subjected to corona discharge treatment, a substrate coated on its surface with a hydrophilic compound such as a collagen gel or a hydrophilic polymer, further, collagen And extracellular matrices such as fibronectin, laminin, vitronectin, proteoglycan, and glycosaminoglycan, and substrates coated on the surface with cell adhesion factors such as cadherin family, selectin family, and integrin family. Further, such substrates are commercially available ^{(e.g., Corning (R) TC-Treated} Culture Dish, Corning , etc.). In the case of spheroid formation, for example, temperature-responsive gel (commercial name: meviol gel) obtained by crosslinking soft agar, poly (N-isopropylacrylamide) (PIPAAm) with polyethylene glycol (PEG), polyhydroxyethyl methacrylate ( A substrate coated with a non-cell-adhesive compound such as a hydrogel such as poly (HEMA) or 2-methacryloyloxyethylphosphorhoscholine (MPC) polymer and / or a substrate having a uniform uneven structure on the surface. Can be Such substrates are also commercially available (e.g., EZSPHERE ^(R), etc.). The culture substrate may be entirely or partially transparent or opaque.

The culture substrate may be coated on its surface with a material whose 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, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfuryl methacryl Amide), N, N-dialkyl-substituted (meth) acrylamide derivatives (eg, N, N-dimethyl (meth) acrylamide, N, N-ethylmethylacrylamide, N, N-diethyl (Meth) acrylamide derivatives having a cyclic group (eg, 1- (1-oxo-2-propenyl) -pyrrolidine, 1- (1-oxo-2-propenyl) -piperidine, 4- (1-oxo) -2-propenyl) -morpholine, 1- (1-oxo-2-methyl-2-propenyl) -pyrrolidine, 1- (1-oxo-2-methyl-2-propenyl) -piperidine, 4- (1-oxo -2-methyl-2-propenyl) -morpholine or a homopolymer or copolymer of a vinyl ether derivative (eg, methyl vinyl ether), a light-absorbing polymer having an azobenzene group, triphenylmethane leucohydro A copolymer of a vinyl derivative of an oxide and an acrylamide monomer, and spirobenzopyra Can be used to include N- and isopropyl acrylamide gels known, such as photoresponsive materials (e.g., JP-A-2-211865, see JP-2003-33177). By applying a predetermined stimulus to these materials, their physical properties, for example, hydrophilicity or hydrophobicity, can be changed, and the detachment of the cell culture adhered on the materials can be promoted. Culture dishes coated with a temperature-responsive materials are commercially available (e.g., UpCell of CellSeed Inc. ^(R)), they can be used in the production method of the present disclosure.

The culture substrate may be in various shapes. The area is not particularly limited, but may be, for example, about 1 cm ² to about 200 cm ² , about 2 cm ² to about 100 cm ² , about 3 cm ² to about 50 cm ² , and the like. For example, a circular culture dish having a diameter of 10 cm is used as a culture substrate. In this case, the area is 56.7 cm ² . The culture surface may be flat or may have an uneven structure. In the case of having an uneven structure, it is preferable to have a uniform uneven structure.

The culture substrate may be coated (coated or coated) with serum. By using a culture substrate coated with serum, a higher-density sheet-shaped cell culture can be formed. “Coated with serum” means a state in which serum components are attached to the surface of the culture substrate. Such a state is not limited, and can be obtained, for example, by treating a culture substrate with serum. The treatment with serum includes contacting the serum with a culture substrate and, if necessary, incubating for a predetermined period.

As serum, heterologous serum and / or allogeneic serum can be used. Xenogeneic serum means serum derived from an organism of a different species from the recipient when a sheet-shaped cell culture is used for transplantation. For example, when the recipient is a human, serum derived from cattle or horse, such as fetal calf serum (FBS, FCS), calf serum (CS), horse serum (HS), etc., corresponds to the heterologous serum. Further, “homogeneous serum” means serum derived from an organism of the same species as the recipient. For example, when the recipient is human, human serum corresponds to allogeneic serum. Allogeneic serum includes autologous serum (also called autologous serum), ie, serum from the recipient and allogeneic serum from an individual other than the recipient. In the present specification, sera other than autologous serum, that is, heterologous serum and allogeneic serum may be collectively referred to as non-self serum.
Serum for coating the culture substrate is commercially available or can be prepared by a routine method from blood collected from a desired organism. Specifically, for example, a method of leaving the collected blood at room temperature for about 20 minutes to about 60 minutes to coagulate, centrifuging the blood at about 1000 × g to about 1200 × g, and collecting the supernatant And the like.

