JP6670040B2 - Method for producing sheet-shaped cell culture - Google Patents

Description

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

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

As part of such attempts, cell structures formed using a scaffold 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-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 and the like are underway.

  In order for the sheet-shaped cell culture to survive on the transplanted tissue and perform a desired function, it is necessary to supply oxygen, nutrients, and the like to the culture, and for this purpose, 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-shaped cell culture can be improved, it is thought that the engraftment rate and functionality of the sheet-shaped cell culture can be increased. In addition, since these cytokines are known to be useful for tissue regeneration (Non-Patent Document 3), cytokines produced from a sheet-shaped cell culture act on a tissue to be transplanted, whereby the tissue is regenerated. It is also conceivable to promote the regeneration of coconut. However, a low-cost and simple method for improving the cytokine-producing ability of a sheet-shaped cell culture is not known.

JP 2007-528755 Gazette WO 2006/080434 JP 2005-229869 A JP 2010-81829 A JP 2010-226991 A

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 production ability, a method for producing the same, a composition containing the sheet-shaped cell culture, a graft and a medical product, and the sheet-shaped cell culture. It is intended to provide a method for treating a disease.

  The present inventors have been conducting intensive research to solve the above-mentioned problems, and at the time of producing a sheet-shaped cell culture, providing a step of freezing and thawing the cells, thereby producing cytokines in the sheet-shaped cell culture. It has been found that the activity such as the ability can be enhanced, and the present invention has been completed.

That is, the present invention relates to the following.
(1) 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. .
(2) The method according to the above (1), wherein a step of growing the cells is not included between the step of thawing the mesenchymal stem cells and the step of forming the sheet-shaped cell culture.
(3) The method according to the above (1) or (2), 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 the sheet-shaped cell culture is performed in a cell culture solution containing serum.
(5) The method according to any one of the above (1) to (4), comprising a step of freezing the mesenchymal stem cells before the step of thawing the mesenchymal stem cells.
(6) The method according to the above (5), comprising a step of expanding the mesenchymal stem cells before the step of freezing the mesenchymal stem cells.

(7) Any of the above (1) to (6), including 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-shaped cell culture. The method according to any one of the above.
(8) Between the step of thawing the mesenchymal stem cells and the step of forming the sheet-shaped cell culture, a cytokine-producing ability and engraftment from a sheet-shaped cell culture produced by a method comprising a step of growing cells. The method according to any one of the above (2) to (7), wherein a sheet-shaped cell culture having a high activity selected from the group consisting of ability, blood vessel inducing ability and tissue regeneration ability is obtained.
(9) The method according to (8), 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 cells are autologous cells.
(11) A sheet-shaped mesenchymal stem cell culture produced by the method according to any one of the above (1) to (10).
(12) A composition comprising the sheet-shaped mesenchymal stem cell culture according to (11).

(13) A method for treating a disease associated with tissue abnormality in a subject, comprising the step of: administering an effective amount of the sheet-shaped mesenchymal stem cell culture according to (11) or the composition according to (12). A method comprising administering to a subject in need thereof.
(14) The method according to (13) above, wherein the tissue is selected from the group consisting of myocardium, 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, muscular disease and middle ear disease. The method according to (13), wherein the method is selected from a group.
(16) A frozen mesenchymal stem cell for use in the method according to any one of (1) to (10).
(17) A culture substrate for use in the method according to any one of (1) to (10).
(18) A serum for use in the method according to any one of (3) to (10).

  The sheet-shaped cell culture produced by the method of the present invention has high activity such as cytokine production ability, and is excellent in engraftment and subsequent survival and functionality in tissues, persistence of functions, etc. Can be efficiently treated. Furthermore, the cytokine produced by the sheet-shaped cell culture produced by the method of the present invention acts on the tissue to be transplanted, whereby the regeneration of the tissue can be promoted. In addition, since the freezing and thawing operations in the present invention have high affinity with the conventional method for producing a sheet-shaped cell culture, and the labor and cost are minimal, the production method of the present invention is suitable for producing a sheet-shaped cell culture. Can be widely used.

5 is a graph showing the concentrations of VEGF and HGF secreted into the culture solution in the step of culturing sheets. It is the graph which showed the density | concentration of VEGF which the sheet-shaped cell culture after manufacture secreted in the culture solution. It is the graph which showed the density | concentration of HGF which the sheet-shaped cell culture after manufacture secreted in the culture solution. FIG. 9 is a photograph showing a sheet-shaped mesenchymal stem cell culture produced by the production method of Example 5. Sheet cell cultures are found in the middle 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 referred to herein are hereby incorporated by reference in their entirety.

  One aspect of the present invention provides a method for producing a sheet-shaped cell culture, which comprises the steps of: thawing frozen cells; and forming a sheet-shaped cell culture using the thawed cells (hereinafter referred to as “the present invention, 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. While not wishing to be bound by any particular theory, in the production method of the present invention, by thawing the frozen cells, weakly active cells are dropped off, while strongly active cells remain, and sheet cell culture is performed. It is thought that the activity as a whole increases.

