Decellularized cell aggregate and preparation method thereof

Abstract

The purpose of the present invention is to provide a decellularized graft which can be produced easily. The technical problem can be solved by decellularizing a cell aggregate, a decellularized cell aggregate obtained by decellularizing a cultured cell aggregate according to the present invention and having 3-45 surface pores having a pore diameter of 5 [mu] m or more per 11088 [mu] m2 on the aggregate surface, and / or a decellularized cell aggregate which is obtained by decellularizing a cultured cell aggregate and which contains 10 pg / mg or more of nucleofibrin relative to the decellularized cell aggregate.

Description

Technical Field

[0001] The present invention relates to a decellularized cell condensate and a method for preparing the same. Background Art

[0002] When transplanting a graft of a biological tissue derived from another person or a xenogeneic animal, there is a problem that the tissue of the recipient produces a rejection reaction against the graft. Therefore, in order to improve the compatibility with the biological tissue, a technique has been developed in which a decellularized tissue composed of a supporting tissue (extracellular matrix, ECM) remaining after removing cells from the biological tissue is used as a graft. Decellularization means removing cell components such as nucleic acids that are antigenic to the recipient, thereby avoiding immune rejection (Patent Documents 1 and 2).

[0003] Prior Art Documents

[0004] Patent Documents

[0005] Patent Document 1: Japanese Patent Application Laid-Open No. 2005-514971

[0006] Patent Document 2: International Publication No. 2016 / 136633 Summary of the Invention

[0007] (1) Technical Problem to be Solved

[0008] The decellularized tissue is useful in that a graft with less rejection reaction can be obtained by decellularizing a biological tissue. However, it is necessary to process a biological tissue derived from an animal and it is difficult to prepare, so a graft that can be more easily prepared is desired.

[0009] Therefore, an object of the present invention is to provide a decellularized graft that can be easily prepared.

[0010] (2) Technical Solution

[0011] The inventors of the present application conducted in-depth research on a graft that can be easily prepared, and surprisingly found that a graft can be easily obtained by decellularizing a cell condensate.

[0012] The present invention is based on such an insight.

[0013] Therefore, the present invention relates to:

[0014] [1] A decellularized cell condensate, which is a decellularized cell condensate obtained by decellularizing a cultured cell condensate, and has 3 to 45 surface pores with a pore diameter of 5 μm or more per 11088 μm on the condensate surface; 2 having 3 to 45 surface pores with a pore diameter of 5 μm or more;

[0015] [2] A decellularized cell aggregate obtained by decellularizing a cultured cell aggregate, the decellularized cell aggregate comprising 10 pg / mg or more of a lamin protein relative to the decellularized cell aggregate;

[0016] [3] A decellularized cell aggregate obtained by decellularizing a cultured cell aggregate, wherein the cell aggregate has a surface area of ​​11088 μm 2 The invention has 3 to 45 surface pores with a pore diameter of 5 μm or more, and contains 10 pg / mg or more of lamin protein relative to the decellularized cell aggregate;

[0017] [4] The decellularized cell aggregate according to any one of [1] to [3], wherein the cells cultured in the cell aggregate are immortalized cells;

[0018] [5] The decellularized cell aggregate according to any one of [1] to [3], wherein the cells in the cultured cell aggregate are a mixture of two or more types of cells;

[0019] [6] The decellularized cell aggregate according to any one of [1] to [3], wherein the DNA content per unit dry mass of the decellularized cell aggregate is 1.000 mass % or less;

[0020] [7] A cell culture substrate comprising the decellularized cell aggregate described in any one of [1] to [3];

[0021] [8] A transplant for a living body, comprising the decellularized cell aggregate described in any one of [1] to [3];

[0022] [9] A composition comprising the decellularized cell aggregate according to any one of [1] to [3] and cells;

[0023]

[10] A method for preparing a decellularized cell aggregate, comprising a step of forming a cultured cell aggregate by culturing cells to form a cultured cell aggregate, and a step of decellularizing the cultured cell aggregate, wherein the decellularized cell aggregate has a surface area of ​​11088 μm 2 It has 3 to 45 surface pores with a pore diameter of 5 μm or more;

[0024]

[11] A method for preparing a decellularized cell aggregate, comprising a step of forming a cultured cell aggregate by culturing cells to form a cultured cell aggregate, and a step of decellularizing the cultured cell aggregate, wherein the decellularized cell aggregate contains 10 pg / mg or more of lamin protein relative to the decellularized cell aggregate;

[0025]

[12] The method for preparing a decellularized cell aggregate according to

[10] or

[11] , wherein the cell is an immortalized cell;

[0026]

[13] The method for preparing a decellularized cell aggregate according to

[10] or

[11] , wherein the culturing of the cells is the culturing of two or more types of cells; and

[0027]

[14] The method for preparing a decellularized cell aggregate according to

[10] or

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

[0028] (III) Beneficial effects

[0029] According to the method for preparing a decellularized cell aggregate of the present invention, a graft can be easily obtained. In addition, the decellularized cell aggregate of the present invention exhibits excellent cell viability as a scaffold for cells due to its specific physical properties. Brief description of the drawings

[0030] Figure 1 A graph showing the area retention rates in Examples 1 to 8 and Comparative Example 1 based on the method for preparing a decellularized cell aggregate of the present invention.

[0031] Figure 2 SEM photographs showing the surface states of the decellularized cell aggregates of Examples 1 to 8 and Comparative Example 1.

[0032] Figure 3 A graph showing the results obtained by counting the surface pores of the decellularized cell aggregates of Examples 1 to 8 and Comparative Examples 1 to 2.

[0033] Figure 4 A graph obtained by culturing cells using the decellularized cell aggregates of Examples 1 to 8 and Comparative Examples 1 to 2 and measuring the cell viability.

[0034] Figure 5 Photographs obtained by immunohistochemically staining the decellularized cell aggregates of Examples 1 to 8 and Comparative Example 1 with an antibody against lamin B1.

[0035] Figure 6 A graph obtained by quantifying lamin B1 in the decellularized cell aggregates of Examples 1 to 6 and 8 using an enzyme immunoassay.

[0036] Figure 7 A graph obtained by using image processing software (ImageJ) to acquire images of immunohistochemically stained sections of Examples 1 to 8 and calculating the positive area of lamin B1. Detailed implementation mode

[0037] [1] Decellularized cell aggregates

[0038] One embodiment of the decellularized cell aggregates of the present invention is a decellularized cell aggregate obtained by decellularizing a cultured cell aggregate, with 3 to 45 surface pores having a pore diameter of 5 μm or more per 11088 μm on the aggregate surface. 2 It has 3 to 45 surface pores with a pore diameter of 5 μm or more.

[0039] One embodiment of the decellularized cell aggregates of the present invention is a decellularized cell aggregate obtained by decellularizing a cultured cell aggregate, which contains more than 10 pg / mg of laminin.

[0040] The decellularized cell aggregates of the present invention, per 11088 μm on the aggregate surface 2 Have 3 to 45 surface pores with a pore diameter of 5 μm or more, and contain more than 10 pg / mg of laminin.

[0041] Preferably, the cells of the cultured cell aggregate are immortalized cells. It is speculated that by using immortalized cells to produce decellularized cell aggregates, more laminin described later can be contained, and the area retention rate and the number of surface pores can be effectively maintained at specified values. Thus, the cell viability can be improved when cells are seeded on the decellularized cell aggregates.

[0042] Preferably, the cells of the cultured cell aggregate are a mixture of two or more types of cells. It is speculated that by co-culturing cells to produce decellularized cell aggregates, more laminin described later can be contained, and the area retention rate and the number of surface pores can be effectively maintained at specified values. Thus, the cell viability can be improved when cells are seeded on the decellularized cell aggregates.

[0043] 《Cultured cell aggregates》

[0044] The cultured cell aggregate is not particularly limited as long as it is a cell aggregate obtained by in vitro culture. Specifically, through three-dimensional culture, cells produce extracellular matrix (ECM) outside the cells, and thus the cells adhere to maintain the shape of a three-dimensional aggregate, such as an ellipsoid. In addition, the cultured cell aggregate can be a cell mass formed by combining multiple ellipsoids.

[0045] The shape of the cultured cell aggregates is not particularly limited, and examples include spherical, ellipsoidal, cubic, cuboid, cylindrical, or quadrangular prism shapes, etc. When the cultured cell aggregates are ellipsoids, they are mostly cylindrical or ellipsoidal. The size of the cultured cell aggregates is not particularly limited. When they are ellipsoids, the shortest length is 80 μm or more, preferably 90 μm or more, more preferably 95 μm or more, and further preferably 100 μm or more. The longest length is also not limited, being 600 μm or less, preferably 500 μm or less, more preferably 300 μm or less. When they are cell clumps, the shortest length is 90 μm or more, preferably 100 μm or more, more preferably 150 μm or more, and further preferably 200 μm or more. The longest length is also not limited, being 2100 μm or less, preferably 2000 μm or less, more preferably 1700 μm or less, further preferably 1400 μm or less, further more preferably 1200 μm or less, and most preferably 1000 μm. The upper and lower limits can be combined as the size range of the cultured cell aggregates. Thereby, the effects of the present invention can be exerted well.

[0046] The ellipsoids can be produced by three-dimensionally culturing cells and aggregating them into a spherical shape or the like. Although the ellipsoids are formed by cells and extracellular matrix (ECM), they contain more extracellular matrix compared to cell aggregates obtained by two-dimensional culture (monolayer culture).

[0047] As a three-dimensional culture method for ellipsoids, there is no limitation, and examples include a non-adhesive surface culture method, a hanging drop culture method, or a rotary culture method.

