WO2020067443A1

WO2020067443A1 - Somatic cell sheet-forming method

Info

Publication number: WO2020067443A1
Application number: PCT/JP2019/038197
Authority: WO
Inventors: 賢二大山; 文哉大橋
Original assignee: テルモ株式会社
Priority date: 2018-09-27
Filing date: 2019-09-27
Publication date: 2020-04-02
Also published as: JPWO2020067443A1

Abstract

The purpose of the present invention is to provide: a method for producing a high quality transplant including adherent cells such as skeletal muscle cells, while maintaining the functions thereof at high-levels; a transplant produced by using said method; and a disease treatment method or the like using said transplant. This method for producing a transplant comprises: (a) a step for plating a cell population including adherent cells on a culture substrate; and (b) a step for culturing the plated cell population by using a medium containing a platelet lysate.

Description

How to make somatic cells into sheets

The present disclosure relates to a method for producing an implant containing various biological cells, for example, myoblasts, a sheet-like cell culture produced using the method, and a method for treating a disease using the sheet-like cell culture. And so on.

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

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

JP-T 2007-528755

The present disclosure relates to a method for producing a high-quality sheet-shaped cell culture, including various living organism-derived cells, for example, myoblasts while maintaining the function of the cells at a high level, and a sheet-shaped cell produced using the method. It is an object to provide a cell culture, a method for treating a disease using the sheet-shaped cell culture, and the like.

When preparing a cell-containing graft, it must be prepared in a xeno-free environment. Therefore, such an implant is usually prepared using human-derived, preferably recipient-derived serum, instead of xenogenic serum such as fetal bovine serum, so as to not contain impurities derived from the manufacturing process. However, there is a problem that using a large amount of human serum is costly, and using a serum derived from a recipient further imposes a heavy burden on the recipient.

The present inventors have attempted to prepare a sheet-shaped cell culture for living body transplantation using myoblasts having sufficient functions even when prepared in a xeno-free environment. Even when lysate was used, a new finding was found that a sheet-shaped cell culture could be produced which was comparable to the case where serum was used. Further research based on such findings and the use of a platelet lysate can produce high-quality grafts that can withstand clinical applications in the preparation of grafts containing various biological cells. This led to the completion of the present invention.

That is, the present invention relates to the following:
[1] A method for producing a graft containing adherent cells;
(A) inoculating a cell population containing the adherent cells on a culture substrate, and
(B) incubating the seeded cell population with a medium containing platelet lysate;
The above method, comprising:
[2] The method of [1], wherein the adherent cells are cells into which no gene has been introduced.
[3] The method of [1] or [2], wherein the medium does not contain serum different from the species from which the cells contained in the graft are derived.
[4] The method of [1] to [3], wherein the medium further contains a cell adhesive component.
[5] The method of [1] to [4], wherein the culture substrate is coated with a cell adhesive component and / or a platelet lysate.
[6] The method of [1] to [5], wherein the adherent cells are myoblasts.
[7] The method of [1] to [6], wherein the transplant is a sheet-shaped cell culture.

According to the present invention, a graft such as a sheet-shaped cell culture can be produced with high quality and high efficiency from a cell population containing various biological cells, for example, myoblasts. In particular, in the preparation of grafts, such as sheet-shaped myoblast cell cultures, which exhibit an effect when transplanted into a living body, it is possible to prepare a graft having desired properties such as cytokine production at a high level. It is possible to provide a graft or the like very suitably.

FIG. 1 is a photograph showing the results of a test for forming a sheet-shaped skeletal myoblast culture. A is a sheet-shaped skeletal myoblast cell culture formed in an FBS-containing medium, and BD are sheet-shaped skeletal myoblast cell cultures formed in a PL-containing medium. B is for UltraGro, C is for UltraGRO @ PURE, and D is for UltraGRO @ ADVANCE.

