JP2016096732A - Production method of sheet-like cell culture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sheet-like cell culture with high activities, such as cytokine production ability, and to provide a production method thereof.SOLUTION: The invention relates to a production method of a sheet-like cell culture comprising thawing frozen cells, and forming a sheet-like cell culture; to a sheet-like cell culture with high activity produced by the production method; and to a treatment method of diseases associated with an aberrant tissue by the sheet-like cell culture.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing a highly active sheet-like cell culture, a sheet-like cell culture produced by the production method, a composition containing the sheet-like cell culture, a graft and a medical product, and the sheet-like cell. The present invention relates to a method for treating a disease using a culture.

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

As part of such attempts, cell structures formed using scaffolds and sheet-shaped cell cultures in which cells are formed into sheets have been developed (Patent Documents 1 to 5, Non-Patent Document 2). .
For the application of sheet cell culture to the treatment, use of cultured epidermis sheet for skin damage caused by burns, use of corneal epithelial sheet cell culture for corneal injury, oral mucosa sheet for endoscopic resection of esophageal cancer Studies such as the use of cell cultures are ongoing.

  In order for a sheet-like cell culture to be engrafted in a transplanted tissue and perform a desired function, it is necessary to supply oxygen, nutrients, and the like to the culture. For this reason, HGF (Hepatocyte) having an angiogenic action is required. It is known that the action of various cytokines such as growth factor) and VEGF (Vascular endothelial growth factor) is useful (Patent Document 1). Therefore, if the ability to produce these cytokines by the sheet-like cell culture can be improved, it is considered possible to increase the engraftment rate and functionality of the sheet-like cell culture. In addition, since these cytokines are known to be useful for tissue regeneration (Non-patent Document 3), the cytokines produced from the sheet-like cell culture act on the tissue of the transplant destination, so that the tissue It is also possible to promote the regeneration of However, there is no known low-cost and simple method for improving the cytokine-producing ability of the sheet-like cell culture.

Special Table 2007-528755 WO 2006/080434 Publication JP 2005-229869 JP 2010-81829 JP 2010-226991

Haraguchi et al., Stem Cells Transl Med. 2012 Feb; 1 (2): 136-41 Ohashi et al., Transplant Proc. 2011 Nov; 43 (9): 3188-91 Nakagami et al., J Atheroscler Thromb. 2006 Apr; 13 (2): 77-81

  The present invention uses a sheet-shaped cell culture having high activity such as cytokine-producing ability, a production method thereof, a composition containing the sheet-shaped cell culture, a graft and a medical product, and the sheet-shaped cell culture. It aims at providing the treatment method of a disease.

  While the present inventors are diligently researching to solve the above-mentioned problems, by producing a step of freezing and thawing the cells when producing the sheet-like cell culture, the cytokine production of the sheet-like cell culture is achieved. The present invention has been completed by finding that the activity such as the ability can be enhanced.

That is, the present invention relates to the following.
(1) A method for producing a sheet-shaped cell culture, comprising a step of thawing frozen mesenchymal stem cells and a step of forming a sheet-shaped cell culture on a culture substrate using the thawed mesenchymal stem cells. .
(2) The method according to (1) above, wherein the method does not include a step of growing cells between the step of thawing mesenchymal stem cells and the step of forming a sheet-like cell culture.
(3) The method according to (1) or (2) above, wherein the culture substrate is coated with serum.
(4) The method according to any one of (1) to (3) above, wherein the step of forming a sheet-shaped cell culture is performed in a cell culture medium containing serum.
(5) The method according to any one of (1) to (4) above, which comprises a step of freezing mesenchymal stem cells before the step of thawing mesenchymal stem cells.
(6) The method according to (5) above, comprising a step of proliferating mesenchymal stem cells before the step of freezing mesenchymal stem cells.

(7) Any of the above (1) to (6), comprising a step of washing the mesenchymal stem cells after the step of thawing the mesenchymal stem cells and before the step of forming the sheet-like cell culture The method as described in one.
(8) Cytokine-producing ability and engraftment from a sheet-shaped cell culture produced by a method comprising a step of proliferating cells between the step of thawing mesenchymal stem cells and the step of forming a sheet-shaped cell culture The method according to any one of (2) to (7) above, wherein a sheet-like cell culture having a high activity selected from the group consisting of a potency, a blood vessel induction ability and a tissue regeneration ability is obtained.
(9) The method according to (8) above, wherein the cytokine is selected from the group consisting of HGF and VEGF.
(10) The method according to any one of (1) to (9) above, wherein the cell is an autologous cell.
(11) A sheet-like mesenchymal stem cell culture produced by the method according to any one of (1) to (10) above.
(12) A composition comprising the sheet-like mesenchymal stem cell culture according to (11) above.

(13) A method for treating a disease associated with a tissue abnormality in a subject, comprising an effective amount of the sheet-like mesenchymal stem cell culture according to (11) or the composition according to (12), Administering to a subject in need thereof.
(14) The tissue according to (13), wherein the tissue is selected from the group consisting of heart muscle, cornea, retina, esophagus, skin, joint, cartilage, liver, pancreas, gingiva, kidney, thyroid, skeletal muscle, and middle ear. Method.
(15) The disease comprises heart disease, corneal disease, retinal disease, esophageal disease, skin disease, joint disease, cartilage disease, liver disease, pancreatic disease, dental disease, kidney disease, thyroid disease, muscle disease and middle ear disease. The method according to (13) above, which is selected from the group.
(16) A frozen mesenchymal stem cell for use in the method according to any one of (1) to (10) above.
(17) A culture substrate for use in the method according to any one of (1) to (10) above.
(18) Serum for use in the method according to any one of (3) to (10) above.

  The sheet-shaped cell culture produced by the method of the present invention has high activities such as cytokine production ability, and is excellent in engraftment in the tissue and subsequent survival, functionality, function sustainability, etc. It is possible to perform an efficient treatment. Furthermore, when the cytokine produced by the sheet-shaped cell culture produced by the method of the present invention acts on the tissue of the transplant destination, it becomes possible to promote the regeneration of the tissue. In addition, the freezing / thawing operation in the present invention has a high affinity with the conventional method for producing a sheet-shaped cell culture, and the labor and cost are small. Therefore, the production method of the present invention is suitable for the production of a sheet-shaped cell culture. Can be widely used.

It is the graph which showed the density | concentration of VEGF and HGF secreted in the culture solution in the sheet | seat culture | cultivation step. It is the graph which showed the density | concentration of VEGF secreted in the culture solution by the sheet-like cell culture after manufacture. It is the graph which showed the density | concentration of HGF which the sheet-like cell culture after manufacture secreted in the culture solution. 6 is a photograph showing a sheet-like mesenchymal stem cell culture produced by the production method of Example 5. FIG. Sheet-like cell cultures are found in the central four consecutive wells.

  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 referenced herein are hereby incorporated by reference in their entirety.

  One aspect of the present invention is a method for producing a sheet-shaped cell culture (hereinafter referred to as “the present invention”) comprising the steps of thawing frozen cells and forming the sheet-shaped cell culture using the thawed cells. Sometimes referred to as "manufacturing method"). The production method of the present invention may include a step of freezing the cells before the step of thawing the frozen cells. Although not wishing to be bound by a specific theory, in the production method of the present invention, by thawing frozen cells, weakly active cells are removed, while strongly active cells remain, and sheet-like cell culture is performed. It is thought that the activity as a whole increases.

  The cells in the production method of the present invention are not particularly limited as long as they can form a sheet-like cell culture, 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 cells, gingival cells, periosteum cells, skin Cells, synovial cells, chondrocytes, etc.) and stem cells (eg tissue stem cells such as myoblasts, cardiac stem cells, embryonic stem cells, pluripotent stem cells such as iPS (induced pluripotent stem) cells, mesenchymal stem cells, etc.) Etc. Somatic cells may be differentiated from stem cells, particularly iPS cells. Non-limiting examples of cells in the production method of the present invention include, for example, myoblasts (for example, skeletal myoblasts), mesenchymal stem cells (for example, 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, retina) Pigment epithelial cells, nasal mucosal epithelial cells), endothelial cells (eg, vascular endothelial cells), hepatocytes (eg, liver parenchymal cells), pancreatic cells (eg, islet cells), kidney cells, adrenal cells, tooth roots Examples include membrane cells, gingival cells, periosteum cells, skin cells, and the like.

