Decellularized support composition

Abstract

The purpose of the present invention is to provide a useful application for a decellularized cell aggregate or a decellularized cell structure.　A decellularized support composition according to the present invention is obtained by decellularizing a cultured cell aggregate or a cultured cell structure and is for use in dedifferentiation, maintenance of an undifferentiated state, or differentiation of cells.

Description

Decellularized support composition

[0001] The present invention relates to a decellularized support composition. According to the present invention, cells can be dedifferentiated, maintained in an undifferentiated state, differentiated into nerve cells, differentiated into muscle cells, formed angiogenesis, or differentiated into chondrocytes.

[0002] When transplanting grafts derived from the living tissue of another person or animal of a different species, rejection of the graft by the recipient's tissue is a problem. To improve compatibility with living tissue, techniques have been developed to remove cells from living tissue and use decellularized tissue, consisting of the remaining supportive tissue (extracellular matrix, ECM), as a graft. Decellularization refers to the removal of cellular components, such as nucleic acids, that are antigenic to the recipient, thereby avoiding immune rejection (Patent Documents 1 and 2).

[0003] Patent Publication No. 2005-514971 International Publication No. 2016 / 136633 International Publication No. 2022 / 270569

[0004] Drug Delivery System, 2021 (Japan), Vol. 36, pp. 18-27

[0005] The present inventors have found that by decellularizing a cultured cell structure obtained by culturing, a decellularized cell structure having any thickness and maintaining hardness can be obtained (Patent Document 3). The development of useful applications for such decellularized cell structures is anticipated. Therefore, an object of the present invention is to provide useful applications for decellularized cell aggregates or decellularized cell structures.

[0006] The present inventors have conducted extensive research into useful uses of decellularized cell aggregates or decellularized cell structures, and as a result have surprisingly found that decellularized cell aggregates or decellularized cell structures are useful for cellular dedifferentiation, maintenance of undifferentiation, neural differentiation, muscle cell differentiation, angiogenesis, or chondrocyte differentiation. The present invention is based on this finding. Accordingly, the present invention comprises: [1] a decellularized scaffold composition obtained by decellularizing a cultured cell aggregate or a cultured cell structure, the decellularized scaffold composition being for cellular dedifferentiation, maintenance of undifferentiation, or differentiation; [2] the decellularized scaffold composition according to [1], wherein the differentiation is for neural differentiation, muscle cell differentiation, angiogenesis, or chondrocyte differentiation; [3] a decellularized scaffold composition derived from the cultured cell aggregate, the decellularized scaffold composition comprising surface pores having a pore diameter of 5 μm or more, the surface pores being formed by pore formation within a range of 11088 μm on the aggregate surface. ２[4] A decellularized support composition derived from the cultured cell structure, the decellularized support composition according to [1], having a porosity of 40% to 70%; [5] The decellularized support composition according to [1], wherein the decellularized support composition is in a protease-treated liquid state; [6] The decellularized support composition according to [5], wherein the protease is pepsin; [7] The decellularized support composition according to any of [1] to [6], wherein the decellularized support composition has a DNA content of 1.000 mass% or less per dry mass; [8] An extracellular matrix composition for dedifferentiation, maintenance of undifferentiation, or differentiation of cells, comprising at least one selected from proteins, ribosomes, and extracellular vesicles having a molecular weight of 5,000 to 80,000; [9] A cell culture substrate comprising the decellularized support composition according to any of [1] to [6], or the extracellular matrix composition according to [8].

[10] A biological graft comprising the decellularized support composition according to any one of [1] to [6] or the extracellular matrix composition according to [8];

[11] A cell composition comprising the decellularized support composition according to any one of [1] to [6] or the extracellular matrix composition according to [8] and cells;

[12] A method for producing a decellularized support composition for dedifferentiation, maintenance of undifferentiation, or differentiation of cells, comprising a cultured cell aggregate formation step of forming cell aggregates by culturing cells, and a step of decellularizing the cultured cell aggregates;

[13] A method for producing a decellularized support composition for dedifferentiation, maintenance of undifferentiation, or differentiation of cells, comprising a cultured cell aggregate formation step of forming cell aggregates by culturing cells, a cultured cell structure production step of further culturing the cultured cell aggregates to produce a cell structure, and a step of decellularizing the cultured cell structure;

[14] A method for producing a decellularized support composition according to

[12] , wherein cells are cultured on a non-adhesive surface in the cultured cell aggregate formation step;

[15] The method for producing a decellularized support composition according to

[13] , wherein the cultured cell aggregates are cultured in a space surrounded by a mesh-like or comb-like support in the cultured cell structure production step.

[16] The method for producing a decellularized support composition according to any one of

[12] to

[15] , wherein the decellularization is performed by high hydrostatic pressure treatment.

[17] A decellularized support composition for dedifferentiating, maintaining the undifferentiated state, or differentiating cells, obtained by the manufacturing method according to any one of

[12] to

[15] ;

[18] A method for dedifferentiating, maintaining the undifferentiated state, or differentiating cells, in which cells are cultured using a decellularized support composition obtained by decellularizing a cultured cell aggregate or a cultured cell structure, or the extracellular matrix composition according to [8];

[19] A method for differentiating cells into neurons, in which cells are cultured using a decellularized support composition obtained by decellularizing a cultured cell aggregate or a cultured cell structure, or the extracellular matrix composition according to [8];

[20] A method for differentiating cells into muscle cells, in which cells are cultured using a decellularized support composition obtained by decellularizing a cultured cell aggregate or a cultured cell structure, or the extracellular matrix composition according to [8];

[21] A method for angiogenesis, in which cells are cultured using a decellularized support composition obtained by decellularizing a cultured cell aggregate or a cultured cell structure, or the extracellular matrix composition according to [8];

[22] A method for differentiating cells into chondrocytes, comprising culturing cells using a decellularized support composition obtained by decellularizing a cultured cell aggregate or a cultured cell structure, or the extracellular matrix composition according to [8];

[23] A method for producing a decellularized support composition solution, comprising: a liquefaction step of liquefying the decellularized support composition obtained by the production method according to any one of

[12] to

[15] using a protease or an acidic solution; a separation step of treating the liquefied decellularized support composition by centrifugation; and a neutralization step of neutralizing the supernatant obtained by the centrifugation to a pH of 7.0 to 8.0;

[24] A method for producing a decellularized support composition solution according to

[23] , wherein the protease used in the liquefaction step is pepsin;

[25] A method for producing a decellularized support composition solution according to

[23] , wherein the liquefaction step is carried out at a pH of 1.0 to 5.0 and at a temperature range of 4 to 37°C; and

[26] The method for producing a decellularized support composition solution according to

[23] , wherein the decellularized support composition from which the cultured cell aggregates or cultured cell structures have been decellularized is in powder form.

[0007] The decellularized support composition of the present invention can induce cellular dedifferentiation, maintenance of undifferentiated state, neuronal differentiation, muscle cell differentiation, angiogenesis, or chondrocyte differentiation.

[0008] Figure 1 shows a diagram of molecular weight analysis of proteins contained in the decellularized cell aggregate composition of Example 1 and the decellularized cell structure composition of Example 2, measured using a microchip-type electrophoresis device. Figure 2 shows photographs of ribosome immunostaining of sections of the decellularized cell aggregate composition of Example 1 and the decellularized cell structure composition of Example 2. Figure 3 shows photographs of staining of extracellular vesicles of sections of the decellularized cell aggregate composition of Example 1 and the decellularized cell structure composition of Example 2. Figure 4 shows graphs analyzing mRNA of NANOG (A), SOX2 (B), and POU5F1 (C), which are pluripotency marker genes of cultured cells from the decellularized cell aggregate composition of Example 1, the decellularized cell structure composition of Example 2, and the bovine-derived collagen sheet of Comparative Example 1. 1 shows graphs analyzing the mRNA of the neuronal differentiation marker GDNF (A) and the neurotransmitter precursor TAC1 (B) in cultured cells of the decellularized cell aggregate composition of Example 1, the decellularized cell structure composition of Example 2, and the bovine-derived collagen sheet of Comparative Example 1; the decellularized cell aggregate composition of Example 3; and the bovine-derived collagen sheet of Comparative Example 1.

[0033] Fig. 1 shows graphs analyzing the mRNA of the nerve regeneration marker GAP43 (C) and the synapse marker Synapsin-1 (D) in the collagen powder of Comparative Example 3 and the gelatin fiber substrate for cell culture of Comparative Example 4; and graphs analyzing the mRNA of the nerve regeneration marker GAP43 (E) and the synapse marker Synapsin-1 (F) in the decellularized cell aggregate composition solution of Example 4 and the solubilized collagen of Comparative Example 2. Graph (A) shows an analysis of vascular endothelial cell differentiation marker PECAM1 (CD31) mRNA in the decellularized cell aggregate composition of Example 1, the decellularized cell structure composition of Example 2, and cultured cells on the bovine-derived collagen sheet of Comparative Example 1, and graph (B) shows an analysis of PECAM1 (CD31) mRNA in the decellularized cell aggregate composition solution of Example 4, the bovine-derived collagen sheet of Comparative Example 1, and the solubilized collagen of Comparative Example 2. Photographs of immunostaining of α-SMA (a muscle cell differentiation marker) of muscle cells detected in the decellularized cell structure composition of Example 2 and the cultured cells on the bovine-derived collagen sheet of Comparative Example 1. Graph (B) shows the results of calculating the positive staining area in immunostaining of α-SMA (a muscle cell differentiation marker) of muscle cells detected in the decellularized cell structure composition of Example 2 and the cultured cells on the bovine-derived collagen sheet of Comparative Example 1.1 is a photograph of hematoxylin-eosin (HE) staining of the decellularized cell aggregate composition of Example 1 and the decellularized cell structure composition of Example 2. 2 is a graph analyzing the mRNA of CD73 (an undifferentiation marker) and CD90 (an undifferentiation marker) of the decellularized cell aggregate composition of Example 3 and the bovine-derived collagen sheet of Comparative Example 1. 3 is a graph analyzing the mRNA of the chondrocyte marker SOX9 of the decellularized cell aggregate composition solution of Example 4 and the bovine-derived collagen sheet of Comparative Example 1.

[0009] [1] Decellularized support composition The decellularized support composition of the present invention is a decellularized support composition obtained by decellularizing a cultured cell aggregate containing an extracellular matrix or a cultured cell structure further containing an extracellular matrix, and is used for cell dedifferentiation, neuronal differentiation, muscle cell differentiation, or angiogenesis. In this specification, a decellularized support composition derived from a cultured cell aggregate may be referred to as a decellularized cell aggregate composition (decellularized cell aggregate), and a decellularized support composition derived from a cultured cell structure may be referred to as a decellularized cell structure composition (decellularized cell structure).

[0010] Decellularized Cell Aggregate Composition The cultured cell aggregate is not particularly limited as long as it is a cell aggregate obtained by in vitro culture. Specifically, the cells adhere to each other by producing extracellular matrix (ECM) outside the cells during three-dimensional culture (Figure 9), maintaining the shape of a three-dimensional aggregate, such as a spheroid. The shape of the cultured cell aggregate is not particularly limited, and examples include spherical, ellipsoidal, cuboidal, rectangular, cylindrical, and rectangular prism-like shapes. When the cultured cell aggregate is a spheroid, it is often cylindrical or ellipsoidal. The size of the cultured cell aggregate is also not particularly limited, but in the case of a spheroid, the shortest length when observed with a scanning electron microscope (SEM) is 80 μm or more, preferably 90 μm or more, more preferably 95 μm or more, and even more preferably 100 μm or more. The maximum length is not limited, but is 600 μm or less, preferably 500 μm or less, and more preferably 300 μm or less. The upper and lower limits can be combined to define a size range for the cultured cell aggregate. This allows the effects of the present invention to be effectively exerted.

[0011] The spheroids can be produced by three-dimensionally culturing cells and aggregating them into a spherical shape or the like. Spheroids are formed by cells and extracellular matrix (ECM), and contain more extracellular matrix than cell populations produced by two-dimensional culture (monolayer culture). Three-dimensional cell culture methods for producing spheroids include, but are not limited to, the non-adhesive surface culture method, the hanging drop culture method, and the rotary culture method.

[0012] The non-adhesive surface culture method is a culture method for obtaining spheroids using a non-adhesive culture plate with an improved hydrophilic surface. Cells contained in the non-adhesive culture plate float in the medium and form spheroids by adhering to each other. By enlarging the culture plate, it is possible to culture a large number of spheroids.

[0013] In the hanging drop culture method, suspended cells are dropped onto the inside of the lid of a culture plate, and the droplets rise due to surface tension and are then cultured. The cells gather downward due to gravity, adhere to each other, and form spheroids. In the hanging drop culture method, it is relatively easy to uniform the number of cells contained in each spheroid.

[0014] The rotary culture method forms spheroids by rotating the culture chamber. Rotation allows the suspended cells to come into even contact with each other, resulting in the production of spheroids with a relatively uniform cell number and size. Furthermore, the rotary culture method allows for large-scale culture.

[0015] The decellularized cell aggregate composition of the present invention can be obtained by decellularizing cultured cell aggregates.

[0016] (Surface Pores) The decellularized cell aggregate composition of the present invention has surface pores with a pore diameter of 5 μm or more on the surface of the aggregate. ２ The decellularized cell aggregate composition of the present invention has 5 to 45 pores per cell. As used herein, surface pores refer to pores present on the surface of the decellularized cell aggregate composition when the decellularized cell aggregate is observed with a scanning electron microscope (SEM). Furthermore, the term "surface" refers to the surface that can be observed with the SEM. The decellularized cell aggregate composition of the present invention has a specific number of surface pores of 5 μm or more. By having a certain number of surface pores, the composition exhibits excellent effects as a scaffold for cell culture. The diameter of the surface pores to be counted is not particularly limited as long as it is 5 μm or more, but the lower limit is preferably 5 μm or more and the upper limit is preferably 50 μm or less. The diameter of the surface pores can be measured mechanically or visually from the SEM photograph. Cultured cell aggregate surface 11088 μm ２The number of surface pores per aggregate is not particularly limited as long as it is between 5 and 45, but the lower limit is preferably 5 or more, in some embodiments 6 or more, in some embodiments 7 or more, in some embodiments 8 or more, in some embodiments 9 or more, in some embodiments 10 or more, in some embodiments 12 or more, and in some embodiments 15 or more. The upper limit of the number of surface pores is preferably 45 or less, in some embodiments 43 or less, in some embodiments 41 or less, in some embodiments 39 or less, in some embodiments 37 or less, in some embodiments 35 or less, in some embodiments 33 or less, in some embodiments 31 or less, and in some embodiments 30 or less. The upper and lower limits can be combined to form a range for the number of surface pores. By keeping the number within this range, the number of viable cells can be increased. The aggregate surface area for measuring surface pores is 11088 μm ２ Although there is no particular limitation on how to specify the number of surface pores, for example, the number of surface pores in an area of ​​88 μm×126 μm may be counted on an SEM photograph.

[0017] Decellularized Cell Structure Composition: A cultured cell structure is a cell structure obtained by in vitro culture. The cultured cell structure is not particularly limited, as long as the cells adhere and maintain a three-dimensional shape by producing extracellular matrix (ECM) outside the cells (Figure 9). Examples of such a structure include a cell block formed by bonding multiple spheroids. The shape of the cultured cell structure is not particularly limited, and examples include a sheet, tube, or block, and may also be any of the shapes listed for cultured cell aggregates. The size of the cultured cell structure is also not particularly limited, but in the case of a cell block, the shortest length, when observed with a scanning electron microscope (SEM), is 90 μm or more, preferably 100 μm or more, more preferably 150 μm or more, and even more preferably 200 μm or more. The maximum length is not limited, but is preferably 2100 μm or less, preferably 2000 μm or less, more preferably 1700 μm or less, even more preferably 1400 μm or less, even more preferably 1200 μm or less, and most preferably 1000 μm or less. The upper and lower limits can be combined to define the size range of the cultured cell aggregate. This allows for efficient production of decellularized cell structures and the effects of the present invention to be effectively achieved. The strength of the cultured cell structure is also not particularly limited, as long as it maintains a certain three-dimensional shape, but it is preferable that it has strength that allows it to be handled with tweezers and sutured in a wet state. This makes it easier to create decellularized cell structures for various purposes, such as transplants and cell therapy.

[0018] A cultured cell structure can be obtained by further culturing a plurality of cultured cell aggregates. Specifically, the cultured cell structure can be produced by the "cultured cell structure formation step" in the method for producing a decellularized support composition described below.

[0019] The decellularized cell structure composition of the present invention can be obtained by decellularizing a cultured cell structure.