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

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

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

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

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

細胞 The cells constituting the graft of the present disclosure are not particularly limited as long as they can form the graft, and include, for example, adherent cells (adherent cells). In a preferred embodiment, the cells constituting the graft are cells into which no gene has been introduced. Adherent cells include, for example, adherent somatic cells (eg, cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin Cells, synovial cells, chondrocytes, etc.) and stem cells (eg, tissue stem cells such as myoblasts, cardiac stem cells, embryonic stem cells, mesenchymal stem cells, etc.). Somatic cells may be those differentiated from stem cells. Non-limiting examples of cells constituting the graft include, for example, myoblasts (eg, skeletal myoblasts), mesenchymal stem cells (eg, bone marrow, adipose tissue, peripheral blood, skin, hair root, muscle tissue, Endometrial, placenta, cord blood-derived cells), cardiomyocytes, fibroblasts, cardiac stem cells, embryonic stem cells, synovial cells, chondrocytes, epithelial cells (eg, oral mucosal epithelial cells, retinal pigment epithelial cells, Nasal mucosal epithelial cells, etc.), endothelial cells (eg, vascular endothelial cells), hepatocytes (eg, liver parenchymal cells), pancreatic cells (eg, pancreatic islet cells), kidney cells, adrenal cells, periodontal ligament cells, gingiva Cells, periosteal cells, skin cells and the like.

Non-limiting examples of stem cell-derived adherent cells include stem cell-derived cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, and skin Cells, synovial cells, chondrocytes and the like.

細胞 The cells constituting the graft can be derived from any organism that can be treated with the graft. Such organisms include, without limitation, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents (eg, mice, rats, hamsters, guinea pigs, etc.), rabbits, etc. Is included. The number of types of cells constituting the graft is not particularly limited, and may be composed of only one type of cell, or may be a type using two or more types of cells. When there are two or more types of cells forming the graft, the content ratio (purity) of the most abundant cells is, for example, at least 50%, preferably at least 60%, more preferably at least 70% at the end of the formation of the sheet cell culture. %, More preferably 75% or more.

The culture substrate is not particularly limited as long as cells can form an implant thereon, and includes, for example, containers of various materials and / or shapes, solid or semi-solid surfaces in the containers, and the like. The container is preferably made of a structure / material that does not allow the passage of a liquid such as a culture solution. Examples of such a material include, but are not limited to, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, and polydimethyl. Acrylamide, metal (for example, iron, stainless steel, aluminum, copper, brass) and the like can be mentioned. Also, the container preferably has at least one flat surface. Examples of such a container include, without limitation, a culture container having a bottom surface formed of a culture substrate capable of forming a cell culture and a liquid impermeable side surface. Specific examples of such culture vessels include, but are not limited to, cell culture dishes, cell culture bottles, and the like. The bottom of the container may be transparent or opaque. If the bottom surface of the container is transparent, observation and counting of cells can be performed from the back side of the container. Further, the container may have a solid or semi-solid surface inside. Examples of the solid surface include plates and containers made of various materials as described above, and examples of the semi-solid surface include a gel and a soft polymer matrix. The culture substrate may be prepared using the above materials, or a commercially available substrate may be used.

Preferred culture substrates include, but are not limited to, for example, a substrate having an adhesive surface suitable for forming a sheet-shaped cell culture, and a substrate having a low adhesive surface suitable for forming a spheroid. And / or a substrate having a uniform well-like structure. Specifically, in the case of forming a sheet-shaped cell culture, for example, corona discharge-treated polystyrene, a substrate coated on the surface with a hydrophilic compound such as a collagen gel or a hydrophilic polymer, further, collagen, Examples include extracellular matrices such as fibronectin, laminin, vitronectin, proteoglycan, and glycosaminoglycan, and substrates coated on the surface with cell adhesion factors such as cadherin family, selectin family, and integrin family. Further, such substrates are commercially available ^{(e.g., Corning (R) TC-Treated} Culture Dish, Corning , etc.). In the case of spheroid formation, for example, temperature-responsive gel (commercial name: meviol gel) obtained by crosslinking soft agar, poly (N-isopropylacrylamide) (PIPAAm) with polyethylene glycol (PEG), polyhydroxyethyl methacrylate ( A substrate coated with a non-cell-adhesive compound such as a hydrogel such as poly (HEMA) or 2-methacryloyloxyethylphosphorhoscholine (MPC) polymer and / or a substrate having a uniform uneven structure on the surface. Can be Such substrates are also commercially available (e.g., EZSPHERE ^(R), etc.). The culture substrate may be entirely or partially transparent or opaque.