  In the present invention, the “sheet-shaped cell culture” refers to a product in which cells are connected to each other to form a sheet. The cells may be linked to each other directly (including those via cell elements such as adhesion molecules) and / or via intervening substances. The intervening substance is not particularly limited as long as it is a substance that can connect cells at least physically (mechanically), and examples thereof include an extracellular matrix. The intervening substance is preferably derived from cells, particularly derived from cells constituting the sheet-shaped cell culture. The cells are at least physically (mechanically) connected, but may be further functionally, for example, chemically or electrically connected. The sheet-shaped cell culture is composed of one cell layer (single layer) or composed of two or more cell layers (stacked (multilayer), for example, two layers, three layers, four layers) Layer, 5 layers, 6 layers, etc.).

  The sheet-shaped cell culture of the present invention preferably does not contain a scaffold (support). Scaffolds may be used in the art to attach cells on and / or within its surface and maintain the physical integrity of sheet-like cell cultures, for example, polyvinylidene difluoride ( PVDF) membranes and the like are known, but the sheet-like cell culture of the present invention can maintain its physical integrity without such a scaffold. In addition, the sheet-shaped cell culture of the present invention is preferably composed only of cells derived from the cells constituting the sheet-shaped cell culture, and does not contain other substances.

  The cells in the present invention can be derived from any organism that can be treated with a sheet-like cell culture. Examples of such organisms include, but are not limited to, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents (eg, mice, rats, hamsters, guinea pigs, etc.), rabbits, and the like. Is included.

  Only one type of cell may be used for forming the sheet-shaped cell culture, but two or more types of cells may be used. In addition, in cells obtained by isolation from living organisms, the cells often contain multiple types of cells, and it may be difficult to specify all cell types. Sometimes only the most common cells in a cell population. For example, when a certain cell population includes a predetermined ratio of cells A and one or more cells other than cell A, and the ratio of cells A is the highest, the cell population is referred to as cell A for convenience, The sheet-shaped cell culture composed of the cell population may be referred to as “sheet-shaped cell culture composed of cells A”. In a preferred embodiment of the present invention, when there are two or more types of cells forming a sheet-shaped cell culture, the most cell ratio (purity) is determined at the start of sheet culture or at the end of production of the sheet-shaped cell culture. About 60% or more, preferably about 70% or more, more preferably about 75% or more.

  The cell may be a xenogeneic cell or a homologous cell. The term “heterologous cell” as used herein means a cell derived from an organism of a species different from the recipient when the sheet-shaped cell culture is used for transplantation. For example, when the recipient is a human, cells derived from monkeys or pigs correspond to xenogeneic cells. The “same species-derived 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 the allogeneic cell. The allogeneic cells include autologous cells (also referred to as autologous cells or autologous cells), that is, cells derived from the recipient, and allogeneic non-autologous cells (also referred to as allogeneic cells). Autologous cells are preferred in the present invention because no rejection occurs even after transplantation. However, it is also possible to use heterologous cells or 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, heterologous cells and allogeneic nonautologous cells may be collectively referred to as nonautologous cells. In one embodiment of the invention, the cells are autologous cells or allogeneic cells. In one embodiment of the present invention, the cell is an autologous cell. In another embodiment of the invention, the cell is an allogeneic cell.

The step of freezing the cells in the production method of the present invention can be performed by any known technique. Such techniques include, but are not limited to, for example, subjecting the cells in the container to a freezing means such as a freezer, a deep freezer, or a low-temperature medium (for example, liquid nitrogen). The temperature of the freezing means is not particularly limited as long as it is a temperature at which a part of the cell population in the container, preferably the whole can be frozen, but is typically about 0 ° C. or lower, preferably about −20 ° C. or lower, more preferably. Is about −40 ° C. or lower, more preferably about −80 ° C. or lower. The cooling rate in the freezing operation is not particularly limited as long as it does not significantly impair the viability and function of the cells after freezing and thawing. Typically, the cooling rate is about 1 until cooling starts from 4 ° C. and reaches about −80 ° C. The cooling rate is from about 2 hours to about 5 hours, preferably from about 2 hours to about 4 hours, especially about 3 hours. Specifically, for example, cooling can be performed at a rate of about 0.46 ° C./min. Such a cooling rate can be achieved by providing the container containing the cells directly or in a freezing treatment container in a freezing means set to a desired temperature. The freezing treatment container may have a function of controlling the temperature lowering speed in the container to a predetermined speed. As such a freezing container, any known container such as BICELL ^(R) (Japan Freezer) can be used.

  The freezing operation may be performed while the cells are immersed in a culture solution or physiological buffer solution, but a cryoprotectant for protecting the cells from freezing and thawing operations is added to the culture solution, or the culture solution is cryoprotected. You may perform after performing the process of replacing with the cryopreservation liquid containing an agent. Therefore, the production method of the present invention may further include a step of adding a cryoprotectant to the culture solution or a step of replacing the culture solution with a cryopreservation solution. When replacing the culture solution with a cryopreservation solution, if the solution in which cells are immersed during freezing contains an effective concentration of cryoprotectant, remove the culture solution before adding the cryopreservation solution. Alternatively, the cryopreservation solution may be added while leaving a part of the culture solution. Here, the “effective concentration” means that the cryoprotectant exhibits a cryoprotective effect without exhibiting toxicity, for example, the viability, vitality, and function of the cell after freeze-thawing compared to the case where the cryoprotectant is not used. This means a concentration that exhibits a decrease suppressing effect such as Such a concentration is known to those skilled in the art or can be appropriately determined by routine experimentation.

  The cryoprotectant used in the production method of the present invention is not particularly limited as long as it exhibits a cryoprotective action on cells. For example, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, sericin, propane Examples include diol, dextran, polyvinylpyrrolidone, polyvinyl alcohol, hydroxyethyl starch, chondroitin sulfate, polyethylene glycol, formamide, acetamide, adonitol, perseitol, raffinose, lactose, trehalose, sucrose, mannitol and the like. Cryoprotectants may be used alone or in combination of two or more.

  The concentration of the cryoprotectant added to the culture solution or the concentration of the cryoprotectant in the cryopreservation solution is not particularly limited as long as it is an effective concentration as defined above. About 2% to about 20% (v / v) with respect to the whole stock solution. However, although outside this concentration range, alternative use concentrations known or experimentally determined for each cryoprotectant may be employed, and such concentrations are within the scope of the present invention.

  In the production method of the present invention, the step of thawing frozen cells can be performed by any known cell thawing technique. Typically, for example, the frozen cells are heated to a thawing means, for example, higher than the freezing temperature. Use in a solid, liquid or gaseous medium (eg, water), water bath, incubator, incubator, etc., or immerse the frozen cells in a medium (eg, culture solution) at a temperature higher than the freezing temperature. However, the present invention is not limited to this. The temperature of the thawing means or the immersion medium is not particularly limited as long as it is a temperature at which cells can be thawed within a desired time, but typically about 4 ° C to about 50 ° C, preferably about 30 ° C to about 40 ° C, more Preferably, it is about 36 ° C to about 38 ° C. The thawing time is not particularly limited as long as it does not significantly impair the viability and function of the cells after thawing, but it is typically within about 2 minutes, and particularly within about 20 seconds, The decrease can be greatly suppressed. The thawing time can be adjusted, for example, by changing the temperature of the thawing means or the immersion medium, the volume or composition of the culture solution or cryopreservation solution at the time of freezing. The frozen cells include cells frozen by any technique, and non-limiting examples thereof include, for example, cells frozen by the step of freezing the above cells. In one embodiment, the frozen cell is a cell that has been frozen in the presence of a cryoprotectant. In one embodiment, the frozen cells are for use in the production method of the present invention.