[0020] (Porosity) The porosity of the decellularized cell structure composition of the present invention is 40% to 70%. The lower limit of the porosity is preferably 42%, more preferably 44%, even more preferably 47%, and most preferably 50%. The upper limit of the porosity is preferably 68%, more preferably 66%, even more preferably 64%, and most preferably 55%. By keeping the porosity within this range, a graft can be obtained that has sufficient strength and is free from rejection. Furthermore, a decellularized graft of any thickness or specific shape can be provided. In this specification, porosity is calculated as follows: A section of the decellularized cell structure is stained with Sirius Red, which stains the extracellular matrix. Microscopic tissue images are analyzed using image processing software, and areas not stained with Sirius Red are determined to be voids, and the porosity is calculated. Sirius Red staining is performed as follows: A section sample of the decellularized cell structure is deparaffinized. Next, the section sample is treated with iron hematoxylin. Next, the decellularized cell structure is treated with a hydrochloric acid / ethanol solution and a phosphomolybdic acid aqueous solution. The structure is then treated with Sirius Red (Direct Red 80), dehydrated, and mounted. The resulting Sirius Red-stained sections are observed under a microscope, and the porosity is calculated using image processing software (ImageJ). The sections are photographed at 10x magnification, and the area of ​​the entire decellularized cell structure and the area of ​​the Sirius Red-stained portion are measured. The porosity is calculated using the following formula: Porosity (%) = (area of ​​the entire decellularized cell structure - area of ​​the Sirius Red-stained portion) / area of ​​the entire decellularized cell structure x 100

[0021] The decellularized support composition of the present invention may be in a wet or dry state. A decellularized support composition provided in a wet state can be used immediately for transplantation. On the other hand, a decellularized support composition provided in a dry state can be stored for a long period of time, is easy to transport, and can be restored and used at the time of use. The decellularized support composition of the present invention may be in a liquid state. The term "liquid" is not limited, and may be a liquid obtained by treating the decellularized cell aggregate or decellularized cell structure with, for example, a protease or an acidic solution. Even in a liquid state, the effects of the present invention, such as dedifferentiation, maintenance of undifferentiated cells, and differentiation (e.g., neuronal differentiation, muscle cell differentiation, angiogenesis, or chondrocyte differentiation), can be exerted by the action of components contained in the liquid. The solvent used in the liquid decellularized support composition is not limited as long as the effects of the present invention can be obtained, and a medium suitable for the cells used can be used without limitation, for example, PBS (phosphate-buffered saline, pH 7.4).

[0022] Cells used in the decellularized scaffold composition include, but are not limited to, suspension cells and adhesive cells. However, from the viewpoint of ease of preparation of the decellularized scaffold composition, adhesive cells are preferred. The adhesive cells are not particularly limited and can be selected appropriately depending on the purpose of use of the decellularized scaffold composition. For example, differentiated adhesive cells include hepatocytes, stellate cells, Kupffer cells, vascular endothelial cells, meatus endothelial cells, endothelial cells, osteoclasts, periodontal ligament-derived cells, epidermal cells, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells, mammary gland cells, pericytes, smooth muscle cells, cardiac muscle cells, muscle cells, kidney cells, pancreatic islet cells of Langerhans, peripheral nerve cells, nerve cells, chondrocytes, and bone cells. Examples of differentiation-competent adherent cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, hematopoietic stem cells, neural stem cells, cardiac progenitor cells, vascular endothelial progenitor cells, neural progenitor cells, preadipocytes, fibroblasts, dermal fibroblasts, skeletal myoblasts, osteoblasts, and odontoblasts. From the viewpoint of ease of preparation of the decellularized support composition, fibroblasts, mesenchymal stem cells, and cardiac progenitor cells are preferred, and dermal fibroblasts are more preferred. Furthermore, immortalized cells can also be used as cells for the decellularized support composition. Immortalized cells refer to cells whose proliferation does not cease even after repeated cell division. Immortalized cells may be prepared (immortalized) by artificially introducing a gene into the cells. From the viewpoint of achieving the effects of the present invention, the immortalized cells are preferably immortalized by artificially introducing a gene into the cells. Immortalized cells are not particularly limited, but examples include myeloma cell lines, immortalized fibroblasts, and immortalized vascular endothelial cells, with immortalized fibroblasts being preferred from the viewpoint of exerting the effects of the present invention. Examples of myeloma cell lines include NSI-Ag4 / 1 and Sp2 / O-Agl4. Examples of immortalized fibroblasts include OUMS-36T-1 and TelCOFS02MA, with OUMS-36T-1 being preferred from the viewpoint of exerting the effects of the present invention. Examples of immortalized vascular endothelial cells include HUEhT-1.The origin of the cells is not particularly limited, and includes eukaryotic cells, prokaryotic cells, cells of multicellular organisms, and cells of unicellular organisms. Examples of eukaryotic cells include animal cells, insect cells, plant cells, fungi, algae, and protozoa, with animal cells being preferred from the viewpoint of exerting the effects of the present invention. Examples of animal cells include, but are not limited to, cells derived from humans, monkeys, dogs, cats, rabbits, ferrets, sheep, goats, cattle, pigs, horses, camels, mice, rats, hamsters, guinea pigs, and gerbils, with humans, rabbits, cattle, pigs, mice, and rats being preferred from the viewpoint of exerting the effects of the present invention.

[0023] The decellularized support composition of the present invention is preferably, but not limited to, adhered by an extracellular matrix. Adhesion to the extracellular matrix allows the cultured cell construct to obtain a certain level of strength. Examples of the extracellular matrix include collagen, laminin, fibronectin, chondroitin sulfate, heparin sulfate, keratan sulfate, hyaluronic acid, vitronectin, elastin, tenascin, entactin, fibrillin, and proteoglycan, but the type of extracellular matrix varies depending on the type of cell and is not particularly limited.

[0024] <<DNA Content>> The decellularized support composition has a DNA content per dry mass of 1.000% by mass or less, but is not limited thereto. The DNA content per dry mass of the decellularized support composition is 0.900% by mass or less, preferably 0.800% by mass or less, more preferably 0.700% by mass or less, even more preferably 0.600% by mass or less, even more preferably 0.500% by mass or less, even more preferably 0.400% by mass or less, even more preferably 0.300% by mass or less, and even more preferably 0.250% by mass or less. This allows for the production of a decellularized support composition that is appropriately decellularized and exhibits minimal rejection upon transplantation. The DNA content can be measured using the Picogreen method. A dried test piece of the decellularized support composition (hereinafter sometimes referred to as a sample) is immersed in a protease solution and dissolved, followed by treatment with phenol / chloroform to remove proteins and recover DNA. After treatment with phenol / chloroform to remove proteins, ethanol precipitation may be performed. The recovered DNA is fluorescently stained with Picogreen (Life Technologies) and the fluorescence intensity is measured to quantify the DNA, and the DNA content (mass) of the sample is calculated. Quantification is performed using a calibration curve created using the standard DNA provided with the Picogreen. The DNA ratio is calculated from the dry mass and DNA content of the sample according to the following formula: (DNA content per dry mass (hereinafter sometimes referred to as decellularized DNA ratio) = (DNA content of the dry test piece of the decellularized support composition) / (mass of the dry test piece of the decellularized support composition) x 100 (mass%)

[0025] <<Containing Protein>> The decellularized support composition of the present invention preferably contains a protein, although this is not limited thereto. The molecular weight of the protein can be measured, for example, by subjecting the protein to electrophoresis. Electrophoresis can be performed, for example, by polyacrylamide electrophoresis (Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis, SDS-PAGE). From the viewpoint of exerting the effects of the present invention, the molecular weight of the contained protein is preferably 5,000 to 80,000, more preferably 5,500 to 78,000, even more preferably 5,700 to 76,000, even more preferably 10,000 to 74,000, even more preferably 13,000 to 73,000, and most preferably 15,000 to 71,000. Specific proteins contained therein include, but are not limited to, insulin (molecular weight 5,800), hepatocyte growth factor (HGF) (molecular weight 70,000), transforming growth factor-β1 (TGF-β1) (molecular weight 25,000), transforming growth factor-β2 (TGF-β2) (molecular weight 25,000), transforming growth factor-β3 (TGF-β3) (molecular weight 25,000), transforming growth factor-α (TGF-α) (molecular weight 5,500), insulin-like growth factor binding protein-1 (IGFBP-1) (molecular weight 25,000), insulin-like growth factor binding protein-2 (IGFBP-2) (molecular weight 31,500), insulin-like growth factor binding protein-3 (IGFBP-4) (molecular weight 31,500), insulin-like growth factor binding protein-4 (IGFBP-5) (molecular weight 31,500), insulin-like growth factor binding protein-5 (IGFBP-6) (molecular weight 31,500), insulin-like growth factor binding protein-6 (IGFBP-7) (molecular weight 31,500), insulin-like growth factor binding protein-8 (IGFBP-9) (molecular weight 31,500), insulin-like growth factor binding protein-9 (IGFBP-10) (molecular weight 31,500), insulin-like growth factor binding protein-1 (IGFBP-11) (molecular weight 31,500), insulin-like growth factor binding protein-1 (IGFBP-12) (molecular weight 31,500), insulin-like growth factor binding protein-1 (IGFBP-13) (molecular weight 31,500), insulin-like growth factor binding protein-1 (IGFBP-14) (molecular weight 31,500), insulin-like growth Synthetic protein-3 (IGFBP-3) (molecular weight 29,000), insulin-like growth factor binding protein-4 (IGFBP-4) (molecular weight 29,000), insulin-like growth factor binding protein-6 (IGFBP-6) (molecular weight 23,900), bone morphogenetic protein-4 (BMP-4) (molecular weight 26,000), bone morphogenetic protein-5 (BMP-5) (molecular weight 15,600), bone morphogenetic protein-7 (BMP-7) (molecular weight 36,000), vascular endothelial growth factor (VEGF) (molecular weight 16,000-45,000), vascular endothelial growth factor-D (VEGF-D) (molecular weight 20,000), endocrine gland-derived vascular endothelial growth factor (EGF-VEGF) (molecular weight 9,600), amphiregulin (molecular weight 11,000), basic fibroblast growth factor (b-FGF) (molecular weight 18,000), fibroblast growth factor-4 (FGF-4) (molecular weight 17,000), fibroblast growth factor-7 (FGF-7) (molecular weight 18,000), growth differentiation factor-15 (GDF-15) (molecular weight 17,000), osteoprotegerin (molecular weight 60,000), platelet-derived growth factor (PDGF) (molecular weight 35,000-45,000), epidermal growth factor (EGF) (molecular weight 6,200), heparin-binding epidermal growth factor (HB-EGF) (molecular weight 9,700), neurotrophin-3 (NT-3) (molecular weight 13,600 / 27,200), neurotrophin-4 (NT-4) (molecular weight 28,000), beta nerve growth factor (β-NGF) (molecular weight 13,500), brain-derived nerve growth factor (BDNF) (molecular weight 13,600), glial cell line-derived neurotrophic factor (GDNF) (molecular weight 30,400), stem cell factor (SCF) (molecular weight 35,000-45,000), placental growth factor (PIGF) (molecular weight 50,000), growth hormone (GH) (molecular weight 22,000), insulin-like growth factor-1 (IGF-1) (molecular weight 7,600), granulocyte-macrophage colony-stimulating factor (GM-CSF) (molecular weight 15,000-36,000), Noggin (molecular weight 65,000), and / or neurogenin 2 (molecular weight 55,000). As shown in FIG. 1, the decellularized support composition of the present invention contains the above-mentioned proteins with a molecular weight of 5,000 to 80,000. It is presumed that the inclusion of the above-mentioned protein enables the decellularized support composition of the present invention to be used for a variety of purposes, although this is not limited thereto.

[0026] Among the above proteins, beta-nerve growth factor (β-NGF) (molecular weight 13,500), brain-derived nerve growth factor (BDNF) (molecular weight 13,600), glial cell line-derived neurotrophic factor (GDNF) (molecular weight 30,400), neurotrophin-3 (NT-3) (molecular weight 13,600 / 27,200), neurotrophin-4 (NT-4) (molecular weight 28,000), basic fibroblast growth factor (b-FGF) (molecular weight 18,000), fibroblast growth factor-4 (FGF-4) (molecular weight 17,000), fibroblast growth factor-7 (FGF-7) (molecular weight 18,000), Noggin (molecular weight 65,000), or neurogenin 2 (molecular weight 55,000) are presumed to be particularly useful for neural differentiation.

[0027] Among the above proteins, transforming growth factor-β1 (TGF-β1) (molecular weight 25,000), transforming growth factor-β2 (TGF-β2) (molecular weight 25,000), transforming growth factor-β3 (TGF-β3) (molecular weight 25,000), and transforming growth factor-α (TGF-α) (molecular weight 5,500) are presumed to be particularly useful for muscle cell differentiation.

[0028] Among the above proteins, vascular endothelial growth factor (VEGF) (molecular weight 16,000-45,000), vascular endothelial growth factor-D (VEGF-D) (molecular weight 20,000), endocrine gland-derived vascular endothelial growth factor (EG-VEGF) (molecular weight 9,600), basic fibroblast growth factor (b-FGF) (molecular weight 18,000), fibroblast growth factor-4 (FGF-4) (molecular weight 17,000), fibroblast growth factor-7 (FGF-7) (molecular weight 18,000), and fibroblast growth factor-8 (FGF-8) (molecular weight 17,000) are particularly well known. FGF-7 (molecular weight 18,000), platelet-derived growth factor (PDGF) (molecular weight 35,000-45,000), transforming growth factor-β1 (TGF-β1) (molecular weight 25,000), transforming growth factor-β2 (TGF-β2) (molecular weight 25,000), or transforming growth factor-α (TGF-α) (molecular weight 5,500) are presumed to be particularly useful for angiogenesis.

[0029] <<Ribosomes>> The decellularized support composition of the present invention preferably contains, but is not limited to, ribosomes. Ribosomes are contained in cells and perform biological protein synthesis (mRNA translation). In ribosomes, polypeptide chains are formed from mRNA. Ribosomes consist of two major components: the small ribosomal subunit (30S) and the large ribosomal subunit (50S). Each subunit is composed of one or more ribosomal RNA (rRNA) molecules and numerous ribosomal proteins. The decellularized support composition of the present invention may be used for a variety of purposes, but is presumed to be particularly useful for dedifferentiation, by including, but not limited to, ribosomes.

[0030] <Extracellular Vesicles> The decellularized support composition of the present invention preferably contains, but is not limited to, extracellular vesicles. Examples of extracellular vesicles include exosomes, microvesicles, apoptotic vesicles, matrix vesicles, and matrix-bound vesicles. Exosomes are membrane-bound extracellular vesicles (EVs) formed in the endosomal compartment of most eukaryotic cells. They contain proteins, lipids, and nucleic acids and have a size of 30 to 150 nm. Repeated budding into the inward direction of the endosomal membrane results in the formation of multivesicular bodies. Multivesicular bodies fuse with the plasma membrane, releasing the exosomes contained therein into the extracellular fluid. Molecules can be transported from one cell to another via membrane vesicle transport, potentially affecting the immune system, such as dendritic cells and B cells, and potentially influencing adaptive immune responses to pathogens and tumors. Microvesicles are derived from the plasma membrane and are particles with a diameter of 100 nm to 1 μm that are secreted by direct budding from the plasma membrane after a portion of the EV is constricted. These include cytoskeletal proteins, heat shock proteins, integrins, and proteins involved in the cell fusion machinery. They arise through the transport of molecular cargo to the plasma membrane, where the contractile machinery induces budding and ultimately pinch-off of these vesicles. Furthermore, the proteins found on the surface of microvesicles resemble those of the parent cell. Apoptotic bodies are particles approximately 50 nm to 5 μm in size derived from dying (apoptotic) cells and formed late in apoptosis. They contain nuclei, mitochondria, endoplasmic reticulum, Golgi apparatus, large modified proteins, entire organelles, or chromatin fragments. They are biosynthesized by the release of plasma membrane fragments as cells shrink and hydrostatic forces separate lipid membrane fragments from the cell cytoskeleton. Cells are rapidly fragmented by apoptotic bodies and phagocytosed by macrophages. They can also contain much larger cargo than other extracellular vesicles. Matrix vesicles are 100-300 nm vesicles derived from chondrocytes and osteoblasts. orphan phosphatase, sphingomyelin phosphodiesterase 3, and the like.Like myovesicles, they are thought to arise through the transport of molecular cargo to the plasma membrane, where a contractile mechanism induces sprouting and ultimately pinch-off of these vesicles. They also support bone formation in both endochondral and membranous ossification. Matrix-bound vesicles are 100-300 nm vesicles derived from tissue-resident cells. They contain proteins, miRNA, or bioactive lipids. Matrix-bound vesicles are secreted by tissue-resident cells and incorporated into the solid fibrous structure of the ECM. They can be separated from ECM materials by enzymatic digestion. Secreted extracellular vesicles are taken up by surrounding cells or distant cells via the bloodstream, etc., and are involved in intercellular signaling, such as immune cell activation and the transmission of morphogenetic signals during development. It is believed that the decellularized support composition of the present invention may be used for a variety of purposes, including, but not limited to, the inclusion of extracellular vesicles.