  Cells in the production method of the present invention are not particularly limited as long as they can form a sheet-shaped 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 ligament cells, gingival cells, periosteal 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.) Including. The somatic cells may be those differentiated from stem cells, especially iPS cells. Non-limiting examples of cells in the production method of the present invention include, for example, myoblasts (eg, skeletal myoblasts), mesenchymal stem cells (eg, bone marrow, adipose tissue, peripheral blood, skin, hair root, muscle tissue) , Endometrial, 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, etc.), endothelial cells (eg, vascular endothelial cells, etc.), hepatocytes (eg, liver parenchymal cells, etc.), pancreatic cells (eg, pancreatic islet cells, etc.), kidney cells, adrenal cells, roots of teeth Examples include membrane cells, gingival cells, periosteal cells, and skin cells.

  In the present invention, the “sheet-shaped cell culture” refers to a cell in which cells are connected to each other to form a sheet. 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 the sheet-shaped cell culture. 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 (stacked (multilayer), for example, two layers, three layers, Layers, five layers, six layers, etc.).

  The sheet-shaped cell culture of the present invention preferably does not contain 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 a membrane made of PVDF) is known, the sheet-shaped cell culture of the present invention can maintain its physical integrity without such a scaffold. Further, the sheet-shaped cell culture of the present invention is preferably composed only of a cell-derived substance constituting the sheet-shaped cell culture, and does not contain any other substance.

  The cells in the present invention can be derived from any organism that can be treated with a sheet-shaped cell culture. 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.

  Only one type of cells may be used for forming the sheet-shaped cell culture, but two or more types of cells may be used. Also, in cells and the like obtained by isolating from a living body, the cells often include a plurality of types of cells, and it may be difficult to identify all types of cells. Sometimes only the most prominent cells in a cell population are confined. For example, when a certain cell population contains a predetermined ratio of cells A and one or more cells other than the cells A, and the ratio of the cells A is the largest, the cell population is referred to as a cell A for convenience. The sheet-shaped cell culture composed of the cell population may be referred to as a “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 ratio (purity) of the largest number of cells is determined at the start of sheet-forming culture or at the end of sheet-shaped cell culture production. , About 60% or more, preferably about 70% or more, more preferably about 75% or more.

  The cells may be xenogenic cells or allogeneic cells. 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 invention because rejection does not occur even after transplantation. 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 aspect of the invention, the cells are autologous cells or allogeneic cells. In one aspect of the invention, the cells are autologous cells. In another aspect of the invention, the cells are allogeneic cells.

The step of freezing the cells in the production method of the present invention can be performed by any known technique. Such a technique includes, but is not limited to, for example, subjecting the cells in the container to a freezing means, for example, a freezer, a deep freezer, or a low-temperature medium (for example, liquid nitrogen or the like). The temperature of the freezing means is not particularly limited as long as it can freeze a part, preferably the whole, of the cell population in the container, but is typically about 0 ° C. or lower, preferably about −20 ° C. or lower, and more preferably. Is about −40 ° C. or less, more preferably about −80 ° C. or less. The cooling rate in the freezing operation is not particularly limited as long as the viability and function of the cells after freezing and thawing are not significantly impaired. The cooling rate is in the order of hours to about 5 hours, preferably about 2 hours to about 4 hours, especially about 3 hours. Specifically, for example, the cooling can be performed at a rate of about 0.46 ° C./min. Such a cooling rate can be achieved by providing a container containing cells directly to a freezing means set to a desired temperature or by providing the container containing the cells in a freezing treatment container. The freezing treatment container may have a function of controlling the rate of temperature decrease in the container to a predetermined speed. Such freezing vessel of a known arbitrary, for example, can be used as BICELL ^{(R) (Nippon} freezer).

  The freezing operation may be performed while the cells are immersed in a culture solution or physiological buffer, but a cryoprotectant to protect the cells from freezing and thawing operations may be added to the culture solution, or the culture solution may be cryoprotected. It may be performed after treatment such as replacement with a cryopreservation solution containing the 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 liquid in which the cells are immersed during freezing contains an effective concentration of a cryoprotectant, virtually all of the culture solution is removed 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 does not show toxicity and has a cryoprotective effect, for example, cell viability, vitality, and function after cryothawing as compared with the case where no cryoprotectant is used. Etc. means a concentration which exhibits a reduction suppressing effect. Such a concentration is known to those skilled in the art or can be appropriately determined by routine experiments and the like.

  The cryoprotectant used in the production method of the present invention is not particularly limited as long as it exhibits a cryoprotective effect on cells. For example, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, sericin, propane Including diol, dextran, polyvinylpyrrolidone, polyvinyl alcohol, hydroxyethyl starch, chondroitin sulfate, polyethylene glycol, formamide, acetamide, adonitol, perseitol, raffinose, lactose, trehalose, sucrose, mannitol and the like. The 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. It is about 2% to about 20% (v / v) based on the whole storage solution. However, although outside this concentration range, alternative use concentrations known or experimentally determined for each cryoprotectant may be employed, and such concentrations are also within the scope of the invention.

