WO2018168983A1

WO2018168983A1 - Method for producing sheet-shaped cell culture

Info

Publication number: WO2018168983A1
Application number: PCT/JP2018/010122
Authority: WO
Inventors: 枝莉野口
Original assignee: テルモ株式会社
Priority date: 2017-03-15
Filing date: 2018-03-15
Publication date: 2018-09-20
Also published as: JPWO2018168983A1

Abstract

The purpose of the present invention is to provide a sheet-shaped cell culture having a high mechanical strength and a method for producing the same. This problem is solved by providing a method for producing a sheet-shaped cell culture, said method comprising (i) a step for, in a cell population containing myoblasts and fibroblasts, controlling the ratio of the both cell species, (ii) a step for seeding the cell population obtained in step (i) on a culture base material, (iii) a step for sheeting, in a cell culture liquid, the cell population seeded in step (ii) and thus forming a sheet-shaped cell culture, and (iv) a step for stripping the sheet-shaped cell culture formed in step (iii) from the culture base material, wherein, in step (ii), the cell population is seeded at such a seeding density that, immediately after the cells, which have been seeded on the culture base material, settle down on the culture base material, 90% or more, relative to the total cells, of the cells are in contact with each other on the culture base material.

Description

Method for producing sheet cell culture

The present disclosure relates to a method for producing a sheet-like cell culture containing myoblasts and fibroblasts, a sheet-like cell culture produced by the production method, a composition containing the sheet-like cell culture, a graft, and The present invention relates to a medical product, a disease treatment method using the sheet-shaped cell culture, a kit for producing the sheet-shaped cell culture, and the like.

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

As part of such attempts, cell structures formed using scaffolds and sheet-shaped cell cultures in which cells are formed into sheets have been developed (Patent Document 1).
However, cell sheets are generally fragile, and are prone to wrinkles and tears during isolation from culture substrates and during subsequent operations, requiring considerable skill in operations such as transfer, storage, and transplantation. .

Special Table 2007-528755

The present disclosure relates to a method for producing a sheet-like cell culture containing myoblasts and fibroblasts, a sheet-like cell culture produced by the production method, a composition containing the sheet-like cell culture, a graft, and It is intended to provide a medical product, a method for treating a disease using the sheet-shaped cell culture, a kit for producing the sheet-shaped cell culture, and the like.

While the present inventors are diligently researching to solve the above problems, when producing a sheet-shaped cell culture containing myoblasts and fibroblasts, the number of myoblasts and fibroblasts It has been found that by providing a step for adjusting the ratio, a sheet-shaped cell culture having favorable properties such as high mechanical strength can be obtained, and the present invention has been completed.

Some aspects of the present disclosure relate to:
<1> (i) adjusting the ratio of the number of cells of both cell types in a cell population containing myoblasts and fibroblasts;
(Ii) seeding the cell population obtained in step (i) on a culture substrate;
(Iii) sheeting the cell population seeded in step (ii) in a cell culture medium to form a sheet-like cell culture; and
(Iv) peeling the sheet-shaped cell culture formed in step (iii) from the culture substrate
And the seeding density of the cell population in step (ii) is 90% of the total number of cells in contact with each other on the culture substrate immediately after seeding on the culture substrate after seeding on the culture substrate. A method for producing a sheet-shaped cell culture having a density of at least%.
<2> The production method according to the above <1>, wherein the cell culture solution contains allogeneic serum.
<3> The production method according to <1> or <2>, wherein the culture substrate is coated with serum.
<4> The production method according to any one of <1> to <3>, wherein the culture substrate is coated with a temperature-responsive material.
<5> In the step (i), the cell population is adjusted so as to contain 60% to 99% myoblasts and 1% to 40% fibroblasts on a cell number basis. 5>. The production method according to any one of 5>.
<6> In step (i), the cell population is adjusted to include myoblasts and fibroblasts in a ratio of myoblast: fibroblast number of 1.5: 1 to 99: 1. The production method according to any one of <1> to <5> above.
<7> The production according to any one of <1> to <6>, wherein the seeding density of the cell population is 7.5 × 10 ⁵ cells / cm ² to 3.0 × 10 ⁶ cells / cm ^2. Method.
<8> A sheet-like cell culture produced by the production method according to any one of <1> to <7> above.
<9> a culture substrate, serum, and a cell population containing 60% to 99% myoblasts and 1% to 40% fibroblasts on a cell number basis, and the cell number of the cell population is <1> above, which is a number that provides a density at which the proportion of cells in contact with each other on the culture substrate is 90% or more of the total cells immediately after the cells are seeded on the culture substrate after seeding on the culture substrate. A kit for use in the production method according to any one of to <7>.
<10> A method for treating a disease ameliorated by application of a sheet-shaped cell culture, the method comprising applying an effective amount of the sheet-shaped cell culture according to <8> above to a subject in need thereof Said method.

The sheet-shaped cell culture produced by the method of the present disclosure has advantages such as high mechanical strength, easy to handle integrally, difficult to tear, difficult to tear, and difficult to chip edges. Therefore, since the sheet-shaped cell culture of the present disclosure is excellent in operability and has little operational difference depending on the skill level of the operator, the sheet-shaped cell culture is peeled from the culture substrate, transferred, stored, and further Not only can operations such as transplantation be facilitated and reliable treatment of the disease becomes possible, but also the spread of regenerative medicine using the sheet-like cell culture can be expected.
In addition, the myoblasts and fibroblasts contained in the sheet-shaped cell culture of the present disclosure have different sizes and properties, so that gaps between the cells can be filled with each other, and the sheet-shaped cell culture of the present disclosure Compared with a sheet-like cell culture composed of only myoblasts or only fibroblasts, the gap between cells is reduced, thereby forming a surface having moderate sliding properties. For this reason, when the sheet-shaped cell culture is applied to the affected area, it is easy to adjust the position after being applied to the affected area, and the sheet-shaped cell culture is less likely to slip after being applied to the affected area. is there.
Furthermore, since myoblasts and fibroblasts have different adhesive forces, the sheet-shaped cell culture of the present disclosure has been detached from the culture substrate as compared to a sheet-shaped cell culture formed of a single type of cell. There is an advantage that it is easy to extend later and hard to curl.

It is the photograph figure which showed the state after progress for 10 minutes after seed | inoculating the cell population containing a skeletal myoblast and a fibroblast on a culture | cultivation base material. Arrowheads indicate skeletal myoblasts and arrows indicate fibroblasts. The length of the scale bar is 30 μm. It is the photograph figure which showed the size of the skeletal myoblast and the fibroblast which are seed | inoculated on the hemocytometer and which has not spread. Arrowheads indicate skeletal myoblasts and arrows indicate fibroblasts.

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 and other publications referenced herein are hereby incorporated by reference in their entirety.

One aspect of the present disclosure includes (i) adjusting the ratio of cell numbers of both cell types in a cell population comprising myoblasts and fibroblasts;
(Ii) seeding the cell population obtained in step (i) on a culture substrate;
(Iii) sheeting the cell population seeded in step (ii) in a cell culture medium to form a sheet-like cell culture; and
(Iv) peeling the sheet-shaped cell culture formed in step (iii) from the culture substrate
And the seeding density of the cell population in step (ii) is 90% of the total number of cells in contact with each other on the culture substrate immediately after seeding on the culture substrate after seeding on the culture substrate. The present invention relates to a method for producing a sheet-shaped cell culture (hereinafter, sometimes referred to as “the production method of the present disclosure”).

