JPWO2019177135A1 - Method for producing sheet-shaped cell culture - Google Patents

Abstract

An object of the present invention is to inoculate a part of the discharge device while contacting the culture substrate or the cell suspension on the culture substrate, so that the cell suspension does not scatter and the culture substrate falls. It is an object of the present invention to provide a method for producing a uniform sheet-like cell culture without any risk such as unevenness. One aspect of the present invention is a method for producing a sheet-shaped cell culture, in which a cell suspension is cultured from the discharge medium while a part of the discharge device is brought into contact with the cell suspension on the culture substrate. The present invention relates to a method including a step of discharging onto a material.

Description

The present disclosure relates to a method for producing a sheet-like cell culture, which comprises a method of intermittently seeding a stationary culture substrate at a plurality of sites in a step of seeding a cell population containing sheet-forming cells.

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

As a part of such an attempt, a cell structure formed by using a scaffold and a sheet-shaped cell culture in which cells are formed in a sheet shape have been developed (Patent Document 1 and Non-Patent Document 2).

Special Table 2007-528755

Haraguchi et al., Stem Cells Stem Med. 2012 Feb; 1 (2): 136-41 Sawa et al., Surg Today. 2012 Jan; 42 (2): 181-4

In the case of producing a homogeneous and even sheet-like cell culture, in the step of seeding a cell population containing sheet-forming cells, in order to seed the cell population on the culture substrate, a conventional method such as shaking the culture substrate is performed. However, there is a risk that the cell suspension may scatter and the culture medium may be dropped. Further, there is a problem that labor (uniformization operation) is required and the operation is complicated.
An object of the present invention is to be able to produce a uniform and uniform sheet-shaped cell culture by a simpler operation.

While studying sheet-like cell cultures of skeletal myoblasts, the present inventors conducted intensive studies and found that the cell population was uniformly seeded at a plurality of locations on a stationary culture substrate. We have found a method for producing a uniform and uniform sheet-shaped cell culture that can omit the chemistry operation and simplify the operation, and have arrived at the present invention.
That is, with respect to the following in this disclosure:
(1) A method for producing a sheet-shaped cell culture, in which a cell suspension is cultivated from the discharge medium while a part of the discharge device is brought into contact with the culture substrate or the cell suspension on the culture substrate. A method that includes a step of discharging onto a material.
(2) The method according to (1), further comprising a step of positioning the discharge device substantially perpendicular to the culture medium.
(3) The method according to (1) or (2), further comprising a step of discharging the cell suspension discharged from the discharge device so as not to move from the discharge position.
(4) The method according to (1) to (3), further comprising a step of completing the discharge before the cells of the cell suspension to be discharged settle on the culture substrate.
(5) The method according to (1) to (4), further comprising the step of discharging the cell suspension on one culture medium a plurality of times.
(6) The method according to (1) to (5), further comprising a step of discharging the cell suspension to be discharged onto the culture substrate so as not to generate a flow.
(7) The method according to (1) to (6), further comprising the step of forming a sheet after discharging the cell culture solution.

(8) The method according to (1) to (7), further comprising a step of forming a sheet and then peeling it off.
(9) The method according to 1 to 8, which is performed in an automated device.
(10) (i) A step of seeding cells containing sheet-forming cells on a culture substrate, and (ii) The cell population seeded in step (i) is sheeted in a cell culture medium to form a sheet-like cell culture. (Iii) A method for producing a sheet-shaped cell culture, which comprises a step of peeling the sheet-shaped cell culture formed in step (iii) from the culture medium, wherein step (i) is static. The production method, which is a step of intermittently seeding the placed culture medium at a plurality of locations.
(11) The production method according to (1) to (10), wherein the cells include myoblasts, fibroblasts or cardiomyocytes.
(12) Sheet-shaped cell cultures produced by the production methods according to (1) to (11).
(13) A method for treating a disease improved by application of a sheet-shaped cell culture, which requires a sheet-shaped cell culture produced by the production methods described in (1) to (12). The method comprising applying the steps to the subject.
We will replenish as soon as the complaint is confirmed. )

By the method of the present invention, by omitting the homogenization operation, it becomes possible to easily produce a uniform sheet-like cell culture without spilling the cell suspension, and it is also possible to scale up, automate, etc. Can be.

FIG. 1 is a matrix showing the relationship between the method of seeding cells on a culture medium and the state of the sheet after seeding.

Hereinafter, the present disclosure will be described in detail.
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as those 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. In the event of a conflict between the publications referred to herein and the description herein, the description herein shall prevail.

One aspect of the present disclosure is a method for producing a sheet-shaped cell culture, in which a cell suspension is cultured from the discharge medium while a part of the discharge device is brought into contact with the cell suspension on the culture substrate. The present invention relates to a method including a step of continuously discharging onto a material.
Another aspect of the present invention is i) seeding cells containing sheet-forming cells on a culture medium, (ii) sheeting the cell population seeded in step (i) in a cell culture medium, and sheet-like cells. A method for producing a sheet-shaped cell culture, which comprises a step of forming a culture and a step of exfoliating the sheet-shaped cell culture formed in (iii) step (iii) from a culture medium, wherein the sheet-shaped cell culture is produced in step (i). ) Is a step of intermittently inoculating a stationary culture medium at a plurality of locations, according to the above-mentioned production method.

In the present disclosure, "sheet-shaped cell culture" refers to cells 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 capable of at least physically (mechanically) connecting cells to each other, and examples thereof include an extracellular matrix. The mediator is preferably derived from cells, particularly from the cells that make up the cell culture. The cells are at least physically (mechanically) connected, but may be more functionally, for example, chemically or electrically connected. The sheet-like cell culture may be composed of one cell layer (single layer) or two or more cell layers (laminated (multilayer) body, for example, two layers, three layers, etc. It may be 4 layers, 5 layers, 6 layers, etc.). Further, the sheet-shaped cell culture may have a three-dimensional structure having a thickness exceeding the thickness of one cell without showing a clear layered structure of the cells. For example, in the vertical cross section of a sheet-shaped cell culture, cells may be present in a non-uniformly (for example, mosaic-like) arrangement without being uniformly aligned in the horizontal direction.
"Mura" in the sheet-shaped cell culture means that there are parts having different thicknesses (parts having uneven thickness) in the sheet-shaped cell culture, and such parts are scattered in the sheet-shaped cell culture. If this is the case, the sheet-like cell culture may be torn or damaged due to uneven thickness (unevenness) between the sheet peeling and use. It is necessary to avoid the occurrence.