[0031] The decellularized support composition of the present invention contains the above-mentioned contained proteins, ribosomes, and / or extracellular vesicles, and therefore can be effectively used for dedifferentiation, neural differentiation, muscle cell differentiation, or angiogenesis.

[0032] Dedifferentiation: Dedifferentiation refers to differentiated cells losing their function or morphology, or returning to an undifferentiated state. In other words, it refers to cells losing the characteristics of the tissue they constitute. Markers of dedifferentiation include pluripotency markers such as NANOG, SOX2, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-54, alkaline phosphatase, and POU5F1 (Oct4).

[0033] Neuronal differentiation refers to the differentiation of cells into neurons, astrocytes, oligodendrocytes, or the like that constitute the nervous system. Examples of markers of neuronal differentiation include GDNF, TUBB3, MAP2, NeuN, Neurofilament-H, Neurofilament-L, Neurofilament-M, Neuron Specific Enolase, Tau, Thy1, UCHL1, GFAP, ALDH1L1, GAP-43, synapsin-1, and TAC1.

[0034] "Muscle cell differentiation" means differentiation into myoblasts, myofibroblasts, myotubes, and further into fast-twitch muscle cells, slow-twitch muscle cells, cardiomyocytes, etc. Examples of muscle cell markers include α-SMA, skeletal muscle troponin-I, myoglobin, FABP, MLC-1, CK-MM, MHC, cardiac troponin-I, cardiac myosin light chain-1, etc.

[0035] "Angiogenesis" refers to the formation of new blood vessels. Examples of angiogenesis markers include PECAM1 (also known as CD31), CD34, ICAM-1 / CD54, Tie-2 / Tek, CD144 (also known as VE-cadherin), VEGF-R2, and von Willeber factor.

[0036] "Maintaining an undifferentiated state" means that cells having pluripotency to differentiate into various cells can divide while remaining in an undifferentiated state. As an indicator of the maintenance of an undifferentiated state, it is sufficient to confirm that undifferentiation markers such as CD73, CD90, CD105, NANOG, SOX2, POU5F1 (Oct4), or Oct3 do not decrease or increase.

[0037] Chondrocyte differentiation refers to the differentiation of cells into chondrocytes. Examples of chondrocyte markers include SOX9, COL2A1, COL11A2, and Aggrecan.

[0038] [2] Extracellular matrix composition The extracellular matrix (ECM) composition of the present invention contains at least one selected from a protein having a molecular weight of 5,000 to 80,000, a ribosome, and an extracellular vesicle, and is used for dedifferentiation or differentiation of cells. The "extracellular matrix," "protein having a molecular weight of 5,000 to 80,000," "ribosome," and "extracellular vesicle" in the extracellular matrix composition of the present invention are the same as those described in the section "[1] Decellularized support composition" above.

[0039] The extracellular matrix composition of the present invention also contains an extracellular matrix component. The extracellular matrix component is an assembly of extracellular matrix molecules formed by a plurality of extracellular matrix molecules. Examples of extracellular matrix molecules include collagen, laminin, fibronectin, chondroitin sulfate, heparin sulfate, keratan sulfate, hyaluronic acid, vitronectin, elastin, tenascin, entactin, fibrillin, and proteoglycan, with collagen being preferred. These extracellular matrix components may be used alone or in combination.

[0040] Examples of collagen include fibrous collagen and non-fibrous collagen. Fibrous collagen refers to collagen that is the main component of collagen fibers, and specific examples include type I collagen, type II collagen, and type III collagen. Examples of non-fibrous collagen include type IV collagen.

[0041] Examples of proteoglycans include chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, keratan sulfate proteoglycans, and dermatan sulfate proteoglycans.

[0042] The extracellular matrix component may be derived from an animal. The animal species from which the extracellular matrix component is derived may be mammals, birds, reptiles, fish, etc., and is preferably a mammal from the viewpoint of achieving the effects of the present invention. Examples of animal species from which the extracellular matrix component is derived include humans, monkeys, dogs, cats, rabbits, ferrets, sheep, goats, cattle, pigs, horses, camels, mice, rats, hamsters, guinea pigs, and gerbils. However, from the viewpoint of achieving the effects of the present invention, humans, rabbits, cattle, pigs, mice, or rats are preferred. The extracellular matrix component may be derived from a single animal, or may be derived from multiple animal species in combination. The animal species from which the extracellular matrix component is derived may be the same as or different from the origin of the cells to be dedifferentiated or differentiated.

[0043] The extracellular matrix composition of the present invention can be obtained by adding at least one selected from the group consisting of proteins having a molecular weight of 5,000 to 80,000, ribosomes, and extracellular vesicles from the outside to a plurality of extracellular matrix molecules.

[0044] An example of an extracellular matrix composition is a decellularized support composition.

[0045] Because the extracellular matrix composition of the present invention contains the above-mentioned contained proteins, ribosomes, and / or extracellular vesicles, it can be effectively used for dedifferentiation, maintenance of undifferentiation, neural differentiation, muscle cell differentiation, angiogenesis, or chondrocyte differentiation.

[0046] <<Cell Culture Substrate>> The cell culture substrate of the present invention comprises the decellularized support composition or the extracellular matrix composition. That is, the decellularized support composition or the extracellular matrix composition of the present invention can be used as a substrate for cell culture. As a method for culturing cells to be used on the cell culture substrate, known methods can be used, except that the decellularized support composition of the present invention or the extracellular matrix composition is used. For example, a predetermined amount of medium or culture reagent is placed in a cell culture plate or cell culture well, the decellularized support composition of the present invention or the extracellular matrix composition is added, cells are seeded, and the culture is incubated at a predetermined temperature and CO ２ The cells can be cultured under the concentration conditions. The medium or culture reagent may be selected appropriately depending on the cells to be cultured. The cells to be cultured are not particularly limited, and examples thereof include the same cells as those used in the "decellularized support composition."

[0047] <<Graft>> The graft of the present invention comprises the decellularized support composition or the extracellular matrix composition. That is, the decellularized support composition or the extracellular matrix composition of the present invention can be used as a graft for a living organism. The decellularized support composition or the extracellular matrix composition of the present invention has excellent biocompatibility, and when transplanted into a living organism, it can serve as, for example, a suitable scaffold for cell growth. Examples of living organisms into which the decellularized support composition or the extracellular matrix composition of the present invention can be transplanted include humans, monkeys, dogs, cats, rabbits, ferrets, sheep, goats, cows, pigs, horses, camels, mice, rats, hamsters, guinea pigs, chickens, and gerbils. Diseases into which the graft of the present invention can be transplanted include cytopenic diseases (cell loss diseases). Examples of cells that decrease (are lost) in vivo include hepatocytes, stellate cells, Kupffer cells, vascular endothelial cells, meatal endothelial cells, endothelial cells, osteoclasts, periodontal ligament-derived cells, epidermal cells, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells, mammary gland cells, pericytes, smooth muscle cells, cardiac muscle cells, muscle cells, kidney cells, pancreatic islet cells, peripheral nerve cells, neurons, chondrocytes, and bone cells. Examples of differentiated adhesive cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, hematopoietic stem cells, neural stem cells, cardiac progenitor cells, vascular endothelial progenitor cells, neural progenitor cells, preadipocytes, fibroblasts, skin fibroblasts, skeletal muscle myoblasts, osteoblasts, and odontoblasts. Transplantation of the graft of the present invention can effectively increase the number of cells in diseases that cause a decrease in these cells. The cells may be transplanted into a living body simultaneously with the transplantation of the graft of the present invention. Furthermore, the graft of the present invention can be transplanted into an individual in which the cells have not been reduced in vivo. By administering the graft, various cells can be proliferated.

[0048] The cell composition of the present invention comprises the decellularized support composition or the extracellular matrix composition and cells. Examples of the cells include hepatocytes, stellate cells, Kupffer cells, vascular endothelial cells, meatal endothelial cells, endothelial cells, osteoclasts, periodontal ligament-derived cells, epidermal cells, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells, mammary gland cells, pericytes, smooth muscle cells, cardiac muscle cells, muscle cells, kidney cells, pancreatic islet cells, peripheral nerve cells, neurons, chondrocytes, and bone cells. Examples of differentiation-competent adhesive cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, hematopoietic stem cells, neural stem cells, cardiac progenitor cells, vascular endothelial progenitor cells, neural progenitor cells, preadipocytes, fibroblasts, skin fibroblasts, skeletal muscle myoblasts, osteoblasts, and odontoblasts. Examples of cells include SH-SY5Y and SK-N-SH derived from human neuroblastoma, Neuro 2a and N1E-115 derived from mouse neuroblastoma, NTERA-2 derived from human pluripotent embryonal carcinoma, and PC12 derived from rat adrenal pheochromocytoma. From the viewpoint of exerting the effects of the present invention, fibroblasts, mesenchymal stem cells, cardiac precursor cells, and rat adrenal pheochromocytoma are preferred, and skin fibroblasts, mesenchymal stem cells, and rat adrenal pheochromocytoma are more preferred.

[0049] The ratio of cells to the decellularized support composition or the extracellular matrix composition is not particularly limited, as long as it is equal to or less than the number of cells that can be physically contained in the decellularized support composition or the extracellular matrix composition. For example, ３ ~1.0 x 10 ７ In one embodiment, the lower limit of the number of cells is 2.0 x 10 ３ or more, and in one embodiment, 5.0 × 10 ３ or more, and in one embodiment, 1.0 × 10 ４ or more, and in one embodiment, 2.0 × 10 ４or more, and in one embodiment, 5.0 × 10 ４ In one embodiment, the upper limit of the number of cells is 5.0 × 10 ６ is less than or equal to 1.0 × 10 ６ is less than or equal to 5.0 × 10 ５ or less, and in one embodiment, 2.0×10 ５ or less, and in one embodiment, 1.0×10 ５ The lower limit and upper limit can be arbitrarily combined to set a range of the ratio of cells to the decellularized support composition or the extracellular matrix composition. By keeping the ratio within the range, the number of viable cells can be increased.

[0050] The cell composition of the present invention is not particularly limited as long as it contains a decellularized support composition or the extracellular matrix composition and cells. It can be used for in vitro cell culture. It can also be administered to a living body and used for autologous or allogeneic transplantation in regenerative medicine or cell therapy, for example.

[0051] [3] Method for producing a decellularized support composition The method for producing a decellularized support composition of the present invention includes a method for producing a decellularized cell aggregate composition and a method for producing a decellularized cell structure composition.

[0052] <<Method for Producing Decellularized Cell Aggregate Composition>> The method for producing a decellularized cell aggregate composition of the present invention includes a cultured cell aggregate formation step of culturing cells to form cell aggregates, and a step of decellularizing the cultured cell aggregates to form surface pores having a pore diameter of 5 μm or more on the aggregate surface. ２ The decellularized cell aggregate composition produced by the production method of the present invention is used for dedifferentiation of cells, maintenance of undifferentiation, neural differentiation, muscle cell differentiation, angiogenesis, or chondrocyte differentiation.

[0053] (Cultured Cell Aggregate Formation Step (1)) In the cultured cell aggregate formation step (1), cells are cultured to form cultured cell aggregates. The cultured cell aggregates are not particularly limited as long as they are cell aggregates obtained by in vitro culture, and examples include the "cultured cell aggregates" described in the section "[1] Decellularized Support Composition" above. Cultured cell aggregates can be formed when cells produce extracellular matrix (ECM) outside the cells, causing cells to adhere and bond to each other. In the case of spheroids, which are one type of cultured cell aggregate, three-dimensional culture methods include, for example, the non-adhesive surface culture method, the hanging drop culture method, and the rotary culture method. The formation of spheroids in the cultured cell aggregate formation step (1) is specifically described below.

[0054] Cultured cell aggregates (e.g., spheroids) can be produced as follows. Adherent cells cannot survive for long periods of time if they are suspended in a solution. When adherent cells are placed in a non-adhesive environment, the cells seek a foothold and adhere to each other, forming cell aggregates. Specifically, cultured cell aggregates (e.g., spheroids) can be produced by aggregating multiple cells. After monolayer culture, cells are transferred to a water-repellent or non-cell-adhesive round-bottom multiwell or U-shaped plate (dimple plate) and incubated, resulting in cell aggregation and formation of cultured cell aggregates. From the standpoint of efficiency, the incubation time required to form cultured cell aggregates is preferably 6 to 48 hours, more preferably 6 to 24 hours. However, methods for producing cultured cell aggregates are not limited to the above methods. Other methods that can be used include a rotational culture method in which a cell suspension is placed in a rotating solution, a method in which a cell suspension is placed in a test tube and precipitated using a centrifuge, and an alginate bead method. From the viewpoint of processing a large amount of uniform cultured cell aggregates, a method of placing a cell suspension in a water-repellent or cell-nonadhesive multi-well is efficient and preferable. By adjusting the incubation time of the cultured cell aggregates (e.g., spheroids), cultured cell aggregates of any thickness can be cultured. Furthermore, by connecting cultured cell aggregates (e.g., spheroids) to each other, cultured cell aggregates of any thickness and shape can be cultured.

[0055] (Decellularization step (2)) In the decellularization step (2), the cultured cell aggregates are decellularized. The decellularization treatment is not particularly limited as long as the effects of the present invention can be obtained, and examples include high hydrostatic pressure treatment, freeze-thaw treatment, a method using a surfactant, ultrasonic treatment, a method using an enzyme, or a method of treatment with a hypertonic electrolyte solution, a method using physical stirring, a hypertonic solution-hypotonic solution method, an enzyme treatment method using protease or nuclease, or the like, and treatment with an alcohol solvent; two or more of these may be combined; and a method using high hydrostatic pressure treatment is preferred in order to efficiently obtain decellularized cell aggregates and to exert the effects of the present invention.

[0056] (High Hydrostatic Pressure Treatment) When the decellularized cell aggregates are obtained by the high hydrostatic pressure treatment method, a hydrostatic pressure of 50 to 1500 MPa is applied to the obtained cultured cell aggregates in a medium. From the viewpoint of sufficient decellularization, the applied hydrostatic pressure is preferably 50 MPa or more. In order to avoid the need for a pressure vessel that can withstand the pressure, to avoid the need for significant energy, and to prevent damage to the cultured cell aggregates caused by ice generated when the medium used for application is an aqueous medium, a hydrostatic pressure of 1500 MPa or less is preferred. The applied hydrostatic pressure is more preferably 80 to 1300 MPa, more preferably 90 to 1200 MPa, even more preferably 95 to 1100 MPa, even more preferably 95 to 700 MPa, and most preferably 400 to 700 MPa, from the viewpoint of exerting decellularization, sterilization, and viral inactivation effects and being easy to apply.

[0057] Media used for applying hydrostatic pressure include media in which serum has been added to a basal medium, serum-free media, water, physiological saline, water for injection, propylene glycol or an aqueous solution thereof, glycerin or an aqueous solution thereof, and aqueous sugar solutions. Buffer solutions include acetate buffer, phosphate buffer, citrate buffer, borate buffer, tartrate buffer, Tris buffer, HEPES buffer, and MES buffer. These media may contain a surfactant.

[0058] The temperature for the high hydrostatic pressure treatment is not particularly limited as long as it does not produce ice and does not cause thermal damage to the cultured cell aggregates, but since decellularization is carried out smoothly and has little effect on the cultured cell aggregates, it is preferably 0 to 45° C., more preferably 4 to 37° C., and most preferably 15 to 36° C. If the high hydrostatic pressure treatment time is too short, cells will not be sufficiently destroyed, and if it is too long, it will result in wasted energy, so the time for which the desired applied pressure is maintained in the high hydrostatic pressure treatment is preferably 1 to 120 minutes, more preferably 5 to 60 minutes, and even more preferably 7 to 30 minutes.

[0059] The cultured cell aggregates treated with high hydrostatic pressure are preferably treated with a nuclease. The nuclease removes nucleic acid components from the cultured cell aggregates to which hydrostatic pressure has been applied, and is not particularly limited, and examples thereof include pancreatic-, spleen-, or Escherichia coli-derived DNases (e.g., DNase I and DNase II). The nuclease can be added to the medium used in the high hydrostatic pressure treatment (e.g., water, physiological saline, injection solution, buffer solution, etc.) and allowed to act. The amount of enzyme added varies depending on the type of enzyme and the definition of the number of units (U), but can be appropriately determined by those skilled in the art. For example, DNase I may be used at 50 to 2000 U / mL. The treatment temperature also varies depending on the nuclease used, but may be set to, for example, 1 to 40°C. The treatment time is also not particularly limited, and may be, for example, 1 to 120 hours (preferably 1 to 96 hours, more preferably 1 to 72 hours). Longer treatment times are required at lower temperatures, and shorter treatment times are required at higher temperatures.