場合 When incubating on a culture substrate, the serum may be used as a stock solution or diluted. Dilution can be performed in any medium, such as, but not limited to, water, saline, various buffers (eg, PBS, HBSS, etc.), various liquid media (eg, DMEM, MEM, F12, DMEM / F12, DME, RPMI 1640, MCDB (MCDB 102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC 80-7, etc. can be used. The dilution concentration is not particularly limited as long as the serum component can adhere to the culture substrate. For example, about 0.5% to about 100% (v / v), preferably about 1% to about 60% (v / V), more preferably from about 5% to about 40% (v / v).

The incubation time is not particularly limited as long as the serum component can adhere to the culture substrate. For example, about 1 hour to about 72 hours, preferably about 2 hours to about 48 hours, more preferably about 2 hours to about 48 hours. 24 hours, more preferably about 2 hours to about 12 hours. The incubation temperature is not particularly limited as long as the serum component can adhere to the culture substrate, and is, for example, about 0 ° C. to about 60 ° C., preferably about 4 ° C. to about 45 ° C., and more preferably room temperature to about 40 ° C. It is.

血清 The serum may be discarded after incubation. As a method of discarding the serum, a conventional liquid discarding method such as suction with a pipette or decantation can be used. In a preferred embodiment of the present disclosure, after discarding the serum, the culture substrate may be washed with a serum-free washing solution. The serum-free washing solution is not particularly limited as long as it is a liquid medium that does not contain serum and does not adversely affect serum components attached to the culture substrate, and includes, for example, without limitation, water, saline, and various buffers. Liquid (eg, PBS, HBSS, etc.), various liquid culture media (eg, DMEM, MEM, F12, DMEM / F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15 , SkBM, RITC80-7, etc.). As a washing method, a conventional culture substrate washing method, for example, without limitation, a method of adding a serum-free washing solution onto a culture substrate, stirring for a predetermined time (for example, about 5 seconds to about 60 seconds), and then discarding Etc. can be used.

に お い て In the present disclosure, the culture substrate may be coated with a growth factor. Here, “growth factor” means any substance that promotes cell proliferation compared to the absence thereof, for example, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast Cell growth factor (FGF) and the like. In the method of coating, discarding, and washing the culture substrate with the growth factor, the dilution concentration during the incubation is, for example, about 0.0001 μg / mL to about 1 μg / mL, preferably about 0.0005 μg / mL to about 0.5 μg / mL. It is basically the same as serum except that it is from 05 μg / mL, more preferably from about 0.001 μg / mL to about 0.01 μg / mL.

に お い て In the present disclosure, the culture substrate may be coated with a steroid agent. Here, the “steroid agent” refers to a compound having a steroid nucleus that can have an adverse effect on the living body such as adrenocortical dysfunction and Cushing's syndrome. Such compounds include, but are not limited to, for example, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone, and the like. In the method of coating, discarding, and washing the culture substrate with 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, as dexamethasone. It is basically the same as serum except that it is about 40 μg / mL, more preferably about 1 μg / mL to about 10 μg / mL.

The culture substrate may be coated with any one of serum, growth factor and steroid, or any combination thereof, ie, serum and growth factor, serum and steroid, serum and growth factor and steroid, Alternatively, it may be coated with a combination of a growth factor and a steroid. When coating with a plurality of components, these components may be mixed and coated simultaneously, or may be coated in separate steps.

<1> Cryopreservation method of the present disclosure
One aspect of the present disclosure relates to a method for cryopreserving cells for transplantation. The cryopreservation method of the present disclosure is characterized by freezing a cell suspension containing cells for transplantation on a culture substrate. Conventionally, when cells are cryopreserved, the cell suspension is sealed in a cryopreservation container such as a cryopreservation vial and cryopreserved by freezing it. Instead, the cells for transplantation are cryopreserved by freezing the cell suspension directly on the culture substrate. Therefore, the cryopreserved cells produced by the cryopreservation method of the present disclosure, typically, cryopreserved cells frozen on a cell culture substrate are also encompassed by the presently disclosed invention.

方法 The method of freezing the cell suspension may be any method known in the art, such as immersion in liquid nitrogen or cooling with a program freezer. The time until the cell suspension is frozen is not particularly limited, but it is preferable that the cell damage is small and the state of the cell does not change. For example, when cryopreserving adherent cells on a cell-adhesive culture substrate, it is preferable to freeze the adherent cells before they start to adhere to the culture substrate. Therefore, the time from the start to the end of freezing is not limited to this, for example, within about 24 hours, within about 12 hours, within about 10 hours, within about 8 hours, within about 6 hours, within about 5 hours, It can be within about 4 hours, within about 3 hours, within about 2 hours, within about 1 hour, within about 30 minutes, and the like.