Sheet cell cultures preferably do not contain scaffolds. Scaffolds may be used in the art to attach cells to and / or inside the surface and maintain the physical integrity of sheet cell cultures, such as polyvinylidene difluoride. Although membranes made of PVDF) and the like are known, the sheet-shaped cell cultures of the present disclosure can maintain their physical integrity even in the absence of such scaffolds. In addition, the sheet-shaped cell culture of the present disclosure preferably comprises only the cell-derived substances constituting the sheet-shaped cell culture, and does not contain any other substances.

The cells constituting the sheet-like cell culture are not particularly limited as long as they can form the sheet-like cell culture, and include, for example, adherent cells (adherent cells). Adhesive cells include, for example, adherent somatic cells (eg, myocardial cells, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, renal cells, adrenal cells, root membrane cells, gingival cells, bone membrane cells, skin. 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 pulipotent stem) cells, mesenchymal stem cells, etc.) And so on. Somatic cells may be stem cells, particularly those differentiated from iPS cells (iPS cell-derived adherent cells). Non-limiting examples of cells constituting the sheet-like cell culture include, for example, myoblasts (eg, skeletal myoblasts), mesenchymal stem cells (eg, bone marrow, adipose tissue, peripheral blood, skin, hair roots, etc.). Muscle tissue, endometrial membrane, placenta, umbilical cord blood, etc.), myocardial cells, fibroblasts, heart stem cells, embryonic stem cells, iPS cells, synovial cells, chondrocytes, epithelial cells (eg, oral mucosal epithelial cells) , Retinal pigment epithelial cells, nasal mucosal epithelial cells, etc.), endothelial cells (eg, vascular endothelial cells, etc.), hepatocytes (eg, hepatic parenchymal cells, etc.), pancreatic cells (eg, pancreatic islet cells, etc.), renal cells, adrenal cells , Dental membrane cells, gingival cells, epithelial cells, skin cells and the like. Non-limiting examples of iPS cell-derived adherent cells include iPS cell-derived myocardial cells, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, renal cells, adrenal cells, root membrane cells, gingival cells, and bone membrane cells. , Skin cells, synovial cells, cartilage cells and the like. The cultured cell may be a gene-introduced cell.

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

In the present disclosure, "myocardial blast" means a myoblast present in the myocardium. Myocardial blasts are well known in the art and can be identified by markers such as Isl1. Myocardial blasts are any organism with myocardium, including, but not limited to, humans, non-human primates, rodents (mouse, rat, hamster, guinea pig, etc.), rabbits, dogs, cats, pigs, horses, etc. It may be derived from mammals such as cows, goats and sheep. In one embodiment, the myocardial blast is a mammalian myocardial blast. In certain embodiments, the myocardial blast is a human myocardial blast.
In the present disclosure, "cardiomyocyte" means a cell having the characteristics of cardiomyocytes, and the characteristics of cardiomyocytes include, without limitation, for example, expression of cardiomyocyte markers, presence of autonomous pulsation, and the like. .. Non-limiting examples of cardiomyocyte markers include, for example, c-TNT (cardiac troponin T), CD172a (also known as SIRPA or SHPS-1), KDR (also known as CD309, FLK1 or VEGFR2), PDGFRA, EMILIN2, VCAM and the like. ..

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

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

The sheet-shaped cell culture can be produced by any known method (see, for example, Patent Document 1, Patent Document 2, Japanese Patent Application Laid-Open No. 2010-081829, Japanese Patent Application Laid-Open No. 2011-10368, etc.). The method for producing a sheet-shaped cell culture is typically a step of seeding cells on a culture substrate, a step of sheeting the seeded cells, and peeling the formed sheet-shaped cell culture from the culture substrate. Includes, but is not limited to, steps. Prior to the step of seeding the cells on the culture medium, a step of freezing the cells and a step of thawing the cells may be performed. In addition, a step of washing the cells may be performed after the step of thawing the cells. Each of these steps can be performed by any known technique suitable for the production of sheet cell cultures. The production method of the present disclosure may include a step of producing a sheet-shaped cell culture, in which case the step of producing the sheet-shaped cell culture is a step relating to the above-mentioned method for producing a sheet-shaped cell culture as a sub-step. It may contain 1 or 2 or more of. In one embodiment, it does not include the step of growing the cells after the step of thawing the cells and before the step of seeding the cells on the culture medium.

The culture substrate is not particularly limited as long as the cells can form a cell culture on the cells, and includes, for example, containers of various materials, solid or semi-solid surfaces in the containers, and the like. The container preferably has a structure / material that does not allow a liquid such as a culture solution to permeate. Such materials include, without limitation, for example, polyethylene, polypropylene, Teflon®, polyethylene terephthalate, polymethylmethacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, polydimethyl. Acrylamide, metals (eg, iron, stainless steel, aluminum, copper, brass) and the like can be mentioned.

Also, the container preferably has at least one flat surface. Examples of such a container include, without limitation, a culture container having a bottom surface made of a culture substrate capable of forming a cell culture and a liquid-impermeable side surface. Specific examples of such a culture vessel include, but are not limited to, a cell culture dish, a cell culture bottle, and the like. The bottom surface of the container may be transparent or opaque. If the bottom surface of the container is transparent, cells can be observed and counted from the back side of the container. Further, the container may have a solid or semi-solid surface inside the container. Examples of the solid surface include plates and containers of various materials as described above, and examples of the semi-solid surface include gels and soft polymer matrices. The culture substrate may be prepared using the above-mentioned materials, or a commercially available one may be used. Preferred culture substrates include, but are not limited to, for example, substrates with an adhesive surface suitable for the formation of sheet-like cell cultures. Specifically, a base material having a hydrophilic surface, for example, a base material coated with a hydrophilic compound such as corona discharge-treated polystyrene, collagen gel or hydrophilic polymer, and further, collagen, vitronectin, laminin. , Vitronectin, proteoglycan, glycosaminoglycan and other extracellular matrices, and base materials coated with cell adhesion factors such as cadoherin family, selectin family and integrin family on the surface. In addition, such substrates are commercially available (eg, Corning ^(R) TC-Treated Culture Dish, Corning, etc.). The culture medium may be transparent or opaque in whole or in part.