  The production method of the present invention may include a step of washing cells after the step of thawing frozen cells and before the step of forming a sheet-like cell culture. Washing of cells can be performed by any known technique. Typically, for example, cells are washed with a washing solution (for example, serum or serum component (serum albumin, etc.), or a culture solution (for example, a medium). Etc.) or a physiological buffer (eg, PBS, HBSS, etc.), centrifuged, and the supernatant is discarded, and the precipitated cells are collected, but not limited thereto. In the step of washing the cells, the suspension, centrifugation, and recovery cycle may be performed once or a plurality of times (for example, 2, 3, 4, 5 times, etc.). In one embodiment of the invention, the step of washing the cells is performed immediately after the step of thawing the frozen cells.

  The step of forming a sheet-shaped cell culture in the production method of the present invention can be performed by any known technique. Examples of such a technique include, but are not limited to, those described in Patent Documents 1, 4, 5, and the like. The step of forming a sheet cell culture can be performed on a culture substrate. In addition, the step of forming the sheet-shaped cell culture may include a step of seeding the cells on a culture substrate and a step of forming the seeded cells into a sheet. In one aspect, the production method of the present invention does not include the step of growing cells between the step of thawing the cells and the step of forming a sheet-like cell culture.

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, solid or semi-solid surfaces in containers, and the like. The container preferably has a structure / material that does not allow permeation of a liquid such as a culture solution. Examples of such materials 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, polydimethyl. Examples include acrylamide and metals (for example, iron, stainless steel, aluminum, copper, brass). The container preferably has at least one flat surface. Examples of such containers include, but are not limited to, cell culture dishes and cell culture bottles. Further, the container may have a solid or semi-solid surface therein. Examples of solid surfaces include plates and containers of various materials as described above, and examples of semi-solid surfaces include gels and soft polymer matrices. The culture substrate may be prepared using the above materials, or commercially available materials may be used. Preferable culture substrates include, but are not limited to, substrates having an adhesive surface suitable for the formation of sheet cell cultures. Specifically, a substrate having a hydrophilic surface, for example, a substrate coated with a hydrophilic compound such as polystyrene subjected to corona discharge treatment, collagen gel or hydrophilic polymer, and further, collagen, fibronectin, laminin , Substrates coated with an extracellular matrix such as vitronectin, proteoglycan and glycosaminoglycan, and cell adhesion factors such as cadherin family, selectin family and integrin family. Such base materials are commercially available (for example, Corning ^(R) TC-Treated Culture Dish, Corning, etc.).

The surface of the culture substrate may be coated with a material whose physical properties change in response to stimulation, for example, temperature or light. Examples of such materials include, but are not limited to, (meth) acrylamide compounds, N-alkyl substituted (meth) acrylamide derivatives (for example, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylate Amides), N, N-dialkyl-substituted (meth) acrylamide derivatives (eg, N, N-dimethyl (meth) acrylamide, N, N-ethylmethylacrylamide, N, N-diethyl) Chloramide and the like), (meth) acrylamide derivatives having a cyclic group (for example, 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 vinyl ether derivative (for example, methyl vinyl ether) homopolymer or copolymer, a temperature-responsive material, a light-absorbing polymer having an azobenzene group, triphenylmethane leucohydro Copolymer of vinyl derivative of oxide and acrylamide monomer, and spirobenzopyra It 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 giving a predetermined stimulus to these materials, the physical properties, for example, hydrophilicity and hydrophobicity can be changed, and peeling 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)) can be used, these the production method of the present invention.

The culture substrate may have various shapes, but is preferably flat. The area 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 , more preferably about 3 cm ² to about 50 cm ² .

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

  As the serum, heterologous serum and allogeneic serum can be used. Xenogeneic serum refers to serum derived from a different species of organism than the recipient when a sheet cell culture is used for transplantation. For example, when the recipient is a human, serum derived from bovine or horse, for example, fetal calf serum (FBS, FCS), calf serum (CS), horse serum (HS), etc. corresponds to the heterologous serum. “Allogeneic serum” means serum derived from the same species of organism as the recipient. For example, when the recipient is a human, human serum corresponds to allogeneic serum. Allogeneic serum includes autoserum (also called autologous serum), ie, serum derived from the recipient, and allogeneic serum derived from allogeneic individuals other than the recipient. In the present specification, sera other than autoserum, that is, heterologous serum and allogeneic sera are sometimes collectively referred to as non-self serum.

  Serum for coating the culture substrate is commercially available or can be prepared from blood collected from a desired organism by a conventional method. Specifically, for example, the collected blood is allowed to coagulate by standing at room temperature for about 20 minutes to about 60 minutes, centrifuged at about 1000 × g to about 1200 × g, and the supernatant is collected. Etc.

  When incubating on a culture substrate, serum may be used as a stock solution or diluted. Dilution can be any medium such as, without limitation, water, saline, various buffers (eg, PBS, HBSS, etc.), various liquid media (eg, DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12, 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, the dilution concentration is 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 also not particularly limited as long as the serum component can adhere to the culture substrate. For example, the incubation time is about 1 hour to about 72 hours, preferably about 4 hours to about 48 hours, and more preferably about 5 hours to about 48 hours. 24 hours, more preferably about 6 hours to about 12 hours. The incubation temperature is not particularly limited as long as serum components can adhere to the culture substrate. For example, the incubation temperature is about 0 ° C to about 60 ° C, preferably about 4 ° C to about 45 ° C, more preferably room temperature to about 40 ° C. It is.

  Serum may be discarded after incubation. As a method for discarding serum, a conventional liquid disposal method such as suction with a pipette or decantation can be used. In a preferred embodiment of the present invention, the culture substrate may be washed with a serum-free washing solution after serum is discarded. 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 the serum components attached to the culture substrate. For example, without limitation, water, physiological saline, various buffers Liquid (eg, PBS, HBSS, etc.), various liquid media (eg, DMEM, MEM, F12, DMEM / F12, DME, RPMI 1640, 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 on the culture substrate, stirring for a predetermined time (for example, about 5 seconds to about 60 seconds), and then discarding the solution. Etc. can be used.

  In the present invention, the culture substrate may be coated with a growth factor. As used herein, “growth factor” means any substance that promotes cell proliferation as compared to the case without it, such as epithelial cell growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast, and the like. Cell growth factor (FGF) and the like. In the coating method, discarding method, and washing method of the culture substrate with the growth factor, the dilution concentration at the time of incubation is, for example, about 0.0001 μg / mL to about 1 μg / mL, preferably about 0.0005 μg / mL to about 0.00. It is basically the same as serum except that it is 05 μg / mL, more preferably about 0.001 μg / mL to about 0.01 μg / mL.

  In the present invention, the culture substrate may be coated with a steroid agent. Here, the “steroid agent” refers to a compound having a steroid nucleus that can adversely affect a living body such as adrenal cortex dysfunction and Cushing's syndrome. Such compounds include, but are not limited to, for example, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone and the like. The culture substrate coating method, the discarding method, and the washing method using a steroid agent have a dilution concentration at the time of incubation of, for example, about 0.1 μg / mL to about 100 μg / mL, preferably about 0.4 μg / mL to 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 agent, any combination of these: serum and growth factor, serum and steroid agent, serum and growth factor and steroid agent, 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.

  The culture substrate may be seeded with cells immediately after coating with serum or the like, or may be stored after coating and then seeded with cells. The coated substrate can be stored for a long 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.

  Cell seeding on the culture substrate can be performed by any known technique and condition. The seeding of the cells on the culture substrate may be performed, for example, by injecting a cell suspension obtained by suspending the cells in the culture solution into the culture substrate (culture vessel). For the injection of the cell suspension, an apparatus suitable for the operation of injecting the cell suspension such as a dropper or a pipette can be used.