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

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

In the present disclosure, "pluripotent stem cells" is a term well known in the art and has the ability to differentiate into cells of all lineages belonging to the three germ layers, i.e., endoderm, mesoderm and ectoderm. Means cell. Non-limiting examples of pluripotent stem cells include, for example, embryonic stem cells (ES cells), nuclear transfer embryonic stem cells (ntES cells), and the like. A pluripotent stem cell of the present disclosure particularly means a pluripotent stem cell into which no gene has been introduced. Usually, when inducing differentiation of a pluripotent stem cell into a specific cell, first, a suspension culture of the pluripotent stem cell is carried out to form an aggregate of any of the above three germ layers, and then a cell forming an aggregate Induce differentiation into specific cells of interest.

において In the present disclosure, “pluripotent stem cell-derived differentiation-inducing cell” means any cell that has been subjected to differentiation-inducing treatment so as to differentiate from a pluripotent stem cell into a specific type of cell. Non-limiting examples of differentiation-inducing cells include muscular cells such as cardiomyocytes and skeletal myoblasts, neuronal cells such as neuronal cells, oligodendrocytes and dopamine-producing cells, retinal cells such as retinal pigment epithelial cells, and blood cells. Cells, hematopoietic cells such as bone marrow cells, immune cells such as T cells, NK cells, NKT cells, dendritic cells, B cells, cells constituting organs such as hepatocytes, pancreatic β cells, kidney cells, In addition to chondrocytes, germ cells, and the like, progenitor cells and somatic stem cells that differentiate into these cells are included. Typical examples of such progenitor cells and somatic stem cells include, for example, mesenchymal stem cells in cardiomyocytes, pluripotent heart progenitor cells, unipotent heart progenitor cells, neural stem cells in nervous system cells, hematopoietic cells and immune cells. Related cells include hematopoietic stem cells and lymphoid stem cells. Induction of differentiation of pluripotent stem cells can be performed using any known technique. For example, induction of differentiation from pluripotent stem cells into cardiomyocytes can be performed based on the method described in Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015, and WO 2014/185358.

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

One aspect of the present disclosure relates to a method of producing a high-quality implant comprising adherent cells, particularly non-transgenic adherent cells. The method of the present disclosure includes the following steps (a) and (b):
(A) inoculating a cell population containing the adherent cells on a culture substrate, and
(B) incubating the seeded cell population with a medium containing platelet lysate.

において In the present disclosure, “a cell into which a gene has not been introduced” means a cell into which a foreign gene has not been introduced in the process of obtaining the cell. When the cell is a cell derived from a stem cell, gene transfer has not been performed in any of the steps of stem cell isolation and differentiation induction. For example, when a certain somatic cell is used, a primary cell obtained from a living tissue, a subcultured cell obtained by subculturing the primary cell, a cell obtained by differentiating a precursor cell of the somatic cell obtained from the living tissue, or the like can be used.

In the step (a), the seeding on the culture substrate may be performed, for example, by injecting a cell suspension in which cells are suspended in a medium into a culture container provided with the culture substrate. For injection of the cell suspension, a device suitable for the operation of injecting the cell suspension, such as a dropper or pipette, can be used. The seeding density of the cells is set at a density at which a graft can be formed, and such a density may vary depending on the type of the graft and the desired cells. However, those skilled in the art can use an appropriate method based on techniques known in the art. Density can be selected. For example, in the case of a sheet-shaped cell culture containing skeletal myoblasts, it may be 2.0 × 10 ⁵ cells / cm ² or more, for example, but may be seeded at higher density.