The cells cryopreserved by the method of the present disclosure are not particularly limited as long as they are cells to be used for transplantation, and any cells for transplantation can be used. The cells for transplantation are as described in detail above. In a preferred embodiment, the transplant cells are adherent cells, and in a more preferred embodiment, the transplant cells are myoblasts or cardiomyocytes.
As a medium for suspending cells to be cryopreserved, any medium may be used as long as it is used for cryopreservation of cells in the art, and is not limited thereto. For example, DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDB 102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, basal medium such as DMEM / F12, Hank's balanced salt solution (HBSS), Earl's balanced salt solution (EBSS), a buffer such as a phosphate buffer (PBS), and the like.

移植 The transplant cells cryopreserved by the method of the present disclosure are then thawed and used as they are for culture. Therefore, the culture substrate used in the method of the present disclosure is not particularly limited as long as it is a culture substrate that can withstand freezing treatment and can suitably culture cells for transplantation. For example, if the transplant cell is an adherent cell, the culture substrate preferably has a cell-adhesive culture surface. Further, the culture substrate may vary depending on the use of the cells for transplantation. For example, for the purpose of producing a sheet-shaped cell culture from cells for transplantation, the culture substrate preferably has a culture surface coated with a cell-adhesive, preferably temperature-responsive material, and forms spheroids. If so, the culture substrate is preferably a culture surface having a culture surface for spheroid formation, for example, a culture surface having a low-adhesion surface and / or a uniform well-like structure. A person skilled in the art can select a culture substrate having appropriate properties in view of the cryopreserved cells for transplantation and its use.

凍結 When the cells for transplantation are cryopreserved by the method of the present disclosure, they may be cryopreserved together with a support, a scaffold, and the like (hereinafter, collectively referred to as “support etc.”). Accordingly, the cell suspension of the cells for transplantation may include a support or the like. Such a support or the like may be any as long as it is advantageous for forming an implant. The material of the support or the like is preferably a material that does not have an adverse effect when implanted in a living body, more preferably a biodegradable material, such as polylactic acid and fibrin gel. Therefore, examples of such a support include films and supports made of biodegradable polymers such as polylactic acid, polyglycolic acid, and polycaprolactone, and supports made of biological components such as fibrin gel.

(4) The cell suspension cryopreserved on the culture substrate preferably contains a cryoprotectant to prevent frost damage. Cryoprotective agents that can be used in the cryopreservation method of the present disclosure include those commonly used in the art, as long as they do not contain manufacturing process impurities such as serum in view of preservation of cells for transplantation. Examples thereof include, but are not limited to, dimethyl sulfoxide (DMSO), polyhydric alcohols such as glycerol, saccharides such as trehalose, and polyamino acids such as polylysine. The content of the cryoprotectant is not particularly limited as long as the cells can be protected from damage during freezing and does not adversely affect the cells, and is not limited thereto. For example, about 1 to 20%, about 5% -15%, about 7-12%, and the like.

The amount of the cell suspension to be cryopreserved is not particularly limited as long as it can be suitably frozen on the culture substrate. If the amount of the cell suspension is too large, the freezing takes a long time and frost damage is likely to occur. Therefore, an appropriate amount or more of the cell suspension is not preferable. The amount of the cell suspension is preferably about 5 to 1000 μL / cm ² , more preferably about 10 to 300 μL / cm ² , based on the area of the culture substrate.

The amount of cells to be cryopreserved can vary depending on the size of the culture substrate, and can be determined in view of the purpose of culture after thawing. When the cells are subjected to growth culture after thawing, the amount of cells in the cell suspension is, for example, about 7.5 × 10 ⁵ cells / cm ² to 3.0 × 10 ⁶ cells based on the area of the culture substrate. / Cm ² .

に お い て In one embodiment, the cryopreserved cells are thawed and then subjected to transplant formation culture. In the present disclosure, “graft formation culture” means a culture for forming cultured cells as a transplant, and particularly when the transplant is a sheet-shaped cell culture, is referred to as “sheet culture”. Sheeting of cells can be performed by any known technique and conditions. Non-limiting examples of such a method are described in, for example, JP-A-2010-081829, JP-A-2010-226991, JP-A-2011-110368, JP-A-2011-172925, WO 2014/185517, and the like. It is believed that cell graft formation (eg, sheeting) is achieved by cells adhering to each other via intercellular adhesion mechanisms, such as adhesion molecules and extracellular matrix. Therefore, graft formation culture can be achieved, for example, by culturing cells under conditions that form cell-cell adhesion. Such conditions may be any as long as they can form cell-cell adhesion, but usually, cell-cell adhesion can be formed under the same conditions as general cell culture conditions. Such conditions include, for example, culturing at about 37 ° C., 5% CO 2. The cultivation can be performed under normal pressure (atmospheric pressure, non-pressurized). In the explant formation culture (sheet culture), the cells do not necessarily need to proliferate since cell-cell adhesion only needs to be formed. In a preferred embodiment, the explant formation culture (sheet culture) is performed without growing the cells.