In a preferred embodiment of the present invention, seeding is performed at a density that allows a sheet-like cell culture to form without substantial growth of the cells. “The density at which cells can form a sheet-shaped cell culture without substantial growth” means that the sheet-shaped cell culture is expressed when cultured in a non-proliferating medium that does not substantially contain growth factors. It means the cell density that can be formed. For example, in the case of skeletal myoblasts, in a method using a culture solution containing a growth factor, cells are seeded on a culture substrate at a density of about 6,500 cells / cm ² to form a sheet-like cell culture. However, even if cells having such a density are cultured in a culture solution containing no growth factor, a sheet-shaped cell culture is not formed. Therefore, the seeding density in this embodiment is higher than that in the method using a culture solution containing a growth factor. Specifically, such density is typically about 1.0 × 10 ⁵ cells / cm ² or more, for example, for skeletal myoblasts. The upper limit of the seeding density is not particularly limited as long as the formation of the sheet-shaped cell culture is not impaired and the cells do not shift to differentiation, but for skeletal myoblasts, the upper limit of seeding density is less than about 3.4 × 10 ⁶ cells / cm ² . is there.

Accordingly, the “density capable of forming a sheet-shaped cell culture without substantial proliferation” in skeletal myoblasts is about 1.0 × 10 ⁵ to about 3.4 × 10 ⁶ cells / cm ^{2 in} some embodiments. In another embodiment, about 3.0 × 10 ⁵ to about 3.4 × 10 ⁶ pieces / cm ² , and in another embodiment about 3.5 × 10 ⁵ to about 3.4 × 10 ⁶ pieces / cm ² , In yet another aspect, from about 1.0 × 10 ⁶ to about 3.4 × 10 ⁶ pieces / cm ² , and in yet another aspect, from about 3.0 × 10 ⁵ to about 1.7 × 10 ⁶ pieces / cm ² , In another embodiment, from about 3.5 × 10 ⁵ to about 1.7 × 10 ⁶ pieces / cm ² , and in another embodiment, from about 1.0 × 10 ⁶ to about 1.7 × 10 ⁶ pieces / cm ² . . As long as the upper limit is less than about 3.4 × 10 ⁶ pieces / cm ² , the above range may include both or both of the upper limit and the lower limit. Therefore, the density is, for example, about 3.0 × 10 ⁵ pieces / cm ² or more and less than about 3.4 × 10 ⁶ pieces / cm ² (including the lower limit and not including the upper limit), about 3.5 × 10 ⁵ pieces / cm ² or more and less than about 3.4 × 10 ⁶ pieces / cm ² (including the lower limit, not including the upper limit), about 1.0 × 10 ⁶ pieces / cm ² or more and about 3.4 × 10 ⁶ Less than pieces / cm ² (including lower limit, not including upper limit), more than about 1.0 × 10 ⁶ pieces / cm ² and less than about 3.4 × 10 ⁶ pieces / cm ² (including neither lower limit nor upper limit), It may be about 1.0 × 10 ⁶ pieces / cm ^{2 or} more and about 1.7 × 10 ⁶ pieces / cm ² or less (not including the lower limit but including the upper limit). A person skilled in the art can appropriately determine the cell density suitable for the present invention for cells other than skeletal myoblasts by experiments according to the teaching of the present specification. In one embodiment, the “density at which cells can form a sheet-like cell culture without substantial growth” is at or above the density at which the cells reach confluence, or at or above the confluent density.

The step of forming the seeded cells into a sheet can also be performed by any known technique and condition. Non-limiting examples of such techniques are described in, for example, Patent Documents 1, 4, 5 and the like. It is considered that the formation of a cell sheet is achieved when cells adhere to each other via an adhesion molecule or an intercellular adhesion mechanism such as an extracellular matrix. Therefore, the step of forming the seeded cells into a sheet can be achieved, for example, by culturing the cells under conditions that form cell-cell adhesion. Such conditions may be any as long as cell-cell adhesion can be formed, but cell-cell adhesion can usually be formed under the same conditions as general cell culture conditions. Examples of such conditions include culture at about 37 ° C. and 5% CO ₂ . Further, the culture can be performed under normal pressure (atmospheric pressure, non-pressurized). A person skilled in the art can select optimal conditions according to the type of cells to be seeded. In the present specification, the culture for forming the seeded cells into a sheet may be referred to as “sheet culture”.

  In one embodiment of the present invention, cell culture is performed within a predetermined period, preferably within a period in which cells do not shift to differentiation. Thus, in this embodiment, the cells are maintained in an undifferentiated state during the culture period. The transition to cell differentiation can be evaluated by any method known to those skilled in the art. For example, in the case of skeletal myoblasts, MHC expression, creatine kinase (CK) activity, cell multinucleation, myotube formation, etc. can be used as indicators of differentiation. In a preferred embodiment of the invention, the culture period is within about 48 hours, more preferably within about 40 hours, and even more preferably within about 24 hours.

The cell culture medium used for the culture (sometimes simply referred to as “culture medium” or “medium”) is not particularly limited as long as it can maintain the survival of the cells. Typically, amino acids, vitamins, electrolytes are used. Can be used. In one embodiment of the present invention, the culture solution is based on a basal medium for cell culture. Such a basal medium is not limited, for example, DMEM, MEM, F12, DMEM / F12, DME, RPMI 1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80 -7 and the like are included. Many of these basal media are commercially available, and their compositions are also known.
The basal medium may be used in a standard composition (for example, as it is commercially available), or the 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 one in which one or more components are added, removed, increased or decreased.

Examples of amino acids contained in the basal medium include, but are not limited to, L-arginine, L-cystine, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and the like are not limited to vitamins, for example, calcium D-pantothenate, choline chloride, folic acid, i - inositol, niacinamide, riboflavin, thiamine, pyridoxine, biotin, lipoic acid, vitamin B12, adenine, thymidine and, and, as the electrolyte, without limitation, for _{example, CaCl 2, KCl, MgSO 4} , NaCl, NaH _{2 PO 4, NaHCO 3, Fe} (NO 3) 3, FeS _{4, CuSO 4, MnSO 4,} Na 2 SiO 3, include _{(NH 4) 6 Mo 7 O} 24, NaVO 3, NiCl 2, etc. ZnSO _4, respectively. In addition to these components, the basal medium may contain sugars such as D-glucose, pH indicators such as sodium pyruvate and phenol red, putrescine and the like.

In one embodiment of the present invention, the concentration of the amino acid contained in the basal medium is as follows: L-arginine: about 63.2 mg / L to about 84 mg / L, L-cystine: about 35 mg / L to about 63 mg / L, L-glutamine : About 4.4 mg / L to about 584 mg / L, glycine: about 2.3 mg / L to about 30 mg / L, L-histidine: about 42 mg / L, L-isoleucine: about 66 mg / L to about 105 mg / L, L-leucine: about 105 mg / L to about 131 mg / L, L-lysine: about 146 mg / L to about 182 mg / L, L-methionine: about 15 mg / L to about 30 mg / L, L-phenylalanine: about 33 mg / L To about 66 mg / L, L-serine: about 32 mg / L to about 42 mg / L, L-threonine: about 12 mg / L to about 95 mg / L, L-tryptophan: about 4.1 mg / L About 16mg / L, L- Tyrosine: about 18.1mg / L~ about 104mg / L, L- Valine: is about 94mg / L~ about 117mg / L.
In one embodiment of the present invention, the concentration of the vitamin preparation contained in the basal medium is as follows: calcium D-pantothenate: about 4 mg / L to about 12 mg / L, choline chloride: about 4 mg / L to about 14 mg / L, folic acid : About 0.6 mg / L to about 4 mg / L, i-inositol: about 7.2 mg / L, niacinamide: about 4 mg / L to about 6.1 mg / L, riboflavin: about 0.0038 mg / L to about 0 .4 mg / L, thiamine: about 3.4 mg / L to about 4 mg / L, pyridoxine: about 2.1 mg / L to about 4 mg / L.

  In addition to the above, the cell culture medium may contain one or more additives such as serum, growth factor, steroid component, and selenium component. However, if these components are not self-derived, they may be impurities derived from the manufacturing process that cannot be ruled out as side effects such as anaphylactic shock to the recipient in clinical practice. It is desirable to eliminate derived components. Accordingly, in a preferred embodiment of the invention, the cell culture medium does not contain an effective amount of at least one of these non-autologous additives. In a more preferred embodiment of the present invention, the cell culture medium is substantially free of at least one of these non-self-derived additives. Furthermore, in a particularly preferred embodiment of the present invention, the cell culture medium is substantially free of non-autologous additives. In one embodiment, the cell culture medium may contain only a basal medium.