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

The culture substrate may have various shapes, but is preferably flat. The area thereof is not particularly limited, but may be, for example, about 1 cm ² to about 200 cm ² , about 2 cm ² to about 100 cm ² , about 3 cm ² to about 50 cm ² .
The culture substrate may be coated (coated or coated) with serum. By using a culture substrate coated with serum, a higher density sheet-like cell culture can be formed. "Coated with serum" means a state in which serum components are attached to the surface of the culture medium. Such a state can be obtained without limitation, for example, by treating the culture substrate with serum. Treatment with serum involves contacting the serum with the culture medium and, if necessary, incubating for a predetermined period of time.

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

Serum for coating the culture substrate can be commercially available or prepared by routine from blood collected from the desired organism. Specifically, for example, a method in which the collected blood is left at room temperature for about 20 minutes to about 60 minutes to coagulate, and this is centrifuged at about 1000 × g to about 1200 × g, and the supernatant is collected. And so on.

When incubating on a culture medium, the serum may be used as a stock solution or diluted. Dilution can be made in any medium, such as, but not limited to, water, saline, various buffers (eg, PBS, HBSS, etc.), various liquid media (eg, DMEM, MEM, F12, DMEM / F12, etc.). It can be performed in DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, etc.). The dilution concentration is not particularly limited as long as the serum component can adhere to the culture substrate, and is, 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 also not particularly limited as long as the serum component can adhere to the culture substrate, for example, about 1 hour to about 72 hours, preferably about 2 hours to about 48 hours, more preferably about 2 hours to about. It is 24 hours, more preferably about 2 hours to about 12 hours. The incubation temperature is also 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., more preferably room temperature to about 40 ° C. Is.

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

In the present disclosure, the culture substrate may be coated with a growth factor. Here, "growth factor" means any substance that promotes cell growth as compared to its absence, eg, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblasts. Includes cell growth factor (FGF) and the like. In the coating method, disposal method and washing method of the culture substrate by 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. 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 disclosure, the culture substrate may be coated with a steroid agent. Here, the "steroid agent" refers to a compound having a steroid nucleus that can adversely affect the living body such as adrenocortical insufficiency and Cushing's syndrome. Such compounds include, but are not limited to, for example, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone and the like. In the coating method, disposal method and washing method of the cultured substrate with a steroid agent, the dilution concentration at the time of incubation is, for example, about 0.1 μg / mL to about 100 μg / mL, preferably about 0.4 μg / mL as dexamethasone. It is basically the same as serum except that it is about 40 μg / mL, more preferably about 1 μg / mL to about 10 μg / mL.

The culture medium may be coated with any one of serum, growth factors and steroids and any combination of these, ie serum and growth factors, serum and steroids, serum and growth factors and steroids, Alternatively, it may be coated with a combination of growth factors and steroids. When coating with a plurality of components, these components may be mixed and coated at the same time, or may be coated in separate steps.

The culture substrate may be seeded with cells immediately after being coated with serum or the like, or may be stored after being coated and then seeded with cells. The coated substrate can be stored for a long period of time, for example, by keeping it at about 4 ° C. or lower, preferably about −20 ° C. or lower, more preferably about −80 ° C. or lower.
Seeding of cells into the culture medium can be performed by any known method and conditions. Seeding of cells into a culture medium may be carried out, for example, by injecting a cell suspension in which cells are suspended in a culture medium into a culture medium (culture container). For injecting the cell suspension, an instrument suitable for the operation of injecting the cell suspension, such as a dropper or a pipette, can be used.

In (i), a cell population containing sheet-forming cells is seeded on a culture medium. The sheet-forming cells are not particularly limited as long as they are the cells described above as cells that can constitute a sheet-like cell culture. The cell population includes at least one type of sheet-forming cell, but may include two or more types of sheet-forming cells, or may include cells other than sheet-forming cells. In one aspect of the present disclosure, the at least one sheet-forming cell contained in the cell population is a myoblast, preferably a skeletal myoblast. In such an embodiment, the cell population may further include fibroblasts. In another aspect of the disclosure, at least one sheet-forming cell included in a cell population is a cardiomyocyte. In yet another aspect of the disclosure, the at least one sheet-forming cell included in the cell population is a mesenchymal stem cell. In such an embodiment, the cell population may further include vascular endothelial cells.

The cell density to be seeded is not particularly limited as long as it can form a sheet-like cell culture, but in a preferred embodiment, the cell population is seeded at a density reaching confluence or higher. In the present disclosure, the “density reaching confluence” refers to a density at which it is assumed that when cells are seeded, the seeded cells cover the entire adhesive surface of the culture vessel without gaps. For example, the density at which cells are expected to come into contact with each other when seeded, the density at which contact inhibition occurs, or the density at which cell proliferation is substantially stopped by contact inhibition.

Non-limiting examples of seeding density of the cell population are about 7.1 × 10 ⁵ cells / cm ² to about 3.0 × 10 ⁶ cells / cm ² , about 7.3 × 10 ⁵ cells / cm ² to about 2. 8 x 10 ⁶ pieces / cm ² , about 7.5 x 10 ⁵ pieces / cm ² to about 2.5 x 10 ⁶ pieces / cm ² , about 7.5 x 10 ⁵ pieces / cm ² to about 3.0 x 10 ⁶ pieces / cm ² , about 7.8 x 10 ⁵ pieces / cm ² to about 2.3 x 10 ⁶ pieces / cm ² , about 8.0 x 10 ⁵ pieces / cm ² to about 2.0 x 10 ⁶ Pieces / cm ² , about 8.5 x 10 ⁵ pieces / cm ² to about 1.8 x 10 ⁶ pieces / cm ² , about 9.0 x 10 ⁵ pieces / cm ² to about 1.6 x 10 ⁶ pieces / cm Includes densities such as cm ^2. Unless otherwise specified, these densities are the densities of all cells contained in a cell population.