[0048] The non-adhesive surface culture method is a culture method for obtaining ellipsoids by using a non-adhesive culture plate with enhanced hydrophilicity on the plate surface. The cells contained in the non-adhesive culture plate are suspended in the culture medium and form ellipsoids by adhering to each other. By increasing the culture plate, a large number of ellipsoids can be cultured.

[0049] In the hanging drop culture method, suspended cells are dropped inside the lid of the culture plate, and the droplets that bulge due to surface tension are cultured. The cells that gather below due to gravity adhere to each other to form ellipsoids. In the hanging drop culture method, it is relatively easy to make the number of cells contained in one ellipsoid consistent.

[0050] The rotary culture method forms an ellipsoid by rotating the culture chamber. Through rotation, the cells in the suspended state come into uniform contact with each other, and an ellipsoid with relatively uniform cell number or size can be produced. In addition, a large amount of culture can be carried out by the rotary culture method.

[0051] Cells for culturing cell aggregates generally include suspension cells and adherent cells. The cells for culturing cell aggregates are not particularly limited. From the perspective of easily producing cell aggregates, adherent cells are preferred.

[0052] Adherent cells are not particularly limited and can be appropriately selected according to the purpose of use of the cultured cell aggregates. For example, as differentiated adherent cells, hepatocytes, astrocytes, Kupffer cells, vascular endothelial cells, sinusoidal endothelial cells, endothelial cells, osteoblasts, 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, cardiomyocytes, muscle cells, renal cells, pancreatic islet cells, peripheral nerve cells, nerve cells, chondrocytes or bone cells can be cited. For example, as adherent cells with differentiation potential, 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, adipose progenitor cells, fibroblasts, skin fibroblasts, skeletal muscle myoblasts, osteoblasts or odontoblasts can be cited. Two or more types of cells can be used. From the perspective of easily producing cell aggregates, fibroblasts, endothelial cells, mesenchymal stem cells, and cardiac progenitor cells are preferred, and skin fibroblasts, vascular endothelial cells, mesenchymal stem cells, and cardiac progenitor cells are preferred.

[0053] In addition, immortalized cells can also be used as cells for culturing cell aggregates. Immortalized cells refer to cells that do not stop proliferating even after repeated cell division. Immortalized cells can be produced (immortalized) by artificially introducing genes into the above cells. From the perspective of exerting the effects of the invention of the present application, immortalized cells are preferably immortalized by artificially introducing genes into the above cells.

[0054] As the immortalized cells, there is no particular limitation, and examples thereof include cell lines of myeloma cells, immortalized fibroblasts, and immortalized vascular endothelial cells. From the perspective of achieving the effects of the invention of the present application, immortalized fibroblasts and immortalized vascular endothelial cells are preferred. As the cell lines of myeloma cells, for example, NSI-Ag4 / 1 and Sp2 / O-Agl4 can be mentioned. As the immortalized fibroblasts, for example, OUMS-36T-1 and TelCOFS02MA can be mentioned. From the perspective of achieving the effects of the invention of the present application, OUMS-36T-1 is preferred. As the immortalized vascular endothelial cells, HUEhT-1 can be mentioned.

[0055] As the cells for culturing cell aggregates, for example, from the perspective of preparing a decellularized cell aggregate by using the method for preparing a decellularized cell aggregate described below and being able to improve cell viability when cells are seeded on the decellularized cell aggregate, immortalized cells are preferably used, and immortalized fibroblasts and immortalized endothelial cells are more preferably used, and immortalized fibroblasts are further preferably used.

[0056] The source of the cells is not particularly limited, and includes eukaryotic cells, prokaryotic cells, multi-cellular organism cells, or single-cellular organism cells. As the eukaryotic cells, animal cells, insect cells, plant cells, fungi, algae, or protozoa can be mentioned. As the animal cells, there is no limitation, and cells derived from humans, monkeys, dogs, cats, rabbits, ferrets, sheep, goats, cows, pigs, horses, camels, mice, rats, hamsters, guinea pigs, or gerbils can be mentioned.

[0057] For example, from the perspective of preparing a decellularized cell aggregate by using the method for preparing a decellularized cell aggregate described below and being able to improve cell viability when cells are seeded on the decellularized cell aggregate, it is preferred to prepare the ellipsoid by co-culturing different types of cells. For the co-culture of cells during the preparation of the ellipsoid, two or more types of cells can be used. From the perspective of further achieving the effects of the invention of the present application, three types of cells are preferably used, and two types of cells are more preferably used. The cells used in the co-culture of cells during the preparation of the ellipsoid are not particularly limited and can be selected from the cells for culturing the above-mentioned cell aggregates. From the perspective of further achieving the effects of the invention of the present application, it is preferably selected from fibroblasts and endothelial cells, and more preferably skin fibroblasts and vascular endothelial cells. The cells used in the co-culture of cells during the preparation of the ellipsoid can be immortalized. From the perspective of further achieving the effects of the invention of the present application, skin fibroblasts and immortalized vascular endothelial cells are most preferably used.

[0058] The cultured cell aggregates are not limited, and preferably adhered using an extracellular matrix. By adhering using an extracellular matrix, the cultured cell aggregates can thereby obtain a certain strength. Examples of the extracellular matrix include collagen, laminin, fibronectin, chondroitin sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, etc., which vary depending on the type of cells and are not particularly limited.

[0059] The decellularized cell aggregates of the present invention are not limited. For example, they can be obtained by decellularizing the above-mentioned cultured cell aggregates using the method for preparing decellularized cell aggregates described later. In particular, by decellularizing an ellipsoid, a decellularized cell aggregate with uniform physical properties can be obtained. The decellularized cell aggregates of the present invention can be in a wet state or a dry state. The decellularized cell aggregates provided in a wet state can be immediately used in transplantation. On the other hand, the decellularized cell aggregates provided in a dry state can be stored for a long time, are easy to transport, and can be restored and used when in use.

[0060] 《Surface Pores》

[0061] The decellularized cell aggregates of the present invention have 3 to 45 surface pores with a pore diameter of 5 μm or more per 11088 μm on the surface of the aggregate. In this specification, the surface pores refer to the pores present on the surface of the decellularized cell aggregates when observing the decellularized cell aggregates using a scanning electron microscope (SEM). In addition, the surface refers to the surface that can be observed using the SEM. As 2 shown, the decellularized cell aggregates of the present invention have a specific number of surface pores of 5 μm or more. By having a certain number of surface pores, they exhibit excellent effects as a scaffold for cell culture. Figure 2 The diameter of the surface pores to be counted is not particularly limited as long as it is 5 μm or more, and preferably the lower limit is 5 μm or more and the upper limit is 50 μm or less. The diameter of the surface pores can be counted physically based on the SEM photograph or by the naked eye.

[0062] As long as there are 3 to 45 surface pores with a pore diameter of 5 μm or more per 11088 μm on the surface of the cultured cell aggregates

[0063] 2 ​If the number of surface pores is 3 to 45, there is no particular limitation. Preferably, in a certain aspect, the lower limit is 3 or more, in a certain aspect, 4 or more, in a certain aspect, 5 or more, in a certain aspect, 6 or more, in a certain aspect, 7 or more, in a certain aspect, 8 or more, in a certain aspect, 9 or more, in a certain aspect, 10 or more, in a certain aspect, 11 or more, in a certain aspect, 12 or more, in a certain aspect, 13 or more, in a certain aspect, 14 or more, in a certain aspect, 15 or more. The upper limit of the number of surface pores is 45 or less, preferably in a certain aspect, 43 or less, in a certain aspect, 41 or less, in a certain aspect, 39 or less, in a certain aspect, 37 or less, in a certain aspect, 35 or less, in a certain aspect, 33 or less, in a certain aspect, 31 or less, in a certain aspect, 30 or less, in a certain aspect, 28 or less, in a certain aspect, 26 or less, in a certain aspect, 24 or less, in a certain aspect, 22 or less, in a certain aspect, 20 or less, in a certain aspect, 18 or less. The upper and lower limits can be combined as the range of the number of surface pores. By being within the said range, the survival number of cells can be increased.

[0064] For the 11088 μm of the condensate surface for counting surface pores 2 There is no particular limitation on the definition. For example, the number of surface pores in an area of 88 μm × 126 μm can be counted on an SEM photograph.

[0065] "Lamin"

[0066] The decellularized cell condensate of the present invention contains 10 pg / mg or more of lamin with respect to the decellularized cell condensate. There are lamin A, lamin B1, lamin B2, and lamin C in lamin, but there is no limitation on the lamin contained in the decellularized cell condensate of the present invention. Preferably, it is lamin B1 (SEQ ID NO: 1). However, the decellularized cell condensate of the present invention may contain lamin A, lamin B2, and lamin C.

[0067] Lamin B1 is encoded by the LMNB1 (alias LMN2, or LMNB) gene located on human chromosome 5q23, and it is a homodimeric protein synthesized together with a short propeptide (aa584 - 586). Lamin is a fibrous protein that maintains the structure and regulates transcription in the cell nucleus, and together with membrane proteins, forms a nuclear lamina on the inner side of the nuclear membrane. The nuclear lamina regulates the cell cycle and the decomposition and formation of the nuclear membrane. During mitosis (somatic cell division), the protein of lamin is phosphorylated, and the nuclear lamina decomposes reversibly.