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

Cardiomyocyte means a myoblast present in the myocardium. Myocardial blasts are well known in the art and can be identified by markers such as Isl1. Cardiomyocytes can be any organism with a heart muscle, including but not limited to, humans, non-human primates, rodents (mouse, rats, hamsters, guinea pigs, etc.), rabbits, dogs, cats, pigs, horses, It may be derived from mammals such as cows, goats and sheep. In one embodiment, the cardiac myoblast is a mammalian cardiac myoblast. In certain embodiments, the cardiac myoblast is a human cardiac myoblast.

Fibroblasts are also well known in the art, and include TE-7 (eg Rosendaal et al., J Cell Sci. 1994; 107 (Pt 1): 29-37, Goodpaster et al., J Histochem Cytochem. 2008; 56 (4): 347-58 and the like). Fibroblasts are present in various tissues, and fibroblasts derived from any tissue can be used in the present disclosure. In certain embodiments, fibroblasts are derived from muscle tissue. In more specific embodiments, the fibroblasts are derived from skeletal muscle tissue.

The myoblasts and fibroblasts contained in the cell population may be derived from the same tissue, from the same type of tissue, or from different types of tissue. . Here, the same tissue means the same tissue both histologically and locally, and the same type of tissue means a tissue that is regarded as the same type histologically. Means tissues that are considered histologically different types. For example, the right quadriceps muscle and biceps brachii belong to the same type of tissue called skeletal muscle, but are not the same tissue because the parts are different. On the other hand, the right quadriceps muscle and the skin on the front of the thigh are different types of tissues. Accordingly, in the present disclosure, for example, both myoblasts and fibroblasts may be derived from striated muscle tissue, particularly skeletal muscle tissue, and myoblasts may be striated muscle tissue (e.g., The fibroblast may be derived from a tissue other than the striated muscle tissue such as a skin tissue. In certain embodiments, the myoblasts and fibroblasts are derived from skeletal muscle tissue. In a more specific embodiment, the myoblast and fibroblast are derived from the same skeletal muscle tissue.

Adjustment of the ratio of the cell numbers of both cell types in the cell population containing myoblasts and fibroblasts can be performed by increasing or decreasing one or both of the myoblast number and the fibroblast number. For example, the number of cells can be increased or decreased by simultaneously culturing cells of both cell types. Since myoblasts have a longer doubling time than fibroblasts, the ratio of fibroblasts can be increased by culturing both cells simultaneously. When myoblasts become confluent, proliferation substantially stops due to contact inhibition, but even if fibroblasts become confluent, the proliferation rate decreases, but they can still proliferate, so the difference in doubling time is confluent. It becomes larger by culture in the state.
The number of myoblasts and / or fibroblasts can be increased or decreased by any known cell purification method, for example, flow cytometry method using cell surface marker, affinity separation method (for example, affinity column method, magnetic cell separation method). , Immunopanning, etc.), filtration using a filter (for example, nylon mesh filter) or a micro-channel using the difference in cell size, centrifugation, flow cytometry, and the like. In addition, fibroblasts tend to have a lower survival rate after freezing / thawing than myoblasts, and thus the ratio of fibroblasts can be reduced by freezing / thawing operations (a particular theory). Although it is not desired, it is considered that fibroblasts have a larger cell size than myoblasts, and it is difficult to obtain the effect of a cryoprotectant). Non-limiting examples of freeze / thaw operations are described below. When a sheet-like cell culture is used for transplantation, a technique in which impurities derived from production processes such as antibodies do not adhere to cells is preferable.

The ratio between the number of myoblasts and the number of fibroblasts is determined by any known method, for example, counting under a microscope, flow cytometry using an antibody against a cell marker (eg, anti-CD56 antibody, etc.), etc. be able to. The ratio of the number of myoblasts and fibroblasts is determined before step (i), during step (i), or both before step (i) and during step (i). be able to. When the quality of the cell population subjected to step (i) is stable and the ratio of myoblast number to fibroblast number is small, the desired ratio can be determined only during step (i). If it is possible to stably predict how much the number of cells will fluctuate by the method of increasing / decreasing the number of myoblasts and / or fibroblasts, the determination is made only before step (i). The desired ratio may be obtained. If the desired ratio is not obtained as a result of determining the ratio of the number of cells in the step (i), a method for increasing / decreasing the number of myoblasts and / or fibroblasts is applied to the cell population. Further application may be made one or more times until a ratio is obtained. Moreover, even if cells other than myoblasts and fibroblasts are not included in the cell population, even if they are included, the ratio is negligible (for example, less than 1%, less than 0.5%, Less than 0.3%), or if it can be reasonably inferred, determine the ratio of the number of myoblasts and / or fibroblasts and The cell number ratio may be considered the remainder. In this case, for example, if the ratio of the number of myoblasts to the total number of cells is 70%, the ratio of the number of fibroblasts to the total number of cells can be regarded as 30%. Furthermore, it is possible to determine the ratio of the number of cells of only one of myoblasts and fibroblasts and not to determine the ratio of the number of cells of the other cell. In certain embodiments, the ratio of the number of myoblasts alone is determined and the ratio of the number of fibroblasts is not determined.

The ratio of the number of myoblasts to the total number of cells is, for example, about 51 to about 99%, about 55 to about 99%, about 60 to about 99%, about 65 to about 99%, about 70 to about 99%, about May range from 75 to about 99%, from about 80 to about 99%, from about 85 to about 99%, from about 90 to about 99%, or from about 95 to about 99%, etc., relative to the total number of fibroblasts The ratio can be, for example, about 1 to about 49%, about 1 to about 45%, about 1 to about 40%, about 1 to about 35%, about 1 to about 30%, 1 to about 25%, about 1 to about 20%, about 1 to about 15%, about 1 to about 10%, or about 1 to about 5%, etc., and the ratio of myoblast number to fibroblast number is the number of myoblasts: As the number of fibroblasts, for example, 51:49 to 99: 1, 55:45 to 99: 1, 60:40 to 99: 1, 65:35 to 99: 1, 70:30 to 99: 1, 5:25 to 99: 1, 80: 20-99: 1,85: 15 ~ 99: 1,90: 10-99: 1 or 95: 5-99: may range, such as 1.

Fibroblasts have higher ability to produce extracellular matrix such as laminin than myoblasts, and cell populations containing more fibroblasts tend to have higher ability to produce extracellular matrix.

In the present disclosure, the “sheet-shaped cell culture” refers to a sheet-like cell connected to each other. The cells may be linked to each other directly (including those via cell elements such as adhesion molecules) and / or via intervening substances. The intervening substance is not particularly limited as long as it is a substance that can connect cells at least physically (mechanically), and examples thereof include an extracellular matrix. The intervening substance is preferably derived from cells, particularly derived from cells constituting the sheet-shaped cell culture. The cells are at least physically (mechanically) connected, but may be further functionally, for example, chemically or electrically connected. The sheet-shaped cell culture is composed of one cell layer (single layer) or composed of two or more cell layers (stacked (multilayer), for example, two layers, three layers, four layers) Layer, 5 layers, 6 layers, etc.). Further, the sheet-shaped cell culture may have a three-dimensional structure having a thickness exceeding the thickness of one cell without the cells showing a clear layer structure. For example, in the vertical cross section of the sheet-shaped cell culture, the cells may be present in a non-uniform (for example, mosaic) arrangement without being uniformly aligned in the horizontal direction.