  In the production method of the present invention, the step of thawing the frozen cells can be performed by any known cell thawing method. Use in a solid, liquid or gaseous medium at a temperature (eg, water), water bath, incubator, incubator, etc., or immerse frozen cells in a medium at a temperature higher than the freezing temperature (eg, a culture solution) But is not limited to this. The temperature of the thawing means or the immersion medium is not particularly limited as long as the cells can be thawed within a desired time, but is typically about 4 ° C to about 50 ° C, preferably about 30 ° C to about 40 ° C, Preferably it is from about 36C to about 38C. 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 is typically about 2 minutes or less, and particularly about 20 seconds or less. The decrease can be greatly suppressed. The thawing time can be adjusted by, for example, changing the temperature of the thawing means or the immersion medium, and the volume or composition of the culture solution or the cryopreservation solution at the time of freezing. The frozen cells include cells frozen by any technique, and non-limiting examples include, for example, cells frozen by the above-described cell freezing step. In one embodiment, the frozen cells are cells that have 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 the cells after the step of thawing the frozen cells and before the step of forming the sheet-shaped cell culture. Washing of the cells can be performed by any known technique. Typically, for example, the cells are washed with or without a washing solution (eg, serum or serum components (such as serum albumin), and a culture solution (eg, a medium). Or by suspending in a physiological buffer (eg, PBS, HBSS, etc.), centrifuging, discarding the supernatant, and collecting the precipitated cells, but is not limited thereto. In the step of washing the cells, such a cycle of suspension, centrifugation, and collection may be performed once or plural times (for example, 2, 3, 4, 5, etc.). In one aspect 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, techniques described in Patent Documents 1, 4, 5, and the like. The step of forming a sheet-shaped 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 sheeting the seeded cells. In one embodiment, the production method of the present invention does not include a step of growing the cells between the step of thawing the cells and the step of forming the sheet-shaped cell culture.

The culture substrate is not particularly limited as long as the cells can form a cell culture thereon, and includes, for example, containers of various materials, solid or semi-solid surfaces in the containers, 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 (for example, iron, stainless steel, aluminum, copper, brass) and the like can be mentioned. Also, the container preferably has at least one flat surface. Examples of such containers include, without limitation, cell culture dishes, cell culture bottles, and the like. Further, the container may have a solid or semi-solid surface inside. 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, substrates having an adhesive surface that are suitable for forming sheet cell cultures. Specifically, a substrate having a hydrophilic surface, for example, corona discharge-treated polystyrene, a substrate having a hydrophilic compound such as a collagen gel or a hydrophilic polymer coated on the surface, further, collagen, fibronectin, laminin And extracellular matrices such as vitronectin, proteoglycan, and glycosaminoglycan, and substrates coated on the surface with cell adhesion factors such as cadherin family, selectin family, integrin family, and the like. Further, such substrates are commercially available ^{(e.g., Corning (R) TC-Treated} Culture Dish, Corning , etc.).

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. Examples of such materials include, but are not limited to, (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 Acrylamide), (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 the like, a temperature-responsive material comprising 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)) can be used, these the production method of the present invention.

The culture substrate may have various shapes, but is preferably flat. Although the area is not particularly limited, typically, from about 1 cm ² ~ about 200 cm ^2, preferably about 2 cm ² ~ about 100 cm ^2, more preferably from about 3 cm ² ~ about 50 cm ^2.

  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 homologous 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 collected blood at about 1000 × g to about 1200 × g, and collecting the supernatant And the like.

  When incubating on a culture substrate, the serum may be used as a stock solution or diluted. Dilution may 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, DME, RPMI 1640, MCDB (MCDB 102, 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, 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 4 hours to about 48 hours, 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 the serum component can adhere to the culture substrate. 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.

  After incubation, the serum may be discarded. 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 invention, 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 in which a serum-free washing solution is added onto a culture substrate, stirred for a predetermined time (for example, about 5 seconds to about 60 seconds), and then discarded. Etc. can be used.

  In the present invention, 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 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 0.5 μg / mL. It is basically the same as serum except that the amount is from 05 μg / mL, more preferably from 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 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 the culture substrate with a steroid agent, the method of discarding, and the method of washing, the dilution concentration during incubation is, 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, 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.

  The culture substrate may be inoculated with cells immediately after being coated with serum or the like, or may be preserved after being coated, and then inoculated with cells. The coated substrate can be stored for a long period of time, for example, by keeping it at about 4 ° C or less, preferably about -20 ° C or less, more preferably about -80 ° C or less.

  Seeding of the cells into the culture substrate can be performed by any known method and conditions. Seeding of the cells into the culture substrate may be performed, for example, by injecting a cell suspension in which the cells are suspended in a culture solution into the culture substrate (culture container). For injection of the cell suspension, a device 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 invention, the seeding is performed at a density that allows the cells to form a sheet cell culture without substantial proliferation. "The density at which cells can form a sheet-shaped cell culture without substantial proliferation" means that the sheet-shaped cell culture is cultured in a non-proliferating culture medium substantially free of growth factors. 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-shaped 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 (see, for example, Patent Document 1). Therefore, the seeding density in this embodiment is higher than that in the method using a culture solution containing a growth factor. Such densities are, for example, typically about 1.0 × 10 ⁵ cells / cm ² or more 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 undergo differentiation, but for skeletal myoblasts, it is less than about 3.4 × 10 ⁶ cells / cm ² . is there.