[0068] The content of laminin in the decellularized cell aggregates is not particularly limited as long as it is 10 pg / mg or more. Preferably, the lower limit is 15 pg / mg or more in a certain aspect, 18 pg / mg or more in a certain aspect, 20 pg / mg or more in a certain aspect, 25 pg / mg or more in a certain aspect, 30 pg / mg or more in a certain aspect, 35 pg / mg or more in a certain aspect, 40 pg / mg or more in a certain aspect, 45 pg / mg or more in a certain aspect, 50 pg / mg or more in a certain aspect, 60 pg / mg or more in a certain aspect, 80 pg / mg or more in a certain aspect, 100 pg / mg or more in a certain aspect, 110 pg / mg or more in a certain aspect, 120 pg / mg or more in a certain aspect, 130 pg / mg or more in a certain aspect, 135 pg / mg or more in a certain aspect. The upper limit is also not particularly limited. Preferably, it is 5000 pg / mg or less in a certain aspect, 4000 pg / mg or less in a certain aspect, 3000 pg / mg or less in a certain aspect, 2000 pg / mg or less in a certain aspect, 1000 pg / mg or less in a certain aspect, 900 pg / mg or less in a certain aspect, 800 pg / mg or less in a certain aspect, 700 pg / mg or less in a certain aspect, 600 pg / mg or less in a certain aspect, 500 pg / mg or less in a certain aspect, 450 pg / mg or less in a certain aspect, 400 pg / mg or less in a certain aspect, 350 pg / mg or less in a certain aspect, 300 pg / mg or less in a certain aspect, 250 pg / mg or less in a certain aspect, 200 pg / mg or less in a certain aspect, 150 pg / mg or less in a certain aspect. The upper limit and the lower limit can be appropriately combined as the range of the content of laminin.

[0069] By making the content of laminin within the above range, excellent cell viability is thereby demonstrated.

[0070] In this specification, for example, the detection and quantification of laminin can be carried out as follows. Immunohistochemical staining of laminin is performed on sections of the decellularized cell aggregates to detect laminin. Then, the content of laminin in the decellularized cell aggregates is quantified using an enzyme immunoassay (ELISA; Enzyme Linked Immunosorbent Assay).

[0071] For example, immunohistochemical staining can be performed as described below. A section sample of the decellularized cell aggregate is treated with a primary antibody and a secondary antibody. Then, it is developed with DAB (3,3'-diaminobenzidine tetrahydrochloride) / hydrogen peroxide, dehydrated, and mounted.

[0072] For example, an enzyme immunoassay can be performed as described below. Using the extract of the decellularized cell aggregate, the lamin protein content of the decellularized cell aggregate is quantified by ELISA.

[0073] 《DNA Content》

[0074] The decellularized cell aggregate is not limited, and the DNA content per unit dry mass is 1.000 mass% or less. The DNA content per unit dry mass of the decellularized cell aggregate is 0.900 mass% or less, preferably 0.800 mass% or less, more preferably 0.700 mass% or less, further preferably 0.600 mass% or less, further preferably 0.500 mass% or less, further preferably 0.400 mass% or less, further preferably 0.300 mass% or less, further preferably 0.250 mass% or less. Thus, by appropriately performing decellularization, a decellularized cell aggregate with less rejection during transplantation can be obtained.

[0075] The DNA content can be measured by the PicoGreen method. After immersing a dry test piece of the decellularized cell aggregate (hereinafter sometimes referred to as a sample) in a proteolytic enzyme solution and dissolving it, it is treated with phenol / chloroform to remove proteins, and the DNA is recovered. After treating with phenol / chloroform to remove proteins, an ethanol precipitation method can be performed. The recovered DNA is fluorescently stained with PicoGreen (Life Technologies) and the fluorescence intensity is measured, thereby quantifying the DNA and calculating the DNA content (mass) of the sample. Quantification can utilize a standard curve prepared using the standard DNA attached to PicoGreen. Based on the dry mass of the sample and the DNA content, the DNA ratio is calculated according to the following formula.

[0076] (DNA content per unit dry mass (hereinafter sometimes referred to as decellularized DNA ratio) = (DNA content of the dry test piece of the decellularized cell aggregate) / (mass of the dry test piece of the decellularized cell aggregate) × 100 (mass%)

[0077] 《Cell Culture Substrate》

[0078] The acellularized cell aggregates of the present invention can be used as a substrate for cell culture. As a culture method, in addition to using the acellularized cell aggregates of the present invention, known methods can be used. For example, a specified amount of a culture medium or a culture reagent can be added to a cell culture plate or a cell culture well, the acellularized cell aggregates of the present invention can be added, cells can be seeded, and the cells can be cultured under specified temperature and CO2 concentration conditions. The culture medium or the culture reagent can be appropriately selected according to the cells to be cultured.

[0079] The cells to be cultured are not particularly limited. For example, the same cells as those used for the "cultured cell aggregates" can be mentioned.

[0080] (Cell viability)

[0081] When the acellularized cell aggregates of the present invention are used as a substrate for cell culture, excellent cell viability is exhibited ( Figure 4 ). The cell viability can be analyzed by counting the number of cells after culturing the acellularized cell aggregates and the cells, but as shown in the examples, it can also be analyzed by measuring ATP.

[0082] 《Transplant piece》

[0083] The acellularized cell aggregates of the present invention can be used as a transplant piece for an organism. The acellularized cell aggregates of the present invention have excellent biocompatibility, and by being transplanted into an organism, for example, they can become a suitable scaffold for cell proliferation.

[0084] Examples of the organism into which the acellularized cell aggregates of the present invention are transplanted include humans, monkeys, dogs, cats, rabbits, ferrets, sheep, goats, cows, pigs, horses, camels, mice, rats, hamsters, guinea pigs, chickens, or gerbils.

[0085] Diseases that are the transplantation targets of the graft of the present invention include cytopenic diseases (cell loss diseases). Examples of cells that are reduced (lost) in a living body include hepatocytes, stellate cells, Kupffer cells, vascular endothelial cells, sinusoidal endothelial cells, endothelial cells, fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, epidermal cells, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells, mammary cells, pericytes, smooth muscle cells, cardiomyocytes, muscle cells, renal cells, pancreatic islet cells, peripheral nerve cells, nerve cells, chondrocytes or bone cells. For example, as undifferentiated adherent cells, there can be mentioned embryonic stem cells (ES cells), embryonic germ cells (EG cells), germ line 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, adipose progenitor cells, skin fibroblasts, skeletal muscle myoblasts, osteoblasts or odontoblasts. By transplanting the graft of the present invention into the diseases with reduced cells as described above, cells can be effectively increased. In addition, the above cells can be transplanted into a living body simultaneously with the transplantation of the graft of the present invention.

[0086] In addition, the graft of the present invention can also transplant the composition to an individual in which the cells are not reduced in a living body. By administering the graft, various cells can be proliferated.

[0087] 《Composition》

[0088] The composition of the present invention comprises a decellularized cell condensate and cells. Examples of the cells include hepatocytes, stellate cells, Kupffer cells, vascular endothelial cells, sinusoidal endothelial cells, endothelial cells, fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, epidermal cells, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells, mammary cells, pericytes, smooth muscle cells, cardiomyocytes, muscle cells, renal cells, pancreatic islet cells, peripheral nerve cells, nerve cells, chondrocytes or bone cells. For example, as undifferentiated adherent cells, there may be mentioned embryonic stem cells (ES cells), embryonic germ cells (EG cells), germ line stem cells (GS cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, hematopoietic stem cells, neural stem cells, cardiac progenitors, vascular endothelial progenitors, neural progenitors, adipose progenitors, skin fibroblasts, skeletal muscle myoblasts, osteoblasts, or odontoblasts. Two or more types of cells may be used. Immortalized cells as described in the cells for the cultured cell condensate may also be used for the cells.

[0089] The ratio of the cells to the decellularized cell condensate is not particularly limited as long as it is a cell that can be physically contained relative to the decellularized cell condensate. For example, relative to 1 mg of the decellularized cell condensate, 1.0×10 3 ~1.0×10 7 cells can be contained. The lower limit of the cell number is 2.0×10 3 or more in some embodiments, 5.0×10 3 or more in some embodiments, 1.0×10 4 or more in some embodiments, 2.0×10 4 or more in some embodiments, 5.0×10 4 or more. The upper limit of the cell number is 5.0×10 6 or less in some embodiments, 1.0×10 6 or less in some embodiments, 5.0×10 5 or less, 2.0×10 5 or less in some embodiments, 1.0×10 5 or less in some embodiments. The lower limit and the upper limit can be arbitrarily combined as the range of the ratio of the cells to the decellularized cell condensate. By being within the said range, the survival number of the cells can be increased.

[0090] The composition of the present invention is not particularly limited as long as it contains a decellularized cell aggregate and cells, and can be used for culturing cells in vitro. In addition, it can be administered to an organism, for example, for autologous transplantation or allogeneic transplantation such as regenerative medicine or cell therapy.

[0091] [2] Method for preparing decellularized cell aggregate

[0092] One embodiment of the method for preparing the decellularized cell aggregate of the present invention includes a cultured cell aggregate formation step of forming a cultured cell aggregate by culturing cells, and a step of decellularizing the cell aggregate. And, on the surface of the aggregate, every 11088 μm 2 has 3 to 45 surface pores with a pore diameter of 5 μm or more.

[0093] One embodiment of the method for preparing the decellularized cell aggregate of the present invention includes a cultured cell aggregate formation step of forming a cultured cell aggregate by culturing cells, and a step of decellularizing the cell aggregate. And, it contains 10 pg / mg or more of laminin with respect to the decellularized cell aggregate.