When the cryopreserved cells are thawed and then subjected to explant formation culture, the amount of cryopreserved cells (ie, cells for transplantation) is not limited to this, but may be, for example, based on the area of the culture surface of the culture substrate. , About 5.0 × 10 ⁵ / cm ² to about 1.0 × 10 ⁷ / cm ² , about 5.0 × 10 ⁵ / cm ² to about 5.0 × 10 ⁶ / cm ² , about 5.0 × 10 ⁵ / cm ² to about 3.0 × 10 ⁶ / cm ² , about 1.0 × 10 ⁶ / cm ² to about 1.0 × 10 ⁷ / cm ² , about 1. 0 × 10 ⁶ / cm ² to about 5.0 × 10 ⁶ / cm ² , about 1.0 × 10 ⁶ / cm ² to about 3.0 × 10 ⁶ / cm ² , about 1.5 × 10 ⁶ / cm ² to about 1.0 × 10 ⁷ / cm ² , about 1.5 × 10 ⁶ / cm ² to about 5.0 × 10 ⁶ / cm ² , about 1.5 × 10 ⁶ Pieces / cm ² to about 3.0 × 10 ⁶ / cm ² , about 2.0 × 10 ⁶ / cm ² to about 1.0 × 10 ⁷ / cm ² , about 2.0 × 10 ⁶ / cm ² to about 5. It may be 0 × 10 ⁶ / cm ² , about 2.0 × 10 ⁶ / cm ² to about 3.0 × 10 ⁶ / cm ² , and the like. In one preferred embodiment, from about 7.5 × 10 ⁵ / cm ² to 3.0 × 10 ⁶ / cm ² , and in another preferred embodiment, from about 1.76 × 10 ⁶ / cm ² It is about 2.33 × 10 ⁶ pieces / cm ² .

The volume of the cell suspension is not particularly limited as long as it is a dose capable of suspending the above cell amount, but if it is too large, the size of the culture substrate also needs to be relatively large, so that it is not too large. preferable. Non-limiting examples of cell suspension volumes include about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL , About 23 mL, about 24 mL, about 25 mL, about 26 mL, about 27 mL, about 28 mL, about 29 mL, about 30 mL, and the like.

容量 The capacity of the culture substrate for cryopreserving the cells is not particularly limited as long as the cells can be accommodated, but is preferably larger than the capacity of the cell suspension in view of the subsequent addition of the medium. In one aspect, the volume of the culture substrate can be about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times the cell suspension.

凍結 The cryopreserved cells of the present disclosure may be subjected to culturing as it is on a culture substrate after thawing. In the case of culturing as it is after thawing, the medium used for culturing is added to the cell suspension obtained by thawing, and then used for culturing. The details of the culture are as described in the culture method below. In a preferred embodiment, the step of culturing the explant does not include a step of washing the cells (replacement of the cell culture medium) after thawing the frozen cells and before culturing the explant.

<2> Culture method of the present disclosure
One aspect of the present disclosure relates to a method for culturing cells for transplantation, which comprises incubating cells with a medium containing a cryoprotectant. In the culture method of the present disclosure, the cryoprotectant and the cells for transplantation are as described in detail in <1> above. Such a culture method can be typically performed after thawing cells cryopreserved by the cryopreservation method described in <1> above.

Hereinafter, the present invention will be described in detail by taking as an example the case of culturing cells cryopreserved by the cryopreservation method <1>.
The culture method of the present disclosure, in one exemplary embodiment, includes the following steps:
(A) thawing the cell population cryopreserved on the culture substrate to obtain a cell suspension containing a cryoprotectant;
(B) adding a culture medium to the cell suspension obtained in (a) to dilute the cryoprotectant; and
(C) a step of incubating the cell suspension obtained in (b) on the culture substrate.

に お い て In step (a), thaw the cryopreserved cells cryopreserved on the culture substrate. Thawing may be performed by any method known in the art, as long as the method does not cause undue damage to the cryopreserved cells.For example, a water bath, a hot water bath, spontaneous thawing, and rapid addition of a heated medium may be used. It can be carried out by a method such as thawing. By such a step, a cell suspension dispersed in the cryopreservation solution can be obtained.