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

The cell may be a xenogeneic cell or a homologous cell. The term “heterologous cell” as used herein means a cell derived from an organism of a species different from the recipient when the sheet-shaped cell culture is used for transplantation. For example, when the recipient is a human, cells derived from monkeys or pigs correspond to xenogeneic cells. The “same species-derived cell” means a cell derived from an organism of the same species as the recipient. For example, when the recipient is a human, the human cell corresponds to the allogeneic cell. The allogeneic cells include autologous cells (also referred to as autologous cells or autologous cells), that is, cells derived from the recipient, and allogeneic non-autologous cells (also referred to as allogeneic cells). Autologous cells are preferred in the present disclosure because they do not cause rejection even after transplantation. However, it is also possible to use heterologous cells or allogeneic non-autologous cells. When using heterologous cells or allogeneic non-autologous cells, immunosuppressive treatment may be required to suppress rejection. In the present specification, cells other than autologous cells, that is, heterologous cells and allogeneic nonautologous cells may be collectively referred to as nonautologous cells. In one embodiment of the present disclosure, the cell is an autologous cell or an allogeneic cell. In one aspect of the present disclosure, the cell is an autologous cell. In another aspect of the present disclosure, the cell is an allogeneic cell.

The culture substrate is not particularly limited as long as cells can form a cell culture thereon, and includes, for example, containers of various materials, solid or semi-solid surfaces in containers, and the like. The container preferably has a structure / material that does not allow permeation of a liquid such as a culture solution. Examples of such materials include, but are not limited to, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, polydimethyl. Examples include acrylamide and metals (for example, iron, stainless steel, aluminum, copper, brass). The container preferably has at least one flat surface. Examples of such a container include, but are not limited to, 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 culture vessels include, but are not limited to, cell culture dishes, cell culture bottles, and the like. The bottom surface of the container may be transparent or opaque. When the bottom surface of the container is transparent, it is possible to observe and count cells from the back side of the container. Further, the container may have a solid or semi-solid surface therein. Examples of solid surfaces include plates and containers of various materials as described above, and examples of semi-solid surfaces include gels and soft polymer matrices. The culture substrate may be prepared using the above materials, or commercially available materials may be used. Preferable culture substrates include, but are not limited to, substrates having an adhesive surface suitable for the formation of sheet cell cultures. Specifically, a substrate having a hydrophilic surface, for example, a substrate coated with a hydrophilic compound such as polystyrene subjected to corona discharge treatment, collagen gel or hydrophilic polymer, and further, collagen, fibronectin, laminin , Substrates coated with an extracellular matrix such as vitronectin, proteoglycan and glycosaminoglycan, and cell adhesion factors such as cadherin family, selectin family and integrin family. Such base materials are commercially available (for example, Corning ^(R) TC-Treated Culture Dish, Corning, etc.). The whole or part of the culture substrate may be transparent or opaque.

The surface of the culture substrate may be coated with a material whose physical properties change in response to stimulation, for example, temperature or light. Examples of such materials include, but are not limited to, (meth) acrylamide compounds, N-alkyl-substituted (meth) acrylamide derivatives (eg, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylate Amide), N, N-dialkyl-substituted (meth) acrylamide derivatives (eg, N, N-dimethyl (meth) acrylamide, N, N-ethylmethylacrylamide, N, N-diethyl) Chloramide and the like), (meth) acrylamide derivatives having a cyclic group (for example, 1- (1-oxo-2-propenyl) -pyrrolidine, 1- (1-oxo-2-propenyl) -piperidine, 4- (1-oxo -2-propenyl) -morpholine, 1- (1-oxo-2-methyl-2-propenyl) -pyrrolidine, 1- (1-oxo-2-methyl-2-propenyl) -piperidine, 4- (1-oxo -2-methyl-2-propenyl) -morpholine etc.) or a vinyl ether derivative (eg methyl vinyl ether) homopolymer or copolymer, temperature-responsive material, light-absorbing polymer having azobenzene group, triphenylmethane leucohydro Copolymer of vinyl derivative of oxide and acrylamide monomer, and spirobenzopyra It can be used to include N- and isopropyl acrylamide gels known, such as photoresponsive materials (e.g., JP-A-2-211865, see JP-2003-33177). By giving a predetermined stimulus to these materials, the physical properties, for example, hydrophilicity and hydrophobicity can be changed, and peeling of the cell culture adhered on the materials can be promoted. Culture dishes coated with a temperature-responsive materials are commercially available (e.g., UpCell of CellSeed Inc. ^(R)), they can be used in the production method of the present disclosure.

The culture substrate may have various shapes, but is preferably flat. The area is not particularly limited, and may be, for example, about 1 cm ² to about 200 cm ² , about 2 cm ² to about 100 cm ² , about 3 cm ² to about 50 cm ² , and the like.

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

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

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

When incubating on a culture substrate, serum may be used as a stock solution or diluted. Dilution can be any medium such as, without limitation, water, saline, various buffers (eg, PBS, HBSS, etc.), various liquid media (eg, DMEM, MEM, F12, DMEM / F12, DME, RPMI 1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, etc.) can be used. The dilution concentration is not particularly limited as long as the serum component can adhere to the culture substrate. For example, the dilution concentration is about 0.5% to about 100% (v / v), preferably about 1% to about 60% (v / V), more preferably from about 5% to about 40% (v / v).

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

Serum may be discarded after incubation. As a method for discarding serum, a conventional liquid disposal method such as suction with a pipette or decantation can be used. In a preferred embodiment of the present disclosure, the culture substrate may be washed with a serum-free washing solution after serum is discarded. The serum-free washing solution is not particularly limited as long as it is a liquid medium that does not contain serum and does not adversely affect the serum components attached to the culture substrate. For example, without limitation, water, physiological saline, various buffers Liquid (eg, PBS, HBSS, etc.), various liquid media (eg, DMEM, MEM, F12, DMEM / F12, DME, RPMI 1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15 , SkBM, RITC80-7, etc.). As a washing method, a conventional culture substrate washing method, for example, without limitation, a method of adding a serum-free washing solution on the culture substrate, stirring for a predetermined time (for example, about 5 seconds to about 60 seconds), and then discarding it. Etc. can be used.

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

In the present 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 a living body such as adrenal cortex dysfunction and Cushing's syndrome. Such compounds include, but are not limited to, for example, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone and the like. In the coating method, disposal method and washing method of the culture substrate with a steroid agent, the dilution concentration at the time of incubation is, for example, about 0.1 μg / mL to about 100 μg / mL, preferably about 0.4 μg / mL to dexamethasone. It is basically the same as serum except that it is about 40 μg / mL, more preferably about 1 μg / mL to about 10 μg / mL.

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

The culture substrate may be seeded with cells immediately after 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 time, for example, by keeping it at about 4 ° C. or lower, preferably about −20 ° C. or lower, more preferably about −80 ° C. or lower.