[0060] The cultured cell aggregates treated with high hydrostatic pressure are washed with a washing solution. The washing solution may be the same as or different from the medium used in the high hydrostatic pressure treatment. The washing solution may contain an organic solvent or a chelating agent. The organic solvent can improve the efficiency of lipid removal, and the chelating agent can inactivate calcium ions and magnesium ions in the decellularized cell aggregates, thereby preventing calcification when the decellularized cell aggregates of the present invention are applied to a diseased area. As the organic solvent, a water-soluble organic solvent is preferred because of its high lipid removal effect, and ethanol, isopropanol, acetone, and dimethyl sulfoxide are preferred. Examples of the chelating agent include iminocarboxylic acid chelating agents such as ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraminehexaacetic acid (TTHA), 1,3-propanediaminetetraacetic acid (PDTA), 1,3-diamino-2-hydroxypropanetetraacetic acid (DPTA-OH), hydroxyethyliminodiacetic acid (HIDA), dihydroxyethylglycine (DHEG), glycol ether diaminetetraacetic acid (GEDTA), dicarboxymethylglutamic acid (CMGA), 3-hydroxy-2,2'-iminodisuccinic acid (HIDA), and dicarboxymethylaspartic acid (ASDA), or salts thereof; and hydroxycarboxylic acid chelating agents such as citric acid, tartaric acid, malic acid, and lactic acid, or salts thereof. Examples of the salts of these chelating agents include sodium salts and potassium salts. The washing temperature is not particularly limited as long as it does not cause thermal damage to the cultured cell aggregates, but is preferably 0 to 45°C, more preferably 1 to 40°C, and most preferably 2 to 35°C, because it provides good washability and has little effect on the cultured cell aggregates. When washing, the washing solution may be shaken or stirred as needed. The cultured cell aggregates obtained in step (1) are decellularized by high hydrostatic pressure treatment under the above-mentioned conditions, thereby forming surface pores with a pore diameter of 5 μm or more on the aggregate surface. ２ Decellularized cell aggregates having 5 to 45 cells per cell can be obtained.

[0061] (Surface Pores) The decellularized cell aggregate composition obtained by the production method of the present invention has surface pores with a pore diameter of 5 μm or more on the aggregate surface. ２ The decellularized cell aggregate composition has 5 to 45 pores per pore. As used herein, surface pores refer to pores present on the surface of the decellularized cell aggregate composition when the decellularized cell aggregate composition is observed with a scanning electron microscope (SEM). Furthermore, the surface refers to the surface that can be observed with the SEM. By having a certain number of surface pores, the composition exhibits excellent effects as a scaffold for cell culture. The diameter of the surface pores to be counted is not particularly limited as long as it is 5 μm or more, but the lower limit is preferably 5 μm or more and the upper limit is preferably 50 μm or less. The diameter of the surface pores can be measured mechanically or visually from the SEM photograph. Aggregate surface 11088 μm ２ The number of surface pores per aggregate is not particularly limited as long as it is between 5 and 45, but the lower limit is preferably 5 or more, in some embodiments 6 or more, in some embodiments 7 or more, in some embodiments 8 or more, in some embodiments 9 or more, in some embodiments 10 or more, in some embodiments 12 or more, and in some embodiments 15 or more. The upper limit of the number of surface pores is preferably 45 or less, in some embodiments 43 or less, in some embodiments 41 or less, in some embodiments 39 or less, in some embodiments 37 or less, in some embodiments 35 or less, in some embodiments 33 or less, in some embodiments 31 or less, and in some embodiments 30 or less. The upper and lower limits can be combined to form a range for the number of surface pores. By keeping the number within this range, the number of viable cells can be increased. The aggregate surface area for measuring surface pores is 11088 μm ２The determination of the surface pore density can be performed by, but is not limited to, measuring the number of surface pores in an area of ​​88 μm x 126 μm on an SEM photograph. The number of surface pores increases with increasing the intensity of the high hydrostatic pressure treatment (e.g., hydrostatic pressure, time, temperature), and decreases with decreasing the intensity. The number of surface pores also increases with increasing the number of freeze-thaw cycles, and decreases with decreasing the number of cycles. Furthermore, the number of surface pores increases with increasing the intensity of the ultrasonic treatment (e.g., intensity, frequency, time), and decreases with decreasing the intensity. Therefore, from the description herein, one skilled in the art can obtain decellularized cell aggregates with various numbers of surface pores by adjusting the intensity of the decellularization treatment.

[0062] The obtained decellularized cell aggregate composition may be subjected to, but is not limited to, a freeze-drying process. The freeze-drying process may be omitted. The obtained decellularized cell aggregate composition may also be sterilized by gamma irradiation, UV irradiation, or the like.

[0063] The size of the decellularized cell aggregate composition is not particularly limited, but when observed with a scanning electron microscope (SEM), the shortest length is preferably 80 μm or more, more preferably 90 μm or more, more preferably 95 μm or more, and more preferably 100 μm or more. The upper limit is preferably 600 μm or less, more preferably 500 μm or less, and more preferably 300 μm or less. The upper and lower limits can be combined to form a size range for the decellularized cell aggregate composition. The effects of the present invention can be achieved by being within the above range.

[0064] <<Method for producing a decellularized cell structure composition>> The method for producing a decellularized cell structure composition of the present invention comprises a cell structure production step of producing a cell structure by adhering cells, and a decellularization step of decellularizing the cell structure to a porosity of 40% to 70%. The decellularized cell structure composition produced by the production method of the present invention is for cell dedifferentiation, maintenance of undifferentiation, neural differentiation, muscle cell differentiation, angiogenesis, or chondrocyte differentiation.

[0065] <<Cultured Cell Structure Formation Step (1)>> In the cultured cell structure formation step (1), cells are cultured to form a cultured cell structure. The cells used are not particularly limited as long as they are culturable in vitro. For example, they may be primary cells collected directly from tissues or organs, or passaged cells obtained by passage of primary cells collected directly from tissues or organs for several generations. They may also be passaged cells that have been passaged for a long period of time. Specifically, any of the cells described in the above “[1] Decellularized Support Composition” can be used without limitation. The cultured cell structure can be formed by cells secreting extracellular matrix (ECM) outside the cells, adhering and bonding the cells together, and maintains a three-dimensional shape. The method for forming the cultured cell structure is not particularly limited as long as it utilizes adhesion via the extracellular matrix, and examples include the net mold method, cell sheet method, bioprinting method, and pin-mountain method. All of these methods for producing structures utilize the extracellular matrix secreted by cells to adhere cells together and form a cultured cell structure. Examples of extracellular matrices include those described in the above section "[1] Decellularized support composition" or "Cell composition," but are not particularly limited thereto.

[0066] (Net Mold Method) In the net mold method, a cultured cell structure can be formed, for example, by culturing cultured cell aggregates (spheroids) as follows. A suitable culture medium can be selected depending on the cells used. The cultured cell aggregates are placed in a cavity surrounded by a mesh-like or comb-like support with gaps smaller than the size of the cultured cell aggregates. The cavity is formed into a desired shape by a mesh-like or comb-like support and a plate, and the support has numerous openings. The medium can contact the cultured cell aggregates through these openings. By shaking the shaking culture vessel while the cultured cell aggregates are held in the cavity, the medium can effectively contact the cultured cell aggregates, allowing the cultured cell aggregates to be cultured well. The cultured cell aggregates produce an extracellular matrix, and the cultured cell aggregates begin to adhere to each other, allowing a cultured cell structure with a desired three-dimensional structure to be produced. In this way, by further culturing the cultured cell aggregates (e.g., spheroids), the cells further secrete extracellular matrix, allowing the cells to adhere to each other and forming a structural cultured cell structure that maintains a three-dimensional shape. Furthermore, by culturing the cultured cell aggregates (e.g., spheroids) by introducing them into a cavity as described above, multiple spheroids bond together, allowing the accurate production of a cultured cell structure (e.g., a cell block) with the desired three-dimensional shape.

[0067] (Cell Sheet Method) In the cell sheet method, a cultured cell structure is formed by stacking multiple cell sheets (sheet-like cells) and culturing the stacked cell sheets. The cell sheet culture can be performed according to the cell sheet production method typically performed in this field using a culture plate or the like. By stacking and culturing the obtained cell sheets, cells can be cultured to any thickness and shape. After this, the cells secrete extracellular matrix, which causes the stacked cell sheets to adhere to each other, resulting in a cultured cell structure that maintains its three-dimensional shape.

[0068] (Bioprinting Method) The bioprinting method is a method in which cells are arranged three-dimensionally one by one together with a small amount of gel using a 3D printer (Non-Patent Document 1).

[0069] (Sword-holder method) The sword-holder method is a method in which a needle shaped like a sword is inserted into spheroids, and after organization, the needle is removed (Non-Patent Document 1).

[0070] Decellularization Step (2) In the decellularization step (2), the cultured cell structure is decellularized to a porosity of 40% to 70%. That is, the porosity of the cultured cell structure after decellularization is 40% to 70%. The lower limit of the porosity is preferably 42%, more preferably 44%, even more preferably 47%, and most preferably 50%. The upper limit of the porosity is preferably 68%, more preferably 66%, even more preferably 64%, and most preferably 55%. By achieving a porosity within this range, a graft with sufficient strength and no rejection reaction can be obtained. Furthermore, a decellularized graft with any thickness or specific shape can be provided. Herein, porosity is calculated as follows: A section of the decellularized cell structure is stained with Sirius Red, which stains the extracellular matrix. Microscopic tissue images are analyzed using image processing software, and areas not stained with Sirius Red are determined to be voids, and the porosity is calculated. Sirius Red staining was performed as follows. Section samples of the decellularized cell structure are deparaffinized. Next, the section samples are treated with iron hematoxylin. Next, they are treated with a hydrochloric acid / ethanol solution and a phosphomolybdic acid aqueous solution. Subsequently, they are treated with Sirius Red (Direct Red 80), dehydrated, and mounted. The obtained Sirius Red-stained sections are observed under a microscope, and the porosity is calculated using image processing software (ImageJ). The sections are photographed at 10x magnification, and the area of ​​the entire decellularized cell structure and the area of ​​the Sirius Red-stained portion are measured. The porosity is calculated using the following formula: Porosity (%) = (area of ​​the entire decellularized cell structure - area of ​​the Sirius Red-stained portion) / area of ​​the entire decellularized cell structure x 100

[0071] The decellularization step can be performed using conventionally known methods. The decellularization method is not particularly limited as long as it achieves the effects of the present invention, and examples include high hydrostatic pressure treatment, freeze-thaw treatment, surfactant treatment, ultrasonic treatment, enzyme treatment, treatment with a hypertonic electrolyte solution, physical stirring, hypertonic / hypotonic solution treatment, enzyme treatment with protease, nuclease, or the like, and treatment with an alcohol solvent. Two or more of these may be combined, and high hydrostatic pressure treatment is preferred in order to efficiently obtain a decellularized cell structure and to achieve the effects of the present invention.

[0072] (High Hydrostatic Pressure Treatment) When the decellularized structure is obtained by the high hydrostatic pressure treatment method, a hydrostatic pressure of 50 to 1500 MPa is applied to the obtained cell structure in a medium. From the viewpoint of sufficient decellularization, the applied hydrostatic pressure is preferably 50 MPa or more. It does not require a pressure vessel that can withstand the pressure, does not require a large amount of energy, and, if the medium used for application is an aqueous medium, ice is generated. From the viewpoint of preventing damage to the cultured cell structure by the generated ice, a hydrostatic pressure of 1500 MPa or less is preferred. The applied hydrostatic pressure is more preferably 80 to 1300 MPa, more preferably 90 to 1200 MPa, even more preferably 95 to 1100 MPa, even more preferably 95 to 700 MPa, and most preferably 400 to 700 MPa, from the viewpoint of exerting decellularization effects, sterilization effects, and viral inactivation effects, and from the viewpoint of ease of application.

[0073] Media used for applying hydrostatic pressure include media in which serum has been added to a basal medium, serum-free media, water, physiological saline, water for injection, propylene glycol or an aqueous solution thereof, glycerin or an aqueous solution thereof, and aqueous sugar solutions. Buffer solutions include acetate buffer, phosphate buffer, citrate buffer, borate buffer, tartrate buffer, Tris buffer, HEPES buffer, and MES buffer. These media may contain a surfactant.

[0074] The temperature for the high hydrostatic pressure treatment is not particularly limited as long as it does not produce ice and does not cause thermal damage to the cell structure, but since decellularization is carried out smoothly and has little effect on the cultured cell structure, it is preferably 0 to 45° C., more preferably 4 to 37° C., and most preferably 15 to 36° C. If the high hydrostatic pressure treatment time is too short, cells will not be sufficiently destroyed, and if it is too long, it will result in a waste of energy, so the time for which the intended applied pressure is maintained in the high hydrostatic pressure treatment is preferably 1 to 120 minutes, more preferably 5 to 60 minutes, and even more preferably 7 to 30 minutes.

[0075] The cultured cell structure treated with high hydrostatic pressure is preferably subjected to a nuclease treatment. The nuclease removes nucleic acid components from the cultured cell structure to which hydrostatic pressure has been applied, and is not particularly limited, and examples thereof include pancreatic-, spleen-, or Escherichia coli-derived DNases (e.g., DNase I and DNase II). The nuclease can be added to the medium used in the high hydrostatic pressure treatment (e.g., water, physiological saline, injection solution, buffer solution, etc.) and allowed to act. The amount of enzyme to be added varies depending on the type of enzyme and the definition of the number of units (U), but can be appropriately determined by those skilled in the art. For example, DNase I may be used at 50 to 2000 U / mL. The treatment temperature also varies depending on the nuclease used, but may be set to, for example, 1 to 40°C. The treatment time is also not particularly limited, and may be, for example, 1 to 120 hours (preferably 1 to 96 hours, more preferably 1 to 48 hours). Longer treatment times are required at lower temperatures, and shorter treatment times are required at higher temperatures.

[0076] The cultured cell structure treated with high hydrostatic pressure is washed with a washing solution. The washing solution may be the same as or different from the medium used in the high hydrostatic pressure treatment. The washing solution may contain an organic solvent or a chelating agent. The organic solvent can improve the efficiency of lipid removal, and the chelating agent can inactivate calcium ions and magnesium ions in the decellularized cell structure, thereby preventing calcification when the decellularized cell structure of the present invention is applied to a diseased area. As the organic solvent, a water-soluble organic solvent is preferred because of its high lipid removal effect, and ethanol, isopropanol, acetone, and dimethyl sulfoxide are preferred. Examples of the chelating agent include iminocarboxylic acid chelating agents such as ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraminehexaacetic acid (TTHA), 1,3-propanediaminetetraacetic acid (PDTA), 1,3-diamino-2-hydroxypropanetetraacetic acid (DPTA-OH), hydroxyethyliminodiacetic acid (HIDA), dihydroxyethylglycine (DHEG), glycol ether diaminetetraacetic acid (GEDTA), dicarboxymethylglutamic acid (CMGA), 3-hydroxy-2,2'-iminodisuccinic acid (HIDA), and dicarboxymethylaspartic acid (ASDA), or salts thereof; and hydroxycarboxylic acid chelating agents such as citric acid, tartaric acid, malic acid, and lactic acid, or salts thereof. Examples of the salts of these chelating agents include sodium salts and potassium salts. The washing temperature is not particularly limited as long as it does not cause thermal damage to the cultured cell structure, but in order to provide good washing properties and little effect on the cultured cell structure, it is preferably 0 to 45°C, more preferably 1 to 40°C, and most preferably 2 to 35°C. When washing, the washing solution may be shaken or stirred as necessary.

[0077] The obtained decellularized cell structure composition may be subjected to, but is not limited to, a freeze-drying process. The freeze-drying process may be omitted. The obtained decellularized cell structure composition may also be sterilized by gamma irradiation, UV irradiation, or the like.

[0078] The size of the decellularized cell structure composition is not particularly limited, but when observed with a scanning electron microscope (SEM), the shortest length is preferably 90 μm or more, more preferably 100 μm or more, more preferably 150 μm or more, and more preferably 200 μm or more. The upper limit is preferably 2100 μm or less, more preferably 2000 μm or less, more preferably 1700 μm or less, more preferably 1400 μm or less, more preferably 1200 μm or less, and more preferably 1000 μm or less. The upper and lower limits can be combined to form a size range for the decellularized cell structure composition. The effects of the present invention can be achieved by being within the above range.