Thus, the "density at which a sheet-shaped cell culture can be formed without substantial proliferation" in skeletal myoblasts is in some embodiments from about 1.0 x 10 < ⁵ > to about 3.4 x 10 < ⁶ > cells / cm < ² >. in one aspect, from about 3.0 × 10 ⁵ ~ about 3.4 × 10 ⁶ cells / cm ^2, in yet another embodiment about 3.5 × 10 ⁵ ~ about 3.4 × 10 ⁶ cells / ^cm 2, In yet another aspect, about 1.0 × 10 ⁶ to about 3.4 × 10 ⁶ / cm ² , in yet another aspect, about 3.0 × 10 ⁵ to about 1.7 × 10 ⁶ / cm ² , In another aspect, from about 3.5 × 10 ⁵ to about 1.7 × 10 ⁶ / cm ² , and in yet another aspect from about 1.0 × 10 ⁶ to about 1.7 × 10 ⁶ / cm ² . . The above range may include both or both of the upper and lower limits, as long as the upper limit is less than about 3.4 × 10 ⁶ / cm ² . Therefore, the density is, for example, not less than about 3.0 × 10 ⁵ / cm ^{2 and} less than about 3.4 × 10 ⁶ / cm ² (including the lower limit, not including the upper limit), and about 3.5 × 10 5 ⁵ / cm ² or more and less than about 3.4 × 10 ⁶ / cm ² (including the lower limit but not the upper limit), about 1.0 × 10 ⁶ / cm ² or more and about 3.4 × 10 ⁶ pieces / cm less than ² (including the lower and the upper limit is not included), about 1.0 × 10 ⁶ cells / cm ² ultra and about 3.4 × 10 than ⁶ / cm ² (lower limit also contains no upper limit), It may be more than about 1.0 × 10 ⁶ / cm ² and about 1.7 × 10 ⁶ / cm ² or less (not including the lower limit, but including the upper limit). Those skilled in the art can appropriately determine the cell density suitable for the present invention for cells other than skeletal myoblasts by experiments in accordance with the teachings of the present specification. In one embodiment, the "density at which cells can form a sheet-shaped cell culture without substantially growing" is a density at which cells reach confluence or higher, or a confluence density or higher.

The step of sheeting the seeded cells can also be performed by any known method and conditions. Non-limiting examples of such techniques are described in, for example, Patent Documents 1, 4, 5, and the like. It is believed that cell sheeting is achieved by cells adhering to each other via intercellular adhesion mechanisms such as adhesion molecules and 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 intercellular 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, culture at about 37 ° C., 5% CO ₂ . The cultivation can be performed under normal pressure (atmospheric pressure, non-pressurized). Those skilled in the art can select optimal conditions according to the type of cells to be seeded. In this specification, the culture for forming the seeded cells into sheets may be referred to as “sheet culture”.

  In one embodiment of the present invention, the culturing of the cells is performed within a predetermined period, preferably within a period during which the cells do not enter into differentiation. Thus, in this embodiment, the cells are maintained in an undifferentiated state during the culture period. The transition of a cell to differentiation can be assessed by any method known to those skilled in the art. For example, in the case of skeletal myoblasts, MHC expression, creatine kinase (CK) activity, multinucleation of cells, myotube formation, and the like can be used as indicators of differentiation. In a preferred embodiment of the present 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 solution (sometimes simply referred to as “culture solution” or “medium”) used for the culture is not particularly limited as long as it can maintain the survival of the cells, but typically includes amino acids, vitamins, and electrolytes. Can be used. In one aspect of the present invention, the culture solution is based on a basal medium for cell culture. Examples of such a basal medium include, but are not limited to, DMEM, MEM, F12, DMEM / F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80. -7 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.

Examples of the 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, Examples of vitamins include L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and the like, without limitation, 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. The basal medium may contain, in addition to these components, sugars such as D-glucose, pH indicators such as sodium pyruvate and phenol red, and putrescine.

In one embodiment of the present invention, the concentration of amino acids contained in the basal medium is about 63.2 mg / L to about 84 mg / L for L-arginine, about 35 mg / L to about 63 mg / L for L-cystine, and 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 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 contained in the basal medium is about 4 mg / L to about 12 mg / L for calcium D-pantothenate, about 4 mg / L to about 14 mg / L for choline chloride, and 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 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 solution may contain one or more additives such as serum, growth factors, steroid components, and selenium components. However, if these components are not of autologous origin, they can be undeniable impurities in the manufacturing process that can be a side effect factor such as anaphylactic shock to the recipient in clinical practice. It is desirable to eliminate the origin components. Thus, in a preferred embodiment of the invention, the cell culture 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 solution is substantially free of at least one of these non-autologous additives. Further, in a particularly preferred embodiment of the present invention, the cell culture is substantially free of non-autologous additives. In one aspect, the cell culture may comprise only a basal medium.

  In one aspect of the invention, the cell culture 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 has no adverse effect when the sheet-shaped cell culture is applied to a living body (for example, in a sheet-shaped cell culture. Serum albumin content of less than about 50 ng), preferably not actively adding these substances to the culture. In the present invention, in order to avoid side effects at the time of transplantation, the cell culture solution preferably contains substantially no heterologous serum, and more preferably contains substantially no non-self serum.