In step (b), a culture medium is added to the cell suspension obtained in step (a). Thereby, the cryoprotectant contained in the cell suspension obtained in (a) is diluted. The culture medium is not particularly limited as long as it is commonly used in the art, and examples thereof include physiological saline, various physiological buffers (eg, PBS, HBSS, etc.), various cell culture base media. For example, a device based on the above may be used. Examples of such a basal medium include, but are not limited to, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12 and the like. Many of these basal media are commercially available and their compositions are also known. The basal medium may be used with its standard composition (for example, as it is commercially available), or its composition may be appropriately changed depending on the cell type and cell conditions. Therefore, the basal medium used in the present invention is not limited to those having a known composition, and includes those in which one or more components have been added, removed, increased or reduced in weight. The graft forming medium may contain additives such as normal serum (eg, bovine serum such as fetal bovine serum, horse serum, human serum, etc.) and various growth factors (eg, FGF, EGF, VEGF, HGF, etc.). Preferably, when the sheet-shaped cell culture is produced under xeno-free conditions, it is particularly preferable that the cell-free cell culture does not contain heterologous serum such as bovine serum and horse serum. The present disclosure is characterized in that graft forming culture is performed using a graft forming medium containing a cell adhesive component, whereby a high quality graft can be formed even when the graft forming medium is serum-free. It is effective. Thus, in one preferred embodiment, the graft forming medium does not contain serum.

The amount of the medium to be added is not particularly limited as long as the cryoprotectant can be diluted to a concentration sufficient for culturing the cryopreserved cells. For example, when about 7.5 × 10 ⁵ cells / cm ² to about 3.0 × 10 ⁶ cells / cm ² are cryopreserved in a circular culture dish having a diameter of 10 cm, the amount of medium to be added is about 5 It can be up to 30 mL, and in one preferred embodiment it can be 5-10 mL. The dilution ratio of the cryopreservation solution can be 2 to 100 times, and in a preferred embodiment, 5 to 15 times.

に お い て In step (c), the cell suspension to which the culture medium has been added in (b) is incubated on a culture substrate. The culturing in the culturing method of the present disclosure is not limited to culturing for growing general cells, but also, for example, explant forming culture for forming a transplant from a cell population (typically, sheet culture). A culture in which the cell number does not substantially change is also included.

培養 The culture method of the present disclosure can be used for any cell population, but in a preferred embodiment, the cell population to be cultured includes cells for transplantation. The cells for transplantation used in the culture method of the present disclosure are as described in detail above. Thus, in one preferred embodiment, the cell population comprises adherent cells, and in one more preferred embodiment, the cell population comprises myoblasts or cardiomyocytes.

In the culture method of the present disclosure, a medium (cell suspension) containing a cryoprotectant may be added to the culture medium, or a culture medium may be added to the medium containing the cryoprotectant. Typically, as described above, a culture medium is added to a medium (cell suspension) containing a cryoprotectant. The cryoprotectant is as described in detail in <1> above. In one preferred embodiment, the cryoprotectant is DMSO.

培養 In the culture method of the present disclosure, typically, a cell population cryopreserved by the method <1> is used as it is for culture. Therefore, the culture substrate used in the culture method of the present disclosure is the same as that described in detail in <1> above. That is, the culture substrate used in the culture method of the present disclosure has a cell-adhesive culture surface in a preferred embodiment, and has a culture surface coated with a temperature-responsive material in a more preferred embodiment. In another preferred embodiment, the culture substrate has a culture surface for spheroid formation. Such culture substrates are known in the art, as described above, and are commercially available.

As mentioned above, the incubation may be a substantially unchanged cell number incubation, such incubation being typically an explant forming culture. Therefore, in one particularly preferred embodiment of the present disclosure, step (c) is a step of culturing explants. If step (c) is a graft forming culture, the resulting cell culture will be a graft. Accordingly, one preferred embodiment of the present disclosure comprises the following steps:
(A) a step of thawing a cell population containing cells for transplant cryopreserved on a culture substrate to obtain a cell suspension containing a cryoprotectant;
(B) adding a culture medium to the cell suspension obtained in (A);
(C) a step of explant-culturing the cell suspension obtained in (B) on the culture substrate;
And a method for producing an implant.

In the method for producing an implant of the present disclosure, steps (A) and (B) are as described in detail for steps (a) and (b) in the culture method.
The explant formation culture in the step (C) is typically a sheet culture. The details of the sheet culture are as described above. Therefore, in one preferred embodiment of the method for producing an implant of the present disclosure, the implant is a sheet-shaped cell culture. The transplant formation culture in step (C) may be a culture for forming spheroids. After the transplant formation culture in the step (C), the method may further include a step of further diluting the cryoprotectant in the transplant formation culture or a washing step for removing the cryoprotectant from the transplant formation culture. .

に お い て In such a method for producing a graft, the cell for transplantation is not particularly limited as long as it can form a graft, and is typically an adherent cell. In one preferred embodiment, the cells for transplantation are myoblasts or cardiomyocytes. The cardiomyocytes may be iPS cell-derived cardiomyocytes.