[0079] The decellularized support composition of the present invention for dedifferentiation, neural differentiation, muscle cell differentiation, or angiogenesis is preferably, but not limited to, one obtained by the method for producing a decellularized cell aggregate composition or the method for producing a decellularized cell structure composition. In particular, one decellularized by high hydrostatic pressure treatment is preferred. By decellularizing by high hydrostatic pressure treatment, the contained proteins, ribosomes, and / or extracellular vesicles are contained, and the composition can be effectively used for dedifferentiation, maintenance of undifferentiation, neural differentiation, muscle cell differentiation, angiogenesis, or chondrocyte differentiation.

[0080] <<Method for Producing Decellularized Support Composition Solution (Decellularized Cell Aggregate Composition Solution)>> The method for producing a decellularized support composition solution of the present invention comprises a liquefaction step of liquefying a decellularized support composition using a protease or an acidic solution, a separation step of treating the liquefied decellularized support composition by centrifugation, and a neutralization step of neutralizing the supernatant obtained by the centrifugation to a pH of 7.0 to 8.0. The decellularized support composition solution of the present invention can be produced by treating the decellularized support composition with a protease or the like.

[0081] (Liquefaction step) In the liquefaction step, the decellularized support composition is treated with a protease or an acidic solution. Examples of proteases include, but are not limited to, acidic proteases and metalloproteases. Examples of acidic proteases that can be used include pepsin, trypsin, and chymotrypsin, and examples of metalloproteases include collagenase. Treatment buffers that are commonly used for each protease can be used without any restrictions. In the case of acidic proteases, for example, a 0.01 M HCl solution can be used. The pH of the buffer for acidic proteases is not particularly limited as long as it is acidic, and the reaction can be carried out at, for example, a pH of about 1.0 to 5.0. In the case of collagenase, Ca ２＋ A buffer solution or medium containing the above can be used. The reaction temperature is not particularly limited as long as it is a temperature at which the protease can act, but the reaction can be carried out at, for example, 4 to 37°C. The reaction time is also not particularly limited, but the reaction can be carried out for, for example, 10 minutes to 48 hours. When the reaction temperature is low, the reaction time can be extended, and when the reaction temperature is high, the reaction time can be shortened. Acids used to prepare the acidic solution include, for example, hydrochloric acid, sulfuric acid, nitric acid, etc., and hydrochloric acid is more preferred. The acid concentration can be adjusted as appropriate. The pH of the acidic solution can be, for example, about 1.0 to 5.0, and the decellularized support composition can be reacted with the acidic solution. The temperature and time for liquefying the decellularized support composition using the acidic solution are not particularly limited as long as they are sufficient to liquefy the decellularized support composition.

[0082] (Separation step) In the separation step, the liquefied decellularized support composition is centrifuged. Solid matter can be removed by centrifugation. Centrifugation can be performed by a method commonly used in the field, for example, at 5,000 to 10,000 rpm for 3 to 10 minutes.

[0083] (Neutralization Step) In the pre-neutralization step, the supernatant obtained by centrifugation is neutralized to a pH of 7.0 to 8.0. However, if a reaction is carried out at neutral pH using a metalloprotease in the liquefaction step, the neutralization step can be omitted. When an acidic protease is used, the supernatant is neutralized from a pH of 1.0 to 5.0 to a pH of 7.0 to 8.0 using an alkaline solution. The alkaline solution is not particularly limited, but examples thereof include 0.1 M NaOH, NaHCO ３ The temperature of the neutralization step is not particularly limited, but it can be carried out at, for example, 4 to 37°C.

[0084] <<Cell Dedifferentiation Method>> In the cell dedifferentiation method of the present invention, cell dedifferentiation is induced by culturing cells using the decellularized support composition or extracellular matrix composition of the present invention. The cells used in the cell dedifferentiation method of the present invention are not particularly limited, and include suspension cells and adhesive cells. Differentiated cells and pre-differentiated cells can also be used, and from the perspective of exerting the effects of the present invention, differentiated cells are preferred. Examples of differentiated adhesive cells include hepatocytes, stellate cells, Kupffer cells, vascular endothelial cells, meatus endothelial cells, endothelial cells, osteoclasts, periodontal ligament-derived cells, epidermal cells, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells, mammary gland cells, pericytes, smooth muscle cells, cardiac muscle cells, muscle cells, kidney cells, pancreatic islet cells of Langerhans, peripheral nerve cells, neurons, chondrocytes, and bone cells. Examples of differentiated suspension cells include blood cells such as T cells and B cells. Examples of pre-differentiation adhesive cells include differentiation-competent adhesive cells, i.e., embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, hematopoietic stem cells, neural stem cells, cardiac progenitor cells, vascular endothelial progenitor cells, neural progenitor cells, preadipocytes, fibroblasts, skin fibroblasts, skeletal muscle myoblasts, osteoblasts, odontoblasts, and neural crest cells. From the viewpoint of exerting the effects of the present invention, mesenchymal stem cells, cardiac progenitor cells, and fibroblasts are preferred, and mesenchymal stem cells and skin fibroblasts are more preferred. Examples of pre-differentiation suspension cells include undifferentiated suspension cells, i.e., dedifferentiated fat cells (DFAT).

[0085] <<Method for Neuronal Differentiation of Cells>> In the method for neuronal differentiation of cells of the present invention, cells are differentiated into neurons by culturing them using the decellularized support composition or extracellular matrix composition of the present invention. The cells used in the method for neuronal differentiation of cells are not particularly limited, and suspension cells and adherent cells can be used. Differentiated cells and pre-differentiated cells can also be used. Examples of differentiated adherent cells include hepatocytes, stellate cells, Kupffer cells, vascular endothelial cells, meatus endothelial cells, endothelial cells, osteoclasts, periodontal ligament-derived cells, epidermal cells, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells, mammary gland cells, pericytes, smooth muscle cells, cardiac muscle cells, muscle cells, kidney cells, pancreatic islet cells of Langerhans, peripheral nerve cells, nerve cells, chondrocytes, and bone cells. Examples of pre-differentiation adhesive cells include differentiation-competent adhesive cells, i.e., embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, hematopoietic stem cells, neural stem cells, cardiac progenitor cells, vascular endothelial progenitor cells, neural progenitor cells, preadipocytes, fibroblasts, skin fibroblasts, skeletal myoblasts, osteoblasts, odontoblasts, and neural crest cells. From the viewpoint of exerting the effects of the present invention, mesenchymal stem cells, cardiac progenitor cells, and fibroblasts are preferred, and mesenchymal stem cells and skin fibroblasts are more preferred. Examples of differentiated suspension cells include blood cells such as T cells and B cells. Examples of pre-differentiation suspension cells include undifferentiated suspension cells, i.e., dedifferentiated fat cells (DFAT). Examples of cells include SH-SY5Y and SK-N-SH derived from human neuroblastoma, Neuro 2a and N1E-115 derived from mouse neuroblastoma, NTERA-2 derived from human pluripotent embryonal carcinoma, and PC12 derived from rat adrenal pheochromocytoma, and from the viewpoint of exerting the effects of the present invention, rat adrenal pheochromocytoma is preferred.

[0086] <<Method for Differentiating Cells into Muscle Cells>> In the method for differentiating cells into muscle cells of the present invention, cells are cultured using the decellularized support composition or extracellular matrix composition of the present invention, thereby differentiating them into muscle cells. The cells used in the method for differentiating cells into muscle cells are not particularly limited, and suspension cells and adherent cells can be used. Differentiated cells and pre-differentiated cells can also be used. Examples of differentiated adherent cells include hepatocytes, stellate cells, Kupffer cells, vascular endothelial cells, meatus endothelial cells, endothelial cells, osteoclasts, periodontal ligament-derived cells, epidermal cells, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells, mammary gland cells, pericytes, smooth muscle cells, cardiac muscle cells, muscle cells, kidney cells, pancreatic islet cells of Langerhans, peripheral nerve cells, nerve cells, chondrocytes, and bone cells. Examples of pre-differentiation adhesive cells include differentiation-competent adhesive cells, i.e., embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, hematopoietic stem cells, neural stem cells, cardiac progenitor cells, vascular endothelial progenitor cells, neural progenitor cells, preadipocytes, fibroblasts, skin fibroblasts, skeletal myoblasts, osteoblasts, odontoblasts, and neural crest cells. From the viewpoint of exerting the effects of the present invention, mesenchymal stem cells, cardiac progenitor cells, and fibroblasts are preferred, and mesenchymal stem cells and skin fibroblasts are more preferred. Examples of differentiated suspension cells include blood cells such as T cells and B cells. Examples of pre-differentiation suspension cells include undifferentiated suspension cells, i.e., dedifferentiated fat cells (DFAT).

[0087] <<Angiogenesis Method>> In the angiogenesis method of the present invention, cells are cultured using the decellularized support composition or extracellular matrix composition of the present invention to generate blood vessels. More specifically, the cells are differentiated into vascular cells. The cells used in the angiogenesis method are not particularly limited, and suspension cells and adherent cells can be used. Differentiated cells and pre-differentiated cells can also be used. Examples of differentiated adherent cells include hepatocytes, stellate cells, Kupffer cells, vascular endothelial cells, meatus endothelial cells, endothelial cells, osteoclasts, periodontal ligament-derived cells, epidermal cells, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells, mammary gland cells, pericytes, smooth muscle cells, cardiac muscle cells, muscle cells, kidney cells, pancreatic islet cells of Langerhans, peripheral nerve cells, nerve cells, chondrocytes, and bone cells. Examples of pre-differentiation adhesive cells include differentiation-competent adhesive cells, i.e., embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, hematopoietic stem cells, neural stem cells, cardiac progenitor cells, vascular endothelial progenitor cells, neural progenitor cells, preadipocytes, fibroblasts, skin fibroblasts, skeletal myoblasts, osteoblasts, odontoblasts, and neural crest cells. From the viewpoint of exerting the effects of the present invention, mesenchymal stem cells, cardiac progenitor cells, and fibroblasts are preferred, and mesenchymal stem cells and skin fibroblasts are more preferred. Examples of differentiated suspension cells include blood cells such as T cells and B cells. Examples of pre-differentiation suspension cells include undifferentiated suspension cells, i.e., dedifferentiated fat cells (DFAT).

[0088] <<Method for Maintaining Undifferentiated Cells>> In the method for maintaining undifferentiated cells of the present invention, cells are cultured using the decellularized support composition or extracellular matrix composition of the present invention, thereby inducing the maintenance of an undifferentiated state of cells. The cells used in the method for maintaining undifferentiated cells of the present invention are not particularly limited, and suspension cells and adhesive cells can be used. Furthermore, from the viewpoint of exerting the effects of the present invention, it is preferable to use pre-differentiated cells. Examples of pre-differentiated adhesive cells include differentiation-competent adhesive cells, i.e., embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, hematopoietic stem cells, neural stem cells, cardiac progenitor cells, vascular endothelial progenitor cells, neural progenitor cells, preadipocytes, fibroblasts, skin fibroblasts, skeletal myoblasts, osteoblasts, odontoblasts, or neural crest cells. From the viewpoint of exerting the effects of the present invention, mesenchymal stem cells are preferred. Pre-differentiated floating cells include, for example, undifferentiated floating cells, that is, dedifferentiated fat cells (DFAT).

[0089] <<Method for Differentiating Cells into Chondrocytes>> In the method for differentiating cells into chondrocytes of the present invention, cells are cultured using the decellularized support composition or extracellular matrix composition of the present invention, thereby differentiating them into chondrocytes. The cells used in the method for differentiating cells into chondrocytes are not particularly limited, and suspension cells and adherent cells can be used. Differentiated cells and pre-differentiated cells can also be used. Examples of differentiated adherent cells include hepatocytes, stellate cells, Kupffer cells, vascular endothelial cells, meatus endothelial cells, endothelial cells, osteoclasts, periodontal ligament-derived cells, epidermal cells, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells, mammary gland cells, pericytes, smooth muscle cells, cardiac muscle cells, muscle cells, kidney cells, pancreatic islet cells of Langerhans, peripheral nerve cells, nerve cells, chondrocytes, and bone cells. Examples of pre-differentiation adhesive cells include differentiation-competent adhesive cells, i.e., embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, hematopoietic stem cells, neural stem cells, cardiac progenitor cells, vascular endothelial progenitor cells, neural progenitor cells, preadipocytes, fibroblasts, skin fibroblasts, skeletal myoblasts, osteoblasts, odontoblasts, and neural crest cells. From the viewpoint of exerting the effects of the present invention, mesenchymal stem cells, cardiac progenitor cells, and fibroblasts are preferred, and mesenchymal stem cells and skin fibroblasts are more preferred. Examples of differentiated suspension cells include blood cells such as T cells and B cells. Examples of pre-differentiation suspension cells include undifferentiated suspension cells, i.e., dedifferentiated fat cells (DFAT).

[0090] <<Action>> In the present invention, the mechanism by which cellular dedifferentiation, maintenance of undifferentiation, and differentiation occur using the decellularized support composition or extracellular matrix composition has not been analyzed in detail, but can be presumed as follows. However, the present invention is not limited by the following presumption. It is believed that the decellularized support composition or extracellular matrix composition of the present invention produces proteins, ribosomes, and extracellular vesicles useful for dedifferentiation, maintenance of undifferentiation, or differentiation through cell culture. Typically, these proteins, ribosomes, and extracellular vesicles are removed during decellularization. However, according to the decellularized support composition or extracellular matrix composition of the present invention and the method for producing a decellularized support composition, these proteins, ribosomes, and extracellular vesicles remain. Therefore, it is presumed that these proteins, ribosomes, and extracellular vesicles promote dedifferentiation, maintenance of undifferentiation, and differentiation, and that dedifferentiation, maintenance of undifferentiation, or differentiation occur using the decellularized support composition or extracellular matrix composition of the present invention.

[0091] The present disclosure includes the following aspects: [1] A decellularized support composition obtained by decellularizing a cultured cell aggregate or a cultured cell structure, the decellularized support composition being used for dedifferentiation, maintaining undifferentiated cells, or differentiating cells; [2] The decellularized support composition according to [1], wherein the differentiation is for neural differentiation, muscle cell differentiation, angiogenesis, or chondrocyte differentiation; [3] A decellularized support composition derived from the cultured cell aggregate, the decellularized support composition being used by forming surface pores having a pore diameter of 5 μm or more on the surface of the aggregate, the surface pores having a pore diameter of 11088 μm or more on the surface of the aggregate; ２[4] A decellularized support composition derived from the cultured cell structure, the decellularized support composition according to [1] or [2], having a porosity of 40% to 70%; [5] The decellularized support composition according to any of [1] to [4], wherein the decellularized support composition is in a protease-treated liquid form; [6] The decellularized support composition according to [5], wherein the protease is pepsin; [7] The decellularized support composition according to any of [1] to [6], wherein the decellularized support composition has a DNA content of 1.000 mass% or less per dry mass; [8] An extracellular matrix composition for dedifferentiation, maintenance of undifferentiation, or differentiation of cells, comprising at least one selected from proteins, ribosomes, and extracellular vesicles having a molecular weight of 5,000 to 80,000; [9] A cell culture substrate comprising the decellularized support composition according to any of [1] to [7], or the extracellular matrix composition according to [8].

[10] A biological graft comprising the decellularized support composition according to any one of [1] to [7] or the extracellular matrix composition according to [8];

[11] A cell composition comprising the decellularized support composition according to any one of [1] to [7] or the extracellular matrix composition according to [8] and cells;

[12] A method for producing a decellularized support composition for dedifferentiation, maintenance of undifferentiation, or differentiation of cells, comprising a cultured cell aggregate formation step of forming cell aggregates by culturing cells, and a step of decellularizing the cultured cell aggregates;

[13] A method for producing a decellularized support composition for dedifferentiation, maintenance of undifferentiation, or differentiation of cells, comprising a cultured cell aggregate formation step of forming cell aggregates by culturing cells, a cultured cell structure production step of further culturing the cultured cell aggregates to produce a cell structure, and a step of decellularizing the cultured cell structure;

[14] A method for producing a decellularized support composition according to

[12] , wherein cells are cultured on a non-adhesive surface in the cultured cell aggregate formation step;

[15] The method for producing a decellularized support composition according to

[13] , wherein the cultured cell aggregates are cultured in a space surrounded by a mesh-like or comb-like support in the cultured cell structure production step.