Examples of higher densities include, for example, densities that reach confluence, i.e., densities at which cells are expected to cover the entire adhesive surface of the culture vessel upon seeding, e.g., upon seeding, cells contact each other The density at which contact inhibition occurs, or the density at which cell growth is substantially stopped by contact inhibition. The upper limit of the seeding density is not particularly limited. However, if the seeding density is excessively high, the number of dead cells increases, resulting in inefficiency. In one aspect of the present disclosure, the seeding density is, for example, from about 1.0 × 10 ⁶ / cm ² to about 1.0 × 10 ⁷ / cm ² , from about 1.0 × 10 ⁶ / cm ² to about 5 0.0 × 10 ⁶ / cm ² , about 1.0 × 10 ⁶ / cm ² to about 3.0 × 10 ⁶ / cm ² , about 1.5 × 10 ⁶ / cm ² to about 1.0 × 10 ⁷ pieces / cm ² , about 1.5 × 10 ⁶ pieces / cm ² to about 5.0 × 10 ⁶ pieces / cm ² , about 1.5 × 10 ⁶ pieces / cm ² to about 3.0 × 10 ⁶ pieces / cm ² , about 2.0 × 10 ⁶ pieces / cm ² to about 1.0 × 10 ⁷ pieces / cm ² , about 2.0 × 10 ⁶ pieces / cm ² to about 5.0 × 10 ⁶ pieces / Cm ² , about 2.0 × 10 ⁶ / cm ² to about 3.0 × 10 ⁶ / cm ² , and the like. In one preferred embodiment, the seeding density is from about 1.76 × 10 ⁶ / cm ² to about 2.33 × 10 ⁶ / cm ² .

The culture substrate to be seeded is as described in detail above, but in one embodiment, a culture substrate having a surface coated with a cell adhesive component such as an extracellular matrix or a cell adhesion factor may be preferable. Cell adhesion components include, but are not limited to, for example, extracellular matrices such as collagen, fibronectin, laminin, vitronectin, proteoglycans, glycosaminoglycans, cadherin family, selectin family, cell adhesion factors such as integrin family In addition, these modifications, such as laminin 511 (a modification of laminin), VTN-N (a modification of vitronectin), and RetroNectin ^(R) (a modification of fibronectin ⁾ may be used.

In step (b), the seeded cells are incubated for graft formation. In the present disclosure, graft formation when the graft is a sheet-shaped cell culture is particularly referred to as “sheeting”, and incubation for forming the seeded cells into a sheet is particularly referred to as “sheeting culture”. is there. Incubation of the seeded cells for graft formation can be performed by any known technique and conditions. Non-limiting examples of such techniques are described in, for example, JP-A-2010-081829, JP-A-2010-226991, JP-A-2011-110368, JP-A-2011-172925, WO 2014/185517, and the like. For example, it is considered that graft formation such as sheeting of cells is achieved by cells adhering to each other via an intercellular adhesion mechanism such as an adhesion molecule or an extracellular matrix. Therefore, it is considered that the step of forming a graft (for example, forming a sheet) of the seeded cells can be achieved by culturing the cells under conditions that form intercellular adhesion. Such conditions may be any as long as they can form cell-cell adhesion, but usually, cell-cell adhesion can be formed under the same conditions as general cell culture conditions. Such conditions include, for example, culture at about 37 ° C., 5% CO ₂ . Incubation can be performed under normal pressure (atmospheric pressure, non-pressurized). Incubation can be performed in containers of any size and shape. The size and shape of the transplant can be adjusted by adjusting the size and shape of the culture vessel, or by placing a mold of the desired size and shape in the culture vessel and culturing cells inside it. It can be adjusted arbitrarily.

Incubation time for graft formation can vary depending on the type of seeded cells and cell density. For example, when skeletal myoblasts are collected from a living body and formed into a sheet, the sheet may be formed by disseminating the skeletal myoblast at a density of, for example, about 3.0 × 10 ⁶ cells / cm ² and incubating for 2 hours or more. When the seeding density reaches a confluent density, that is, when seeding is performed at a higher density, the period of sheet culture can be shortened, and the culture time may be 2 to 12 hours, more preferably 2 to 6 hours. .