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

In one embodiment, seeding is performed on the surface of the culture substrate (for example, the bottom surface of the culture container) immediately after seeding on the culture substrate (in the culture container) after seeding on the culture substrate (for example, the culture container). The density is such that the proportion of cells in contact with each other is about 90% or more of all cells. “Cells settled on the culture substrate” means that the cells descend by the action of gravity in the liquid medium existing on the culture substrate (for example, in the culture vessel), and the surface of the culture substrate ( It means a state in which it cannot be lowered any further due to contact with the bottom surface of the culture vessel or contact with other cells. When the seeding density is low, most of the settled cells are in contact with the surface of the culture substrate (bottom of the culture vessel), but when the seeding density is high, the precipitated cells are in contact with the surface of the culture substrate (bottom of the culture vessel). Can be in contact with other cells without contact. The time required for the cells to settle on the culture substrate depends on the depth and viscosity of a liquid medium such as a cell culture solution existing on the culture substrate, but is, for example, normal culture conditions. About 5 to 10 minutes when a 5 to 3 mm deep cell culture is present on the culture substrate. Therefore, the ratio of the cells in contact with each other on the culture substrate is determined about 5 to 10 minutes after seeding the cell population suspended in the cell culture medium on the culture substrate with a microscope or the like. It can be determined by observation. Whether or not the cells are in contact with each other can be determined based on the relationship between the contour of one cell and the contour of another cell. For example, if at least part of the outline of a certain cell touches or overlaps the outline of another cell, it can be determined that the cells touch each other. Further, when the entire circumference of a contour of a certain cell is away from the contour of another cell, it can be determined that the cells are not in contact with each other. The ratio of cells in contact with each other can be calculated, for example, as the ratio of cells in contact with each other in the total number of cells included in one visual field. In this case, an average value in a plurality of visual fields (for example, 3 to 5 visual fields) can be taken in order to reduce the influence due to the variation of each visual field. Moreover, the influence by the dispersion | variation for every visual field can also be reduced by increasing the number of the cells to count. The number of cells to be counted can be changed by adjusting the magnification. Typically, for example, by counting about 10 or more (for example, about 10 to 20) at a magnification of 200 times, a good counting result can be obtained. Cell counting may be performed manually or mechanically using image processing software or the like. In certain embodiments, the percentage of cells that touch each other on the culture substrate is, for example, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about It may be 97% or more, about 98% or more, about 99% or more, or about 100%. It should be noted that the ratio of cells that are in contact with each other on the culture substrate is determined without distinguishing myoblasts and fibroblasts unless otherwise specified.

In another embodiment, seeding is performed after seeding on a culture substrate (eg, culture vessel) and immediately after the cells settle on the culture substrate (in the culture vessel), about 70% or more of the cells are on the surface of the culture substrate. It is performed at a density that does not contact the bottom of the culture vessel. The percentage of cells not in contact with the surface of the culture substrate is about 5 to 10 minutes after seeding the cell population suspended in the cell culture medium after considering the sedimentation rate of the cells as described above. It can be determined by observing cells on the culture substrate with a microscope or the like. Whether or not the cells are in contact with the surface of the culture substrate can be determined based on, for example, observation with an inverted microscope or phase contrast microscope, observation with a microscope with a shallow depth of focus, observation with a fiberscope, and the like. When using an inverted microscope, the number of cells in contact with the culture substrate surface can be directly counted to determine the ratio to the total number of seeded cells. When using a microscope with a shallow depth of focus, by adjusting the focal length, cells that are closer than the predetermined distance are considered as cells that are not in contact with the culture substrate surface and / or farther than the predetermined distance. Cells can be counted as cells that are in contact with the culture substrate surface. The percentage of cells that do not contact the culture substrate surface is the number of cells that are closer than the predetermined distance and cells that are further than the predetermined distance. It can be determined as a ratio of the number of cells that are closer than a predetermined distance to the sum of the number of cells. The focal distance is set, for example, close to the thickness of one myoblast or fibroblast from the surface of the culture substrate to distinguish cells that are not in contact with the surface of the culture substrate. can do. The depth of focus can be set, for example, to a value that is equal to or close to the thickness of one myoblast or fibroblast (for example, thickness ± 1 to 2 μm). When using a fiberscope, place the fiberscope in a liquid medium containing cells, and observe the seeded cells obliquely from above or horizontally in the culture substrate surface. Cells that are not in contact and / or that are in contact with the culture substrate surface can be counted. The percentage of cells that do not contact the culture substrate surface is, for example, the number of cells that are not in contact with the culture substrate surface and the number of cells that are in contact with the culture substrate surface and the cells that are not in contact with the culture substrate surface. As a percentage of the number of Note that the percentage of cells that do not contact the culture substrate surface is determined without distinguishing myoblasts and fibroblasts unless otherwise specified.

In yet another aspect, the seeding is from about 7.1 × 10 ⁵ pieces / cm ² to about 3.0 × 10 ⁶ pieces / cm ² , about 7.3 × 10 ⁵ pieces / cm ² to about 2.8 ×. 10 ⁶ pieces / cm ² , about 7.5 × 10 ⁵ pieces / cm ² to about 2.5 × 10 ⁶ pieces / cm ² , about 7.8 × 10 ⁵ pieces / cm ² to about 2.3 × 10 ⁶ Pieces / cm ² , about 8.0 × 10 ⁵ pieces / cm ² to about 2.0 × 10 ⁶ pieces / cm ² , about 8.5 × 10 ⁵ pieces / cm ² to about 1.8 × 10 ⁶ pieces / cm ² cm ² , and a density of about 9.0 × 10 ⁵ pieces / cm ² to about 1.6 × 10 ⁶ pieces / cm ² can be used. Note that these densities are the total density of myoblasts and fibroblasts unless otherwise specified.
In yet another embodiment, seeding is performed in a cell culture medium substantially free of growth factors at a density that allows proliferation while myoblasts do not substantially proliferate and fibroblasts undergo growth inhibition. be able to.

Myoblasts and fibroblasts differ in the ease of adhesion to the culture substrate surface. After seeding, cells that are not in contact with the culture substrate surface and are surrounded by other cells on the top and bottom tend to extend in the direction of gravity. After seeding, myoblasts tend to deform easily and tend to be polygonal when attached to the culture substrate surface. Moreover, when a myoblast becomes confluent, proliferation is suppressed by contact inhibition. On the other hand, fibroblasts tend to extend linearly in one direction after seeding, are heavier than myoblasts, and tend to adhere to the culture substrate surface. In addition, fibroblasts can continue to proliferate even if they become confluent and contact inhibition occurs and proliferation is suppressed. Therefore, when myoblasts and fibroblasts are seeded at a high density as described above, myoblasts hardly proliferate, but fibroblasts proliferate, so the doubling time differs.