[16] A method for producing a decellularized support composition according to any one of

[12] to

[15] , wherein the decellularization is carried out by high hydrostatic pressure treatment;

[17] A decellularized support composition for dedifferentiation, maintenance of undifferentiation, or differentiation of cells, obtained by the production method according to any one of

[12] to

[16] ;

[18] A method for dedifferentiation, maintenance of undifferentiation, or differentiation of cells, wherein cells are cultured using a decellularized support composition from which a cultured cell aggregate or a cultured cell structure has been decellularized, or the extracellular matrix composition according to [8];

[19] A method for neuronal differentiation of cells, wherein cells are cultured using a decellularized support composition from which a cultured cell aggregate or a cultured cell structure has been decellularized, or the extracellular matrix composition according to [8];

[20] A method for muscle cell differentiation of cells, wherein cells are cultured using a decellularized support composition from which a cultured cell aggregate or a cultured cell structure has been decellularized, or the extracellular matrix composition according to [8];

[21] A method for angiogenesis, wherein cells are cultured using a decellularized support composition from which a cultured cell aggregate or a cultured cell structure has been decellularized, or the extracellular matrix composition according to [8];

[22] A method for differentiating cells into chondrocytes, comprising culturing cells using a decellularized support composition obtained by decellularizing a cultured cell aggregate or a cultured cell structure, or the extracellular matrix composition according to [8];

[23] A method for producing a decellularized support composition solution, comprising: a liquefaction step of liquefying the decellularized support composition obtained by the production method according to any one of

[12] to

[16] using a protease or an acidic solution; a separation step of treating the liquefied decellularized support composition by centrifugation; and a neutralization step of neutralizing the supernatant obtained by the centrifugation to a pH of 7.0 to 8.0;

[24] A method for producing a decellularized support composition solution according to

[23] , wherein the protease used in the liquefaction step is pepsin;

[25] A method for producing a decellularized support composition solution according to

[23] or

[24] , wherein the liquefaction step is carried out at a pH of 1.0 to 5.0 and at a temperature range of 4 to 37°C; and

[26] The method for producing a decellularized support composition solution according to any one of

[23] to

[25] , wherein the decellularized support composition from which the cultured cell aggregate or cultured cell structure has been decellularized is in powder form.

[0092] The present invention is not limited to the above-described embodiments. The above-described embodiments are merely examples, and any configuration that is substantially identical to the technical idea described in the claims of the present invention and that provides similar effects is included within the technical scope of the present invention.

[0093] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0094] Example 1 In this example, a decellularized cell aggregate composition was prepared. (Preparation of cultured cell aggregates (spheroids)) A T-175 flask (vent cap, 175 cm ２ NHDF-Neo-dermal fibroblasts (CC-2509, manufactured by Lonza) were cultured in a medium containing 100% NHDF-Neo dermal fibroblasts (Biolamo). When the cells reached approximately 90-100% confluence, they were detached, and a cell suspension (20 mL) was prepared (approximately 2.0 × 10 cells) using FKCM medium (serum-free medium for fibroblasts, manufactured by Fukoku). ７ The dimple plate was placed in a 10 cm dish, and the cell suspension was evenly poured into the dimple plate. The plate was then incubated at 37°C and 5% CO ２ The cells were cultured in an incubator (IP400, Yamato Scientific). After culturing overnight, spheroids (aggregations of cells) in the dimple plate were observed using an inverted microscope (CKX31, Olympus). The spheroids were removed from the depressions of the dimple plate by pipetting and transferred to a 10 cm dish. The 10 cm dish was rotated to collect the spheroids in the center and recover them, yielding spheroids.

[0095] (Decellularization of Spheroids) Spheroids and FKCM medium were placed in a nylon poly vacuum bag (Kashibijin, manufactured by Kurilon Chemical Co., Ltd.) and double-sealed on all four sides with a sealer. Using FKCM medium as the medium, high hydrostatic pressure treatment was performed at 600 MPa for 10 minutes using a research and development high-pressure treatment device (Dr. CHEF, manufactured by Kobe Steel, Ltd.). High hydrostatic pressure treatment was performed at a minimum temperature of 23.3°C and a maximum temperature of 35.9°C. The high hydrostatic pressure-treated spheroids were recovered from the nylon poly vacuum bag, transferred to a centrifuge tube, and washed with water for injection. The high hydrostatic pressure-treated spheroids were then immersed in a nuclease DNase I solution (800 U / mL) and washed with shaking at 4°C for at least 18 hours. After washing three times with water for injection and lyophilization, decellularized cell aggregates (decellularized spheroids) were obtained.

[0096] Example 2 In this example, a decellularized cell structure composition was produced using fibroblasts. (Preparation of cultured cell structure (cell block)) The spheroids obtained in Example 1 were placed in a net mold (Net Mold Starter Kit V6, manufactured by Tissue By Net Co., Ltd.). The net mold was transferred to a 60 mL safe container (manufactured by WATSON) containing FKCM medium (80 mL), and incubated at 37°C and 5% CO ２ The cells were placed in a shaker (OS-762, Optima) in an incubator and culture was initiated under shaking (45 rpm). Half of the medium was replaced once or twice a week, and the culture was continued. After approximately three weeks of culture, the net mold was removed, and a cultured cell construct was obtained.

[0097] (Decellularization of cultured cell constructs) The cultured cell constructs removed from the net mold and FKCM medium were placed in a nylon poly vacuum bag (Kashibijin, manufactured by Kurilon Chemical Co., Ltd.) and double-sealed on all four sides with a sealer. Using FKCM medium as a medium, high hydrostatic pressure treatment was performed at 600 MPa for 10 minutes using a research and development high-pressure treatment device (Dr. CHEF, manufactured by Kobe Steel, Ltd.). The high hydrostatic pressure treatment was performed at a minimum temperature of 23.3°C and a maximum temperature of 35.9°C. The high hydrostatic pressure-treated cultured cell constructs were removed from the nylon poly vacuum bag, immersed in water for injection, and washed with shaking at 4°C for at least 5 minutes. The high hydrostatic pressure-treated cultured cell constructs were then immersed in a nuclease DNase I solution (800 U / mL) and washed with shaking at 4°C for at least 18 hours. Furthermore, the cultured cell construct was immersed in water for injection and washed with shaking at 4°C for 5 minutes or more, repeated three times, to obtain a decellularized cell construct (decellularized cell block).

[0098] Example 3 In this example, a decellularized cell aggregate composition was prepared using immortalized fibroblasts. (Preparation of cultured cell aggregates (spheroids)) A 10-layer flask (6360 cm 2 OUMS-36T-1 cells (human immortalized fibroblasts) (JCRB1006.1) were cultured in a medium containing 100% soluble cellulose (Corning Incorporated). When the cells reached approximately 90-100% confluence, they were detached and a cell suspension (30 mL) was prepared (approximately 8.0 × 10 cells) using KBM Fibro Assist medium (Kohjin Bio Co., Ltd.). ７ The cell suspension was evenly poured into an Elplasia (registered trademark) 12K flask (Corning Inc.), and the cells were incubated at 37°C in 5% CO ２ The cells were cultured in an incubator (IP400, Yamato Scientific). After overnight culture, spheroids (aggregations of cells) in the flask were observed using an inverted microscope (CKX31, Olympus). The spheroids were removed from the wells of the flask by pipetting and collected in a 50 mL centrifuge tube to obtain spheroids.

[0099] (Decellularization of Spheroids) Spheroids and KBM Fibro Assist medium were placed in a nylon poly vacuum bag (Kashibijin, manufactured by Kurilon Chemical Co., Ltd.) and double-sealed on all four sides with a sealer. Using KBM Fibro Assist medium as the medium, high hydrostatic pressure treatment was performed for 9 minutes at 500 MPa using a research-grade high-pressure treatment device (Sr. CIP-M, manufactured by Kobe Steel, Ltd.). The high-hydrostatic pressure-treated spheroids were recovered from the nylon poly vacuum bag, transferred to a centrifuge tube, and washed with water for injection. The high-hydrostatic pressure-treated spheroids were then immersed in a nuclease DNase I solution (800 U / mL) and washed with shaking at 4°C for at least 18 hours. After washing three times with water for injection and subsequent lyophilization, decellularized cell aggregates (decellularized spheroids) were obtained.

[0100] Example 4 In this example, a solution of decellularized cell aggregate composition was prepared using immortalized fibroblasts. (Preparation of Decellularized Spheroid Solution) Pepsin was dissolved in 0.01 M HCl at 1 mg / mL. The powdered decellularized spheroids from Example 3 were dissolved in pepsin solution at 10 mg / mL (pH 2.0-4.0, temperature 25°C). The mixture was stirred at 600-1000 rpm at room temperature for several hours or more and then centrifuged in a centrifuge (MX-307, Tomy Kogyo) at 8000 rpm for 5 minutes. The supernatant was collected, and 10x PBS (phosphate-buffered saline, pH 7.4) was added to a final concentration of 1x PBS. 0.1 M NaOH was added until the pH reached 7.2-7.6, yielding a decellularized spheroid solution.

[0101] Comparative Example 1 In Comparative Example 1, a bovine collagen sheet (CCC; Collagen cell carrier, product name: Collagen cell carrier (registered trademark) membrane, manufactured by Viscofan BioEngineering) was used.

[0102] Comparative Example 2 In Comparative Example 2, solubilized collagen (product name: Cellmatrix (registered trademark) Type IC, manufactured by Nitta Gelatin Co., Ltd., 631-00771) was used.

[0103] Comparative Example 3 In Comparative Example 3, collagen powder (product name: Collagen Type I, Powder, Bovine Skin, Acid Soluble, manufactured by Nippi Co., Ltd.) was used.

[0104] Comparative Example 4 In Comparative Example 4, a gelatin fiber substrate for cell culture (product name: Genocel (registered trademark), manufactured by Nikke Medical Co., Ltd.) was used.

[0105] <Calculation of Decellularized DNA Ratio> The masses of Examples 1, 2, and 3 were measured, and these samples were used. After immersion and dissolution in a protease solution, the samples were treated with phenol / chloroform to remove proteins and recover DNA. The recovered DNA was fluorescently stained with Picogreen (Life Technologies) and the fluorescence intensity was measured to quantify the DNA. The DNA content of the sample was calculated from the sample mass and DNA amount. A calibration curve prepared using the standard DNA provided with Picogreen was used to quantify the DNA. (Decellularized DNA ratio) = (DNA content of dried test piece of decellularized spheroid (or decellularized cell block)) / (mass of dried test piece of decellularized spheroid (or decellularized cell block)) × 100

[0106]

[0107] The results are shown in Table 1. It was found that decellularization was performed well in the Examples, and that the Examples can be used as decellularized grafts with reduced rejection.

[0108] <<Measurement of Protein Molecular Weight>> The molecular weight of the protein contained in the compositions of Examples 1 and 2 and the collagen sheet of Comparative Example 1 was measured. The masses of the samples of Example 1, the lyophilized Example 2, and Comparative Example 1 were measured, and the samples were adjusted to 4 mg / mL with ultrapure water. After shredding with scissors, the samples were sonicated on ice for 30 seconds using an ultrasonic homogenizer (UD-201, TOMY Seiko Co., Ltd.) at 60 W output with intermittent oscillation of 0.5 seconds on / 0.5 seconds off to prepare sample suspensions. The resulting suspensions were prepared using a protein preparation kit (Agilent Protein 80 Kit, manufactured by Agilent Technology). Specifically, 1.4 μL of 1 mol / L dithiothreitol (DTT) was mixed with 40 μL of the preparation kit's contents, sodium dodecyl sulfate (SDS) containing an internal standard, to prepare a prepared sample buffer. 4 μL of sample suspension was mixed with 2 μL of prepared sample buffer. The mixed sample suspension and the molecular weight standard ladder included in the kit were each heat-treated at 100°C for 5 minutes. After heat treatment, the mixture was centrifuged, and 84 μL of ultrapure water was added and mixed to obtain the prepared sample and prepared ladder. 6 μL of the prepared sample and prepared ladder were applied to a chip filled with the SDS-polyacrylamide gel included in the preparation kit. The applied chip was analyzed using a microchip electrophoresis device (Bioanalyzer, manufactured by Agilent Technology) to measure the molecular weight of the contained protein.

[0109] The results are shown in Figure 1. It was found that Examples 1 and 2 contained proteins with molecular weights of 5,000 to 80,000. Comparative Example 1 did not contain proteins with molecular weights of 5,000 to 80,000. Although not shown in Figure 1, peaks similar to those in Example 1 were observed in Example 3.

[0110] Paraffin-embedded tissue specimens were prepared using the compositions of Examples 1 and 2. (Sample of Example 1) As a pretreatment, the decellularized spheroids obtained in Example 1 were gelled with IPGell (GenoStuff) and immersed in 4% by weight PFA (4% by weight paraformaldehyde phosphate buffer, for tissue fixation, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) for 24 hours. The gel was then immersed in phosphate buffer (PBS, 0.01 M, pH 7.4) and washed with shaking at 4°C for 30 minutes or more. The gel was then immersed in 70% by weight ethanol, washed with shaking at 4°C for 30 minutes or more, and then immersed again in 70% by weight ethanol to prepare a sample for preparing a paraffin-embedded tissue specimen.

[0111] (Sample of Example 2) As a pretreatment, the decellularized cell structure obtained in Example 2 was immersed in 4% by mass PFA (4% by mass paraformaldehyde phosphate buffer, for tissue fixation, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) for 48 hours or more. Thereafter, the decellularized cell structure was immersed in phosphate buffer (PBS, 0.01 M, pH 7.4) and washed with shaking at 4°C for 30 minutes or more. Subsequently, the decellularized cell structure was immersed in 70% by mass ethanol and washed with shaking at 4°C for 30 minutes or more to prepare a sample for preparing a paraffin-embedded tissue specimen.

[0112] (Preparation of Stained Section Samples) The samples were then washed twice for 20 minutes each in G-Nox (Genostaff), a low-toxicity solvent used as a xylene substitute. The samples were then incubated twice in a 65°C paraffin bath for 30 minutes each. The melted paraffin was poured into a mold, the sample was placed in the mold, and allowed to cool for 15-20 minutes to prepare paraffin-embedded tissue specimens. The paraffin-embedded tissue specimens were then serially sectioned at 5 μm thickness using a microtome and floated in a 37°C water bath containing deionized water. Each section was floated on distilled water placed on a glass slide, heated (30-40°C) on an extension table, and allowed to dry in an incubator (37°C) for 1-2 nights to prepare stained section samples.

[0113] <<Ribosome Immunostaining>> Liposomes in the obtained stained section samples were immunostained. Immunohistochemical staining of Examples 1 and 2 and Comparative Example 1 was performed according to the following procedure to stain S6 Ribosomal Protein, a ribosomal marker protein. First, the stained section samples were deparaffinized and hydrophilized. After washing three times with phosphate buffer (PBS, 0.01 M, pH 7.4), they were immersed in citrate buffer (antigen activation buffer, pH 6.0) and heat-treated to perform antigen retrieval. Subsequently, they were washed three times with phosphate buffer (PBS, 0.01 M, pH 7.4), treated with 0.3% hydrogen peroxide / methanol at room temperature for 30 minutes, and then washed three times with Tris-buffered saline. Then, after treatment with G-Block (GenoStuff) at room temperature for 10 minutes, the sections were treated with Avidin / Biotin Blocking kit (VECTOR LABORATORIES) for blocking. Subsequently, an antibody reaction was carried out overnight at 4°C using Anti-S6 Ribosomal Protein rabbit monoclonal antibody (2217, Santa Cruz Biotechnology) as the primary antibody. Then, the sections were washed twice with Tween 20-containing Tris-buffered saline, followed by further washing with Tris-buffered saline. Subsequently, an antibody reaction was carried out at room temperature for 30 minutes using Anti-Rabbit IgG Biotin (BA-1000, Vector) as the secondary antibody. The sections were then washed twice with Tween 20-containing Tris-buffered saline, followed by another wash with Tris-buffered saline. They were then treated with peroxidase-labeled streptavidin (Nichirei) at room temperature for 5 minutes. They were then washed twice with Tween 20-containing Tris-buffered saline, followed by another wash with Tris-buffered saline. They were then treated with DAB (3,3'-diaminobenzidine tetrahydrochloride) / hydrogen peroxide for color development. Cell nuclei were counterstained with Mayer's hematoxylin (Muto Chemicals) as a marker for observation. They were then dehydrated and cleared, and mounted with mounting medium (xylene-based mounting material: Marinol, Muto Chemicals).

[0114] <Calculation of Positive Area Ratio> The obtained S6 Ribosomal Protein-stained sections were observed under a microscope, and the sections were photographed at 40x magnification. Image processing software (ImageJ) was used to calculate the positive area ratios of Example 1 (decellularized spheroids) and Example 2 (decellularized cell blocks). Images of the stained section samples were imported, and the area of ​​the entire stained section sample and the area of ​​the S6 Ribosomal Protein-stained positive portion (ribosome portion) were measured. The positive area ratios of Examples 1 and 2 were calculated according to the following formula. A rectangular area (500 pixel x 500 pixel) was set as the measurement area, and the average positive area ratio of three locations was calculated. Positive area ratio (%) = Area of ​​S6 Ribosomal Protein-stained portion / Area of ​​entire stained section sample (measurement area) x 100

[0115] The results of ribosome immunostaining are shown in Figure 2. The left image shows ribosome staining, and the right image shows the negative control (stained with rabbit Ig). In the examples, areas stained darker in the left image than in the right image were observed, and ribosomes were detected. The positive area rate in Example 1 was 30.9%, and the positive area rate in Example 2 was 17.3%. In contrast, no ribosomes were detected in the comparative example.