[0094] The decellularized cell aggregate obtained by the method for preparing the decellularized cell aggregate of the present invention, on the surface of the aggregate, every 11088 μm 2 has 3 to 45 surface pores with a pore diameter of 5 μm or more, and contains 10 pg / mg or more of laminin with respect to the decellularized cell aggregate.

[0095] "Cultured cell aggregate formation step (1)"

[0096] In the cultured cell aggregate formation step (1), a cultured cell aggregate is formed by culturing cells. The cultured cell aggregate is not particularly limited as long as it is a cell aggregate obtained by culturing in vitro, and examples thereof include the "cultured cell aggregate" described in the above item "[1] Decellularized cell aggregate". The cultured cell aggregate can be formed by allowing cells to produce extracellular matrix (ECM) outside the cells and causing the cells to adhere and bind to each other.

[0097] In the case of an ellipsoid as one of the cultured cell aggregates, as a three-dimensional culture method, for example, a non-adhesive surface culture method, a hanging drop culture method, or a rotary culture method can be cited. Hereinafter, the formation of the ellipsoid in the specific cultured cell aggregate formation step (1) will be described.

[0098] The production of cultured cell aggregates (such as ellipsoids) can be carried out as follows. Adherent cells cannot survive for a long time in a suspended state in a solution. If adherent cells are placed in a non-adhesive environment, the cells seek a scaffold and adhere to each other to form cell aggregates.

[0099] Specifically, by aggregating a plurality of cells, cultured cell aggregates (such as ellipsoids) can be prepared. After monolayer culturing the cells, they are transferred to a hydrophobic or cell non-adhesive round-bottom multi-well or U-shaped plate (dimple plate) for cultivation, and the cells aggregate to produce cultured cell aggregates. The culture medium used in cell culture for making cultured cell aggregates (such as ellipsoids) can be selected according to the cells used. From the perspective of efficiency, the cultivation time until the production of cultured cell aggregates is preferably 6 to 48 hours, more preferably 6 to 24 hours. However, the method for preparing cultured cell aggregates is not limited to the above method, and a rotary culture method of adding a cell suspension to a rotating solution, a method of adding a cell suspension to a test tube and precipitating it with a centrifuge, or an alginate bead method can be used. From the perspective of being able to handle a large number of uniform cell aggregates, the method of adding a cell suspension to a hydrophobic or cell non-adhesive multi-well is effective and thus preferred.

[0100] By adjusting the cultivation time of the cultured cell aggregates (such as ellipsoids), the cultured cell aggregates can be cultured to any thickness. In addition, by joining the cultured cell aggregates (such as ellipsoids) to each other, cultured cell aggregates of any thickness and shape can be cultured. By further culturing the cultured cell aggregates (such as ellipsoids) obtained in this way, cultured cell aggregates that further secrete extracellular matrix from the cells, adhere to each other, and maintain a three-dimensional shape can be obtained. In addition, by culturing using a method of putting the cultured cell aggregates (such as ellipsoids) into a cavity as described below, a cell mass of cultured cell aggregates in which multiple ellipsoids are combined and have a desired three-dimensional shape can be correctly produced. In addition, a method of preparing a sheet-like three-dimensional structure, a method of laminating multiple cell sheets, etc. can also be used to produce a cell mass.

[0101] For the culture medium used in cell culture for producing cultured cell aggregates (such as cell pellets), a suitable culture medium can be selected according to the cells used. The cultured cell aggregate is placed into a cavity surrounded by a reticular or comb-shaped support with gaps finer than the size of the cultured cell aggregate. The cavity is formed into a desired shape by the reticular or comb-shaped support and a plate, and has a plurality of opening portions on the support. The culture medium can contact the cultured cell aggregate through these opening portions. While the cultured cell aggregate is held in the cavity, the shaking culture container is shaken, so that the culture medium effectively contacts the cultured cell aggregate, and the cultured cell aggregate can be cultured well. The cultured cell aggregate further produces extracellular matrix, and the cell aggregates begin to adhere to each other, enabling the production of a cell pellet with a three-dimensional structure of the desired shape.

[0102] The cells used for producing the cultured cell aggregate are not particularly limited, and the cells described in the above item "[1] Decellularized cell aggregate" can be used.

[0103] The cells in the cultured cell aggregate formation process are not limited, and immortalized cells are preferred. Thus, it is speculated that when producing a decellularized cell aggregate using the preparation method of the decellularized cell aggregate, by containing more lamin proteins, the area retention rate and the number of surface pores can be effectively maintained at specified values. Thereby, the cell viability can be improved when cells are seeded on the decellularized cell aggregate.

[0104] In addition, in another embodiment, the culture of the cells in the cultured cell aggregate formation process is preferably the culture of two or more types of cells. It is speculated that when cells are co-cultured and a decellularized cell aggregate is produced using the preparation method of the decellularized cell aggregate, since it contains more lamin proteins, the area retention rate and the number of surface pores can be effectively maintained at specified values. Thereby, the cell viability can be improved when cells are seeded on the decellularized cell aggregate.

[0105] For the procedure of producing cultured cell aggregates (such as ellipsoids) by co - culture, after monolayer - culturing the same type of cells with each other and then detaching them, different types of cells are mixed in a ratio that results in a specified number of cells to prepare a suspension. Then, in the same manner as for producing the above - mentioned cultured cell aggregates (such as ellipsoids), it is transferred to a hydrophobic or cell - non - adherent round - bottom multi - well or U - shaped plate (concave - well plate) for cultivation, that is, co - culture, thereby proceeding. The cells may or may not form ellipsoids at the start of co - culture. From the perspective of achieving the effects of the invention of the present application, it is preferred that ellipsoids are not formed at the start of co - culture. The co - culture of cells during the production of ellipsoids can use more than two types of cells. From the perspective of further achieving the effects of the invention of the present application, it is preferred to use three types of cells, and more preferably two types of cells. For example, when using two types of cells, the mixing ratio of the two types of cells is 1:99 to 99:1, in a certain embodiment it is 10:90 to 90:10, in a certain embodiment it is 20:80 to 80:20, in a certain embodiment it is 30:70 to 70:30, in a certain embodiment it is 40:60 to 60:40, and in a certain embodiment it is 50:50.

[0106] The cells used in the co - culture during the production of the ellipsoids are not particularly limited and can be selected from the cells used for the above - mentioned cultured cell aggregates. From the perspective of further achieving the effects of the invention of the present application, it is preferred to select from fibroblasts and endothelial cells, and more preferably skin fibroblasts and vascular endothelial cells. The cells used in the co - culture during the production of the ellipsoids can be immortalized. From the perspective of further achieving the effects of the invention of the present application, it is most preferably skin fibroblasts and immortalized vascular endothelial cells. As long as the effects of the present invention can be obtained, the ratio of skin fibroblasts to vascular endothelial cells is not particularly limited. For example, it is 2:8 to 8:2, in a certain embodiment it is 3:7 to 7:3, and in a certain embodiment it is 4:6 to 6:4. In addition, there can also be an embodiment where the number of fibroblasts is more than that of vascular endothelial cells. The ratio of skin fibroblasts to vascular endothelial cells is preferably 2:1 to 15:1, more preferably 4:1 to 12:1, and even more preferably 6:1 to 10:1. The upper and lower limits can be appropriately combined. Although not limited, by being within the above - mentioned range, the invention of the present application can further achieve the effect of higher cell viability during cell seeding.

[0107] 《Decellularization Process (2)》

[0108] In the decellularization step (2), the cultured cell aggregates are decellularized. As long as the effects of the present invention can be obtained, the decellularization treatment is not particularly limited, and examples thereof include high hydrostatic pressure treatment, freeze-thaw treatment, ultrasonic treatment, methods using enzymes, or methods using hypertonic electrolyte solutions, methods based on physical agitation, hypertonic solution hypotonic solution methods, methods using enzyme treatment based on proteolytic enzymes or nuclease enzymes, treatments based on alcohol solvents, etc. Two or more of them can be combined. In order to effectively obtain a decellularized cell structure and to exert the effects of the invention of the present application, a method based on high hydrostatic pressure treatment is preferred.

[0109] "High Hydrostatic Pressure Treatment"

[0110] When obtaining the decellularized aggregates using the method based on the high hydrostatic pressure treatment, a hydrostatic pressure of 50 to 1500 MPa is applied to the obtained cultured cell aggregates in a medium. From the perspective of sufficient decellularization, the applied hydrostatic pressure is preferably 50 MPa or more. There is no need for a pressure vessel that can withstand pressurization, and no extremely large energy is required. At the same time, when the medium for pressurization is an aqueous medium, ice will be generated. From the perspective of preventing damage to the cultured cell aggregates due to the generated ice, the applied hydrostatic pressure is preferably 1500 MPa or less. The applied hydrostatic pressure is more preferably 80 to 1300 MPa, further preferably 90 to 1200 MPa, further more preferably 95 to 1100 MPa, further preferably 95 to 700 MPa, and most preferably 400 to 700 MPa from the perspective of exerting the decellularization effect, sterilization effect, and virus inactivation effect and from the ease of pressurization.

[0111] Examples of the medium used during the pressurization of the hydrostatic pressure include water, physiological saline, water for injection, propylene glycol or its aqueous solution, glycerol or its aqueous solution, sugar aqueous solutions, etc. Examples of the buffer solution include acetic acid buffer solution, phosphate buffer solution, citric acid buffer solution, boric acid buffer solution, tartaric acid buffer solution, Tris buffer solution, HEPES buffer solution, MES buffer solution, etc. These media can contain surfactants.