The step of forming the seeded cells into a sheet can be performed by any known technique and condition. Non-limiting examples of such techniques are described in, for example, Patent Document 1, WO 2014/185517. It is considered that the formation of a cell sheet is achieved when cells adhere to each other via an adhesion molecule or an intercellular adhesion mechanism such as an extracellular matrix. Therefore, the step of forming the seeded cells into a sheet can be achieved, for example, by culturing the cells under conditions that form cell-cell adhesion. Such conditions may be any as long as cell-cell adhesion can be formed, but cell-cell adhesion can usually be formed under the same conditions as general cell culture conditions. Examples of such conditions include culture at about 37 ° C. and 5% CO ₂ . Further, the culture can be performed under normal pressure (atmospheric pressure, non-pressurized). Culturing can be performed in containers of any size and shape. The size and shape of the sheet-shaped cell culture can be adjusted by adjusting the size and shape of the cell adhesion surface of the culture vessel, or by placing a mold of the desired size and shape on the cell adhesion surface of the culture vessel, It can be arbitrarily adjusted by, for example, culturing cells therein. In the present specification, the culture for forming the seeded cells into a sheet may be referred to as “sheet culture”. By sheet culture, the thickness of the sheet-like cell culture on the culture substrate (in the culture vessel) is reduced. That is, after seeding, after the cells settle, the thickness of the cell layer on the culture substrate is reduced by subsequent sheet formation, but the sheet-like cell culture shrinks by peeling from the culture substrate and increases again. . The reduction in thickness due to sheeting is about 90% to about 70%, assuming that the thickness of the cell layer immediately after seeding is 100%.

In one embodiment of the present disclosure, cell culture is performed within a predetermined period, preferably within a period in which myoblasts do not shift to differentiation. Therefore, in this embodiment, myoblasts are maintained in an undifferentiated state during the culture period. The transition to myoblast 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, cell multinucleation, myotube formation, etc. can be used as indicators of differentiation. The culture period can be, for example, about 48 hours, about 40 hours, about 36 hours, about 30 hours, about 26 hours, or about 12 hours. In certain embodiments, the culture period may be about 2 hours to about 36 hours, about 2 hours to about 30 hours, about 2 hours to about 26 hours, about 2 hours to about 12 hours, and the like.

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

The amino acid contained in the basal medium is not limited, and for example, L-arginine, L-cystine, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and the like are not limited to vitamins such as calcium D-pantothenate, choline chloride, folic acid, i Inositol, niacinamide, riboflavin, thiamine, pyridoxine, biotin, lipoic acid, vitamin B12, adenine, thymidine and the like, but are not limited to, for example, CaCl ₂ , KCl, MgSO ₄ , NaCl, NaH _{_{_{_{2 PO 4, NaHCO 3, Fe}}}} (NO 3) 3, FeS _{_{_{4, CuSO 4, MnSO 4,}}} Na 2 SiO 3, include _{_{_{_{(NH 4) 6 Mo 7 O}}}} 24, NaVO 3, NiCl 2, etc. ZnSO _4, respectively. In addition to these components, the basal medium may contain sugars such as D-glucose, pH indicators such as sodium pyruvate and phenol red, putrescine and the like.

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

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

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

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

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

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

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

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

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

The separation 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 (detached) from the culture substrate serving as a scaffold while at least partially maintaining the sheet structure. For example, enzymatic treatment with a proteolytic enzyme (such as trypsin) and / or mechanical treatment such as pipetting. In addition, when cells are cultured on a culture substrate whose surface is coated with a material that changes its physical properties in response to stimulation, for example, temperature or light, a predetermined stimulation is applied. It can also be released non-enzymatically.

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

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

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

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

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

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

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

The production method of the present disclosure may further include a step of growing the cells before the step of adjusting the ratio of the cell numbers of both cell types in the cell population including myoblasts and fibroblasts. The step of growing the cells may be performed by any known technique, and those skilled in the art are familiar with the culture conditions suitable for the growth of various cells.

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

The production method of the present disclosure except that the sheet-like cell culture obtained by the production method of the present disclosure does not include a step of adjusting the ratio of the cell numbers of both cell types in the cell population containing myoblasts and fibroblasts. The mechanical strength is higher than that of the sheet-shaped cell culture produced by the same method (hereinafter sometimes referred to as a control sheet-shaped cell culture). Here, the high mechanical strength is not limited on the basis of the mechanical strength of the control sheet-like cell culture, and is, for example, about 5% or more, about 10% or more, about 20% or more, about 30%. More than about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% or more, which means high mechanical strength. The mechanical strength of the sheet cell culture can be quantified using various techniques. Methods for measuring the mechanical strength of sheet cell cultures are described in, for example, JP 2012-159408, JP 2013-200234, JP 2014-149214, and JP 2016-052272.

In one aspect, all steps of the manufacturing method of the present disclosure are performed in vitro. In another aspect, the production method of the present disclosure includes a step performed in vivo, including, but not limited to, a cell or a source of cells from a subject (eg, striated muscle tissue, particularly skeletal muscle tissue). Including the step of collecting. In one aspect, the manufacturing method of the present disclosure is performed under aseptic conditions in all steps. In one embodiment, the production method of the present disclosure is performed such that the finally obtained sheet-shaped cell culture is substantially sterile. In one embodiment, the production method of the present disclosure is performed such that the finally obtained sheet-shaped cell culture is sterile.

Another aspect of the present disclosure relates to a sheet-shaped cell culture produced by the production method of the present disclosure. The sheet-shaped cell culture of the present disclosure is characterized by having preferable characteristics such as higher mechanical strength than the control sheet-shaped cell culture. Details regarding the high mechanical strength are as described above.

The sheet-shaped cell culture of the present disclosure is useful for the treatment of diseases improved by application of the sheet-shaped cell culture, for example, various diseases related to tissue abnormalities. Accordingly, in one aspect, the sheet-shaped cell culture of the present disclosure is for use in the treatment of diseases improved by application of the sheet-shaped cell culture, particularly diseases related to tissue abnormalities. Since the sheet-shaped cell culture of the present disclosure has the same properties inherent in the constituent cells except that it has a higher mechanical strength than the conventional sheet-shaped cell culture, at least the conventional myoblast It can be applied to tissues and diseases that can be treated with a sheet-like cell culture containing cells or fibroblasts. Examples of the tissue to be treated include, but are not limited to, myocardium, esophagus, skin, pancreas, and skeletal muscle. In addition, the disease to be treated is not limited, for example, heart disease (eg, myocardial injury (myocardial infarction, cardiac injury), cardiomyopathy, etc.), esophageal disease (eg, esophageal surgery (esophageal cancer removal)) Prevention of inflammation and constriction of the esophagus later), skin diseases (eg skin damage (trauma, burn), etc.), pancreatic diseases (eg pancreatic fistula etc.), muscle diseases (eg muscle damage, myositis, etc.) It is done. Usefulness of the sheet-shaped cell culture 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 J Gastroenterol. 2013; 48 (9): 1081-9. Are listed. The sheet-shaped cell culture of the present disclosure can be fragmented into an injectable size, and can be obtained at a higher effect than the injection with a single cell suspension by injecting it into a site requiring treatment (Wang et). al., Cardiovasc Res. 2008; 77 (3): 515-24). Therefore, such a utilization method is also possible for the sheet-shaped cell culture of the present disclosure.

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

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

Another aspect of the present disclosure is a method of treating a disease (eg, a disease associated with tissue abnormality) that is ameliorated by application of a sheet cell culture in a subject, the sheet cell culture of the present disclosure Alternatively, it relates to a method comprising administering an effective amount of a composition or the like to a subject in need thereof (hereinafter sometimes referred to as “treatment method of the present disclosure”). The tissues and diseases to be treated 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, a component that enhances 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 used. It can be used in combination with a cell culture or composition.