  In one embodiment of the present invention, the cell culture medium is substantially free of serum. A cell culture medium substantially free of serum may be referred to herein as “serum-free medium”. Here, “substantially free of serum” means that the serum content in the culture solution does not have an adverse effect when the sheet-shaped cell culture is applied to a living body (for example, in the sheet-shaped cell culture). It means that the serum albumin content is less than about 50 ng), preferably that these substances are not actively added to the culture medium. In the present invention, in order to avoid side effects at the time of transplantation, the cell culture medium preferably contains substantially no heterogeneous serum, and more preferably contains substantially no non-self serum.

  In one embodiment of the present invention, the cell culture medium contains serum. The serum may be homologous serum or heterologous serum. In certain embodiments, the cell culture medium includes autologous serum. When culturing cells on a culture substrate coated with serum, the serum contained in the cell culture medium (serum used for culturing cells) may be the same as the serum used to coat the culture substrate. May be. In one embodiment, the serum contained in the cell culture medium is the same as that used to coat the culture substrate, and in a particular embodiment, the serum is autologous serum. The serum may be for use in the production method of the present invention. For example, the serum may be for use in cell culture or for coating a culture substrate.

  In one embodiment of the invention, the cell culture fluid does not contain an effective amount of growth factor. Here, an “effective amount of growth factor” refers to the amount of growth factor that significantly promotes cell proliferation as compared to the absence of growth factor, or, for convenience, cell proliferation in the art. It means the amount usually added for the purpose. The significance of cell growth promotion can be appropriately evaluated, for example, by any statistical method known in the art, for example, t-test, and the usual addition amount is various in the art. It can be known from known literature. Specifically, the effective amount of EGF in cell culture is, for example, about 0.005 μg / mL or more.

  Therefore, “not containing an effective amount of growth factor” means that the concentration of the growth factor in the culture medium of the present invention is less than the effective amount. For example, the concentration of EGF in the culture medium in cell culture is preferably less than about 0.005 μg / mL, more preferably less than about 0.001 μg / mL. In a preferred embodiment of the present invention, the concentration of the growth factor in the culture solution is less than the normal concentration in the living body. In such embodiments, for example, the concentration of EGF in the culture medium in cell culture is preferably less than about 5.5 ng / mL, more preferably less than about 1.3 ng / mL, and even more preferably about 0.5 ng / mL. Is less than. In a further preferred embodiment, the culture medium in the present invention is substantially free from growth factors. Here, “substantially free” means that the content of the growth factor in the culture solution is such that it does not have an adverse effect when the sheet-shaped cell culture is applied to a living body. Means not actively added. Therefore, in this embodiment, the culture solution does not contain a growth factor at a concentration higher than that contained in other components such as serum.

  In one embodiment of the present invention, the cell culture medium is substantially free of steroid component. Here, the “steroid component” refers to a compound having a steroid nucleus that can adversely affect a living body such as adrenal cortex dysfunction and Cushing's syndrome. Such compounds include, but are not limited to, for example, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone and the like. Therefore, “substantially free of steroid component” means that the content of these compounds in the culture solution is such that it does not have an adverse effect when the sheet-shaped cell culture is applied to a living body, This means that these compounds are not actively added to the culture solution, that is, the culture solution does not contain steroid agent components at a concentration higher than that contained in other components such as serum.

  In one embodiment of the present invention, the cell culture solution is substantially free of a selenium component. Here, the “selenium component” includes a selenium molecule and a selenium-containing compound, in particular, a selenium-containing compound capable of releasing a selenium molecule in vivo, such as selenite. Therefore, “substantially free of selenium component” means that the content of these substances in the culture solution is such that there is no adverse effect when the sheet-shaped cell culture is applied to a living body, This means that these substances are not positively added to the liquid, that is, the culture liquid does not contain selenium components at a concentration higher than that contained in other components such as serum. Specifically, for example, in the case of humans, the selenium concentration in the culture solution is the normal value in human serum (for example, 10.6 μg / dL to 17.4 μg / dL), and the concentration of human serum contained in the medium is Lower than the ratio multiplied (ie, if the human serum content is about 10%, the selenium concentration is, for example, about 1.0 μg / dL to less than about 1.7 μg / dL).

In the above preferred embodiment of the present invention, impurities derived from production processes such as growth factors, steroid components, and heterogeneous serum components, which have been conventionally required when preparing a cell culture to be applied to a living body, are removed by washing or the like. A step becomes unnecessary. Accordingly, one aspect of the method of the present invention does not include the step of removing impurities from this manufacturing process.
Here, “manufacturing process-derived impurities” typically include those listed below, which are derived from each manufacturing process. That is, a substance derived from a cell substrate (for example, host cell-derived protein, host cell-derived DNA), a substance derived from a cell culture medium (for example, inducer, antibiotic, medium component), or a step after cell culture It is derived from the extraction, separation, processing, and purification steps of a certain target substance (see, for example, Pharmaceutical Examination No. 571).

Cell culture can be performed under conditions usually used in the art. For example, typical culture conditions include culture at about 37 ° C. and 5% CO ₂ . Further, the culture can be performed under normal pressure (atmospheric pressure, non-pressurized). The culture period is preferably within about 48 hours, more preferably within about 40 hours, and even more preferably within about 24 hours, from the viewpoint of sufficient formation of a sheet-shaped cell culture and prevention of cell differentiation. Culturing can be performed in containers of any size and shape. The size and shape of the sheet-shaped cell culture can be adjusted by adjusting the size and shape of the cell adhesion surface of the culture vessel, or by placing a mold of the desired size and shape on the cell adhesion surface of the culture vessel, It can be arbitrarily adjusted by, for example, culturing cells therein.

In one embodiment of the production method of the present invention, after the step of thawing the cells, a sheet-like cell culture is formed without substantially growing the cells. This procedure can further enhance the activity of the sheet-like cell culture.
“Substantially does not proliferate” means that the cell does not proliferate beyond the range of measurement error, and whether or not the cell has proliferated is, for example, the number of cells at the time of seeding and the sheet-like cell culture. It can be evaluated by comparing the number of cells after formation. In the present invention, the number of cells after forming the sheet-shaped cell culture is typically about 300% or less, preferably about 200% or less, more preferably about 150% or less, and still more preferably about the number of cells at the time of seeding. 125% or less, particularly preferably about 100% or less.

  Cell growth depends on various conditions such as the number of seeded cells (seeded cell density), the culture environment (eg, culture time, culture temperature, etc.), the composition of the medium, etc. By adjusting these conditions, The cells can be substantially not proliferated. Among these conditions, by increasing the seeded cell density, a sheet-like cell culture can be obtained in a relatively short time while suppressing cell growth. In the present invention, growth is controlled by the seeded cell density. It is preferable. The density at which cells can form a sheet-shaped cell culture without substantial growth is as described above.

  Therefore, in this embodiment, after the step of thawing the cells, the sheet-forming culture is performed under a condition that does not cause the cells to substantially grow without going through a further cell growth step. In this embodiment, after the step of thawing the cells and before the sheet culture, a step without cell proliferation, for example, a step of washing the cells can be performed.

  The production method of the present invention may further include a step of recovering the formed sheet cell culture after the step of forming the sheet cell culture. The recovery of the sheet-shaped cell culture is not particularly limited as long as the sheet-shaped cell culture can be released (peeled) from the culture substrate serving as a scaffold while at least partially maintaining the sheet structure. Enzymatic treatment with an enzyme (for example, trypsin) and / or mechanical treatment such as pipetting can be performed. In addition, when cells are cultured on a culture substrate whose surface is coated with a material that changes its physical properties in response to stimulation, for example, temperature or light, a predetermined stimulation is applied. It can also be released non-enzymatically.