[0112] As long as the temperature does not generate ice and does not cause damage to the cultured cell aggregates due to heat, the temperature of the high hydrostatic pressure treatment is not particularly limited. From the perspective of smoothly performing the decellularization treatment and having little impact on the cultured cell aggregates, the temperature of the high hydrostatic pressure treatment is preferably 0 to 45 °C, further preferably 4 to 37 °C, and most preferably 15 to 36 °C. If the time of the high hydrostatic pressure treatment is too short, the cells will not be sufficiently destroyed. When the time of the high hydrostatic pressure treatment is long, it will result in a waste of energy. Therefore, in the high hydrostatic pressure treatment, the time for maintaining the target applied pressure is preferably 1 to 120 minutes, more preferably 5 to 60 minutes, and further preferably 7 to 30 minutes.

[0113] The cultured cell aggregates that have undergone the high hydrostatic pressure treatment are preferably treated with a nuclease. The nuclease is a substance that removes nucleic acid components from the cultured cell aggregates to which hydrostatic pressure has been applied, and there is no particular limitation thereto. Examples thereof include DNases derived from the pancreas, spleen, or Escherichia coli (for example, DNase I and DNase II).

[0114] The nuclease can be added to the medium used for the high hydrostatic pressure treatment (for example, water, physiological saline, injection solution, or buffer solution, etc.) to exert its effect. The amount of the enzyme added varies depending on the type of the enzyme or the definition of the unit number (U, unit), and those skilled in the art can appropriately set it. For example, if it is DNase I, it can be used at 50 to 2000 U / mL. The treatment temperature also varies depending on the nuclease used. For example, a temperature of 1°C to 40°C can be set. The treatment time is not particularly limited either. For example, it can be 1 to 120 hours (preferably 1 to 96 hours, more preferably 1 to 72 hours). In the case of low temperature, long-term treatment can be carried out, and in the case of high temperature, short-term treatment can be carried out.

[0115] The cultured cell aggregates that have undergone the high hydrostatic pressure treatment are washed with a washing solution. The washing solution can be the same as or different from the medium for the high hydrostatic pressure treatment. The washing solution may contain an organic solvent or a chelating agent. The organic solvent can improve the removal efficiency of lipids, and the chelating agent can prevent calcification when the granulated decellularized cell aggregates of the present invention are applied to the diseased part by inactivating calcium ions or magnesium ions in the decellularized cell aggregates. As the organic solvent, from the perspective of high lipid removal effect, a water-soluble organic solvent is preferred, and ethanol, isopropanol, acetone, and dimethyl sulfoxide are preferred. As the chelating agent, examples include iminocarboxylic acid-based 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), ethylene glycol bis(2-aminoethyl ether) tetraacetic acid (GEDTA), dicarboxymethylglutamic acid (CMGA), 3-hydroxy-2,2'-iminodisuccinic acid (HIDA), dicarboxymethylaspartic acid (ASDA), etc., or salts thereof; hydroxycarboxylic acid-based chelating agents such as citric acid, tartaric acid, malic acid, and lactic acid, or salts thereof. As salts of these chelating agents, sodium salts or potassium salts can be cited.

[0116] The washing temperature is not particularly limited as long as it is a temperature that does not damage the cultured cell aggregates due to heat, but is preferably 0 to 45° C., more preferably 1 to 40° C., and most preferably 2 to 35° C. from the viewpoint of good washing performance and little effect on the cultured cell aggregates. During washing, the washing liquid may be shaken or stirred as needed.

[0117] By decellularizing the cultured cell aggregate obtained in step (1) under the high hydrostatic pressure treatment under the above conditions, it is possible to obtain a cell-free aggregate having a surface area of ​​11088 μm 2 A decellularized cell aggregate having 3 to 45 surface pores with a pore diameter of 5 μm or more, or a decellularized cell aggregate containing 10 pg / mg or more of lamin protein relative to the decellularized cell aggregate.

[0118] 《Freeze-thaw treatment》

[0119] When the decellularized cell aggregate is obtained by a method based on freeze-thaw treatment, it is preferred to repeat the following step once or twice or more (preferably 2 to 5 times): after freezing the cultured cell aggregate at a temperature of -85 to -20°C (preferably -82 to -40°C) for 15 minutes to 48 hours (preferably 20 minutes to 15 hours), it is thawed at a temperature of 20 to 40°C. Then, it is preferably treated with a nucleic acid degrading enzyme, which can be carried out using the same method as the treatment in the high hydrostatic pressure treatment. The cells in the cultured cell aggregate subjected to freeze-thaw treatment are destroyed, and the cells can be removed by a cleaning solution. The cleaning method can be the same method as the cleaning in the high hydrostatic pressure treatment.

[0120] By decellularizing the cultured cell aggregate obtained in step (1) using freeze-thaw treatment under the above conditions, it is possible to obtain a cell-freeze solution per 11088 μm on the aggregate surface. 2 A decellularized cell aggregate having 3 to 45 surface pores with a pore diameter of 5 μm or more, or a decellularized cell aggregate containing 10 pg / mg or more of lamin protein relative to the decellularized cell aggregate.

[0121] Ultrasonic treatment

[0122] When the decellularized cell aggregate is obtained by a method using ultrasonic treatment, for example, the cultured cell aggregate is subjected to ultrasonic treatment (for example, intensity: 10 W / cm 2, Frequency: 10 kHz, Action time: 2 minutes). Then, preferably, a surfactant solution (e.g., 1% by mass Triton X (polyoxyethylene octylphenyl ether) solution) is oscillated at 2 - 10 °C (preferably 4 °C) for 1 - 120 hours (preferably 12 - 120 hours). Further, nuclease treatment is preferably performed, and the nuclease treatment can be carried out by the same method as the treatment in the high hydrostatic pressure treatment. Then, washing is preferably carried out by the same method as the washing in the high hydrostatic pressure treatment.

[0123] By subjecting the cultured cell aggregate obtained in step (1) to decellularization by ultrasonic treatment under the above conditions, a decellularized cell aggregate having 3 - 45 surface pores with a pore diameter of 5 μm or more per 11088 μm on the aggregate surface can be obtained, 2 or a decellularized cell aggregate containing 10 pg / mg or more of lamin in the decellularized cell aggregate.

[0124] 《Surfactant Treatment》

[0125] When obtaining the decellularized cell aggregate by a method using a surfactant, preferably, the cultured cell aggregate is oscillated in a surfactant solution (e.g., RIPA buffer) at 2 - 10 °C (preferably 4 °C) for 1 - 48 hours (preferably 12 - 36 hours). Then, nuclease treatment is preferably performed, and the nuclease treatment can be carried out by the same method as the treatment in the high hydrostatic pressure treatment. Then, washing is preferably carried out by the same method as the washing in the high hydrostatic pressure treatment.

[0126] The surfactant is not limited. For example, RIPA buffer (manufactured by NACALAI TESQUE, INC.), sodium dodecyl sulfate (SDS), alkyl sulfonate, alkyl sulfate, polyoxyethylene alkyl sulfate, α-sulfo fatty acid ester salt, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether (e.g., polyoxyethylene octylphenyl ether, Nonidet (registered trademark) P-40 (NP-40)), alkyl (poly)glycoside, alcohol ethoxylates can be cited. Preferably, it is RIPA buffer, Nonidet (registered trademark) P-40 (NP-40), or alcohol ethoxylates. Although not limited, in order to exert the effects of the invention of the present application, a surfactant with relatively weak detergency is preferred.

[0127] The obtained decellularized cell aggregate is not limited, and freeze-drying treatment can be performed. Freeze-drying can be omitted according to the part of the cell aggregate. In addition, the obtained decellularized cell aggregate can be sterilized by gamma-ray irradiation, UV irradiation, etc.

[0128] "Area retention rate"

[0129] The acellularized cell aggregate obtained by the preparation method of the present invention has a certain area retention rate. The area retention rate refers to the percentage of the acellularized cell aggregate that has been acellularized in step (2) relative to the cultured cell aggregate in step (1).

[0130] As long as the effects of the present invention can be obtained, the area retention rate is not particularly limited. Preferably, the lower limit is 50% or more, in a certain aspect is 55% or more, in a certain aspect is 60% or more, in a certain aspect is 65% or more, in a certain aspect is 70% or more. The upper limit is not particularly limited, and is preferably 99% or less, in a certain aspect is 95% or less, in a certain aspect is 90% or less. The upper limit and the lower limit can be appropriately combined as the range of the area retention rate. In addition, by subjecting the cultured cell aggregate obtained in step (1) to acellularization by high hydrostatic pressure treatment, freeze-thaw treatment or ultrasonic treatment under the above conditions, an area retention rate of 50-99% can be obtained.