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

  In one aspect of the invention, the cell culture medium does not contain an effective amount of a growth factor. As used herein, the term "effective amount of growth factor" refers to the amount of a growth factor that significantly promotes cell growth as compared to the absence of a growth factor, or, for convenience, increases cell growth in the art. It means the amount usually added for the purpose. The significance of promoting cell growth can be appropriately evaluated by, for example, any statistical technique known in the art, for example, a t-test, and the usual amount of addition may be various values 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, "containing no effective amount of growth factor" means that the concentration of the growth factor in the culture solution in the present invention is less than such an effective amount. For example, the concentration of EGF in the culture solution 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 lower than the normal concentration in a living body. In such an embodiment, for example, the concentration of EGF in the culture solution in the cell culture is preferably less than about 5.5 ng / mL, more preferably less than about 1.3 ng / mL, even more preferably about 0.5 ng / mL. Is less than. In a further preferred embodiment, the culture solution of the present invention is substantially free of growth factors. Here, the term "substantially not contained" 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. Thus, in this embodiment, the culture medium does not contain growth factors at concentrations higher than those contained in other components therein, such as serum.

  In one aspect of the invention, the cell culture medium is substantially free of steroid agent components. Here, the “steroid agent component” 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. Therefore, "substantially free of steroid agent components" means that the content of these compounds in the culture solution is such that there is no adverse effect when the sheet-shaped cell culture is applied to a living body, preferably Not actively adding these compounds to the culture solution, that is, the culture solution does not contain other components therein, for example, steroid drug components at concentrations higher than those contained in serum or the like.

  In one aspect of the invention, the cell culture is substantially free of selenium components. 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 a living body, such as selenous acid. Therefore, "substantially free of selenium components" 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, preferably the culture is performed. Not actively adding these substances to the solution, that is, the culture solution does not contain other components therein, for example, selenium components at concentrations higher than those contained in serum or the like. Specifically, for example, in the case of humans, the selenium concentration in the culture medium is set to a normal value (for example, 10.6 μg / dL to 17.4 μg / dL) in human serum, It is lower than the product of the percentages (ie, if the human serum content is about 10%, the selenium concentration is, for example, from 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 the production steps such as growth factors, steroid agent components, and heterologous 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. No steps are required. Therefore, one embodiment of the method of the present invention does not include the step of removing impurities from the manufacturing process.
Here, “manufacturing process-derived impurities” typically include those listed below derived from each manufacturing process. That is, those derived from a cell substrate (eg, a host cell-derived protein, a host cell-derived DNA), those derived from a cell culture solution (eg, an inducer, an antibiotic, a medium component), or These include those derived from the extraction, separation, processing, and purification steps of a certain target substance (for example, see Pharmaceutical Inspection No. 571).

The culturing of the cells can be performed under the conditions usually performed in the art. For example, typical culture conditions include culturing at about 37 ° C., 5% CO ₂ . The cultivation 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. The culture can be performed in a container 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 attachment surface of the culture container, or by installing a mold of the desired size and shape on the cell attachment surface of the culture container, It can be adjusted arbitrarily by culturing the cells therein.

In one embodiment of the production method of the present invention, after the step of thawing the cells, a sheet-shaped cell culture is formed without substantially growing the cells. This procedure can further enhance the activity of the sheet cell culture.
The term "substantially does not proliferate" means that the cells are not proliferated beyond the range of measurement error.Whether the cells proliferated depends on, for example, the number of cells at the time of seeding and the sheet-shaped cell culture. It can be evaluated by comparing the number of cells after product formation. In the present invention, the number of cells after the formation of the sheet-shaped cell culture is typically about 300% or less, preferably about 200% or less, more preferably about 150% or less, even more preferably about 300% of the number of cells at the time of seeding. It is at most 125%, particularly preferably at most about 100%.

  Cell growth depends on various conditions, such as the number of seeded cells (seeding cell density), culture environment (eg, culture time, culture temperature, etc.), composition of the medium, and the like. By adjusting these conditions, Alternatively, the cells may not substantially grow. Among these conditions, by increasing the seeded cell density, a sheet-shaped cell culture can be obtained in a relatively short time while suppressing cell growth, and therefore, in the present invention, the growth is controlled by the seeded cell density. Is preferred. The density at which cells can form sheet cell cultures without substantial proliferation has already been described above.

  Therefore, in the present embodiment, after the step of thawing the cells, the sheet-forming culture is performed under the condition that the cells are not substantially grown, without going through a further cell growing step. Further, in the present embodiment, a step without cell growth, for example, a step of washing the cells, can be performed between the step of thawing the cells and the sheet-forming culture.

  The production method of the present invention may further include a step of collecting the formed sheet-shaped cell culture after the step of forming the sheet-shaped cell culture. The collection of the sheet-shaped cell culture is not particularly limited as long as the sheet-shaped cell culture can be released (peeled off) from the culture substrate serving as a scaffold at least partially while maintaining the sheet structure. It can be performed by an enzyme treatment with an enzyme (for example, trypsin or the like) and / or a mechanical treatment such as pipetting. In addition, when a cell culture is formed by culturing cells on a culture substrate whose surface is coated with a material whose physical properties change in response to temperature or light, for example, a material, the predetermined stimulus is applied. Thus, it can 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 than the upper critical solution temperature (UCST) of water of the temperature-responsive material. The temperature treatment can release the sheet-shaped cell culture non-enzymatically. Such a temperature treatment is not limited. For example, a culture substrate to which the formed sheet-shaped cell culture is adhered is placed in 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 the LCST (for example, a room temperature environment outside the incubator). The transition to the environment below the LCST is not limited. For example, a culture solution at a higher temperature than the LCST where the formed sheet-shaped cell culture is present is transferred to a medium at a temperature below the LCST (eg, a buffer solution (PBS, PBS, etc.)). HBSS or the like) or a liquid such as a culture solution). Therefore, a medium such as the above buffer solution can be used in the production method of the present invention to non-enzymatically release the sheet-shaped cell culture from the culture substrate.