  For example, when cells are cultured in a temperature-responsive culture dish to form a cell culture, the temperature is set to be lower than the lower critical solution temperature (LCST) or higher upper limit critical solution temperature (UCST) for water of the temperature responsive material. By performing the temperature treatment, the sheet-like cell culture can be released non-enzymatically. Such temperature treatment is not limited, and for example, the culture substrate to which the formed sheet-like cell culture is attached can be removed from a culture environment at a temperature higher than LCST (for example, in an incubator at a temperature of about 37 ° C.). It can be achieved by shifting to an environment below LCST (for example, a room temperature environment outside the incubator). Transition to the environment below the LCST is not limited, for example, a medium having a temperature higher than the LCST in which the formed sheet-shaped cell culture is present is transferred to a medium having a temperature below the LCST (for example, a buffer (PBS, PBS, HBSS or the like) or a liquid such as a culture solution). Therefore, the medium such as the buffer solution can be used for non-enzymatic release of the sheet-shaped cell culture from the culture substrate in the production method of the present invention.

  One aspect of the production method of the present invention further includes a step of collecting the sheet-like cell culture after the step of forming the sheet-like cell culture, and the step of thawing the cells collects the sheet-like cell culture. Performed within about 48 hours prior to the step. The time between the step of thawing the cells and the step of recovering the sheet-shaped cell culture is about 48 hours or less, preferably about 36 hours or less, more preferably about 24 hours or less. The activity of the product can be further increased.

  The production method of the present invention may further include a step of growing the cells before the step of freezing the cells. The step of growing the cells may be performed by any known technique, and those skilled in the art are familiar with the culture conditions suitable for the growth of various cells. In the production method of the present invention, after the step of thawing the cells, when forming a sheet-like cell culture without substantially growing the cells, or the step of thawing the cells, If performed within about 48 hours prior to the harvesting step, it is useful to perform the cell growth step prior to the cell freezing step to obtain the desired cell number.

  In one embodiment, the production method of the present invention does not include a step of introducing a gene into a cell. In another embodiment, the production method of the present invention includes a step of introducing a gene into a cell. The gene to be introduced is not particularly limited as long as it is useful for treatment of the target disease, and may be, for example, cytokines such as HGF and VEGF. The gene can be introduced by any known method such as calcium phosphate method, lipofection method, ultrasonic introduction method, electroporation method, particle gun method, adenovirus vector, retrovirus vector or other viral vector method, or microinjection method. Can be used. The introduction of the gene into the cell is not limited and can be performed, for example, before the step of freezing the cell.

  The sheet-like cell culture obtained by the production method of the present invention is a sheet-like cell produced by the same method as the production method of the present invention, except that it does not include a step of freezing cells and a step of thawing frozen cells. The activity is higher than that of the culture (hereinafter sometimes referred to as non-frozen / thawed control sheet-like cell culture). Further, in the production method of the present invention, comprising the step of thawing frozen cells and the step of forming a sheet-like cell culture, between the step of thawing the frozen cells and the step of forming the sheet-like cell culture The sheet-shaped cell culture produced in a mode that does not include the step of proliferating cells in the cell-like cell culture (hereinafter referred to as a growth control sheet-shaped cell culture) produced in a mode that includes the step of growing. More active). Examples of such activity include, but are not limited to, cytokine production ability, engraftment ability, blood vessel induction ability and tissue regeneration ability. Here, the high activity is not limited based on the activity of the non-frozen / thawed and / or expanded control sheet-like cell culture (hereinafter sometimes simply referred to as “control sheet-like cell culture”). For example, about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more or about 100% or more means high activity.

  In the present invention, cytokine means a proteinaceous factor that is released from cells and mediates various cell-cell interactions. Therefore, cytokines in the present invention are not limited to those released from immune cells. In one embodiment of the present invention, the cytokine is useful for engrafting the sheet-like cell culture in the transplanted tissue. In one aspect of the invention, the cytokine is beneficial for angiogenesis. In one embodiment of the invention, the cytokine is beneficial for tissue regeneration. Such cytokines are known in the art, and those skilled in the art can determine appropriate cytokines based on known information. In one aspect of the invention, the cytokine comprises a growth factor. In one embodiment of the invention, the cytokine is selected from the group consisting of HGF and VEGF. Therefore, in one embodiment of the present invention, the cytokine production ability is the growth factor production ability. In one embodiment of the present invention, the cytokine production ability is the production ability of a growth factor selected from the group consisting of HGF and VEGF.

  The activity of the sheet cell culture can be quantified using various techniques. If the activity is cytokine-producing ability, for example, the sheet-shaped cell culture is cultured in the culture solution for a predetermined time, and the amount of cytokine secreted into the culture solution is measured, or the cytokine expressed in the sheet-shaped cell culture Cytokine production ability can be quantified by measuring the amount of gene. Techniques for measuring the amount of a specific protein and the level of gene expression are well known in the art, and the techniques for measuring the amount of protein are not limited and include, for example, electrophoresis, Western blotting, mass Analytical methods, EIA (for example, ELISA, etc.), RIA, immunohistochemical methods, immunocytochemical methods, etc. are not limited as methods for measuring the level of gene expression. For example, Northern blotting method, Southern blotting method , DNA microarray analysis, RNase protection assay, RT-PCR, real-time PCR and other PCR methods, in situ hybridization methods, and the like.

  When the activity is engraftment, the sheet-shaped cell culture is applied to the tissue, and the state of the sheet-shaped cell culture applied after a predetermined time, for example, adhesion to the tissue, remaining sheet-shaped cell culture The engraftment ability can be quantified by observing the size, color, form, etc. of these and scoring them. If the activity is blood vessel inducing ability, apply the sheet-shaped cell culture to the tissue and observe the state of the applied tissue and the tissue at the application site after a predetermined time, such as the presence or absence of angiogenesis Then, the blood vessel inducing ability can be quantified by scoring them. In addition, when the activity is tissue regeneration ability, the sheet-shaped cell culture is applied to the tissue, and after a predetermined time has passed, the tissue state of the application site (transplant destination), for example, the tissue size, the tissue microstructure, Tissue regeneration ability can be quantified by observing the ratio between damaged tissue and normal tissue, tissue function, and scoring them.

  In one embodiment, all steps of the production method of the present invention are performed in vitro. In another embodiment, the production method of the present invention includes a step performed in vivo, including, but not limited to, collecting a cell or a tissue serving as a source of cells from a subject. In one embodiment, the production method of the present invention is performed under aseptic conditions in all steps. In one embodiment, the production method of the present invention is performed so that the finally obtained sheet-shaped cell culture is substantially sterile. In one embodiment, the production method of the present invention is performed such that the finally obtained sheet-shaped cell culture is sterile.

  Another aspect of the present invention relates to a sheet-shaped cell culture produced by the production method of the present invention. The sheet-like cell culture of the present invention is characterized by having higher activity than the control sheet-like cell culture. Details regarding the height of activity are as described above. In one embodiment, the sheet-shaped cell culture of the present invention has a higher activity selected from the group consisting of cytokine-producing ability, engraftment ability, blood vessel induction ability and tissue regeneration ability than that of the control sheet-like cell culture. In one embodiment, the sheet-shaped cell culture of the present invention has a higher ability to produce a cytokine selected from the group consisting of HGF and VEGF than that of the control sheet-shaped cell culture. Since the sheet-like cell culture of the present invention has high activity, it exhibits an effect superior to that of the control sheet-like cell culture in various therapeutic applications. In particular, if the ability to produce HGF and / or VEGF is high, engraftment in the tissue, blood vessel induction, and tissue regeneration are promoted, and the sheet-shaped cell culture functions continuously for a long period of time, thereby enhancing the therapeutic effect. It becomes possible.