In culturing to form a transplant, particularly a sheet-shaped cell culture, the cells have a high density, for example, a density that reaches confluence, that is, the degree that it is assumed that the cells cover the entire adhesion surface of the culture container when seeded. It is preferably present at a density of The "density to reach confluence" is typically the density at which cells are expected to contact each other, the density at which contact inhibition occurs, or the density at which cell growth is substantially stopped by contact inhibition, or It can be more. Although the density is not limited to this, for example, about 5.0 × 10 ⁵ / cm ² to about 1.0 × 10 ⁷ / cm ² , about 5.0 × 10 ⁵ / cm ² About 5.0 × 10 ⁶ pieces / cm ² , about 5.0 × 10 ⁵ pieces / cm ² to about 3.0 × 10 ⁶ pieces / cm ² , about 1.0 × 10 ⁶ pieces / cm ² 1.0 × 10 ⁷ / cm ² , about 1.0 × 10 ⁶ / cm ² to about 5.0 × 10 ⁶ / cm ² , about 1.0 × 10 ⁶ / cm ² to about 3. 0 × 10 ⁶ / cm ² , about 1.5 × 10 ⁶ / cm ² to about 1.0 × 10 ⁷ / cm ² , about 1.5 × 10 ⁶ / cm ² to about 5.0 × 10 ⁶ pieces / cm ² , about 1.5 × 10 ⁶ pieces / cm ² to about 3.0 × 10 ⁶ pieces / cm ² , about 2.0 × 10 ⁶ pieces / cm ² to about 1.0 × 10 ⁷ Pieces / cm ² , about 2.0 × 10 ⁶ pieces / cm ² to about 5.0 × 10 ⁶ / cm ² , about 2.0 × 10 ⁶ / cm ² to about 3.0 × 10 ⁶ / cm ^{2 and the} like. In one preferred embodiment, from about 7.5 × 10 ⁵ / cm ² to 3.0 × 10 ⁶ / cm ² , and in another preferred embodiment, from about 1.76 × 10 ⁶ / cm ² It is about 2.33 × 10 ⁶ pieces / cm ² .

培養 In the culture method and the method for producing a transplant of the present disclosure, as described above, typically, cells cryopreserved by the cryopreservation method described in detail in <1> above are used. Therefore, the culture method and the method for producing a transplant of the present disclosure may include the cryopreservation method described in the above <1> before step (a) or step (A).

In one preferred embodiment, in the culture method and the method for producing a transplant of the present disclosure, at least some of the steps can be performed in a closed container. Typically, the cell population cryopreserved by the method of <1> is aseptically enclosed in a closed system container, and the cryopreserved cell population is thawed in a closed system container. A culture method or a method for producing an explant can be performed. Therefore, preferably, in the culture method and the method for producing a transplant of the present disclosure, all the steps are performed in a closed vessel.

<3> Implant of the present disclosure
Another aspect of the present disclosure relates to an implant manufactured by the manufacturing method of the present disclosure. The implants of the present disclosure are useful for treating diseases that are ameliorated by the application of the implants, such as various diseases associated with tissue abnormalities. Thus, in one aspect, the implant of the present disclosure is for use in treating a disease ameliorated by the application of the implant, particularly a disease associated with tissue abnormalities. Except for having a higher mechanical strength than the conventional graft, the graft of the present disclosure has properties similar to those of the constituent cells, so that at least the conventional myoblast or fibroblast is used. The present invention can be applied to a tissue or a disease that can be treated with a transplant including the same. The tissues to be treated include, but are not limited to, for example, myocardium, cornea, retina, esophagus, skin, joints, cartilage, liver, pancreas, gingiva, kidney, thyroid, skeletal muscle, middle ear, bone marrow, stomach, Gastrointestinal tract such as the small intestine, duodenum, and large intestine. Examples of the disease to be treated include, but are not limited to, heart disease (eg, myocardial injury (myocardial infarction, cardiac trauma), cardiomyopathy, etc.), corneal disease (eg, corneal epithelial stem cell exhaustion, cornea) Injury (thermal / chemical erosion), corneal ulcer, corneal opacity, corneal perforation, corneal scar, Stevens-Johnson syndrome, ocular pemphigus, etc., retinal diseases (eg, retinitis pigmentosa, age-related macular degeneration, etc.) Esophageal disease (eg, prevention of esophageal inflammation / stenosis after esophageal surgery (removal of esophageal cancer)), skin disease (eg, skin damage (trauma, burn), etc.), joint disease (eg, osteoarthritis, etc.) Cartilage disease (eg, cartilage damage), liver disease (eg, chronic liver disease), pancreatic disease (eg, diabetes), dental disease (eg, periodontal disease), kidney disease (eg, renal failure) , Renal anemia, kidney Osteodystrophy, etc.), thyroid disease (eg, hypothyroidism, etc.), muscular disease (eg, muscle damage, myositis, etc.), middle ear disease (eg, otitis media, etc.), bone marrow disease (eg, leukemia, poor regeneration) Anemia, immunodeficiency disease, etc.). The usefulness of the graft of the present disclosure for the above-mentioned diseases is described in, for example, Patent Document 1, Non-patent Document 1, Tanaka et al., J Gastroenterol. 2013; 48 (9): 1081-9. I have. The implants of the present disclosure can also be fragmented to an injectable size and injected at the site of need for treatment to achieve greater efficacy than injection with a single cell suspension (Wang et al., Cardiovasc Res. 2008; 77 (3): 515-24). Thus, such uses are also possible for the implant of the present disclosure.