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

In the present disclosure, the term “subject” means any living individual, preferably an animal, more preferably a mammal, more preferably a human individual. In the present disclosure, the subject may be healthy or may have some kind of disease, but the disease can be improved by the application of the sheet-shaped cell culture (for example, a disease related to tissue abnormality). When treatment is intended, it typically means a subject suffering from or at risk of suffering from the disease.

Also, the term “treatment” is intended to encompass all types of medically acceptable prophylactic and / or therapeutic interventions aimed at healing, temporary remission or prevention of disease. For example, the term “treatment” refers to delaying or stopping the progression of a disease (eg, a disease associated with a tissue abnormality) that is ameliorated by application of a sheet cell culture, regression or disappearance of a lesion, Includes medically acceptable interventions for various purposes, including prevention or prevention of recurrence.

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

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

Another aspect of the present disclosure relates to a kit (set or pack) for producing a sheet-shaped cell culture comprising a cell population containing myoblasts and fibroblasts and serum (hereinafter referred to as “the present disclosure”). Sometimes referred to as a “production kit”). In this specification, the terms “set” and “pack” are used interchangeably with “kit”, and the description relating to “kit” in this specification also applies to “set” and “pack”. The production of the sheet-shaped cell culture by the kit of the present disclosure is performed by using a cell population in which the ratio of the number of myoblasts and the number of fibroblasts is adjusted in the above-described method for producing the sheet-shaped cell culture of the present disclosure as a culture substrate You may carry out by the step after the step of sowing to. Therefore, the kit of the present disclosure includes a step of seeding a sheet-like cell culture on a culture substrate with a cell population in which the ratio of the number of myoblasts and the number of fibroblasts is adjusted, and the seeded cell population is cell culture medium. A sheet-shaped cell culture, and a method for producing the sheet-shaped cell culture, comprising the steps of: forming a sheet-shaped cell culture in the medium; and peeling the formed sheet-shaped cell culture from the culture substrate It may be. The details of each step after the step of seeding on the culture substrate are as described above for the method for producing a sheet-shaped cell culture of the present disclosure.

The cell population contained in the production kit of the present disclosure may be frozen. A frozen cell population is a frozen cell population used for the production of sheet-like cell cultures. Non-limiting examples of the cell population used for the production of the sheet-like cell culture are as described above for the production method of the present disclosure. The cells constituting the cell population may be allogeneic or xenogeneic cells. In one embodiment, the cell is an autologous cell. The frozen cell population may be frozen by any known technique. Non-limiting examples of cell population freezing methods are as described above for the production methods of the present disclosure. In one embodiment, the frozen cell population in the kit of the present disclosure is frozen in the presence of a cryoprotectant. Non-limiting examples of cryoprotectants are as described above for the production method of the present disclosure. In one embodiment, the cell population is frozen in a container suitable for a freezing operation, and the frozen cell population in the kit of the present disclosure is provided in a state accommodated in the container. The container may be sealable. A container for storing a frozen cell population (a cryopreservation container) is not particularly limited as long as it is suitable for a freezing operation, and may be, for example, a tube, a vial, a bottle, a bag, or the like. The material is not particularly limited as long as it is suitable for the freezing operation, and may be, for example, plastic such as polypropylene.

A cell population (frozen or non-frozen) may be contained in one or more containers. The total number of cells per kit (including myoblasts and fibroblasts) is not particularly limited as long as it is suitable for production of a sheet-like cell culture. For example, 1 × 10 ⁶ to 1 × 10 ⁶ ¹¹ pieces, 3 × 10 ⁶ to 3 × 10 ¹⁰ pieces, 5 × 10 ⁶ to 1 × 10 ¹⁰ pieces, 1 × 10 ⁷ to 5 × 10 ⁹ pieces, 3 × 10 ⁷ to 3 × 10 ⁹ pieces, etc. Good. In one embodiment, the cells are obtained by aseptic manipulation. In one aspect, the cells are substantially sterile. In one embodiment, the cells are sterile.

Serum is used as a component of the cell culture medium and for coating the culture substrate. The serum may be homologous or heterologous serum. In addition, the serum may be derived from the same biological species as the cell or may be derived from a different biological species. In one embodiment, the serum is autologous serum. Serum may or may not be mixed with other media (for example, water, physiological saline, physiological buffer, cell culture medium, etc.). The cell culture solution is as described above for the production method of the present disclosure. Serum or a mixture of serum and other media may exist in various storage states, for example, it may be liquid, frozen or lyophilized. In one embodiment, the serum is present in a mixture with cell culture media, particularly serum free media. In certain embodiments, the serum is present in a liquid mixed with the basal medium. In one embodiment, the serum or mixture of serum and other medium is sterilized.

The kit of the present disclosure may include additional components useful for producing a sheet cell culture. Examples of such components include, but are not limited to, for example, a culture substrate, a cell culture solution, a washing solution, a buffer solution, packaging materials, instruments, and production and / or use of a sheet-shaped cell culture (for example, a sheet-shaped cell culture) Instructions regarding treatment of diseases that are improved by the application of products). In one aspect, the production kit of the present disclosure includes the production and / or use of a cell population and serum, as well as a culture substrate, a cell culture solution, a washing solution, a buffer solution, a packaging material, instruments, and a sheet-like cell culture. May further include a component selected from the group consisting of instructions. In certain embodiments, the kit of the present disclosure includes a cell population, serum, culture substrate, cell culture solution, washing solution, buffer solution, packaging material and instruments. In each of the above embodiments, the serum and the cell culture medium may exist in a mixed state.

The culture substrate is used to form a sheet-shaped cell culture. As described above for the production method of the present disclosure, the culture substrate may have various materials, forms, and coatings. In one embodiment, the culture substrate is a temperature responsive culture dish (dish) coated with a temperature responsive material. The culture dish may have a detachable lid. The size of the culture substrate is not particularly limited as long as it is suitable for production of a sheet-shaped cell culture. For example, the culture substrate has a size of about 0.2 cm ² to about 200 cm ² , about 0.5 cm ² to about 150 cm ² , about 1 cm. ² to about 120 cm ² , about 3 cm ² to about 100 cm ² , about 8 cm ² to about 80 cm ² , about 20 cm ² to about 70 cm ² , and the like. Non-limiting examples of the culture substrate size include 96-well plate, 48-well plate, 24-well plate, 12-well plate, 6-well plate, 35 mm dish, 60 mm dish, 100 mm dish and the like. The culture substrate may be a commercially available one or may be prepared by a known method. In one embodiment, the culture substrate is sterilized.

The washing solution is used for washing the thawed cells. The washing solution is not particularly limited as long as it can be used for washing cells, and examples thereof include physiological saline, physiological buffer solution (for example, PBS, HBSS, etc.), culture medium and the like. The medium is as described above with respect to the production method of the present disclosure. HBSS may be HBSS (+) containing cations (Ca ²⁺ and Mg ²⁺ ) or HBSS (−) containing no cations. In one embodiment, the buffer in the kit of the present disclosure is HBSS (−). The washing solution may contain other components useful for washing cells, such as serum albumin. Even if the said component is provided in the state contained in the washing | cleaning liquid, it may be provided without mixing with the washing | cleaning liquid and may be mixed at the time of use. In one embodiment, the cleaning solution is sterilized.