  One embodiment of the production method of the present invention further includes, after the step of forming the sheet-shaped cell culture, the step of collecting the sheet-shaped cell culture, and the step of thawing the cells collects the sheet-shaped cell culture. It takes place within about 48 hours before the step. By setting the time between the step of thawing the cells and the step of collecting the sheet cell culture to about 48 hours or less, preferably about 36 hours or less, more preferably about 24 hours or less, The activity of the object 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 culture conditions suitable for growing various cells. In the production method of the present invention, after the step of thawing the cells, the case where the sheet-shaped cell culture is formed without substantially growing the cells, or the step of thawing the cells, When performed within about 48 hours prior to the harvesting step, it is useful to perform the step of growing the cells before the step of freezing the cells to obtain the desired number of cells.

  In one embodiment, the production method of the present invention does not include a step of introducing a gene into a cell. In another aspect, 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 treating the disease of interest, and may be, for example, a cytokine such as HGF or VEGF. The gene can be introduced by any known method such as the calcium phosphate method, lipofection method, ultrasonic introduction method, electroporation method, particle gun method, adenovirus vector, method using a virus vector such as retrovirus vector, microinjection method, etc. It can be performed using: Introduction of a gene into a cell can be performed without limitation, for example, before the step of freezing the cell.

  The sheet-shaped 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 the step of freezing the cells and the step of thawing the frozen cells are not included. The activity is higher than that of a culture (hereinafter, sometimes referred to as a non-frozen / thawed control sheet-shaped cell culture). Further, in the production method of the present invention including the step of thawing the frozen cells and the step of forming a sheet-shaped cell culture, the step of thawing the frozen cells and the step of forming the sheet-shaped cell culture are performed. The sheet-shaped cell culture manufactured in a mode that does not include the step of growing cells is a sheet-shaped cell culture manufactured in a mode that includes the step of growing (hereinafter, referred to as a growth control sheet-shaped cell culture). A) is more active. Such activities include, but are not limited to, for example, the ability to produce cytokines, the ability to engraft, the ability to induce blood vessels, and the ability to regenerate tissues. Here, the high activity is not limited based on the activity of a non-frozen / thawed and / or expanded control sheet-shaped cell culture (hereinafter, may be simply referred to as “control sheet-shaped 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 refers to a proteinaceous factor released from cells and mediating various cell-cell interactions. Therefore, the cytokine in the present invention is not limited to those released from immune cells. In one embodiment of the present invention, the cytokine is useful for engraftment of the sheet-shaped cell culture into a tissue to be transplanted. In one aspect of the invention, the cytokine is one that is beneficial for angiogenesis. In one aspect 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 an appropriate cytokine based on known information. In one aspect of the invention, the cytokine comprises a growth factor. In one aspect 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-producing ability is a growth-factor producing ability. In one embodiment of the present invention, the cytokine producing ability is a producing 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 a cytokine-producing ability, for example, a sheet-shaped cell culture is cultured in a culture solution for a predetermined time, and the amount of cytokine secreted into the culture solution is measured. The cytokine production ability can be quantified, for example, by measuring the amount of the gene. Techniques for measuring the amount of a specific protein or the level of gene expression are well known in the art, and techniques for measuring the amount of a protein include, but are not limited to, for example, electrophoresis, western blotting, and mass spectrometry. Methods for measuring the level of gene expression, such as analytical methods, EIA (eg, ELISA), RIA, immunohistochemistry, and immunocytochemistry, are not limited, and include, for example, Northern blotting, Southern blotting, and the like. , DNA microarray analysis, RNase protection assay, RT-PCR, PCR such as real-time PCR, in situ hybridization, and the like.

  If the activity is viable, the sheet-shaped cell culture is applied to the tissue, and the state of the sheet-shaped cell culture applied after a predetermined time has elapsed, for example, adhesion to the tissue, the remaining sheet-shaped cell culture The engraftment ability can be quantified by observing the size, color, morphology, and the like of these, and score these. When the activity is vascular inducing ability, the sheet-shaped cell culture is applied to the tissue, and the state of the sheet-shaped cell culture applied after a predetermined time and the tissue at the application site, for example, the presence or absence of angiogenesis is observed. Then, these can be scored to quantify the blood vessel inducing ability. When the activity is tissue regeneration ability, the sheet-shaped cell culture is applied to the tissue, and after a predetermined time, the state of the tissue at the application site (transplantation destination), for example, the size of the tissue, the fine structure of the tissue, By observing the ratio of the damaged tissue to the normal tissue, the function of the tissue, and the like, and scoring them, the tissue regeneration ability can be quantified.