<4> Treatment method of the present disclosure
Another aspect of the present disclosure relates to a method of treating a disease in a subject in need thereof, comprising applying an effective amount of an implant produced by the method of the present disclosure to the subject in need thereof. The disease to be treated is as described above.

に お い て In the present disclosure, the term “treatment” is intended to include all types of medically acceptable prophylactic and / or therapeutic interventions, such as for the cure, temporary remission or prevention of disease. For example, the term "treatment" includes medically acceptable treatments for a variety of purposes, including slowing or stopping the progression of a disease associated with tissue abnormalities, regressing or eliminating lesions, preventing the onset of the disease or preventing its recurrence, and the like. Involve interventions.

In the treatment method of the present disclosure, a component that enhances the survival, engraftment, and / or function of a graft, and other active components that are useful for treating a target disease are used in combination with the graft or the like of the present disclosure. be able to.

処置 The treatment method of the present disclosure may further include a step of manufacturing the implant of the present disclosure according to the manufacturing method of the present disclosure. The treatment method of the present disclosure is a source of cells (eg, skin cells, blood cells, etc., when using iPS cells) or a source of cells for producing a graft from a subject prior to the step of producing the graft. The method may further include a step of collecting a tissue (for example, skin tissue, blood or the like when using iPS cells). In one embodiment, the subject from whom the cells or tissue from which the cells are to be sourced is harvested is the same individual as the subject to whom a cell culture, composition, or explant is administered. In another embodiment, the subject from whom the cells or tissue from which the cells are to be sourced is harvested is a homologous distinct body from the subject to be administered, such as a cell culture, composition, or implant. In another embodiment, the subject from which the cells or the tissue from which the cells are sourced is harvested is an individual that is heterogeneous to the subject receiving the administration, such as a graft.

In the present disclosure, an effective amount is, for example, an amount capable of suppressing the onset or recurrence of a disease, reducing symptoms, or delaying or stopping the progress (for example, the size, weight, and number of sheet-shaped cell cultures). And preferably an amount that prevents the onset and recurrence of the disease or cures the disease. Also preferred is an amount that does not cause adverse effects beyond the benefit of administration. Such an amount can be appropriately determined, for example, by a test in a laboratory animal such as a mouse, a rat, a dog or a pig, or a disease model animal, and such a test method is well known to those skilled in the art. In addition, the size of a tissue lesion to be treated can be an important index for determining an effective amount.

Examples of the administration method include intravenous administration, intramuscular administration, intraosseous administration, intrathecal administration, and direct application to tissues. The frequency of administration is typically once per treatment, but multiple administrations are possible if the desired effect is not obtained. When applied to a tissue, the cell culture, the composition, the sheet-shaped cell culture, or the like of the present invention may be fixed to a target tissue by a locking means such as a suture or staple.

<5> Kit of the present disclosure
One aspect of the present disclosure relates to an implant manufacturing kit for manufacturing an implant using the cryopreservation method and the culture method (method for manufacturing an implant) of the present disclosure. The implant manufacturing kit of the present disclosure includes a storage container 1 in which a cell culture substrate and a cryopreserved cell frozen on the cell culture substrate are enclosed, and a cell culture, which can be closed and connected to the storage container 1. And a storage container 2 in which a culture medium is enclosed. Hereinafter, the kit of the present disclosure and the method of using the same will be described in detail by taking, as an example, the case of producing a sheet-shaped cell culture containing skeletal myoblasts.

キ ッ ト The kit of the present disclosure has a storage container 1 and a storage container 2, and these containers have a structure that can be closed and connected. The term “closeably connectable” means that both containers are connected as one closed system while maintaining the closeability in each container. Therefore, both containers can be connected and detached while maintaining sterility.