[0116] Extracellular Vesicle Immunostaining The extracellular vesicles in the obtained stained section samples were immunostained. Immunohistochemical staining of Examples 1 and 2 and Comparative Example 1 was performed according to the following procedure, and ALG-2-interacting protein X (ALIX), a marker protein for extracellular vesicles, was stained. First, the stained section samples were deparaffinized and hydrophilized. After washing three times with phosphate buffer (PBS, 0.01 M, pH 7.4), the samples were immersed in citrate buffer (antigen activation buffer, pH 6.0) and heat-treated to perform antigen retrieval. Subsequently, the samples were washed three times with phosphate buffer (PBS, 0.01 M, pH 7.4), treated with 0.3% hydrogen peroxide / methanol at room temperature for 30 minutes, and then washed three times with Tris-buffered saline. The sections were then treated with G-Block (GenoStaff) at room temperature for 10 minutes, followed by treatment with Avidin / Biotin Blocking kit (VECTOR LABORATORIES) for blocking. Subsequently, an antibody reaction was carried out overnight at 4°C using Anti-ALIX rabbit polyclonal antibody (ab76608, Abcam) as the primary antibody. The sections were then washed twice with Tween 20-containing Tris-buffered saline, followed by another wash with Tris-buffered saline. Subsequently, an antibody reaction was carried out for 30 minutes at room temperature using Anti-Rabbit IgG Biotin (BA-1000, Vector) as the secondary antibody. The sections were then washed twice with Tween 20-containing Tris-buffered saline, followed by another wash with Tris-buffered saline. The sections were then treated with peroxidase-labeled streptavidin (Nichirei) at room temperature for 5 minutes. Then, they were washed twice with Tween 20-containing Tris-buffered saline, followed by another wash with Tris-buffered saline. They were then treated with DAB (3,3'-diaminobenzidine tetrahydrochloride) / hydrogen peroxide to develop color. Cell nuclei were counterstained with Mayer's hematoxylin (Muto Chemicals) as a marker for observation. They were then dehydrated and cleared, and mounted with mounting medium (xylene-based mounting material: Marinol, Muto Chemicals).

[0117] <Calculation of Positive Area Ratio> The obtained ALIX-stained sections were observed under a microscope, and the sections were photographed at 40x magnification. Image processing software (ImageJ) was used to calculate the positive area ratios of Example 1 (decellularized spheroids) and Example 2 (decellularized cell blocks). Images of the stained section samples were imported, and the area of ​​the entire stained section sample and the area of ​​the ALIX-stained positive portion (extracellular vesicle portion) were measured. The positive area ratios of Examples 1 and 2 were calculated according to the following formula. A rectangular area (500 pixels x 500 pixels) was set as the measurement area, and the average positive area ratio of three locations was calculated. Positive area ratio (%) = Area of ​​ALIX-stained portion / Area of ​​entire stained section sample (measurement area) x 100

[0118] The results are shown in Figure 3. The left image shows staining of extracellular vesicles, and the right image shows the negative control (stained with rabbit Ig). In the examples, areas stained darker in the left image than in the right image were observed, and extracellular vesicles were detected. The positive area ratio in Example 1 was 6.42, and the positive area ratio in Example 2 was 4.30. In contrast, no extracellular vesicles were detected in the comparative example.

[0119] <<Evaluation of Dedifferentiation Potential>> (Cell Culture Method of Example 1) The decellularized spheroids of Example 1 were suspended in DMEM medium to prepare a 1.0 mg / mL suspension, which was then added to a 96-well U-bottom plate (PrimeSurface Plate 96U, Sumitomo Bakelite) in 100 μL portions (0.1 mg / well). ４ 50 μL of a cell suspension of NHDF-Neo-dermal fibroblasts (CC-2509, manufactured by Lonza) was seeded (1.0 × 10 ３ The cells were cultured at 37°C, 5% CO with medium changes every two days. ２ The cells were cultured in an incubator for 14 days.

[0120] (Cell culture method of Example 2) A dedicated adapter was attached to the decellularized cell structure of Example 2, and DMEM medium and FKCM medium (1:1) were added to reach half the height of the net mold. ７NHDF-Neo-dermal fibroblasts (CC-2509, manufactured by Lonza) were seeded at 50 μL / mL and incubated at 37°C, 5% CO ２ The cells were cultured in an incubator for 14 days.

[0121] (Cell culture method of Comparative Example 1) After punching out a circle from a CCC with a 12 mm biopsy trephine, the CCC was sandwiched between a cell insert for a 24-well plate (Z742381-12EA, manufactured by SIGMA-ALDRICH). ４ 200 μL of NHDF-Neo-dermal fibroblasts (CC-2509, manufactured by Lonza) were seeded at 100 cells / mL on the upper side of the cell insert, and 700 μL of DMEM medium and FKCM medium (1:1) was added to the lower side of the cell insert. The cells were incubated at 37°C, 5% CO ２ The cells were cultured in an incubator for 14 days.

[0122] <<Dedifferentiation Marker Gene Analysis>> Total cellular RNA was extracted from the test specimens (Examples and Comparative Examples containing cells after cell culture) using a "FastLane Cell Probe kit" (QIAGEN). cDNA synthesis and real-time PCR were performed using a "Step One Plus" (Applied Biosystems) under a specified cycling protocol (50°C for 30 minutes, 95°C for 15 minutes, 94°C for 15 seconds for 40 cycles, 60°C for 1 minute) from the extracted RNA. The probes used were "TaqMan probes" (Thermo Fisher Scientific Life Technologies Japan). TaqMan probes specific to NANOG (pluripotency marker), SOX2 (pluripotency marker), and POU5F1 (pluripotency marker) were used. Using GAPDH as an endogenous control, the 2-ΔΔCt method was applied to calculate the relative mRNA amount of each marker compared to GAPDH to determine the marker expression level.

[0123] The results of the genetic analysis are shown in Figure 4. It was found that the Examples significantly increased the expression levels of NANOG (pluripotency marker), SOX2 (pluripotency marker), and POU5F1 (pluripotency marker) in the cultured cells compared to the Comparative Examples. It was found that the Examples significantly dedifferentiated the cultured cells compared to the Comparative Examples. It is believed that in the NHDF-Neo-dermal fibroblasts (CC-2509), a portion of the fibroblasts, which are differentiated cells, dedifferentiated.

[0124] <Evaluation of neural differentiation potential> <Seeding of skin fibroblasts> (Cell culture method of Example 1) The decellularized spheroids of Example 1 were suspended in DMEM medium to prepare a 1.0 mg / mL suspension, and 100 μL of the suspension was added to a 96-well U-bottom plate (PrimeSurface Plate 96U, Sumitomo Bakelite) (0.1 mg / well). ５ 50 μL of a cell suspension of NHDF-Neo-dermal fibroblasts (CC-2509, manufactured by Lonza) was seeded (1.0 × 10 ４ The cells were cultured at 37°C, 5% CO with medium changes every two days. ２ The cells were cultured in an incubator for 14 days.

[0125] (Cell culture method of Example 2) A dedicated adapter was attached to the decellularized cell structure of Example 2, and DMEM medium and FKCM medium (1:1) were added to reach half the height of the net mold. ７ NHDF-Neo-dermal fibroblasts (CC-2509, manufactured by Lonza) were seeded at 50 μL / mL and incubated at 37°C, 5% CO ２ The cells were cultured in an incubator for 14 days.

[0126] (Cell culture method of Comparative Example 1) After punching out a circle from a CCC with a 12 mm biopsy trephine, the CCC was sandwiched between a cell insert for a 24-well plate (Z742381-12EA, manufactured by SIGMA-ALDRICH). ４200 μL of NHDF-Neo-dermal fibroblasts (CC-2509, manufactured by Lonza) were seeded at 100 cells / mL on the upper side of the cell insert, and 700 μL of DMEM medium and FKCM medium (1:1) was added to the lower side of the cell insert. The cells were incubated at 37°C, 5% CO ２ The cells were cultured in an incubator for 14 days.

[0127] Neuronal Marker Gene Analysis Total cellular RNA was extracted from the test specimens (Examples and Comparative Examples containing cells after cell culture) using a "FastLane Cell Probe kit" (QIAGEN). cDNA synthesis and real-time PCR were performed using a "Step One Plus" (Applied Biosystems) under a specified cycling protocol (50°C for 30 minutes, 95°C for 15 minutes, 94°C for 15 seconds for 40 cycles, 60°C for 1 minute) from the extracted RNA. TaqMan probes (Thermo Fisher Scientific Life Technologies Japan) were used. TaqMan probes specific for GDNF (a neuronal differentiation marker, a gene encoding glial cell line-derived neurotrophic factor) and TAC1 (a gene encoding a neurotransmitter (precursor), preprotachykinin-1) were used. Using GAPDH as an endogenous control, the 2-ΔΔCt method was applied to calculate the relative mRNA amount of each marker compared to GAPDH to determine the marker expression level.

[0128] The results of the genetic analysis are shown in Figures 5(A) and (B). It was found that the Example significantly increased the expression levels of GDNF (neuron differentiation marker) and TAC1 (neurotransmitter (precursor)) in the cultured cells compared to the Comparative Example. It was found that the Example significantly differentiated the cultured cells into neurons compared to the Comparative Example. The NHDF-Neo-dermal fibroblasts (CC-2509) contained cells that had reached a state where they could differentiate, and it is believed that some of these cells differentiated into neurons.

[0129] <Seeding of Adrenal Pheochromocytoma Cells> (Cell Culture Method of Example 3) The decellularized spheroids of Example 3 were suspended in RPMI medium to prepare a 1.0 mg / mL suspension, which was then added to a 96-well U-bottom plate (PrimeSurface Plate 96U, Sumitomo Bakelite) in 100 μL portions (0.1 mg / well). ４ 100 μL of a cell suspension of PC12 rat adrenal pheochromocytoma cells (EC88022401-F0, ECACC) was seeded (5.0 × 10 ３ The cells were cultured at 37°C, 5% CO with medium changes every two days. ２ The cells were cultured in an incubator for 7 days.

[0130] (Cell Culture Method of Example 4) A cell suspension (1 x 10 ５ 100 μL of the cells (1 × 10 cells / well) were seeded into a 96-well plate (MS-8096F, Sumitomo Bakelite) coated with solubilized collagen (product name: Cellmatrix (registered trademark) Type IC, manufactured by Nitta Gelatin Co., Ltd., 631-00771). ４ The medium was removed, and 100 μL of RPMI medium containing 10% of the decellularized spheroid solution of Example 4 was added to each well. The medium was replaced every two days, and the cells were incubated at 37°C, 5% CO ２ The cells were cultured in an incubator for 7 days.

[0131] (Cell culture method of Comparative Example 1) After punching out a circle from a CCC with a 12 mm biopsy trephine, the CCC was sandwiched between a cell insert for a 24-well plate (Z742381-12EA, manufactured by SIGMA-ALDRICH). ４ 200 μL of PC12 rat adrenal pheochromocytoma cells (EC88022401-F0, manufactured by ECACC) were seeded on the upper side of the cell insert at 37°C, 5% CO ２ The cells were cultured in an incubator for 7 days.

[0132] (Cell Culture Method of Comparative Example 2) A cell suspension (1 × 10) of PC12 rat adrenal pheochromocytoma cells (EC88022401-F0, manufactured by ECACC) was cultured in a 2000 ml / well culture medium. ５ 100 μL of 1 × 10 cells / well was seeded into a 96-well plate (MS-8096F, Sumitomo Bakelite). ４ The medium was removed, and 100 μL of RPMI medium containing the collagen solution of Comparative Example 2 at a ratio of 6.7% (0.2 mg / mL collagen solution) was added to each well. The medium was replaced every two days, and the cells were incubated at 37°C in 5% CO ２ The cells were cultured in an incubator for 7 days.

[0133] (Cell Culture Method of Comparative Example 3) The collagen powder of Comparative Example 3 (product name: Collagen Type I, Powder, Bovine Skin, Acid Soluble, manufactured by Nippi Corporation) was suspended in RPMI medium to prepare a 1 mg / mL suspension, and 100 μL of the suspension was added to a 96-well U-bottom plate (PrimeSurface Plate 96U, Sumitomo Bakelite) (0.1 mg / well). 5.0 × 10 ４ 100 μL of a cell suspension of PC12 rat adrenal pheochromocytoma cells (EC88022401-F0, ECACC) was seeded (5.0 × 10 ３ The cells were cultured at 37°C, 5% CO with medium changes every two days. ２ The cells were cultured in an incubator for 7 days.

[0134] (Cell Culture Method of Comparative Example 4) The gelatin fiber substrate for cell culture of Comparative Example 4 (product name: Genocel (registered trademark), manufactured by Nikke Medical Co., Ltd.) was suspended in RPMI medium to prepare a 1 mg / mL suspension, and 100 μL of the suspension was added to a 96-well U-bottom plate (PrimeSurface Plate 96U, Sumitomo Bakelite) (0.1 mg / well). ４ 100 μL of a cell suspension of PC12 rat adrenal pheochromocytoma cells (EC88022401-F0, ECACC) was seeded (5.0 × 10 ３ The cells were cultured at 37°C, 5% CO with medium changes every two days. ２The cells were cultured in an incubator for 7 days.

[0135] <<Neuron cell marker gene analysis>> Neron cell marker gene analysis was performed in the same manner as in <Dermal fibroblast seeding>> in the above <<Evaluation of neuronal differentiation potential>>, except that TaqMan probes specific to GAP43 (a nerve regeneration marker; a gene encoding a growth-related protein specific to the nervous system) and Synapsin-1 (a gene encoding the most specific marker for synapses in the central and peripheral nervous systems) were used.

[0136] The results of the genetic analysis are shown in Figures 5(C), (D), (E), and (F). It was found that the Example significantly increased the expression levels of GAP43 (a neural regeneration marker) and Synapsin-1 (a synapse marker) in the cultured cells compared to the Comparative Example. It was found that the Example significantly differentiated the cultured cells into neurons compared to the Comparative Example. It is believed that the PC12 rat adrenal pheochromocytoma cells (EC88022401-F0, manufactured by ECACC) differentiated into neurons.

[0137] <<Evaluation of Angiogenesis Induction>> (Cell Culture Method of Example 1) The decellularized spheroids of Example 1 were suspended in DMEM medium to prepare a 1.0 mg / mL suspension, which was then added to a 96-well U-bottom plate (PrimeSurface Plate 96U, Sumitomo Bakelite) in 100 μL portions (0.1 mg / well). ４ 50 μL of a cell suspension of NHDF-Neo-dermal fibroblasts (CC-2509, manufactured by Lonza) was seeded (1.0 × 10 ３ The cells were cultured at 37°C, 5% CO with medium changes every two days. ２ The cells were cultured in an incubator for 14 days.

[0138] (Cell culture method of Example 2) A dedicated adapter was attached to the decellularized cell structure of Example 2, and DMEM medium and FKCM medium (1:1) were added to reach half the height of the net mold. ７ NHDF-Neo-dermal fibroblasts (CC-2509, manufactured by Lonza) were seeded at 50 μL / mL and incubated at 37°C, 5% CO ２The cells were cultured in an incubator for 14 days.

[0139] (Cell Culture Method of Example 4) A cell suspension (1 x 10) of hMSC-AT human adipose-derived mesenchymal stem cells (C-12977, manufactured by Promocell) was prepared. ５ 100 μL of 1 × 10 cells / well was seeded into a 96-well plate (MS-8096F, Sumitomo Bakelite). ４ The medium was removed, and 100 μL of DMEM medium containing 10% of the decellularized spheroid solution of Example 4 was added to each well. The medium was replaced every two days, and the cells were incubated at 37°C, 5% CO ２ The cells were cultured in an incubator for 14 days.

[0140] (Cell culture method of Comparative Example 1) After punching out a circle from a CCC with a 12 mm biopsy trephine, the CCC was sandwiched between a cell insert for a 24-well plate (Z742381-12EA, manufactured by SIGMA-ALDRICH). ４ 200 μL of NHDF-Neo-dermal fibroblasts (CC-2509, manufactured by Lonza) were seeded at 100 cells / mL on the upper side of the cell insert, and 700 μL of DMEM medium and FKCM medium (1:1) was added to the lower side of the cell insert. The cells were incubated at 37°C, 5% CO ２ The cells were cultured in an incubator for 14 days.