In yet another embodiment, seeding can be carried out in a cell culture medium that is substantially free of growth factors at a density at which at least one sheet-forming cell that can be contained in the cell population does not substantially proliferate. In such an embodiment, the other cells that may be included in the cell population may have a proliferative density while undergoing growth inhibition.
The culture medium used in the method of the present disclosure is as described above. In a preferred embodiment, the culture substrate may be coated with serum. In another preferred embodiment, the culture substrate may be coated with a temperature responsive material. In a more preferred embodiment, the culture substrate may be coated with a temperature responsive material and serum.

In the present disclosure, the seeding operation is not particularly limited, but for example, it is preferable to fix the discharge position on the surface of the culture substrate and intermittently sow the culture substrate in a stationary state at a plurality of locations. "Seeding" means releasing the cell suspension from the discharge device and transferring it onto the culture medium. The term "fixing the discharge position" refers to the act of discharging the cell suspension from a pipette or the like without any directionality, and as a result, the cell suspension flows out onto the culture base material on the surface of the culture base material. It is preferable that there is no state. Flow means that the cell suspension begins to flow on the culture medium. When placed on the culture substrate, the cell suspension is preferably placed so that no flow occurs. "Intermittently at a plurality of places" means a state in which a plurality of places for fixing the discharge positions are set on the culture medium and the seeding at each position is not contacted. The distance from the liquid surface to the tip of the pipette is preferably 9 mm or less, but is not limited to this. If the distance is more than 9 mm, the cell suspension may flow out onto the culture medium due to ejection. The discharge speed from a pipette or the like is preferably, but is not limited to, 3 to 10 seconds per 10 mL.

In one aspect of the present disclosure, a method of fixing the discharge position on the surface of the culture substrate and uniformly seeding the culture substrate in a stationary state is such that the suspension is placed in a pipette at about 20 to about 100 mL, preferably about 40 mL. Uniformize the cells by taking ~ about 80 mL, more preferably about 60 mL, and pipetting about 1 to about 10 times, preferably about 3 to about 8 times, more preferably about 5 times, of which 5 to 15 mL. A step of fixing the discharge position on the surface of the culture substrate and discharging 10 mL to the culture substrate so as not to flow may be included.

In (ii), the seeded cell population is incubated in a cell culture medium to be sheeted to form a sheet-like cell culture.
Sheeting of seeded cells can be performed by any known method and condition. Non-limiting examples of such a method are described in, for example, Patent Document 1, WO 2014/185517 and the like. It is believed that cell sheeting is achieved by the cells adhering to each other via adhesion molecules or intercellular adhesion mechanisms such as extracellular matrix. Thus, sheeting of seeded cells can be achieved, for example, by culturing the cells under conditions that form cell-cell adhesions. Such conditions may be any as long as they can form cell-cell adhesion, but usually, cell-cell adhesion can be formed under the same conditions as general cell culture conditions. Such conditions include, for example, culturing at about 37 ° C. and 5% CO _2. In addition, the culture can be carried out under normal pressure (atmospheric pressure, non-pressurization). Culturing can be carried out in containers of any size and shape. For the size and shape of the sheet-shaped cell culture, adjust the size and shape of the cell adhesion surface of the culture vessel, or install a mold of the desired size and shape on the cell adhesion surface of the culture vessel. It can be arbitrarily adjusted by culturing cells inside the cell. In the present specification, the culture for sheeting the seeded cells may be referred to as "sheeted culture". Sheet culture reduces the thickness of the sheet-like cell culture on the culture substrate (in the culture vessel). That is, after seeding, after the cells have settled, the thickness of the cell layer on the culture substrate decreases due to the subsequent sheet formation, but the sheet-like cell culture shrinks due to peeling from the culture substrate and increases in thickness again. .. The decrease in thickness due to sheeting is about 90% to about 70%, assuming that the thickness of the cell layer immediately after seeding is 100%.

The incubation time for sheet formation is not particularly limited as long as the sheet can be formed. The time during which a sheet can be formed can vary depending on the type of cells contained in the seeded cell population (particularly the type of sheet-forming cells) and the state of the cells. For example, cells containing skeletal myoblasts as sheet-forming cells. When the population is sown, sheets can be formed in about 2 hours. Therefore, in one embodiment, the incubation time for sheeting can be 2 hours or more.

As described above, the present inventors release after peeling by artificially peeling after the sheet-shaped cell culture is formed and before the formed sheet-shaped cell culture is naturally peeled from the culture substrate. It has been found that the produced sheet-shaped cell culture can be maintained in high quality by subjecting the obtained sheet-shaped cell culture to an adhesion-preventing treatment. Therefore, the incubation for sheeting is terminated before the sheet cell cultures are spontaneously exfoliated. The time from the start of incubation to the start of spontaneous exfoliation may vary depending on the type of cells contained in the seeded cell population (particularly the type of sheet-forming cells) and the state of the cells. When a cell population containing myoblasts is seeded, spontaneous exfoliation often occurs in about 6 to 12 hours. Therefore, in one aspect of the present disclosure, the upper limit of incubation time for sheeting is 12 hours, 11.5 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours or 4 hours. possible.

Therefore, in the production method of the present disclosure, the incubation time for sheeting is 2 to 12 hours, 2 to 11.5 hours, 2 to 11 hours, 2 to 10 hours, 2 to 9 hours, 2 to 8 hours, 2 It can be ~ 7 hours, 2-6 hours, 2-5 hours or 2-4 hours.

The cell culture medium used for culturing (sometimes referred to simply as "culture medium" or "medium") is not particularly limited as long as it can maintain the survival of cells, but is typically amino acids, vitamins, and electrolytes. Can be used as the main component. In one aspect of the present disclosure, the culture medium is based on a basal medium for cell culture. Such basal medium is not limited to, for example, DMEM, MEM, F12, DMEM / F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80. -7 and so on are included. Many of these basal media are commercially available, and their compositions are also known.
The basal medium may be used as it has a standard composition (for example, as it is on the market), or the composition may be appropriately changed depending on the cell type and cell conditions. Therefore, the basal medium used in the present disclosure is not limited to those having a known composition, and includes those in which one or more components are added, removed, increased or decreased.