[0131] "Surface pores"

[0132] The acellularized cell aggregate obtained by the preparation method of the present invention has 3 to 45 surface pores with a pore diameter of 5 μm or more per 11088 μm on the surface of the aggregate. 2 has 3 to 45 surface pores with a pore diameter of 5 μm or more per 11088 μm on the surface of the aggregate. 2The number of surface pores is not particularly limited as long as it is 3 to 45. Preferably, the lower limit is 3 or more in a certain aspect, 4 or more in a certain aspect, 5 or more in a certain aspect, 6 or more in a certain aspect, 7 or more in a certain aspect, 8 or more in a certain aspect, 9 or more in a certain aspect, 10 or more in a certain aspect, 11 or more in a certain aspect, 12 or more in a certain aspect, 13 or more in a certain aspect, 14 or more in a certain aspect, 15 or more in a certain aspect. The upper limit of the number of surface pores is 45 or less, preferably 43 or less in a certain aspect, 41 or less in a certain aspect, 39 or less in a certain aspect, 37 or less in a certain aspect, 35 or less in a certain aspect, 33 or less in a certain aspect, 31 or less in a certain aspect, 30 or less in a certain aspect, 28 or less in a certain aspect, 26 or less in a certain aspect, 24 or less in a certain aspect, 22 or less in a certain aspect, 20 or less in a certain aspect, 18 or less in a certain aspect. The upper and lower limits can be combined as the range of the number of surface pores. By being within this range, the survival number of cells can be increased. If the intensity of the high hydrostatic pressure treatment (such as hydrostatic pressure, time, temperature) is enhanced, the number of surface pores increases, and if the intensity of the high hydrostatic pressure treatment (such as hydrostatic pressure, time, temperature) is weakened, the number of surface pores decreases. In addition, if the number of freeze-thaw cycles is increased, the number of surface pores increases, and if the number of freeze-thaw cycles is decreased, the number of surface pores decreases. Further, if the intensity of the ultrasonic treatment (such as intensity, frequency, time) is increased, the number of surface pores increases, and if the intensity of the ultrasonic treatment (such as intensity, frequency, time) is weakened, the number of surface pores decreases. Therefore, those skilled in the art can obtain acellular cell aggregates with various numbers of surface pores by adjusting the intensity of the decellularization treatment according to the description in the specification of this application.

[0133] "Residual Laminin"

[0134] The decellularized cell aggregates obtained by the preparation method of the present invention contain more than 10 pg / mg of laminin relative to the decellularized cell aggregates. The content of laminin is not particularly limited as long as it is 10 pg / mg or more. Preferably, the lower limit is 15 pg / mg or more in a certain aspect, 18 pg / mg or more in a certain aspect, 20 pg / mg or more in a certain aspect, 25 pg / mg or more in a certain aspect, 30 pg / mg or more in a certain aspect, 35 pg / mg or more in a certain aspect, 40 pg / mg or more in a certain aspect, 45 pg / mg or more in a certain aspect, 50 pg / mg or more in a certain aspect, 60 pg / mg or more in a certain aspect, 80 pg / mg or more in a certain aspect, 100 pg / mg or more in a certain aspect, 110 pg / mg or more in a certain aspect, 120 pg / mg or more in a certain aspect, 130 pg / mg or more in a certain aspect, 135 pg / mg or more in a certain aspect. The upper limit is also not particularly limited. Preferably, it is 5000 pg / mg or less in a certain aspect, 4000 pg / mg or less in a certain aspect, 3000 pg / mg or less in a certain aspect, 2000 pg / mg or less in a certain aspect, 1000 pg / mg or less in a certain aspect, 900 pg / mg or less in a certain aspect, 800 pg / mg or less in a certain aspect, 700 pg / mg or less in a certain aspect, 600 pg / mg or less in a certain aspect, 500 pg / mg or less in a certain aspect, 450 pg / mg or less in a certain aspect, 400 pg / mg or less in a certain aspect, 350 pg / mg or less in a certain aspect, 300 pg / mg or less in a certain aspect, 250 pg / mg or less in a certain aspect, 200 pg / mg or less in a certain aspect, 150 pg / mg or less in a certain aspect. The upper limit and the lower limit can be appropriately combined as the range of the content of laminin. By making the content of laminin within the said range, excellent cell viability is thus exhibited. If the intensity of the high hydrostatic pressure treatment (such as hydrostatic pressure, time, temperature) is increased, the content of the said laminin decreases. If the intensity of the high hydrostatic pressure treatment (such as hydrostatic pressure, time, temperature) is weakened, the content of the said laminin increases. In addition, if the number of freeze-thaw cycles is increased, the content of the said laminin decreases. If the number of freeze-thaw cycles is decreased, the content of the said laminin increases. Further, if the intensity of the ultrasonic treatment (such as intensity, frequency, time) is enhanced, the content of the said laminin decreases. If the intensity of the ultrasonic treatment (such as intensity, frequency, time) is weakened, the content of the said laminin increases.Therefore, those skilled in the art can obtain decellularized cell aggregates with various lamin protein contents by adjusting the intensity of the decellularization process according to the description in the specification of the present application.

[0135] The size of the decellularized cell aggregate is not particularly limited. In the decellularized ellipsoid, the lower limit is preferably 80 μm or more, preferably 90 μm or more, preferably 95 μm or more, preferably 100 μm or more. The upper limit is preferably 600 μm or less, preferably 500 μm or less, preferably 300 μm or less. In the decellularized cell mass, the lower limit is preferably 90 μm or more, preferably 100 μm or more, preferably 150 μm or more, preferably 200 μm or more. The upper limit is preferably 2100 μm or less, preferably 2000 μm or less, preferably 1700 μm or less, preferably 1400 μm or less, preferably 1200 μm or less, preferably 1000 μm or less. The upper and lower limits can be combined as the size range of the decellularized cell aggregate. By being within this range, the number of surviving cells can be increased.

[0136] In the present disclosure text, the following aspects can be cited.

[0137] [1] A decellularized cell aggregate, which is a decellularized cell aggregate obtained by decellularizing a cultured cell aggregate, and has 3 to 45 surface pores with a pore diameter of 5 μm or more per 11088 μm on the surface of the aggregate. 2

[0138] [2] A decellularized cell aggregate, which is a decellularized cell aggregate obtained by decellularizing a cultured cell aggregate, and contains 10 pg / mg or more of lamin protein relative to the decellularized cell aggregate.

[0139] [3] A decellularized cell aggregate, which is a decellularized cell aggregate obtained by decellularizing a cultured cell aggregate, has 3 to 45 surface pores with a pore diameter of 5 μm or more per 11088 μm on the surface of the aggregate, and moreover, contains 10 pg / mg or more of lamin protein relative to the decellularized cell aggregate. 2

[0140] [4] The decellularized cell aggregate according to any one of [1] to [3], wherein the cells of the cultured cell aggregate are immortalized cells.

[0141] [5] The decellularized cell aggregate according to any one of [1] to [4], wherein the cells of the cultured cell aggregate are a mixture of two or more types of cells.

[0142] [6] The decellularized cell aggregate according to any one of [1] to [5], wherein the DNA content per unit dry mass of the decellularized cell aggregate is 1.000 mass% or less.

[0143] [7] A substrate for cell culture, which comprises the decellularized cell aggregate according to any one of [1] to [6].

[0144] [8] A graft for an organism, which comprises the decellularized cell aggregate according to any one of [1] to [6].

[0145] [9] A composition, which comprises the decellularized cell aggregate according to any one of [1] to [6] and cells.

[0146]

[10] A method for preparing a decellularized cell aggregate, which includes a step of forming a cultured cell aggregate by culturing cells to form a cultured cell aggregate, and a step of decellularizing the cultured cell aggregate. The decellularized cell aggregate has 3 to 45 surface pores with a pore diameter of 5 μm or more per 11088 μm on the aggregate surface. 2 It has 3 to 45 surface pores with a pore diameter of 5 μm or more.

[0147]

[11] A method for preparing a decellularized cell aggregate, which includes a step of forming a cultured cell aggregate by culturing cells to form a cultured cell aggregate, and a step of decellularizing the cultured cell aggregate. The decellularized cell aggregate contains 10 pg / mg or more of lamin in terms of the decellularized cell aggregate.

[0148]

[12] The method for preparing a decellularized cell aggregate according to

[10] or

[11] , wherein the cells are immortalized cells.

[0149]

[13] The method for preparing a decellularized cell aggregate according to any one of

[10] to

[12] , wherein the culturing of the cells is the culturing of two or more types of cells.

[0150]

[14] The method for preparing a decellularized cell aggregate according to any one of

[10] to

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

[0151] In addition, the present invention is not limited to the above embodiments. The above embodiments are illustrative, and technical solutions having a configuration substantially the same as the technical concept described in the claims of the present invention and exhibiting the same effects are all included in the protection scope of the present invention.

[0152] Examples

[0153] Hereinafter, the present invention will be specifically described using examples, but these examples do not limit the scope of the present invention.

[0154] <<Example 1>>

[0155] In this example, decellularized cell aggregates (decellularized ellipsoids) were prepared using fibroblasts.

[0156] (Preparation of cultured cell aggregates (ellipsoids))

[0157] NHDF-Neo-skin fibroblasts (CC-2509, manufactured by Lonza K.K.) were cultured in a T-175 flask (Vent Cap, 175 cm 2 , manufactured by VIOLAMO). When the confluence rate reached about 90 - 100%, the cells were detached, and a cell suspension (20 mL) (about 2.0×10 7 cells) was prepared using FKCM medium (serum-free medium for fibroblasts, manufactured by FUKOKU Co., Ltd.). A well plate was placed in a 10 cm petri dish, and the above cell suspension was added throughout the well plate, and then cultured in a 37°C, 5% CO2 incubator (IP400, manufactured by Yamato Scientific Co., Ltd.). After culturing for more than one night, the ellipsoids (aggregation of cells) in the well plate were confirmed by an inverted microscope (CKX31, manufactured by Olympus Corporation). The ellipsoids were pipetted out from the wells of the well plate and transferred to a 10 cm petri dish. The 10 cm petri dish was rotated to gather the ellipsoids at the center and then recovered to obtain the ellipsoids.