The medium (sometimes simply referred to as a medium) used for the graft formation is not particularly limited as long as it enables formation of cell-cell adhesion. For example, physiological saline, various physiological buffers (for example, PBS, HBSS, etc.) and those based on various basal media for cell culture may be used. Examples of such a basal medium include, but are not limited to, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12 and the like. Many of these basal media are commercially available and their compositions are also known. The basal medium may be used with its standard composition (for example, as it is commercially available), or its composition may be appropriately changed depending on the cell type and cell conditions. Therefore, the basal medium used in the present invention is not limited to those having a known composition, and includes those in which one or more components have been added, removed, increased or reduced in weight. The sheeting medium may contain additives such as normal serum (eg, bovine serum such as fetal calf serum, horse serum, human serum, etc.) and various growth factors (eg, FGF, EGF, VEGF, HGF, etc.). However, when the transplant is produced under xeno-free conditions, it is preferable that the transplant does not contain serum different from the species from which the cells contained in the transplant are derived, such as bovine serum and horse serum. The present disclosure is characterized in that an incubation for forming a graft is performed in a medium containing platelet lysate instead of or in addition to serum or growth factors. In one preferred embodiment, the vehicle contains platelet lysate but does not contain serum.

において In the present disclosure, “platelet lysate (PL)” refers to a composition rich in growth factors and the like, obtained by repeatedly freezing and thawing platelets. In recent years, platelet lysates have been known to promote the growth of mesenchymal stem cells. In the production of a graft containing adherent cells, the present inventors made it possible to form a graft as in the case of using serum by including a platelet lysate in a medium. It has been found for the first time that piece formation can be easily achieved at low cost.

Platelet lysates are commercially available as media additives for cell culture and are known in the art. The platelet lysate can be prepared, for example, by the method described in JP-T-2014-533715, Bieback et al., STEM CELLS, 2009; 27: 2331-2341.
As a specific preparation method, it can be prepared by, for example, dissolving a platelet population and removing contaminants such as platelet particles and a membrane therefrom to obtain a supernatant. Lysis of platelets can be achieved through steps such as chemical means (eg, using CaCl ₂ ), osmotic means (eg, using distilled water), freeze-thaw means, mechanical disruption means, and the like. Removal of contaminants can be achieved by a method such as centrifugation or filtration.

The concentration of the platelet lysate contained in the medium may be any level commonly used in the art, and may be, for example, 1%, 2.5%, 5%, 10%, 15%, 20%, and the like. . In a preferred embodiment, the platelet lysate is contained in the medium in an amount of about 1% to 20%, more preferably about 2% to 10%, and still more preferably about 2.5% to 10%.
The medium may be replaced as appropriate during the incubation period. Further, the composition of the medium may be changed in accordance with the progress of the graft formation.

The medium may further include a cell adhesive component. The cell adhesive component is as described in detail above. When the medium contains a cell adhesive component, the culture substrate may or may not be further coated with a cell adhesive component or a platelet lysate. When the culture substrate is coated with a cell adhesive component and / or a platelet lysate, the cell adhesive component contained in the medium may be the same as the cell adhesive component coating the culture substrate. However, they are preferably the same cell adhesive component.

濃度 The concentration of the cell adhesive component contained in the medium may vary depending on the type of the cell adhesive component contained, the state of the cells forming the graft, and the like. For example, when the sheet is formed using cells having low viability, that is, cells having weak activity, the content of the cell adhesive component is preferably small. The concentration of the cell adhesive component contained in the medium is about 0.1%, about 0.5% based on the concentration (100%) used when the same cell adhesive component is used as a coating agent for the culture substrate. , About 1%, about 5%, about 10%, about 20%, about 25%, about 50%, about 75%, about 100%, and the like. Therefore, in a preferred embodiment, the concentration range of the cell adhesive component contained in the medium is about 0.1% based on the concentration (100%) used when the same cell adhesive component is used as a coating agent for the culture substrate. % To about 100%, about 0.1% to about 50%, about 0.1% to about 25%, about 0.1% to about 20%, about 0.1% to about 10%, about 1% to About 100%, about 0.5% to about 100%, about 0.5% to about 50%, about 0.5% to about 25%, about 0.5% to about 20%, about 0.5% to About 10%, about 1% to about 50%, about 1% to about 25%, about 1% to about 20%, about 1% to about 10%, about 0.5% to about 100%, about 5% to About 100%, about 5% to about 50%, about 5% to about 25%, about 5% to about 20%, about 5% to about 10%, and the like.