The buffer solution is used for washing the sheet-shaped cell culture, peeling the sheet-shaped cell culture from the culture substrate, and storing the sheet-shaped cell culture. The buffer solution is not particularly limited as long as it can be used for the above applications, and examples thereof include physiological buffer solutions such as PBS and HBSS. The HBSS may be HBSS (+) or HBSS (−). In one embodiment, the buffer in the kit of the present disclosure is HBSS (+). In one embodiment, the buffer is sterilized.

The packaging material is used to wrap the completed sheet-like cell culture. The packaging material is useful when the place where the sheet-shaped cell culture is manufactured and the place where the sheet-shaped cell culture is applied to the target are separated, and it is necessary to move the completed sheet-shaped cell culture peeled off from the culture substrate. It is. Non-limiting examples of packaging materials include sealing materials such as packing, caps, and films for sealing the culture substrate containing the completed sheet-shaped cell culture, and containers for storing the completed sheet-shaped cell culture (for example, , Plastic bags that can be sealed, etc.) If the culture substrate has a detachable lid, a fixture for fixing the lid to the culture substrate (eg, clip, band, wire, etc.), sheet-like cell culture Examples include bags (for example, plastic bags and the like) that contain a culture substrate that contains a product or a container that contains a sheet-shaped cell culture. The material of the sealing material is not particularly limited as long as it can seal the culture substrate, and may be, for example, silicone, plastic, rubber or the like. In one embodiment, the packaging material includes a packing that fits over the lid of the culture dish. The packing may be made of silicone, such as silicone rubber. In one aspect, the packaging material is sterilized. The packaging material in this embodiment may include a bag for accommodating a culture substrate containing a clip and / or a sheet-shaped cell culture for fixing the lid of the culture dish on which the packing is mounted to the culture dish.

Instruments are used for various operations such as washing, resuspension and seeding of cells, peeling, shaping, and movement of sheet-like cell cultures, which are involved in the production of sheet-like cell cultures. Thus, the instruments include instruments used for the production of sheet cell cultures. The instrument is not particularly limited as long as it can be used for the above operation, and examples thereof include a pipette, tweezers, a spatula (for example, an intestinal spatula) and the like. In one aspect, the instrumentation is disposable. In one embodiment, the instruments are sterilized.

The instructions relating to the production and / or use of the sheet-shaped cell culture include any medium on which an instruction relating to the production and / or use of the sheet-shaped cell culture or a URL where the instructions can be viewed is recorded. Examples of such media include, but are not limited to, display media such as instructions, and electronic recording media such as flexible disks, CDs, DVDs, Blu-ray disks, memory cards, and USB memories.

Each component of the kit of the present disclosure may be individually packaged, or two or more components may be packaged together. For example, a frozen cell that needs to be kept at a low temperature and other components may be packaged differently.
In one aspect, the kit of the present disclosure is substantially sterile. In one aspect, the kit of the present disclosure is sterile. In one embodiment, the components of the kit of the present disclosure are substantially aseptic or aseptic or are sterilized.

In certain embodiments, the kit of the present disclosure comprises a cell population, a culture substrate (temperature-responsive culture dish with a lid), a serum-containing cell culture solution, a washing solution, a buffer solution, a packaging material (packing, clip, plastic bag) and an instrument. Includes pipettes and tubes.

In certain embodiments, a kit of the present disclosure comprises a frozen cell population, a culture substrate (temperature-responsive culture dish with a lid), a serum-containing cell culture solution, a washing solution, a buffer solution, a packaging material (packing, clip, plastic bag) and Includes instruments (pipette, tube). Hereinafter, the method of using the kit of the present disclosure will be described based on this specific embodiment, but this does not limit the method of using the kit of the present disclosure.

Another aspect of the present disclosure includes a step of seeding a cell culture medium in which the ratio of the number of myoblasts and the number of fibroblasts is adjusted on a culture substrate, sheeting the seeded cell population in a cell culture medium, The present invention relates to a step of forming a cell culture, a step of peeling the formed sheet-shaped cell culture from a culture substrate, and a method of manufacturing a sheet-shaped cell culture using the manufacturing kit of the present disclosure. The production method using the production kit of the present disclosure uses the steps after the step of seeding the cell culture medium with the cell population in which the ratio of the number of myoblasts and the number of fibroblasts is adjusted in the production method of the present disclosure. The details thereof are as described above in the manufacturing method of the present disclosure. In one embodiment, the production method using the production kit of the present disclosure further includes a step of coating the culture substrate with serum before the step of forming the sheet-shaped cell culture. A production method using the production kit of the present disclosure including a frozen cell population includes a step of thawing frozen cells. In one aspect, a production method using a production kit of the present disclosure that includes a frozen cell population comprises the steps of thawing the frozen cells and growing the cells between the step of forming a sheet cell culture. Absent. In one aspect, the production method using the production kit of the present disclosure further includes a step of washing the cells after the step of thawing the frozen cells and before the step of forming the sheet-like cell culture. In one aspect, the production method using the production kit of the present disclosure further includes a step of peeling the formed sheet-shaped cell culture from the culture substrate after the step of forming the sheet-shaped cell culture.

In one embodiment, the production method using the production kit of the present disclosure further includes a step of packaging the formed sheet-shaped cell culture. The step of packaging the formed sheet-shaped cell culture may be performed using a packaging material included in the production kit of the present disclosure. For example, if the packaging material is a sealing material for sealing a culture substrate containing a completed sheet-shaped cell culture, the packaging step may be performed by sealing the culture substrate with a sealing material. If the packaging material is a container that accommodates the completed sheet-shaped cell culture, the packaging step may be performed by accommodating the completed sheet-shaped cell culture in the container. By packaging the formed sheet-shaped cell culture, the sheet-shaped cell culture can be easily transferred. Therefore, the present disclosure also includes a step of seeding a cell population having a ratio of the number of myoblasts and the number of fibroblasts on a culture substrate, sheeting the seeded cell population in a cell culture medium, A production kit according to the present disclosure comprising the steps of forming a culture, peeling the formed sheet-shaped cell culture from the culture substrate, and packaging the sheet-shaped cell culture peeled from the culture substrate. It also relates to a method of using and packaging a sheet cell culture.

Another aspect of the present disclosure is a kit for treating a disease that is ameliorated by application of the sheet-shaped cell culture by the sheet-shaped cell culture (for example, a disease related to a tissue abnormality). Relates to the kit (hereinafter sometimes referred to as “treatment kit of the present disclosure”), which includes a cell population in which the ratio of the number of myoblasts and the number of fibroblasts is adjusted, and serum. The components of the treatment kit of the present disclosure are the same as the manufacturing kit of the present disclosure. Thus, in addition to the cell population and serum, it is selected from the group consisting of culture substrates, cell cultures, washings, buffers, packaging materials, instruments, and instructions for the manufacture and / or use of sheet cell cultures A component may further be included. In a preferred embodiment, the treatment kit of the present disclosure includes instruments used for treatment with a sheet-shaped cell culture (for example, a transfer device for applying the sheet-shaped cell culture to an affected area (for example, a spatula such as an intestinal spatula)) )including. The disease improved by application of the sheet-shaped cell culture in the treatment kit of the present disclosure is as described above for the sheet-shaped cell culture of the present disclosure.