The amino acids contained in the basal medium are not limited, for example, L-arginine, L-cystine, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, and the like. L-phenylalanine, L-serine, L-threonine, L-tryptophane, L-tyrosine, L-valine and the like are not limited as vitamins, for example, calcium D-pantothenate, choline chloride, folic acid, i. -Inositol, niacinamide, riboflavin, thiamine, pyridoxin, biotin, lipoic acid, vitamin B12, adenine, thymidin, etc., and the electrolytes are not limited, for example, CaCl ₂ , KCl, marriage ₄ , NaCl, NaH. ₂ PO ₄ , NaHCO ₃ , Fe (NO ₃ ) ₃ , FeSO ₄ , CuSO ₄ , MnSO ₄ , Na ₂ SiO ₃ , (NH ₄ ) ₆ Mo ₇ O ₂₄ , NaVO ₃ , NiCl ₂ , ZnSO _4, etc. are included, respectively. Is done. 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 aspect of the present disclosure, the concentration of amino acids contained in the basal medium is 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. : Approximately 4.4 mg / L to approximately 584 mg / L, glycine: approximately 2.3 mg / L to approximately 30 mg / L, L-histidine: approximately 42 mg / L, L-isoleucine: approximately 66 mg / L to approximately 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 ~ 66 mg / L, L-serine: about 32 mg / L ~ about 42 mg / L, L-threonine: about 12 mg / L ~ about 95 mg / L, L-tryptophane: about 4.1 mg / L ~ about 16 mg / L, L-tyrosine: about 18.1 mg / L to about 104 mg / L, L-valine: about 94 mg / L to about 117 mg / L.
Further, in one aspect of the present disclosure, the concentration of the vitamin preparation contained in the basal medium is: 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 can be impurities derived from the manufacturing process that cannot be denied that they can cause side effects such as anaphylactic shock to the recipient in clinical practice. It may be desirable to eliminate the derived components. Therefore, in a preferred embodiment of the present disclosure, the cell culture medium does not contain an effective amount of at least one of these non-self-derived additives. Also, in a more preferred embodiment of the present disclosure, the cell culture medium is substantially free of at least one of these non-self-derived additives. Moreover, in a particularly preferred embodiment of the present disclosure, the cell culture medium is substantially free of non-self-derived additives. In one embodiment, the cell culture medium may contain only the basal medium.

In one aspect of the present disclosure, the cell culture medium is substantially free of serum. A cell culture medium that does not substantially contain serum is sometimes referred to as a "serum-free medium" in the present specification. Here, "substantially free of serum" means that the content of serum in the culture medium does not adversely affect the application of the sheet-shaped cell culture 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 disclosure, in order to avoid side effects during transplantation, the cell culture medium preferably contains substantially no heterologous serum, and more preferably does not contain substantially no non-self serum.

In one aspect of the present disclosure, the cell culture medium comprises serum. The serum may be allogeneic serum or heterologous serum. In certain embodiments, the cell culture medium comprises autologous serum. When culturing cells on a serum-coated culture medium, the serum contained in the cell culture medium (serum used for cell culture) is different even if it is the same as the serum used for coating the culture medium. You may. In one embodiment, the serum contained in the cell culture medium is the same as the serum used to coat the culture substrate, and in a particular embodiment, the serum is autologous serum. Serum may be for use in the production methods of the present disclosure. For example, the serum may be for use in culturing cells or for coating a culture substrate.

In one aspect of the present disclosure, the cell culture medium does not contain an effective amount of growth factor. Here, the "effective amount of growth factor" is an amount of growth factor that significantly promotes cell growth as compared with the case without growth factor, or, for convenience, cell growth in the art. It usually means the amount to be added for the purpose. The significance of promoting cell proliferation can be appropriately evaluated by, for example, any statistical method known in the art, such as t-test, and the usual amount of addition can be various in the art. It can be known from publicly known documents. Specifically, the effective amount of EGF in cell culture is, for example, about 0.005 μg / mL or more.

Therefore, "does not contain an effective amount of growth factor" means that the concentration of growth factor in the culture medium in the present disclosure is less than such an effective amount. For example, the concentration of EGF 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 disclosure, the concentration of growth factor in the culture medium is less than the normal concentration in the living body. In such an embodiment, 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, still more preferably about 0.5 ng / mL. Is less than. In a more preferred embodiment, the culture medium in the present disclosure is substantially free of growth factors. Here, "substantially free" means that the content of the growth factor in the culture solution does not adversely affect the application of the sheet-shaped cell culture to a living body, preferably the growth factor in the culture solution. Means not to add positively. Therefore, in this embodiment, the culture broth does not contain growth factors at a concentration higher than that contained in other components thereof, such as serum.

In one aspect of the present disclosure, 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 the living body such as adrenocortical insufficiency and Cushing's syndrome. Such compounds include, but are not limited to, for example, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone and the like. Therefore, "substantially free of steroid component" means that the content of these compounds in the culture medium does not adversely affect the application of the sheet-like cell culture to a living body, preferably. It means that these compounds are not positively added to the culture broth, that is, the culture broth does not contain other components therein, for example, a steroid component having a concentration higher than that contained in serum or the like.

In one aspect of the present disclosure, the cell culture medium is substantially free of selenium components. Here, the "selenium component" includes a selenium molecule and a selenium-containing compound, particularly a selenium-containing compound capable of liberating the selenium molecule in vivo, such as selenous acid. Therefore, "substantially free of selenium component" means that the content of these substances in the culture medium does not adversely affect the application of the sheet-like cell culture to a living body, preferably the culture. It means that these substances are not positively added to the broth, that is, the culture broth does not contain other components thereof, for example, a selenium component having a concentration higher than that 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 in human serum (for example, 10.6 μg / dL to 17.4 μg / dL) of human serum contained in the medium. It is lower than the value multiplied by the ratio (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 disclosure, impurities derived from the production process such as growth factors, steroid agent components, and heterologous serum components, which have been conventionally required when producing a cell culture to be applied to a living body, are removed by washing or the like. No steps are required. Therefore, one aspect of the method of the present disclosure does not include the step of removing impurities derived from this manufacturing process.
Here, the "impurities derived from the manufacturing process" typically include those derived from each manufacturing process and listed below. That is, those derived from a cell substrate (for example, host cell-derived protein, host cell-derived DNA), those derived from a cell culture medium (for example, inducer, antibiotics, medium component), or in a step after cell culture. It is derived from an extraction, separation, processing, or purification process of a certain target substance (see, for example, Pharmaceutical Trial No. 571).