[0158] (Decellularization treatment of ellipsoids)

[0159] The ellipsoids and FKCM medium were placed in a nylon-polyester vacuum bag (manufactured by Kurilon Chemicals, Japan), and the four corners were double-sealed 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.). The high hydrostatic pressure treatment was carried out in a temperature range of minimum temperature: 23.3°C, maximum temperature: 35.9°C. The ellipsoids that had undergone high hydrostatic pressure treatment were recovered from the nylon-polyester vacuum bag and transferred to a centrifuge tube, and then washed with distilled water for injection. Then, the ellipsoids that had undergone high hydrostatic pressure treatment were immersed in a DNaseI solution (800 U / mL) of nuclease and washed with shaking at 4°C for more than 18 hours. Then, after washing 3 times with distilled water for injection, freeze-drying was performed to obtain decellularized ellipsoids (decellularized cell aggregates).

[0160] Example 2

[0161] In this example, a decellularized ellipsoid was prepared by freeze-thaw treatment.

[0162] After washing the ellipsoid obtained in Example 1 with water for injection, it was suspended in phosphate buffer (PBS, 0.01 M, pH 7.4), frozen with dry ice or a refrigerator, stored at -80 °C for 30 minutes or more, and then dissolved in a water bath at 37 °C. The ellipsoid that had undergone the freeze-thaw process three times was immersed in a DNase I solution (800 U / mL) of nuclease and washed with shaking at 4 °C for 18 hours or more. Then, it was washed three times with water for injection and freeze-dried to obtain a decellularized ellipsoid.

[0163] Example 3

[0164] In this example, immortalized cells were used to prepare a decellularized cell aggregate (decellularized ellipsoid).

[0165] (Preparation of cultured cell aggregate (ellipsoid))

[0166] Except for using OUMS-36T-1 immortalized fibroblasts (JCRB1006.1, JCRB cell bank) instead of NHDF-Neo-dermal fibroblasts, the same operations as in Example 1 were repeated to obtain an ellipsoid.

[0167] (Decellularization treatment of ellipsoid)

[0168] Using the same procedure as in Example 1, a decellularized ellipsoid (decellularized cell aggregate) was obtained.

[0169] Example 4

[0170] In this example, fibroblasts and endothelial cells were used to prepare a decellularized cell aggregate (decellularized ellipsoid).

[0171] (Preparation of cultured cell aggregate (ellipsoid))

[0172] NHDF-Neo-dermal fibroblasts (CC-2509, manufactured by Lonza K.K.) and HUEhT-1 immortalized vascular endothelial cells (JCRB1458, JCRB cell bank) were cultured separately in T-175 flasks (vented caps, 175 cm 2 , manufactured by VIOLAMO).

[0173] When reaching a confluence rate of approximately 90 - 100%, perform detachment. Use a culture medium prepared by equally mixing FKCM medium (serum-free medium for fibroblasts, manufactured by FUKOKU Co., Ltd.) and CS-C medium (manufactured by CELL SYSTEMS CORPORATION) to prepare a cell suspension (20 mL) in a ratio of the number of NHDF cells : the number of HUEhT-1 cells of 9:1 (about 1.7×10 7 cells). Add the above cell suspension to cover the wells of a 96-well plate and perform co-culture in a 37°C, 5% CO2 incubator (IP400, manufactured by Yamato Scientific Co., Ltd.). Culture for more than one night and confirm the ellipsoids (cell aggregates) in the wells of the 96-well plate through an inverted microscope (CKX31, manufactured by Olympus Corporation). Pipette to remove the ellipsoids from the wells of the 96-well plate and transfer them to a 10 cm culture dish. Rotate the 10 cm culture dish to gather the ellipsoids at the center and recover them to obtain ellipsoids.

[0174] (Decellularization treatment of ellipsoids)

[0175] Using the same procedure as in Example 1, obtain decellularized ellipsoids (decellularized cell aggregates).

[0176] 《Example 5》

[0177] In this example, use fibroblasts and vascular endothelial cells to prepare decellularized cell aggregates (decellularized ellipsoids).

[0178] (Preparation of cultured cell aggregates (ellipsoids))

[0179] Use T-175 flasks (vented caps, 175 cm 2 , manufactured by VIOLAMO) to culture NHDF-Neo-skin fibroblasts (CC-2509, manufactured by Lonza K.K.) and HUEhT-1 vascular endothelial cells (JCRB1458, JCRB Cell Bank) respectively.

[0180] When reaching a confluence rate of approximately 90 - 100%, perform detachment. Use a culture medium prepared by equally mixing FKCM medium (serum-free medium for fibroblasts, manufactured by FUKOKU Co., Ltd.) and CS-C medium (manufactured by CELL SYSTEMS CORPORATION) to prepare a cell suspension (20 mL) in a ratio of the number of NHDF cells : the number of HUEhT-1 cells of 7:3 (about 1.7×10 7(A certain number of cells). The above cell suspension was added and spread over the wells of the microplate, and co-cultured in an incubator at 37 °C and 5% CO2 (IP400, Yamato Scientific Co., Ltd.). After culturing for more than one night, the ellipsoids (cell aggregates) in the microplate were confirmed by an inverted microscope (CKX31, Olympus Corporation). The ellipsoids were pipetted out from the wells of the microplate and transferred to a 10-cm culture dish. The 10-cm culture dish was rotated to gather the ellipsoids at the center and then collected to obtain the ellipsoids.

[0181] (Decellularization of ellipsoids)

[0182] Using the same procedure as in Example 1, decellularized ellipsoids (decellularized cell aggregates) were obtained.

[0183] 《Example 6》

[0184] In this example, after performing decellularization of the ellipsoids using the same procedure as in Example 1, gamma-ray irradiation (25 kGy) was carried out to prepare decellularized cell aggregates (decellularized ellipsoids).

[0185] (Preparation of cultured cell aggregates (ellipsoids))

[0186] Ellipsoids were obtained using the same procedure as in Example 1.

[0187] (Decellularization of ellipsoids)

[0188] Using the same procedure as in Example 1 until freeze-drying treatment, gamma-ray irradiation (25 kGy) was carried out, whereby decellularized ellipsoids (decellularized cell aggregates) were obtained.

[0189] 《Example 7》

[0190] In this example, decellularization was carried out by surfactant treatment. The ellipsoids obtained in Example 1 were decellularized by the following surfactant treatment.

[0191] The ellipsoids obtained in Example 1 were washed with water for injection. Then, the ellipsoids were treated with a protein extraction buffer containing a surfactant (RIPA buffer, manufactured by NACALAI TESQUE, INC.) at 4 °C for more than 24 hours. Next, the treated ellipsoids were immersed in a DNase I solution of nuclease (800 U / mL) and washed with shaking at 4 °C for more than 18 hours. Then, they were washed 3 times with water for injection and freeze-dried to obtain decellularized ellipsoids (decellularized cell aggregates).

[0192] 《Example 8》

[0193] In this example, a decellularized cell mass is prepared as a decellularized cell aggregate.

[0194] (Production of cell mass as cultured cell aggregate)

[0195] The ellipsoid obtained in Example 1 was placed into a net mold (Net Mold Starter Kit V6, manufactured by TissueByNet Inc.). The net mold was transferred to a 120 mL sterilized 4-ounce container (manufactured by Thermo Fisher Scientific) containing a mixed medium (40 mL) of FKCM medium and DMEM medium, and set in an oscillator (OS-762, manufactured by OPTIMA Co., Ltd.) in an incubator at 37 °C and 5% CO2, and culturing was started under shaking (45 rpm). The medium was changed by half once or twice a week, and culturing was continued. After culturing for about 3 weeks, the net mold was removed to obtain a cell mass as a cultured cell aggregate.

[0196] (Decellularization treatment of cell mass)

[0197] The cell mass taken out from the net mold and the mixed medium were added to a nylon-polyester vacuum bag (manufactured by Kurilon Chemical Co., Ltd., Kashiwara, Japan), and the four corners were double-sealed with a sealer. Using the mixed 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 carried out within a temperature range of a minimum temperature of 23.3 °C and a maximum temperature of 35.9 °C. The cultured cell structure after high hydrostatic pressure treatment was taken out from the nylon-polyester vacuum bag, immersed in injection water, and washed with shaking at 4 °C for 5 minutes or more. Then, the cultured cell structure after high hydrostatic pressure treatment was immersed in a DNaseI solution (800 U / mL) of a nucleic acid degrading enzyme, and washed with shaking at 4 °C for 18 hours or more. Then, the cultured cell structure was immersed in injection water, and washed with shaking at 4 °C three times for 5 minutes or more to obtain a decellularized cell mass as a decellularized cell aggregate.

[0198] <<Comparative Example 1>>

[0199] In this comparative example, decellularization treatment was performed by surfactant treatment. The ellipsoid obtained in Example 1 was decellularized by the following surfactant treatment.

[0200] The ellipsoids obtained in Example 1 were washed with water for injection. Then, the ellipsoids were treated with a 0.25% by mass sodium dodecyl sulfate solution (10 mM Tris, pH 8.0) at 4°C for 24 hours or more. Next, the treated ellipsoids were treated with a 0.5% by mass Triton-X (polyoxyethylene octylphenyl ether) solution (10 mM Tris, pH 8.0) at 4°C for 24 hours or more. Then, after washing once with water for injection, freeze-drying was performed to obtain decellularized ellipsoids.

[0201] <<Comparative Example 2>>

[0202] As Comparative Example 2, a gelatin fiber substrate for cell culture was used.

[0203] <<Calculation of Decellularized DNA Ratio>>

[0204] The masses of the decellularized cell aggregates of Examples 1-8 and Comparative Example 1 were measured and used as samples. After dissolving them in a proteolytic enzyme solution and treating them with phenol / chloroform to remove proteins, DNA was recovered. The recovered DNA was fluorescently stained with PicoGreen (Life Technologies) and the fluorescence intensity was measured to quantify the DNA. The DNA content per unit mass of the sample was calculated from the mass of the sample and the amount of DNA. In addition, a standard curve prepared using the standard DNA attached to PicoGreen was used for the quantification of DNA.