As described above, myoblasts used in the method of the present disclosure may be directly obtained from a living body or may be derived from other cells, but are preferably obtained directly from a living body. It is a thing. In one aspect, the skeletal myoblasts of the present disclosure are skeletal myoblasts from the thigh, neck, and abdomen. When the skeletal myoblasts are derived from other cells, the other cells to be induced include pluripotent stem cells capable of differentiating into any cells.

Another aspect of the present disclosure treats a disease in a subject in need thereof, comprising applying an effective amount of a graft, preferably a sheet of cell culture, produced by the method of the present disclosure to the subject in need thereof. On how to do it. The disease to be treated is as described above.

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

In the treatment method of the present disclosure, a component that enhances the survival of cells in a graft, the engraftment and / or function of the graft, and other active ingredients useful for treating a target disease, etc. It can be used in combination with a transplant (sheet-like cell culture or the like).

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

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

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

The present invention will be described in more detail with reference to the following examples, which show specific embodiments of the present invention, and do not limit the present invention.
(1) Preparation of cell population
Cells obtained from skeletal muscle tissue aseptically collected from adult thighs were inoculated into culture flasks and grown in MCDB131 medium containing 20% FBS. The grown cells were detached from the culture flask with a protease solution, collected, and concentrated by centrifugation to obtain a cell population containing skeletal myoblasts.

(2) Preparation of sheet-shaped cell culture
Sheeting culture conditions were examined using a DMEM / F12 medium supplemented with 20% fetal bovine serum (FBS) or 20% platelet lysate (PL) as a sheeting medium. As PL, three types of Ultra GRO, UltraGRO Pure, and UltraGRO ADVANCE (AventaCell BioMedical) were used, respectively. The cell population containing the skeletal myoblasts prepared in (1) was inoculated at a density of 1.5 × 10 ⁶ cells / cm ^{2 into} a temperature-responsive culture dish (UpCell®, manufactured by Cell Seed ⁾ , and then seeded at 37 ° C. After culturing for 1 day under the condition of 5% CO ₂ , the state was confirmed. As a result, a sheet-shaped cell culture was formed. Thereafter, the sheet-shaped cell culture was peeled from the culture substrate.
The results are shown in the table below.

When the sheet medium containing laminin and platelet lysate was used, a sheet-like cell culture could be formed as in the case of using the sheet medium containing FBS (FIG. 1).

According to the present invention, when forming a transplant using adherent cells, particularly cells that have not been transfected, such as cells collected from a transplant target, high-quality transplantation that is comparable to conventional cells even in a xeno-free environment You can get a piece. Therefore, in the production of a xeno-free implant used for clinical use, a high-quality implant can be easily formed.

Claims

A method for producing an implant containing adherent cells,
(A) inoculating a cell population containing the adherent cells on a culture substrate, and
(B) incubating the seeded cell population with a medium containing platelet lysate;
The above method, comprising:
方法 The method according to claim 1, wherein the adherent cells are cells into which no gene has been introduced.
The method according to claim 1 or 2, wherein the medium does not contain serum different from the species from which the cells contained in the graft are derived.
方法 The method according to any one of claims 1 to 3, wherein the medium further comprises a cell adhesive component.
The method according to any one of claims 1 to 4, wherein the culture substrate is coated with a cell adhesive component and / or a platelet lysate.
方法 The method according to any one of claims 1 to 5, wherein the adherent cells are myoblasts.
(7) The method according to any one of (1) to (6), wherein the explant is a sheet-shaped cell culture.