[0141] (Cell culture method of Comparative Example 2) A cell suspension (1 × 10) of hMSC-AT human adipose-derived mesenchymal stem cells (C-12977, manufactured by Promocell) was prepared. ５ 100 μL of 1 × 10 cells / well was seeded into a 96-well plate (MS-8096F, Sumitomo Bakelite). ４ The medium was removed, and 100 μL of DMEM medium containing 0.3% of the collagen solution of Comparative Example 2 was added (0.1 mg / mL collagen solution). The medium was replaced every two days and incubated at 37°C in 5% CO ２ The cells were cultured in an incubator for 14 days.

[0142] <<Vascular Endothelial Cell Marker Gene Analysis>> Total cellular RNA was extracted from the test specimens (Examples and Comparative Examples containing cells after cell culture) using a "FastLane Cell Probe kit" (QIAGEN). cDNA synthesis and real-time PCR were performed using a "Step One Plus" (Applied Biosystems) under a specified cycling protocol (50°C for 30 minutes, 95°C for 15 minutes, 94°C for 15 seconds for 40 cycles, 60°C for 1 minute) from the extracted RNA. This was performed using a "TaqMan probe" (Thermo Fisher Scientific Life Technologies Japan). The "TaqMan probe" used was specific for PECAM1 (a gene encoding the vascular endothelial cell differentiation marker, CD31). Using GAPDH as an endogenous control, the 2-ΔΔCt method was applied to calculate the relative mRNA amount of each marker compared to GAPDH to determine the marker expression level.

[0143] The results of the genetic analysis are shown in Figure 6. It was found that the Example significantly increased the expression level of PECAM1 (CD31) (vascular endothelial cell differentiation marker) in the cultured cells compared to the Comparative Example (Figure 6A). It was found that the Example significantly differentiated the cultured cells into vascular endothelial cells compared to the Comparative Example. The NHDF-Neo-dermal fibroblasts (CC-2509) contained cells that were in a differentiation-ready state, and it is believed that some of these cells differentiated into vascular endothelial cells. Furthermore, it is believed that the use of the decellularized spheroid solution of Example 4 caused the hMSC-AT human adipose-derived mesenchymal stem cells (C-12977) to differentiate into vascular endothelial cells (Figure 6B).

[0144] Evaluation of Muscle Cell Differentiation Potential (Preparation of Paraffin-Embedded Tissue Specimens and Stained Section Samples) As a pretreatment for preparing samples containing cells in the Examples and Comparative Examples after cell culture using the method described above, the cells in Example 2 and Comparative Example 1 were cultured in 4% PFA (4% paraformaldehyde phosphate buffer solution for tissue fixation, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) for 48 hours or more. Then, the cells were immersed in phosphate buffer (PBS, 0.01 M, pH 7.4) and washed with shaking at 4°C for 30 minutes or more. The decellularized cell structure was then immersed in 70% ethanol and washed with shaking at 4°C for 30 minutes or more to prepare a sample for preparing a paraffin-embedded tissue specimen. Furthermore, the sample was washed twice for 20 minutes each in the low-toxicity solvent G-Nox (Genostaff) as a xylene substitute. The sample was then incubated twice in a 65°C paraffin bath for 30 minutes each time. The melted paraffin was poured into a mold, the sample was placed in the mold, and allowed to cool for 15-20 minutes to prepare paraffin-embedded tissue specimens. The paraffin-embedded tissue specimens were then serially sectioned at 5 μm thickness using a microtome and floated in a 37°C water bath containing deionized water. Each section was floated on distilled water placed on a glass slide, heated (30-40°C) on an extension table, and allowed to dry in an incubator (37°C) for 1-2 nights to prepare stained section samples.

[0145] (Immunostaining) Immunohistochemical staining of the cell-containing samples of Examples and Comparative Examples after cell culture was performed using the following procedure to stain the muscle cell marker protein α-smooth muscle actin (α-SMA). First, the stained section samples were deparaffinized and hydrophilized. Then, after washing three times with phosphate buffer (PBS, 0.01 M, pH 7.4), they were immersed in citrate buffer (antigen activation buffer, pH 6.0) and heat-treated to perform antigen retrieval. Next, after washing three times with phosphate buffer (PBS, 0.01 M, pH 7.4), they were treated with 0.3% hydrogen peroxide / methanol at room temperature for 30 minutes and then washed three times with Tris-buffered saline. The sections were then treated with G-Block (GenoStaff) at room temperature for 10 minutes, followed by treatment with Avidin / Biotin Blocking kit (VECTOR LABORATORIES) for blocking. Subsequently, an antibody reaction was carried out overnight at 4°C using Anti-α-SMA mouse monoclonal antibody (M0851, Dako) as the primary antibody. The sections were then washed twice with Tween 20-containing Tris-buffered saline, followed by another wash with Tris-buffered saline. Subsequently, an antibody reaction was carried out at room temperature for 30 minutes using Mouse IgG2a (MAB0031, R&D Systems) as the secondary antibody. The sections were then washed twice with Tween 20-containing Tris-buffered saline, followed by another wash with Tris-buffered saline. The sections were then treated with peroxidase-labeled streptavidin (Nichirei) at room temperature for 5 minutes. Then, they were washed twice with Tween 20-containing Tris-buffered saline, followed by another wash with Tris-buffered saline. They were then treated with DAB (3,3'-diaminobenzidine tetrahydrochloride) / hydrogen peroxide to develop color. Cell nuclei were counterstained with Mayer's hematoxylin (Muto Chemicals) as a marker for observation. They were then dehydrated and cleared, and mounted with mounting medium (xylene-based mounting material: Marinol, Muto Chemicals).

[0146] The results of immunostaining are shown in Figure 7. The left image shows stained muscle cells, and the right image shows a negative control (stained with mouse IgG2a). In the example, darker stained areas were observed in the left image compared to the right image, and muscle cells were detected.

[0147] (Calculation of Positive Area) The obtained α-SMA-stained sections were observed under a microscope, and the sections were photographed at 40x magnification. Image processing software (ImageJ) was used to calculate the positive area of ​​the Examples and Comparative Examples containing the cells after cell culture. Images of the stained section samples were imported, and the area of ​​the entire stained section sample and the area of ​​the α-SMA-stained portion were measured. First, the positive area ratio of the Examples and Comparative Examples containing the cells after cell culture was calculated according to the following formula. As the measurement area, a rectangular area (300 pixels x 300 pixels) was set within a range of 100 μm in the depth direction from the cell seeding surface, and the average positive area ratio of six points was calculated. Positive area ratio (%) = Area of ​​α-SMA-stained portion / Area of ​​entire stained section sample (measurement area) x 100. Next, the positive area was calculated as a value when Comparative Example 1 was set as 1.

[0148] The results of calculating the positive area of ​​immunohistochemical staining are shown in Figure 8. It was found that the Example significantly increased the expression level of α-SMA (a muscle cell differentiation marker) in the cultured cells compared to the Comparative Example. It was found that the Example significantly differentiated the cultured cells into muscle cells compared to the Comparative Example. The NHDF-Neo-dermal fibroblasts (CC-2509) contained cells that had reached a state where they could differentiate, and it is believed that some of these cells differentiated into muscle cells.

[0149] <<Evaluation of Ability to Maintain Undifferentiated State>> (Cell Culture Method of Example 3) The decellularized spheroids of Example 3 were suspended in DMEM medium to prepare a 0.1 mg / mL suspension, and 100 μL of the suspension was added to a 96-well U-bottom plate (PrimeSurface Plate 96U, Sumitomo Bakelite) (0.01 mg / well). ４ 100 μL of a cell suspension of hMSC-AT human adipose-derived mesenchymal stem cells (C-12977, manufactured by Promocell) was seeded (5.0 × 10 ３The cells were cultured at 37°C, 5% CO with medium changes every two days. ２ The cells were cultured in an incubator for 14 days.

[0150] (Cell culture method of Comparative Example 1) After punching out a circle from a CCC with a 12 mm biopsy trephine, the CCC was sandwiched between a cell insert for a 24-well plate (Z742381-12EA, manufactured by SIGMA-ALDRICH). ４ 200 μL of hMSC-AT human adipose-derived mesenchymal stem cells (C-12977, manufactured by Promocell) were seeded on the upper side of the cell insert at 100 cells / mL, and 700 μL of DMEM medium was added to the lower side of the cell insert. ２ The cells were cultured in an incubator for 14 days.

[0151] <<Analysis of Undifferentiated Cell Marker Genes>> Total cellular RNA was extracted from the test specimens (Examples and Comparative Examples containing cells after cell culture) using a "FastLane Cell Probe kit" (QIAGEN). cDNA synthesis and real-time PCR were performed using a "Step One Plus" (Applied Biosystems) under a specified cycling protocol (50°C for 30 minutes, 95°C for 15 minutes, 94°C for 15 seconds for 40 cycles, 60°C for 1 minute) from the extracted RNA. This was performed using a "TaqMan probe" (Thermo Fisher Scientific Life Technologies Japan). TaqMan probes specific for CD73 (undifferentiated cell marker) and CD90 (undifferentiated cell marker) were used. Using GAPDH as an endogenous control, the 2-ΔΔCt method was applied to calculate the relative mRNA amount of each marker compared to GAPDH to determine the marker expression level.

[0152] The results of the genetic analysis are shown in Figure 10. It was found that the Examples significantly increased the expression levels of CD73 (an undifferentiation marker) and CD90 (an undifferentiation marker) in the cultured cells compared to the Comparative Examples. It was found that the Examples significantly maintained the undifferentiated state of the cultured cells compared to the Comparative Examples. It is believed that the undifferentiated state of the hMSC-AT human adipose-derived mesenchymal stem cells (C-12977) is maintained.

[0153] Evaluation of chondrocyte differentiation potential (Cell culture method of Example 4) A cell suspension (1 x 10) of hMSC-AT human adipose-derived mesenchymal stem cells (C-12977, manufactured by Promocell) was cultured. ５ 100 μL of 1 × 10 cells / well was seeded into a 96-well plate (MS-8096F, Sumitomo Bakelite). ４ The medium was removed, and 100 μL of DMEM medium containing the decellularized spheroid solution of Example 4 at a ratio of 2.5% was added. The medium was changed every two days while the cells were incubated at 37°C and 5% CO ２ The cells were cultured in an incubator for 14 days.

[0154] (Cell culture method of Comparative Example 1) After punching out a circle from a CCC with a 12 mm biopsy trephine, the CCC was sandwiched between a cell insert for a 24-well plate (Z742381-12EA, manufactured by SIGMA-ALDRICH). ４ 200 μL of hMSC-AT human adipose-derived mesenchymal stem cells (C-12977, manufactured by Promocell) were seeded on the upper side of the cell insert at 100 cells / mL, and 700 μL of DMEM medium was added to the lower side of the cell insert. ２ The cells were cultured in an incubator for 14 days.

[0155] <<Chondrocyte Marker Gene Analysis>> Total cellular RNA was extracted from the test specimens (Examples and Comparative Examples containing cells after cell culture) using a "FastLane Cell Probe kit" (QIAGEN). cDNA synthesis and real-time PCR were performed using a "Step One Plus" (Applied Biosystems) under a specified cycling protocol (50°C for 30 minutes, 95°C for 15 minutes, 94°C for 15 seconds for 40 cycles, 60°C for 1 minute) from the extracted RNA. A "TaqMan probe" (Thermo Fisher Scientific Life Technologies Japan) was used. The "TaqMan probe" used was specific to SOX9 (a chondrocyte marker, a gene encoding a transcription factor essential for chondrocyte differentiation). Using GAPDH as an endogenous control, the 2-ΔΔCt method was applied to calculate the relative mRNA amount of each marker compared to GAPDH to determine the marker expression level.

[0156] The results of the genetic analysis are shown in Figure 11. It was found that the Example significantly increased the expression level of SOX9 (a chondrocyte marker) in the cultured cells compared to the Comparative Example. It was found that the Example significantly differentiated the cultured cells into chondrocytes compared to the Comparative Example. It is believed that by using the decellularized spheroid solution of Example 4, the hMSC-AT human adipose-derived mesenchymal stem cells (C-12977) were differentiated into chondrocytes.

[0157] The decellularized support composition of the present invention can be used in vivo or in vitro for cell dedifferentiation, maintenance of undifferentiated state, neuronal differentiation, muscle cell differentiation, angiogenesis, or chondrocyte differentiation, and can be effectively used as a cell culture substrate or a biological graft.

Claims

1. A decellularized support composition in which cultured cell aggregates or cultured cell structures have been decellularized, the decellularized support composition being used for dedifferentiating, maintaining the undifferentiated state, or differentiating cells.

2. The decellularized support composition according to claim 1, wherein the differentiation is for neural differentiation, muscle cell differentiation, angiogenesis, or chondrocyte differentiation.

3. A decellularized support composition derived from the cultured cell aggregate, comprising surface pores having a pore diameter of 5 μm or more on the aggregate surface. ２ The decellularized support composition according to claim 1, having 5 to 45 per cell.

4. A decellularized support composition according to claim 1, which is a decellularized support composition derived from the cultured cell structure and has a porosity of 40% to 70%.

5. The decellularized support composition according to claim 1, wherein the decellularized support composition is in a protease-treated liquid form.

6. The decellularized support composition according to claim 5, wherein the protease is pepsin.

7. A decellularized support composition according to any one of claims 1 to 6, wherein the DNA content per dry mass of the decellularized support composition is 1.000% by mass or less.

8. An extracellular matrix composition for dedifferentiating, maintaining an undifferentiated state, or differentiating cells, comprising at least one member selected from proteins having a molecular weight of 5,000 to 80,000, ribosomes, and extracellular vesicles.

9. A cell culture substrate comprising the decellularized support composition according to any one of claims 1 to 6, or the extracellular matrix composition according to claim 8.

10. A biological graft comprising the decellularized support composition according to any one of claims 1 to 6, or the extracellular matrix composition according to claim 8.

11. A cell composition comprising a decellularized support composition according to any one of claims 1 to 6, or an extracellular matrix composition according to claim 8, and cells.

12. A method for producing a decellularized support composition for dedifferentiating, maintaining the undifferentiated state, or differentiating cells, comprising: a cultured cell aggregate formation step of forming cell aggregates by culturing cells; and a step of decellularizing the cultured cell aggregates.

13. A method for producing a decellularized support composition for dedifferentiating, maintaining the undifferentiated state, or differentiating cells, comprising: a cultured cell aggregate formation step of forming cell aggregates by culturing cells; a cultured cell structure production step of further culturing the cultured cell aggregates to produce a cell structure; and a step of decellularizing the cultured cell structure.

14. The method for producing a decellularized support composition according to claim 12, wherein in the cultured cell aggregate formation step, cells are cultured on a non-adhesive surface.

15. A method for producing a decellularized support composition as described in claim 13, wherein in the cultured cell structure production process, the cultured cell aggregates are cultured in a space surrounded by a mesh-like or comb-like support.

16. A method for producing a decellularized support composition according to any one of claims 12 to 15, wherein the decellularization is carried out by high hydrostatic pressure treatment.

17. A decellularized support composition for dedifferentiating, maintaining the undifferentiated state, or differentiating cells, obtained by the manufacturing method according to any one of claims 12 to 15.

18. A method for dedifferentiating, maintaining the undifferentiated state, or differentiating cells, comprising culturing cells using a decellularized support composition in which cultured cell aggregates or cultured cell structures have been decellularized, or the extracellular matrix composition described in claim 8.

19. A method for neuronal differentiation of cells, comprising culturing the cells using a decellularized support composition comprising decellularized cultured cell aggregates or cultured cell structures, or the extracellular matrix composition described in claim 8.

20. A method for differentiating cells into muscle cells, comprising culturing the cells using a decellularized support composition comprising decellularized cultured cell aggregates or cultured cell structures, or the extracellular matrix composition described in claim 8.

21. A method for angiogenesis, comprising culturing cells using a decellularized support composition comprising decellularized cultured cell aggregates or cultured cell structures, or the extracellular matrix composition described in claim 8.

22. A method for differentiating cells into chondrocytes, comprising culturing the cells using a decellularized support composition obtained by decellularizing a cultured cell aggregate or a cultured cell structure, or the extracellular matrix composition described in claim 8.

23. A method for producing a decellularized support composition solution, comprising: a liquefaction step of liquefying the decellularized support composition obtained by the production method described in any one of claims 12 to 15 using a protease or an acidic solution; a separation step of treating the liquefied decellularized support composition by centrifugation; and a neutralization step of neutralizing the supernatant obtained by the centrifugation to a pH of 7.0 to 8.

0.

24. The method for producing a decellularized support composition solution according to claim 23, wherein the protease used in the liquefaction step is pepsin.

25. A method for producing a decellularized support composition solution according to claim 23, wherein the liquefaction step is carried out at a pH of 1.0 to 5.0 and at a temperature in the range of 4 to 37°C.

26. A method for producing a decellularized support composition solution according to claim 23, wherein the decellularized support composition from the cultured cell aggregate or cultured cell structure is in powder form.

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