  The sheet-like cell culture of the present invention is useful for the treatment of various diseases associated with tissue abnormalities. Therefore, in one aspect, the sheet-shaped cell culture of the present invention is for use in the treatment of diseases associated with tissue abnormalities. Since the sheet-shaped cell culture of the present invention has the same properties inherent in the constituent cells except that it has a higher activity than the conventional sheet-shaped cell culture, at least the conventional sheet-shaped cell culture It can be applied to tissues and diseases that can be treated with substances. Examples of the tissue to be treated include, but are not limited to, myocardium, cornea, retina, esophagus, skin, joint, cartilage, liver, pancreas, gingiva, kidney, thyroid, skeletal muscle, and middle ear. In addition, the disease to be treated is not limited, and for example, heart disease (eg, myocardial injury (myocardial infarction, cardiac injury), cardiomyopathy, etc.), corneal disease (eg, corneal epithelial stem cell exhaustion, cornea) Injury (heat / chemical corrosion), corneal ulcer, corneal opacity, corneal perforation, corneal scar, Stevens-Johnson syndrome, pemphigoid, etc., retinal diseases (eg retinitis pigmentosa, age-related macular degeneration) , Esophageal diseases (for example, prevention of esophageal inflammation / stenosis after esophageal surgery (esophageal cancer removal)), skin diseases (for example, skin damage (trauma, burn), etc.), joint diseases (for example, 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 Bone like dystrophy), thyroid diseases (e.g., hypothyroidism), muscular disorders (e.g., muscle damage, myositis, etc.), middle ear disease (for example, otitis media) and the like.

  The usefulness of the sheet-shaped cell culture of the present invention for the above-mentioned diseases is, for example, Patent Document 1, Non-Patent Document 1, Arauchi et al., Tissue Eng Part A. 2009 Dec; 15 (12): 3943-9 , Ito et al., Tissue Eng. 2005 Mar-Apr; 11 (3-4): 489-96, Yaji et al., Biomaterials. 2009 Feb; 30 (5): 797-803, Yaguchi et al., Acta Otolaryngol. 2007 Oct; 127 (10): 1038-44, Watanabe et al., Transplantation. 2011 Apr 15; 91 (7): 700-6, Shimizu et al., Biomaterials. 2009 Oct; 30 (30): 5943 -9, Ebihara et al., Biomaterials. 2012 May; 33 (15): 3846-51, Takagi et al., World J Gastroenterol. 2012 Oct 7; 18 (37): 5145-50.

  The sheet-like cell culture of the present invention can be applied to a tissue to be treated and used to repair and regenerate the tissue. However, the sheet-like cell culture is to be treated as a source of a physiologically active substance such as a hormone. It can also be transplanted to a site other than tissue (for example, subcutaneous tissue) (for example, Arauchi et al., Tissue Eng Part A. 2009 Dec; 15 (12): 3943-9, Shimizu et al., Biomaterials. 2009) Oct; 30 (30): 5943-9). It is also possible to obtain a higher effect than injection with a single cell suspension by fragmenting the sheet-like cell culture into an injectable size and injecting it into the site requiring treatment (Wang et al. , Cardiovasc Res. 2008 Feb 1; 77 (3): 515-24). Therefore, such a utilization method is possible also about the sheet-like cell culture of this invention.

  In one embodiment, the sheet cell culture of the present invention is substantially sterile. In one embodiment, the sheet cell culture of the present invention is sterile. In one embodiment, the sheet cell culture of the present invention is not genetically engineered. In another embodiment, the sheet-shaped cell culture of the present invention has been genetically engineered. Genetic manipulation includes, but is not limited to, for example, the introduction of genes that enhance the viability, engraftment, function, etc. of sheet-like cell cultures and / or genes that are useful in the treatment of diseases. Examples of the gene to be introduced include, but are not limited to, cytokine genes such as HGF gene and VEGF gene.

Another aspect of the present invention may be collectively referred to as a composition (for example, a pharmaceutical composition), a graft, a medical product, and the like (hereinafter, “composition etc.”) including the sheet-shaped cell culture of the present invention. )
In addition to the sheet-shaped cell culture of the present invention, the composition of the present invention includes various additional components such as a pharmaceutically acceptable carrier, the viability, engraftment and / or the sheet-shaped cell culture. Or the component which improves a function etc., the other active ingredient useful for treatment of a target disease, etc. may be included. Any known additional components can be used, and those skilled in the art are familiar with these additional components. In addition, the composition of the present invention can be used in combination with components that enhance the viability, engraftment and / or function of the sheet-shaped cell culture, and other active ingredients useful for treating the target disease. . In one embodiment, the composition or the like of the present invention is for use in the treatment of diseases associated with tissue abnormalities. The tissues and diseases to be treated are as described above for the sheet-shaped cell culture of the present invention.

  Another aspect of the present invention is a method of treating a disease associated with a tissue abnormality in a subject, wherein an effective amount of a sheet cell culture or composition of the present invention is administered to the subject in need thereof. (Hereinafter, referred to as “the treatment method of the present invention”). The tissues and diseases to be treated by the treatment method of the present invention are as described above for the sheet-shaped cell culture of the present invention. In addition, in the treatment method of the present invention, components that enhance the viability, engraftment and / or function of the sheet-shaped cell culture, other active ingredients useful for the treatment of the target disease, and the like It can be used in combination with a cell culture or composition.

  The treatment method of the present invention may further include a step of producing a sheet-shaped cell culture according to the production method of the present invention. The treatment method of the present invention may further include a step of collecting a cell for producing a sheet-shaped cell culture from a subject or a tissue serving as a source of the cells before the step of producing the sheet-shaped cell culture. In one embodiment, the subject from which the cells or the tissue that serves as the source of the cells is collected is the same individual as the subject that receives administration of the sheet-shaped cell culture or composition. In another embodiment, the subject from whom the cell or tissue that is the source of the cell is collected is a separate body of the same type as the subject receiving the sheet-like cell culture or composition. In another embodiment, the subject from whom the cell or tissue that serves as the source of the cell is collected is an individual different from the subject receiving the sheet-like cell culture or composition.

  In the context of the present invention, the term “subject” means any living individual, preferably an animal, more preferably a mammal, more preferably a human individual. In the present invention, a subject may be healthy or may have some kind of disease. However, when treatment of a disease associated with a tissue abnormality is intended, Means a subject who is affected or at risk of being affected.

  The term “treatment” is also intended to encompass all types of medically acceptable prophylactic and / or therapeutic interventions intended to cure, temporarily ameliorate or prevent disease. For example, the term “treatment” may be medically acceptable for a variety of purposes, including delaying or stopping the progression of a disease associated with tissue abnormalities, regression or disappearance of a lesion, prevention of the onset of the disease, or prevention of recurrence, etc. Includes interventions.

  In the present invention, the effective amount is, for example, an amount that can suppress the onset or recurrence of a disease, reduce symptoms, or delay or stop progression (for example, the size, weight, number, etc. of sheet-like cell culture). Preferably, it is an amount that prevents the onset and recurrence of the disease or cures the disease. In addition, an amount that does not cause adverse effects exceeding the benefits of administration is preferred. Such an amount can be appropriately determined by, for example, testing in laboratory animals such as mice, rats, dogs or pigs, and disease model animals, and such test methods are well known to those skilled in the art. In addition, the size of the tissue lesion to be treated can be an important index for determining the effective amount.

Administration methods typically include direct application to tissues, but when using sheet cell culture fragments, various routes that can be administered by injection, such as intravenous, intramuscular, Administration may be made by routes such as internal, subcutaneous, local, intraarterial, intraportal, intraventricular, and intraperitoneal.
The frequency of administration is typically once per treatment, but multiple administrations are possible if the desired effect is not obtained.

The present invention will be described in more detail with reference to the following examples, which illustrate specific embodiments of the invention and are not intended to limit the invention.
Example 1
Cryopreserved human skeletal myoblasts (derived from human skeletal muscle samples) were thawed at 37 ° C. and washed twice with a buffer containing 0.5% serum albumin. 5 × 10 ^{6 to} 5 × 10 ⁷ cells were suspended in a 20% serum-containing medium, seeded in a flask, and cultured for 2 to 3 days.
To UpCell ^(R) (3.5 cm dish or 24-well multiwell, CellSeed Inc.), 20% serum-containing medium that covers the entire culture surface is added, and the temperature is 37 ° C., 5% CO _2, normal atmospheric pressure ( For 3 hours to 3 days. After the treatment, the added medium was discarded.
The cultured cells were harvested, suspended in 20% serum-containing medium, seeded at a density of treated ^{UpCell 2 × 10 5 ~20 × 10} 5 cells in ^{(R) / cm 2, 37} ℃, of 5% CO ₂ Sheet culture was performed in the environment for about 1 day.