In (iii), the formed sheet-shaped cell culture is exfoliated from the culture substrate.
The peeling of the sheet-shaped cell culture from the culture substrate 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 maintaining the sheet structure at least partially. , For example, by enzymatic treatment with a proteolytic enzyme (eg trypsin, etc.) and / or mechanical treatment such as pipetting. In addition, when cells are cultured on a culture substrate whose surface is coated with a stimulus, for example, a material whose physical properties change in response to temperature or light to form a cell culture, a predetermined stimulus is applied. It can also be liberated 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 below the lower limit critical solution temperature (LCST) or above the upper limit critical solution temperature (UCST) with respect to water of the temperature responsive material. The sheet-like cell culture can be released non-enzymatically by the temperature treatment. Such temperature treatment is not limited, and for example, the culture substrate to which the formed sheet-shaped cell culture is attached is placed in a culture environment having a temperature higher than LCST (for example, in an incubator having a temperature of about 37 ° C.). This can be achieved by migrating to an environment below the LCST (for example, a room temperature environment outside the incubator). The transition to a sub-LCST environment is not limited to, for example, a culture medium at a temperature higher than the LCST in which the formed sheet cell culture is present, and a medium at a temperature below the LCST (eg, buffer (PBS,). It can be achieved by substituting with (HBSS, etc.) or a liquid such as a culture solution). Therefore, the medium such as the buffer solution can be used in the production method of the present disclosure to non-enzymatically release the sheet-shaped cell culture from the culture substrate.

The sheet-shaped cell culture exfoliated by the step (iii) shrinks and the area becomes smaller than that before exfoliation. The sheet-shaped cell culture produced by the production method of the present disclosure is characterized in that it does not easily shrink after exfoliation and has a larger area. In one aspect of the present disclosure, the stripped sheet cell culture is about 35% or more, eg, about 35%, about the area of the sheet cell culture before stripping (ie, the area of the culture substrate). It has an area of 36%, about 37%, about 38%, about 39%, about 40%.

It is presumed that the contraction after exfoliation of the sheet-shaped cell culture is influenced by the magnitude of the intercellular adhesion force of the cells constituting the sheet-shaped cell culture. Therefore, when the incubation time for sheeting is extended to some extent, for example, when the sheet-shaped cell culture is incubated until the time when spontaneous exfoliation occurs, the post-exfoliation shrinkage rate of the sheet-shaped cell culture reaches the maximum. it is conceivable that. Therefore, the area of the sheet-shaped cell culture may be defined based on the area when the shrinkage rate after peeling is maximized, that is, when the area of the sheet-shaped cell culture after peeling is minimized. In one aspect of the present disclosure, (iii) is the case where the minimum area of the sheet-shaped cell culture after exfoliation (that is, the area of the sheet-shaped cell culture after exfoliation when incubated until a time at which spontaneous exfoliation occurs) is 1. The area of the sheet-like cell culture obtained in) may be about 1.04 to about 1.4, about 1.1 to about 1.4, or about 1.3 to about 1.35.

The production method of the present disclosure may include a step of freezing cells (cell population) and a step of thawing frozen cells before (i). Freezing of cells can be performed by any known method. Such techniques include, but are not limited to, subjecting the cells in the container to a freezing means, such as a freezer, a deep freezer, a cold medium (eg, liquid nitrogen, etc.). The temperature of the freezing means is not particularly limited as long as it can freeze a part, preferably the whole cell population in the container, 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, but typically, cooling is started from 4 ° C. until it reaches about -80 ° C. The cooling rate is about 5 hours, preferably about 2 hours to about 4 hours, particularly about 3 hours. Specifically, for example, it can be cooled at a rate of about 0.46 ° C./min. Such a cooling rate can be achieved by providing the freezing means set to a desired temperature with the container containing the cells directly or by accommodating the container in the freezing treatment container. The freezing treatment container may have a function of controlling the rate of decrease in temperature inside the container to a predetermined speed. As the freezing treatment container, any known container, for example, BICELL ^(R) (Japan Freezer), a program freezer, or the like can be used.

The freezing operation may be performed while the cells are immersed in the culture solution or physiological buffer, but a cryoprotectant for protecting the cells from the freezing / thawing operation may be added to the culture solution, or the culture solution may be cryoprotected. It may be carried out after undergoing a treatment such as replacement with a cryopreservation solution containing an agent. Therefore, the production method of the present disclosure including a freezing step 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 the cells are immersed at the time of freezing contains an effective concentration of cryoprotectant, substantially 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, the survival rate, vitality, and function of cells after freeze-thaw as compared with the case where no cryoprotectant is used. It means a concentration that shows a decrease suppressing effect such as. Such a concentration is known to those skilled in the art or can be appropriately determined by routine experiments or the like.

The cryoprotectant is not particularly limited as long as it exhibits a cryoprotective effect on cells, and is, for example, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, sericin, propanediol, dextran, polyvinylpyrrolidone, and the like. Includes polyvinyl alcohol, hydroxyethyl starch, chondroitin sulfate, polyethylene glycol, formamide, acetamide, adonitol, persetol, raffinose, lactose, trehalose, sucrose, mannitol and the like. The cryoprotectant may be used alone or in combination of two or three 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 the effective concentration defined above, and typically, for example, the culture solution or freezing. It is about 2% to about 20% (v / v) with respect to the entire storage solution. However, although outside this concentration range, alternative use concentrations known or experimentally determined for each cryoprotectant can also be employed, such concentrations are also within the scope of the present disclosure.

The step of thawing frozen cells can be performed by any known cell thawing technique, typically for example, the frozen cells are thawed by means of thawing, eg, solid, liquid or gas at a temperature above the freezing temperature. It is achieved by subjecting the cells to a medium (eg, water), a water bath, an incubator, an incubator, etc., or by immersing the frozen cells in a medium (eg, a culture medium) having a temperature higher than the freezing temperature. , 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. It is preferably from about 36 ° C to about 38 ° C. The thawing time is not particularly limited as long as it does not significantly impair the survival rate and function of the cells after thawing, but is typically within about 2 minutes, and in particular, by setting it within about 20 seconds, the survival rate can be increased. The decrease can be significantly suppressed. The thawing time can be adjusted by changing, for example, the temperature of the thawing means or the immersion medium, the volume or composition of the culture solution or the cryopreservation solution at the time of freezing, and the like. Frozen cells include cells frozen by any method, and non-limiting examples thereof include cells frozen by the above-mentioned step of freezing cells. 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 methods of the present disclosure.