In the storage container 1, a cell culture substrate (for example, a culture dish coated with a temperature-responsive material) and cells for transplantation (for example, skeletal myoblasts) frozen and stored on the culture substrate are stored in the storage container 2. A medium in which cells for transplantation can be cultured (for example, a medium that can be used for sheet culture, such as DMEM / F12) is aseptically enclosed. When the cells for transplantation are cryopreserved, the cells may be cryopreserved on the culture substrate and then aseptically sealed in the storage container 1 or may be sterilely sealed in the storage container 1 (cells). The cells for transplantation on the culture dish) may be frozen together with the storage container.

場合 When using the kit of the present disclosure, first, thaw the cryopreserved cells. Thereby, the cell suspension is thawed on the cell culture substrate (in the cell culture dish). Such cell suspensions typically include a cryoprotectant. Next, the storage container 1 and the storage container 2 are connected in a closed manner, and the medium aseptically sealed in the storage container 2 is added to the cell culture substrate in the storage container 1. As a result, the cell culture substrate is in a state in which the cell population is suspended in the medium containing the cryoprotectant. Next, the suspension is transplanted together with the storage container to form a graft (for example, a sheet culture) to obtain a graft such as a sheet-shaped cell culture. In this way, it is possible to form a graft aseptically.

部 材 As the cryoprotectant, medium, culture substrate, and other members and methods used in the kit of the present disclosure, those described in detail above can be used.

The present disclosure will be described in more detail with reference to the following examples, which show specific examples of the present disclosure, and the present disclosure is not limited to these.

Example 1: Preparation of sheet cell culture
The skeletal myoblasts (including fibroblasts) prepared from human skeletal muscle by a conventional method were each contained in 600, 300, 200, 100, and 50 μL of MCDB131 medium containing 10% DMSO so that 2 × 10 ⁷ cells were contained. It was suspended and stored frozen at -150 ° C. The frozen cells were thawed in warm water at 37 ° C. to obtain a cell suspension, and then diluted by adding 10 mL of DMEM / F12 medium containing 20% human serum (Thermo Fisher Scientific Inc.), followed by temperature responsiveness. The cells were seeded on a culture dish (UpCell ^(R) 3.5 cm, cell seed). At that time, 10 μL of the cell suspension was sampled, stained with trypan blue, and then subjected to cell counting. Thereafter, sheeting culture was performed at 37 ° C. and 5% CO ₂ for 12 to 26 hours. After culturing into a sheet, the medium was removed, 1000 μL of cooled HBSS (+) (Thermo Fisher Scientific Inc.) was added, the mixture was allowed to stand for 10 minutes, and then gently pipetted to completely detach the sheet-shaped cell culture. I let it.

The results are shown in FIG. 1 and FIG.
If the amount of the cell cryopreservation solution was 200 μL or more (that is, the density was 1.0 × 10 ⁷ cells / mL or less), there was a tendency that the recovery rate did not decrease. In addition, since the sheets could be formed under all the conditions, it was found that the sheets could be formed even if the cryopreservation liquid was contained.

According to the method and the kit of the present disclosure, an explant such as a sheet-shaped cell culture that can withstand medical grade can be efficiently produced. In particular, the use of the kit of the present disclosure makes it possible to aseptically transport the graft material and produce the graft aseptically even in a medical institution without a large facility such as a CPC. It is expected to contribute to the spread of regenerative medicine using pieces.

Claims (11)

1. (4) A method for cryopreserving cells, comprising freezing a cell suspension containing cells for transplantation on a culture substrate.
2. 方法 The method according to claim 1, wherein the cells for transplantation are adherent cells.
3. The method according to claim 1 or 2, wherein the cells for transplantation are myoblasts or cardiomyocytes.
4. (4) The method according to any one of (1) to (3), wherein the culture substrate has a cell-adhesive culture surface.
5. (5) The method according to any one of (1) to (4), wherein the culture substrate is coated with a temperature-responsive material.
6. The method according to any one of claims 1 to 3, wherein the culture substrate has a culture surface for spheroid formation.
7. 方法 The method according to any one of claims 1 to 6, wherein the cell suspension contains a cryoprotectant.
8. The method according to any one of claims 1 to 7, wherein the cell suspension comprises 5 to 20% of a cryoprotectant.
9. Cell suspension, the area of the culture substrate, present on the culture substrate at a density of ^{5μL / cm 2 ~ 1000μL / cm} 2, The method according to any one of claims 1-8.
10. The cell for transplantation is present in the cell suspension at a density of 7.5 × 10 ⁵ cells / cm ² to 3.0 × 10 ⁶ cells / cm ² with respect to the area of the culture substrate. 10. The method according to any one of claims 9 to 9.
11. (4) Cryopreserved cells frozen on a cell culture substrate.

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