Example 2
5 × 10 ^{6 to} 5 × 10 ⁷ human skeletal myoblasts (derived from a human skeletal muscle sample) were suspended in a 20% serum-containing medium, seeded in a flask, and cultured for 2 to 3 days. The cultured cells were collected, suspended in a cell freezing preservation solution (basal medium containing 10% DMSO) at a concentration of 1 × 10 ^{7 to} 5 × 10 ⁷ cells / mL, and stored frozen in a liquid nitrogen tank.
To UpCell ^(R) (3.5 cm dish or 24-well multiwell, CellSeed Inc.), 20% serum-containing medium that covers the entire culture surface is added, and the temperature is 37 ° C., 5% CO _2, normal atmospheric pressure ( The treatment was performed for 3 hours to 3 days in an environment of atmospheric pressure. After the treatment, the added medium was discarded.
Cryopreserved skeletal myoblasts were thawed at 37 ° C. and washed twice with a buffer containing 0.5% serum albumin. The washed cells were suspended in 20% serum-containing medium, the treated UpCell ^(R) were plated at a density of 2 × ^{10 5} ~20 × ¹⁰ 5 cells / cm ^2, about at 37 ° C., of 5% CO ₂ environment Sheet culture was performed for 1 day.

Example 3
All the culture supernatants were collected after the sheet culture in Example 1 or Example 2 above, and the concentrations of cytokines (VEGF and HGF) produced in the culture supernatants were measured by ELISA (n = 3). The measurement was performed using a Human VEGF Quantikine ELISA Kit (R & D systems, catalog number DVE00) and RayBio ^(R) Human HGF ELISA Kit (RayBiotech, Inc., catalog number ELH-HGF-001) according to the manufacturer's manual. From the results shown in FIG. 1, it can be seen that VEGF and HGF are produced during the sheet culture in any of the production methods of Examples 1 and 2. Moreover, the production amount of the production method of Example 2 was larger.

Example 4
According to the method of Example 1 or Example 2, the sheet-like cell culture formed into a sheet in UpCell ^(R) (24-well multiwell, CellSeed Inc.) was peeled off by temperature treatment, washed with HBSS (+), 2 400 μL of MCDB medium containing 5% FBS was added to each well and cultured in a CO ₂ incubator for about 24 hours. After the culture, all the supernatant was collected, and the concentrations of cytokines (VEGF and HGF) produced in the culture supernatant were measured by ELISA as in Example 3. 400 μL of 2.5% FBS-containing MCDB medium was again added to each well from which the supernatant was collected, and cultured in a CO ₂ incubator for 48 hours. After the culture, all the supernatant was collected, and the concentrations of cytokines (VEGF and HGF) produced in the culture supernatant were measured by ELISA as in Example 3. From the results shown in FIGS. 2 to 3, it can be seen that the sheet-like cell culture prepared by any of the production methods of Examples 1 and 2 produces VEGF and HGF, and that the production lasts for a long period of 96 hours. In addition, the production amount of the sheet-shaped cell culture prepared by the production method of Example 2 was larger.

Example 5
1 × 10 ^{6 to} 3 × 10 ⁷ human mesenchymal stem cells (human bone marrow-derived) were suspended in a serum-containing medium, seeded in a flask, and then cultured for 2 to 3 days. After the cultured cells were collected, they were suspended in a cell freezing preservation solution (basal medium containing 10% DMSO) at a concentration of 1 × 10 ^{6 to} 5 × 10 ⁶ cells / mL, and stored frozen in a liquid nitrogen tank.
Add serum-containing medium that covers the entire culture surface to UpCell ^(R) (48-well multiwell, CellSeed Inc.), and add 3 in an environment of 37 ° C., 5% CO _{2 and} normal atmospheric pressure (atmospheric pressure). The treatment was performed for a time to 3 days. After the treatment, the added medium was discarded.
The cryopreserved cells were thawed at 37 ° C. and washed twice with a buffer containing 0.5% serum albumin. The washed cells were suspended in serum-containing medium, seeded at a density of treated UpCell ^(R) to 2 × ^{10 5} ~20 × ¹⁰ 5 cells ^{/ cm 2, 37 ℃, 5} % CO 2, normal pressure (atmospheric The sheet culture was performed for about 1 day in an atmosphere of atmospheric pressure.

  As shown in FIG. 4, a sheet-shaped cell culture was formed. The obtained sheet-shaped cell culture had a homogeneous white color, and no defects were observed in appearance inspection. In addition, mesenchymal stem cells are known to produce VEGF and HGF (for example, Nakagami et al., Arterioscler Thromb Vasc Biol. 2005 Dec; 25 (12): 2542-7, Francois et al., Biomed Res Int. 2013; 2013: 151679, Yamahara et al., PLoS One. 2014 Feb 14; 9 (2): e88319, etc.), and the sheet-like mesenchymal stem cell culture is also the sheet-like skeletal muscle of Example 2 Similar to a blast cell culture, from a sheet-like mesenchymal stem cell culture produced by a method comprising a step of thawing frozen cells and a step of growing the cells between the step of forming the sheet-like cell culture VEGF and HGF are considered to have high production ability.

  The various features of the invention described herein can be combined in various ways, and all aspects obtained by such combinations, including combinations not specifically described herein, can Within range. Further, those skilled in the art understand that many various modifications are possible without departing from the spirit of the present invention, and equivalents including such modifications are also included in the scope of the present invention. Accordingly, it should be understood that the embodiments described herein are exemplary only, and are not intended to limit the scope of the present invention.

Claims (18)

A method for producing a sheet-like cell culture, comprising the steps of thawing frozen mesenchymal stem cells and forming a sheet-like cell culture on a culture substrate using the thawed mesenchymal stem cells.   The method according to claim 1, wherein the method does not include the step of growing cells between the step of thawing the mesenchymal stem cells and the step of forming a sheet-like cell culture.   The method according to claim 1 or 2, wherein the culture substrate is coated with serum.   The method according to any one of claims 1 to 3, wherein the step of forming a sheet-shaped cell culture is performed in a cell culture medium containing serum.   The method according to any one of claims 1 to 4, comprising a step of freezing the mesenchymal stem cells before the step of thawing the mesenchymal stem cells.   6. The method of claim 5, comprising the step of expanding the mesenchymal stem cells prior to the step of freezing the mesenchymal stem cells.   The method according to any one of claims 1 to 6, comprising a step of washing the mesenchymal stem cells after the step of thawing the mesenchymal stem cells and before the step of forming the sheet-like cell culture. Method.   Cytokine-producing ability, engraftment ability, blood vessel from sheet-like cell culture produced by a method comprising the step of proliferating cells between the step of thawing mesenchymal stem cells and the step of forming a sheet-like cell culture The method as described in any one of Claims 2-7 which obtains the sheet-like cell culture with high activity selected from the group which consists of induction ability and tissue regeneration ability.   9. The method of claim 8, wherein the cytokine is selected from the group consisting of HGF and VEGF.   The method according to any one of claims 1 to 9, wherein the cell is an autologous cell.   A sheet-like mesenchymal stem cell culture produced by the method according to any one of claims 1 to 10.   A composition comprising the sheet-like mesenchymal stem cell culture according to claim 11.   A method for treating a disease associated with a tissue abnormality in a subject, comprising an effective amount of the sheet-like mesenchymal stem cell culture of claim 11 or the composition of claim 12. A method comprising administering to a subject.   14. The method of claim 13, wherein the tissue is selected from the group consisting of heart muscle, cornea, retina, esophagus, skin, joint, cartilage, liver, pancreas, gingiva, kidney, thyroid, skeletal muscle and middle ear.   The disease is selected from the group consisting of heart disease, corneal disease, retinal disease, esophageal disease, skin disease, joint disease, cartilage disease, liver disease, pancreatic disease, dental disease, kidney disease, thyroid disease, muscle disease and middle ear disease 14. The method of claim 13, wherein:   A frozen mesenchymal stem cell for use in the method according to any one of claims 1 to 10.   A culture substrate for use in the method according to any one of claims 1 to 10.   Serum for use in the method according to any one of claims 3 to 10.