The production method of the present disclosure is to wash the cells after the above-mentioned step of thawing frozen cells and before the step of forming a sheet-like cell culture, preferably the step of seeding the cells on a culture medium. It may include steps. Washing of cells can be performed by any known technique, typically, for example, a culture medium (eg, medium) containing or not containing a wash solution (eg, serum or serum components (eg, serum albumin)). Etc.) or suspension in a physiological buffer (eg, PBS, HBSS, etc.), centrifugation, discarding the supernatant, and collecting the precipitated cells, but not limited to. In the step of washing the cells, such suspension, centrifugation, and recovery cycles may be performed one or more times (for example, 2, 3, 4, 5 times, etc.). In one aspect of the present disclosure, the step of washing cells is performed immediately after the step of thawing frozen cells.

The production method of the present disclosure may further include a step of proliferating cells prior to the step of freezing the cells described above. The step of growing cells may be performed by any known method, and those skilled in the art are familiar with the culture conditions suitable for the growth of various cells.

In one aspect, the production method of the present disclosure does not include the step of introducing a gene into a cell. In another aspect, the production method of the present disclosure comprises the step of introducing a gene into a cell. The gene to be introduced is not particularly limited as long as it is useful for the treatment of the target disease, and may be, for example, a cytokine such as HGF or VEGF. For gene introduction, 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 utilization method, microinjection method, etc. can be used. Can be done using. The introduction of the gene into the cell is not limited, for example, before the step of freezing the cell.

In one aspect, the manufacturing method of the present disclosure is performed in vitro in all steps. In another aspect, the methods of manufacture of the present disclosure are steps performed in vivo, such as, but not limited to, cells from a subject or tissue that is a source of cells (eg, striated muscle tissue, particularly skeletal muscle tissue). Includes steps to collect. In one aspect, the manufacturing method of the present disclosure is carried out under sterile conditions in all steps. In one aspect, the production method of the present disclosure is carried out so that the finally obtained sheet-like cell culture is substantially sterile. In one aspect, the production method of the present disclosure is carried out so that the finally obtained sheet-like cell culture is sterile.

Another aspect of the present disclosure relates to sheet cell cultures produced by the production methods of the present disclosure. The sheet cell cultures of the present disclosure are useful in treating diseases that are ameliorated by the application of sheet cell cultures, such as various diseases associated with tissue abnormalities. Therefore, in one aspect, the sheet cell cultures of the present disclosure are intended for use in the treatment of diseases that are ameliorated by the application of sheet cell cultures, particularly those associated with tissue abnormalities. The sheet-shaped cell cultures of the present disclosure have similar properties peculiar to constituent cells except that they have higher mechanical strength than conventional sheet-shaped cell cultures, and therefore at least conventional myoblasts. It can be applied to tissues and diseases that can be treated with sheet-like cell cultures containing cells or fibroblasts. The tissue to be treated includes, but is not limited to, myocardium, esophagus, skin, pancreas, skeletal muscle and the like. The diseases to be treated include, for example, heart diseases (for example, myocardial infarction (myocardial infarction, heart trauma), myocardial disease, etc.) and esophageal diseases (for example, esophageal surgery (removal of esophageal cancer)). Later esophageal inflammation / stenosis prevention, etc.), skin diseases (eg, skin damage (trauma, burns), etc.), pancreatic diseases (eg, pancreatic fistula, etc.), muscle diseases (eg, muscle damage, myocardial infarction, etc.) Be done. The usefulness of the sheet-shaped cell cultures of the present disclosure for the above-mentioned diseases is described in, for example, Patent Document 1, Non-Patent Document 1, Tanaka et al., J Gastroenterol. 2013; 48 (9): 1081-9. Have been described. The sheet-like cell cultures of the present disclosure can also be fragmented to an injectable size and injected at the site requiring treatment for greater efficacy than injection with a single cell suspension (Wang et. al., Cardiovasc Res. 2008; 77 (3): 515-24). Therefore, such a use is also possible for the sheet-shaped cell culture of the present disclosure.

In one aspect, the sheet cell cultures of the present disclosure are substantially sterile. In one aspect, the sheet cell cultures of the present disclosure are sterile. In one aspect, the sheet cell cultures of the present disclosure are not genetically engineered. In another embodiment, the sheet cell cultures of the present disclosure are genetically engineered. Genetic manipulation includes, but is not limited to, the introduction of, for example, genes that enhance the viability, engraftment, function, etc. of sheet cell cultures and / or genes that are useful in the treatment of disease. The gene to be introduced is not limited, and examples thereof include cytokine genes such as HGF gene and VEGF gene.

Another aspect of the present disclosure may be generically referred to as a composition (eg, pharmaceutical composition, etc.), implant, medical product, etc. (hereinafter, "composition, etc.") containing the sheet-like cell culture of the present disclosure. ).
In addition to the sheet-like cell cultures of the present disclosure, the compositions and the like of the present disclosure include various additional ingredients such as pharmaceutically acceptable carriers and the viability, engraftability and / of sheet-like cell cultures. Alternatively, it may contain an ingredient that enhances the function or the like, another active ingredient that is useful for treating the target disease, or the like. Any known additional ingredient can be used, and those skilled in the art are familiar with these additional ingredients. In addition, the compositions and the like of the present disclosure can be used in combination with components that enhance the viability, engraftment and / or function of sheet-shaped cell cultures, and other active ingredients that are useful for treating a target disease. .. In one aspect, the compositions and the like of the present disclosure are intended for use in the treatment of diseases that are ameliorated by the application of sheet cell cultures (eg, diseases associated with tissue abnormalities). The tissues and diseases to be treated are as described above for the sheet-shaped cell cultures of the present disclosure.

Another aspect of the disclosure is a method of treating a disease that is ameliorated by the application of a sheet cell culture in a subject (eg, a disease associated with a tissue abnormality), the sheet cell culture of the present disclosure. Alternatively, the present invention relates to a method comprising administering an effective amount of a composition or the like to a subject in need thereof (hereinafter, may be referred to as "the treatment method of the present disclosure"). The tissues and diseases covered by the treatment method of the present disclosure are as described above for the sheet-shaped cell culture of the present disclosure. In addition, in the treatment method of the present disclosure, 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 are described in the sheet of the present disclosure. It can be used in combination with a cell culture or a composition.