  In one embodiment, the production method of the present invention is performed in vitro in all steps. In another embodiment, the production method of the present invention includes a step performed in vivo, including, but not limited to, for example, collecting a cell or a tissue that serves as a source of the cell from a subject. In one embodiment, all steps of the production method of the present invention are performed under aseptic conditions. 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 so 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-shaped cell culture of the present invention is characterized by having higher activity than the control sheet-shaped cell culture. Details regarding the activity are as described above. In one embodiment, the sheet-shaped cell culture of the present invention has an activity selected from the group consisting of cytokine-producing ability, engraftment ability, blood vessel inducing ability, and tissue regeneration ability higher than that of the control sheet-shaped cell culture. In one embodiment, the sheet 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 a control sheet cell culture. Since the sheet-shaped cell culture of the present invention has a high activity, the sheet-shaped cell culture is more effective than the control sheet-shaped cell culture in various therapeutic applications. In particular, when the ability to produce HGF and / or VEGF is high, engraftment of the tissue, induction of blood vessels, and tissue regeneration are promoted, and the sheet-shaped cell culture functions continuously for a long period of time to enhance the therapeutic effect. It becomes possible.

  The sheet-shaped cell culture of the present invention is useful for treating various diseases associated with tissue abnormalities. Thus, in one aspect, the sheet cell culture of the present invention is for use in treating a disease associated with tissue abnormalities. Since the sheet-shaped cell culture of the present invention has the same inherent properties as constituent cells except that it has 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 an object. 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, and the like. 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 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, described in 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-shaped cell culture of the present invention can be applied to a tissue to be treated, and used for repairing and regenerating the tissue. However, it can be used as a source of a physiologically active substance such as a hormone. It can also be transplanted to a site other than the tissue (eg, subcutaneous tissue) (eg, Arauchi et al., Tissue Eng Part A. 2009 Dec; 15 (12): 3943-9, Shimizu et al., Biomaterials. 2009). Oct; 30 (30): 5943-9). In addition, fragmenting a sheet-shaped cell culture into an injectable size and injecting the fragment into a site requiring treatment can obtain a higher effect than injection by a single cell suspension (Wang et al. , Cardiovasc Res. 2008 Feb 1; 77 (3): 515-24). Therefore, such a use method is also possible for the sheet-shaped cell culture of the present invention.

  In one aspect, 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 aspect, the sheet cell culture of the present invention is not genetically engineered. In another embodiment, the sheet 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, viability, function, etc. of the sheet cell culture and / or genes that are useful for treating disease. The gene to be introduced is not limited, and includes, for example, cytokine genes such as HGF gene and VEGF gene.

Another aspect of the present invention may include a composition (for example, a pharmaceutical composition or the like), a graft, a medical product or the like (hereinafter, referred to as a “composition or the like”) including the sheet-shaped cell culture of the present invention. ).
The composition or the like of the present invention may contain, in addition to the sheet-shaped cell culture of the present invention, various additional components such as a pharmaceutically acceptable carrier, and the viability, engraftability and / or viability of the sheet-shaped cell culture. Alternatively, it may contain a component that enhances function or the like, another active component useful for treating a target disease, or the like. As such additional components, any known components can be used, and those skilled in the art are familiar with these additional components. Further, the composition and the like 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 components useful for treating the target disease. . In one embodiment, the composition or the like of the present invention is for use in treating a disease associated with a tissue abnormality. The tissue or disease to be treated is 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 tissue abnormalities in a subject, comprising administering to a subject in need thereof an effective amount of a sheet-like cell culture or composition of the present invention. (Hereinafter, sometimes 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. Further, in the treatment method of the present invention, a component for enhancing the viability, engraftment and / or function of the sheet-shaped cell culture, and other active components useful for treatment of a target disease, etc., are added to the sheet of the present invention. It can be used in combination with a dendritic 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, before the step of producing the sheet-shaped cell culture, collecting a cell or a tissue that is a source of the cells for producing the sheet-shaped cell culture from the subject. 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 to which the sheet-like cell culture or the composition or the like is administered. In another embodiment, the subject from which the cells or tissue from which the cells are sourced is harvested is a homologous distinct body from the subject receiving the administration, such as a sheet cell culture or composition. In another embodiment, the subject from which the cells or tissue from which the cells are sourced is harvested is an individual that is heterogeneous to the subject receiving the administration such as a sheet 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, the subject may be healthy or may be suffering from any disease, but when treatment of a disease associated with tissue abnormalities is contemplated, typically the disease is affected by the disease. A subject is suffering or at risk of suffering.

  Also, 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 present invention, 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.

The method of administration typically includes direct application to the tissue, but when using fragments of sheet cell culture, various routes that allow administration by injection, such as intravenous, intramuscular, etc. The drug may be administered through a route such as internal, subcutaneous, local, intraarterial, intraportal, intraventricular, or 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 show specific embodiments of the present invention, and the present invention is not limited thereto.
Example 1
Cryopreserved human skeletal myoblasts (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 medium containing 20% serum, seeded in a flask, and cultured for 2 to 3 days.
To a UpCell® ⁽ 3.5 cm dish or 24-well multiwell, CellSeed Inc.), add a medium containing 20% serum so that the entire culture surface is covered, and add 37% C, 5% CO _{2, and} normal atmospheric pressure ( (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 The sheet culture was performed in the environment for about one day.