Another aspect of the present disclosure relates to a step of producing a sheet-shaped cell culture using the production kit or treatment kit of the present disclosure, and an effective amount of the produced sheet-shaped cell culture. A method for treating a disease that is ameliorated by application of a sheet-shaped cell culture (for example, a disease associated with a tissue abnormality) (hereinafter, “treatment method using the kit of the present disclosure”). May be referred to). The details of each step in the treatment method using the kit of the present disclosure are as described above for the method of producing a sheet-shaped cell culture using the production kit of the present disclosure and the treatment method of the present disclosure. In one aspect, the step of administering an effective amount of the produced sheet cell culture to a subject in need thereof is by transfer of the sheet cell culture or sheet sheet cell culture included in the kit of the present disclosure. It is performed using instruments used for treatment (for example, a spatula such as an intestinal spatula).

The present disclosure will be described in more detail with reference to the following examples, which illustrate specific embodiments of the present disclosure and the present disclosure is not limited thereto.
Example 1 Production of sheet cell culture
(1) Preparation of cell population
MCDB131 medium containing 20% FBS for seeding cells obtained from skeletal muscle tissue aseptically collected from adult thighs in a culture flask and adjusting the ratio of myoblast number to fibroblast number Grown in. The grown cells were detached from the culture flask with a proteolytic enzyme solution, collected, and concentrated by centrifugation.

(2) Examination of myoblast purity
Using a part of the obtained cells, the purity of myoblasts contained in the obtained cells was examined by flow cytometry using an anti-CD56 antibody. The cells pelleted by centrifugation were rinsed by adding PBS solution containing 0.5% BSA, and anti-human CD56 antibody diluted 10-fold with PBS solution containing 0.5% BSA was added and mixed. As a control, a negative control antibody (isotype control) diluted 10-fold with 0.5% BSA-containing PBS solution was added and mixed. After each antibody was mixed, it was immediately reacted in a cool dark place for about 1 hour, and a 0.5% BSA-containing PBS solution was added to rinse the cells. Then, a 0.5% BSA-containing PBS solution was added for analysis. In the analysis, a flow cytometer was used to measure the ratio of antibody-positive cells (myoblasts) contained in cells mixed with each antibody. In the measurement, the positive rate of the negative control was corrected, and 5,000 to 10,000 cells were analyzed. After the analysis, the purity of myoblasts was determined from the difference in the percentage of positive cells in cells mixed with each antibody, and the average was 97 ± 2%.

(3) Production of sheet cell culture
3.0 × 10 ⁶ to 3.1 × 10 ⁶ cells were suspended per 2 mL of MCDB131 medium containing 20% human serum and seeded on a φ3.5 cm temperature-responsive culture dish (UpCell®, manufactured by Cellseed ⁾ . . Ten minutes after seeding, the state of the cells was observed with a microscope from above. A representative photograph is shown in FIG. As is clear from FIG. 1, all the precipitated cells were in contact with each other. Further, when the seeded cells were observed from the bottom of the culture dish with a phase contrast microscope, it was revealed that about 70% of the seeded cells were not in contact with the culture substrate surface. Furthermore, the thickness of the cell layer at this time was about 30 μm. After the observation, the cells seeded on the culture substrate were cultured under the conditions of 37 ° C. and 5% CO ₂ and the state was confirmed after 26 hours. As a result, a sheet-like cell culture was formed. When the sheet-shaped cell culture was peeled from the culture substrate, it was shrunk while maintaining a flat sheet shape without curling. At this time, the thickness of the sheet-shaped cell culture was about 30 μm. This is because the thickness of the cell layer on the culture dish decreases due to sheeting, but the sheet-like cell culture contracts due to peeling from the culture dish, resulting in the same thickness as the seeded cell layer. Conceivable.

(4) Cell size measurement
The cell suspension prepared with a part of the cells obtained in (1) was injected into a hemocytometer and observed under a microscope. As shown in FIG. 2, the diameter of the skeletal myoblast was about 10 μm, and the diameter of the fibroblast was about 30 μm.

Example 2 Measurement of mechanical strength
In the same manner as in Example 1, a cell population in which the ratio of the number of myoblasts to the total number of cells is 55%, 60%, 70%, 80%, 90% or 95% is prepared, and this is 1 × 10 Seed in temperature-responsive culture dish (UpCell®, cell seed ⁾ coated with serum at a density of ⁶ cells / cm ² . After sowing, the cells are cultured under conditions of 37 ° C. and 5% CO ₂ , and after 40 hours, the sheet-like cell culture is detached by temperature treatment.
In a state where the sheet-shaped cell culture was stretched in the liquid, the sheet-shaped cell culture was scooped up with a stainless intestinal spatula (width: 45 mm), and placed out of the liquid with the sheet-shaped cell culture adhered to the surface of the intestinal spatula. A suture with a needle (6-0 proline) is inserted between the sheet-shaped cell culture and the intestinal spatula and penetrates from the lower surface to the upper surface of the sheet-shaped cell culture. Combine both ends of the yarn into a ring and connect it to a gauge (general-purpose digital force gauge, FGC-1B, manufactured by Nidec Sympo). The thread locked to the sheet-shaped cell culture is pulled horizontally through a gauge, and the maximum load (tensile breaking load) until the sheet-shaped cell culture is broken is measured.

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

Claims

(I) adjusting the cell number ratio of both cell types in a cell population comprising myoblasts and fibroblasts;
(Ii) seeding the cell population obtained in step (i) on a culture substrate;
(Iii) sheeting the cell population seeded in step (ii) in a cell culture medium to form a sheet-like cell culture; and
(Iv) peeling the sheet-shaped cell culture formed in step (iii) from the culture substrate
And the seeding density of the cell population in step (ii) is 90% of the total number of cells in contact with each other on the culture substrate immediately after seeding on the culture substrate after seeding on the culture substrate. A method for producing a sheet-shaped cell culture having a density of at least%.
The production method according to claim 1, wherein the cell culture medium contains allogeneic serum.
The production method according to claim 1 or 2, wherein the culture substrate is coated with serum.
The production method according to any one of claims 1 to 3, wherein the culture substrate is coated with a temperature-responsive material.
In step (i), the cell population is adjusted to comprise 60% to 99% myoblasts and 1% to 40% fibroblasts on a cell number basis. The production method according to item.
In step (i), the cell population is adjusted to include 1.5: 1 to 99: 1 myoblasts and fibroblasts as a myoblast: fibroblast cell number ratio; The production method according to any one of claims 1 to 5.
The production method according to any one of claims 1 to 6, wherein the seeding density of the cell population is 7.5 × 10 5 cells / cm 2 to 3.0 × 10 6 cells / cm 2 .
A sheet-shaped cell culture produced by the production method according to any one of claims 1 to 7.
A culture substrate, serum, and a cell population containing 60% to 99% myoblasts and 1% to 40% fibroblasts on a cell number basis, and the number of cells in the cell population is The number of cells according to any one of claims 1 to 7, which is a number that results in a density in which the proportion of cells in contact with each other on the culture substrate is 90% or more of the total cells immediately after the cells are settled on the culture substrate after seeding A kit for use in the production method according to claim 1.
A method of treating a disease that is ameliorated by application of a sheet-shaped cell culture, comprising the step of applying an effective amount of the sheet-shaped cell culture of claim 8 to a subject in need thereof. Method.