The treatment method of the present disclosure may further include the step of producing a sheet-like cell culture according to the production method of the present disclosure. The treatment method of the present disclosure may further include the step of collecting cells for producing a sheet-like cell culture from a subject or a tissue to be a source of the cells before the step of producing the sheet-like cell culture. In one embodiment, the subject from which the cell or tissue to be the source of the cell is collected is the same individual as the subject to whom the sheet-like cell culture or composition is administered. In another embodiment, the subject from which the cell or tissue that is the source of the cell is harvested is a separate entity of the same species as the subject to whom the sheet-like cell culture or composition is administered. In another embodiment, the subject from which the cell or the tissue that is the source of the cell is collected is an individual that is different from the subject to which the sheet-like cell culture or composition is administered.

In the present disclosure, the term "subject" means any individual organism, preferably an animal, more preferably a mammal, even more preferably a human. In the present disclosure, the subject may be healthy or suffer from some disease, but diseases that are ameliorated by the application of sheet cell cultures (eg, diseases associated with tissue abnormalities). When the treatment is intended, it typically means a subject who has or is at risk of developing the disease.

Also, the term "treatment" shall include all types of medically acceptable prophylactic and / or therapeutic interventions aimed at curing, transient remission or prevention of disease. For example, the term "treatment" refers to delaying or stopping the progression of a disease that is ameliorated by the application of sheet cell culture (eg, a disease associated with tissue abnormalities), regression or disappearance of a lesion, or the onset of the disease. Includes medically acceptable interventions for a variety of purposes, including prevention or prevention of recurrence.

In the present disclosure, the effective amount is, for example, an amount capable of suppressing the onset or recurrence of a disease, reducing symptoms, or delaying or stopping the progression (for example, size, weight, number of sheet-shaped cell cultures, etc.). The amount is preferably an amount that prevents the onset and recurrence of the disease or cures the disease. In addition, an amount that does not cause an adverse effect exceeding the benefit of administration is preferable. Such an amount can be appropriately determined by, for example, a test in an experimental 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 the tissue lesion to be treated can be an important index for determining the effective amount.

The method of administration typically includes direct application to tissue, but when using a fragment of sheet-like cell culture, various routes that can be administered by injection, such as intravenous and muscle. It may be administered by a route such as intra-, subcutaneous, local, intra-arterial, intraportal, intraventricular, or intraperitoneal.
The frequency of administration is typically once per treatment, but multiple doses can be administered if the desired effect is not obtained.

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

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

Example: Examination of seeding method Take 40 to 80 mL of the cell suspension in a pipette and homogenize the cells by pipetting 3 to 8 times, and in a 10 cm temperature-responsive culture dish, about 3.0 to 8.0 × When 5 to 10 ^{mL of 10 6} cells / mL cell suspension was discharged, the distance from the liquid surface to the tip of the pipette was changed, and the cell suspension was discharged. The pipette was positioned substantially vertically for discharge as the discharge direction. The discharge speed was 10 mL / 3 sec to 10 mL / 10 sec. The state of the cell suspension immediately after discharge was observed. Further, the temperature-responsive culture dish from which the cell suspension was discharged was cultured at 37 ± 1 ° C. and 5 ± 1% CO ₂ for about 20 hours. The sheet formation state after culturing was observed. The results are shown in FIG.

その結果、液面からピペット先端までの距離が短い（０〜９mm）場合（滴下にならない状態）、より具体的には細胞懸濁液がピペットの先端から培養基材上または培養基材上の細胞懸濁液と連続的につながり、ピペットの先端から培養基材上または培養基材上の細胞懸濁液との間で独立した液滴が生じない状態である場合においてムラのないシートを調製することができた。

As a result, when the distance from the liquid surface to the tip of the pipette is short (0 to 9 mm) (in a state where it does not drip), more specifically, the cell suspension is placed on the culture substrate or the culture substrate from the tip of the pipette. Prepare an even sheet when it is continuously connected to the cell suspension and no independent droplets are generated from the tip of the pipette to the cell suspension on the culture medium or on the culture medium. We were able to.

Since the sheet-like culture produced by the method of the present disclosure can easily produce a homogeneous cell sheet without unevenness by omitting the homogenization operation, it can be scaled up, automated, etc., and has a wide range. It is possible to meet medical needs.

Claims (13)

A method for producing a sheet-like cell culture, in which a part of the discharge device is brought into contact with the culture base material or the cell suspension on the culture base material, and the cell suspension is placed on the culture base material from the discharge device. A method that includes a step of discharging. The method of claim 1, further comprising positioning the discharge device substantially perpendicular to the culture medium. The method according to claim 1 or 2, further comprising a step of discharging the cell suspension to be discharged from the discharge device so as not to move from the discharge position. The method according to any one of claims 1 to 3, further comprising a step of completing the discharge before the cells of the cell suspension to be discharged settle on the culture medium. The method according to any one of claims 1 to 4, further comprising the step of discharging the cell suspension multiple times on one culture medium. The method according to any one of claims 1 to 5, further comprising a step of discharging the cell suspension to be discharged onto the culture substrate so as not to generate a flow. The method according to any one of claims 1 to 6, further comprising a step of forming a sheet after discharging the cell culture solution. The method according to any one of claims 1 to 7, further comprising a step of peeling after forming a sheet. The method according to any one of claims 1 to 8, which is performed in an automated device. (I) A step of seeding cells containing sheet-forming cells on a culture substrate, (ii) a step of sheeting the cell population seeded in step (i) in a cell culture medium to form a sheet-like cell culture. And (iii) A method for producing a sheet-shaped cell culture, which comprises a step of peeling the sheet-shaped cell culture formed in step (ii) from a culture medium, wherein step (i) is a stationary culture. The production method, which is a step of intermittently seeding a substrate at a plurality of locations. The production method according to any one of claims 1 to 10, wherein the cells include myoblasts, fibroblasts or cardiomyocytes. A sheet-shaped cell culture produced by the production method according to any one of claims 1 to 11. A method for treating a disease improved by application of a sheet-shaped cell culture, which requires a sheet-shaped cell culture produced by the production method according to any one of claims 1 to 12. The method comprising applying the steps to the subject.

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