Example 2
5 × 10 ^{6 to} 5 × 10 ⁷ human skeletal myoblasts (derived from a human skeletal muscle sample) were suspended in a medium containing 20% serum, seeded in a flask, and cultured for 2 to 3 days. After recovering the cultured cells, the cells were suspended in a cell freezing storage solution (basal medium containing 10% DMSO) at a concentration of 1 × 10 ^{7 to} 5 × 10 ⁷ cells / mL, and frozen and stored in a liquid nitrogen tank.
To a UpCell® ⁽ 3.5 cm dish or 24-well multiwell, CellSeed Inc.), add a medium containing 20% serum so that the entire culture surface is covered, and add 37% C, 5% CO _{2, and} normal atmospheric pressure ( (Atmospheric pressure) for 3 hours to 3 days. After the treatment, the added medium was discarded.
The cryopreserved skeletal myoblasts were thawed at 37 ° C. and washed twice using 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 The sheet culture was performed for one day.

Example 3
After the sheet-forming culture in Example 1 or Example 2, all culture supernatants were collected, and the concentrations of cytokines (VEGF and HGF) produced in the culture supernatant were measured by ELISA (n = 3). Measurements, Human VEGF Quantikine ELISA Kit (R & D systems, Cat # DVE00) and ^{RayBio (R) Human HGF ELISA Kit} (RayBiotech, Inc., Cat # ELH-HGF-001) was performed using the following the manufacturer's manual. From the results shown in FIG. 1, it can be seen that VEGF and HGF are produced during the sheet-forming culture in any of the production methods of Examples 1 and 2. The production amount of the production method of Example 2 was larger than that of the production method of Example 2.

Example 4
According to the method of Example 1 or Example 2, peeling UpCell ^{(R) (24-well} multi-well, CellSeed Inc.) a sheet-like cell cultures sheeted in a temperature treatment, and washed with HBSS (+), 2 400 μL of MCDB medium containing 0.5% FBS was added to each well, and the cells were cultured in a CO ₂ incubator for about 24 hours. After the culture, all the supernatants were collected, and the concentrations of cytokines (VEGF and HGF) produced in the culture supernatant were measured by ELISA in the same manner as in Example 3. 400 μL of 2.5% FBS-containing MCDB medium was again added to the wells from which the supernatant was collected, and the cells were cultured in a CO ₂ incubator for 48 hours. After the culture, all supernatants were collected, and the concentrations of cytokines (VEGF and HGF) produced in the culture supernatant were measured by ELISA in the same manner as in Example 3. From the results shown in FIGS. 2 and 3, it can be seen that the sheet-shaped cell cultures produced by any of the production methods of Examples 1 and 2 produce VEGF and HGF, and that production is continued for as long as 96 hours. Further, the production amount of the sheet-shaped cell culture produced by the production method of Example 2 was larger.

Example 5
1 × 10 ^{6 to} 3 × 10 ⁷ human mesenchymal stem cells (derived from human bone marrow) were suspended in a serum-containing medium, seeded in a flask, and cultured for 2 to 3 days. After the cultured cells were collected, they were suspended in a cell freezing storage solution (basal medium containing 10% DMSO) at a concentration of 1 × 10 ^{6 to} 5 × 10 ⁶ cells / mL, and frozen and stored in a liquid nitrogen tank.
A serum-containing medium is added to UpCell ^(R) (48-well multiwell, CellSeed Inc.) to such an extent that the whole culture surface is covered, and the cells are added at 37 ° C., 5% CO _{2 and} normal atmospheric pressure (atmospheric pressure). The treatment was performed for a time period of 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 (Atmospheric pressure) for about 1 day.

  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 defect was observed in the 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), and the sheet-like mesenchymal stem cell culture is also the sheet-like skeletal muscle of Example 2. Similar to the blast cell culture, from the step of thawing the frozen cells and the step of growing the cells between the step of forming the sheet-shaped cell culture, a sheet-shaped mesenchymal stem cell culture produced by a method comprising , VEGF and HGF are considered to be high.

  Various features of the present invention described in the present specification can be combined in various ways, and embodiments obtained by such combinations include all of the aspects of the present invention including those not specifically described in the present specification. Within range. In addition, 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. Therefore, it is to be understood that the embodiments described herein are illustrative only and are not intended to limit the scope of the invention.

Claims (11)

Thawing the frozen mesenchymal stem cells, and using the thawed mesenchymal stem cells, forming a sheet-like cell culture on a culture substrate, and thawing the mesenchymal stem cells and the sheet A method for producing a sheet-shaped cell culture, which does not include a step of growing cells between the step of forming the cell culture. The method according to claim 1, wherein the culture substrate is coated with serum. 3. The method according to any one of claims 1 or 2 , wherein the step of forming a sheet cell culture is performed in a cell culture medium containing serum. The method according to any one of claims 1 to 3 , comprising a step of freezing the mesenchymal stem cells before the step of thawing the mesenchymal stem cells. 5. The method of claim 4 , comprising 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 5 , 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-shaped cell culture. Method. Between the step of thawing mesenchymal stem cells and the step of forming a sheet-shaped cell culture, a cytokine-producing ability, a viability, and a blood vessel from a sheet-shaped cell culture produced by a method comprising a step of growing cells. The method according to any one of claims 2 to 6 , wherein a sheet-shaped cell culture having high activity selected from the group consisting of inducibility and tissue regeneration ability is obtained.   The method according to claim 7, wherein the cytokine is selected from the group consisting of HGF and VEGF. The method according to any one of claims 1 to 8 , wherein the cells are autologous cells. A sheet-shaped mesenchymal stem cell culture produced by the method according to any one of claims 1 to 9 . A composition comprising the sheet-shaped mesenchymal stem cell culture according to claim 10 .