[0205] (Decellularized DNA ratio) = (DNA content of the dried test piece of the decellularized ellipsoid or decellularized cell mass (decellularized cell aggregate)) / (mass of the dried test piece of the decellularized ellipsoid or decellularized cell mass (decellularized cell aggregate)) × 100

[0206] [Table 1]

[0207]

[0208] The results are shown in Table 1. It can be seen that compared with the comparative examples, the decellularization was better in the examples. From this, it can be known that the examples can be used as decellularized grafts with less rejection.

[0209] <<Calculation of Area Retention Rate>>

[0210] The ellipsoids or cell masses (before decellularization treatment) and the decellularized ellipsoids or decellularized cell masses of Examples 1-8 and Comparative Example 1 were observed under a microscope and photographed at a magnification of 4 times, and the area was digitized using image processing software (WinROOF2013).

[0211] The area retention rate was calculated from the area of the ellipsoid or cell mass and the area of the decellularized ellipsoid or decellularized cell mass in the examples and comparative examples. In addition, for the area of the ellipsoid or cell mass, the area before treatment was set to 100%.

[0212] Area retention rate (%) = (area of decellularized ellipsoid or decellularized cell mass / area of ellipsoid or cell mass) × 100

[0213] The results of the calculated area retention rate are shown in Figure 1 . For the area retention rate, it was found that, compared with the comparative examples, the examples maintained the area after decellularization relative to before decellularization. In particular, Example 1 maintained the area well before and after decellularization. In addition, especially Examples 3, 4, 5, and 8 maintained the area well before and after decellularization.

[0214] For Example 3, it is speculated that since immortalized cells were used to produce the decellularized cell aggregates, a large amount of lamin B1 described later was contained, whereby the adhesion between cells became strong. In addition, since cells with the same genotype and phenotype were used, the cell size was suppressed and the cells were easily adhered to each other. For Examples 4 and 5, it is speculated that since the decellularized cell aggregates were produced by co-culturing cells, a large amount of lamin B1 described later was contained, whereby the adhesion between cells became strong. For Example 8, it is speculated that since the decellularized cell mass as the decellularized cell aggregate was produced, a large amount of lamin B1 described later was contained through long-term culture, whereby the adhesion between cells became strong. From Example 6, it was found that, compared with the comparative example, the decellularized cell aggregate of Example 1 could maintain the area even when irradiated with gamma rays. Thus, it was found that, compared with the comparative examples, the examples could be used well as a scaffold material or a graft, and it was speculated that the cell activity described later could be promoted.

[0215] 《Measurement of Surface Pores》

[0216] The obtained decellularized ellipsoids, decellularized cell masses, or Comparative Example 2 were observed using a scanning electron microscope (SEM; Scanning Electron Microscope) and photographed at a magnification of 1000 times. Using image processing software (WinROOF2013), the surface pores of the decellularized ellipsoids, decellularized cell masses of Examples 1-8 and Comparative Example 1, or Comparative Example 2 were measured. A measurement area of 88 μm × 126 μm was set, and the number of surface pores with a diameter of 5 μm or more was counted. The surface pores that entered the measurement area in the entire surface pores were measured.

[0217] The state of the surface of the decellularized ellipsoids, decellularized cell masses, or Comparative Example 2 is shown inFigure 2 The results of counting the number of surface pores of the decellularized ellipsoid, the decellularized cell mass, or Comparative Example 2 are shown in Figure 3 . Compared with the comparative examples, the surface pores of the examples are more, and those of Example 1 and Example 8 are particularly numerous. In addition, the surface pores of Example 3, Example 4, and Example 5 are further more.

[0218] For Example 3, immortalized cells were used to prepare a decellularized cell aggregate, thereby containing more lamin B1 described later. In addition, cells with the same genotype and phenotype were used to make the cell sizes consistent. It is speculated that a tightly packed cultured cell aggregate was thus prepared and decellularized. For Example 4 and Example 5, it is speculated that the cells were co-cultured to prepare a decellularized cell aggregate, thereby containing a large amount of lamin B1 described later, and a tightly packed cultured cell aggregate was prepared and decellularized. It can be seen from Example 6 that, compared with the comparative example, the decellularized cell aggregate of Example 1 can maintain the number of surface pores even when irradiated with gamma rays. Thus, it is speculated that, compared with the comparative example, the examples promote the cell activity described later, and thus it is considered that they can be used as a scaffold material or a graft sheet well.

[0219] "Cell Viability Assay"

[0220] The decellularized ellipsoids, decellularized cell masses of the examples and comparative examples or Comparative Example 2 were suspended in DMEM medium to prepare a suspension of 1.0 mg / mL, and 100 μL (0.1 mg / well) was added to each well of a 96-well U-bottom plate (PrimeSurface 96U plate, SumitomoBakelite Co., Ltd.). 50 μL of a cell suspension (1.0×10 5 cells / mL) of NHDF-Neo-human dermal fibroblasts (CC-2509, manufactured by Lonza K.K.) was seeded into each of the wells after addition (1.0×10 4 cells / well). The medium was changed every 2 days, and at the same time, the cells were cultured in an incubator at 37 °C and 5% CO2 for 10 days. After the culture, a CellTiter-Glo 3D Cell Viability Assay Kit (Promega) was used to detect cell viability, and the ATP amount of the cells was measured by the luminescence amount (RLU), thereby measuring cell viability.

[0221] The results of the cell viability assay are shown in Figure 4It can be seen that, compared with the comparative examples, the cell viability in the examples is high, and the effect of nutrient supply to the cells is good. From this, it can be known that by seeding cells in the examples, the proliferation of the cells is promoted. The cell viabilities of Example 1 and Example 8 are particularly high. In addition, the cell viabilities of Example 3, Example 4, and Example 5 are further relatively high. For Example 3, it is speculated that this is because by using immortalized cells to produce decellularized cell aggregates, the area retention rate and the number of surface pores can be effectively maintained at specified values. For Example 4 and 5, it is speculated that this is because by co-culturing cells and producing decellularized cell aggregates, the area retention rate and the number of surface pores can be effectively maintained at specified values. It can be seen from Example 6 that, compared with the comparative examples, the decellularized cell aggregates of Example 1 can maintain cell viability even when irradiated with gamma rays. In addition, it can be confirmed that by culturing the decellularized ellipsoids of the examples and cells, ellipsoids can be produced well again. It can be known that the examples can prepare decellularized grafts in a simple step and can be used as decellularized grafts showing good tissue regeneration.

[0222] Production of Paraffin-Embedded Tissue Specimens and Section-Stained Samples

[0223] As a pretreatment, the decellularized ellipsoids, decellularized cell masses obtained in Examples 1-8 or Comparative Example 1, or Comparative Example 2 were gelated using IPGell (GENOSTAFF CO., LTD.) and immersed in 4% by mass PFA (4% by mass paraformaldehyde phosphate buffer, manufactured by FUJIFILM Wako Pure Chemical Corporation for tissue fixation) for 24 hours. Then, the gel was immersed in phosphate buffer (PBS, 0.01 M, pH 7.4) and washed with shaking at 4°C for 30 minutes or more. Next, the gel was immersed in 70% by mass ethanol and washed with shaking at 4°C for 30 minutes or more, and then immersed in 70% by mass ethanol again, which was called the sample for producing paraffin-embedded tissue specimens.

[0224] The paraffin-embedded tissue specimens were produced from the above samples according to the following steps, and section-stained samples for staining (described later) were produced. The tissue was rinsed with tap water for 30 minutes to 40 minutes to remove formaldehyde. Using a paraffin embedding device (CT-Pro20, manufactured by GENOSTAFF CO., LTD.), the samples were dehydrated in an ethanol bath according to the following steps.

[0225] 70% by mass ethanol for 20 minutes (×1) →

[0226] 95% by mass ethanol for 20 minutes (×2) →

[0227] 100% by mass ethanol for 20 minutes (×2)

[0228] Then, the samples were washed twice in a low-toxicity solvent G-Nox (GENOSTAFF CO., LTD.) as a xylene substitute, for 20 minutes each time. The samples were incubated twice in a paraffin bath at 65 °C, for 30 minutes each time. The dissolved paraffin was poured into a mold, and the samples were placed in the mold and allowed to cool for 15 - 20 minutes to produce paraffin-embedded tissue specimens. Then, using the paraffin-embedded tissue specimens, serial sections were made at a thickness of 5 μm on a microtome and suspended in a water bath at 37 °C containing deionized water. The sections were floated one by one on the distilled water placed on a glass slide, heated (30 - 40 °C) and stretched on a stretching table, and left to dry in an incubator (37 °C) for 1 - 2 nights to produce section-stained samples.

[0229] Detection of Lamin B1 (Immunohistochemical Staining)

[0230] The following steps were carried out for immunohistochemical staining of the decellularized ellipsoids, decellularized cell masses obtained in Examples 1 - 8 or Comparative Example 1, or Comparative Example 2 to stain Lamin B1.

[0231] First, the section-stained samples were dewaxed and hydrophilized. Then, after washing 3 times with phosphate buffer (PBS, 0.01 M, pH 7.4), they were immersed in EDTA buffer (pH 9.0): G-Activate (antigen activation buffer, pH 9.0) for heat treatment to perform antigen activation.

[0232] Next, after washing 3 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 washed 3 times with Tris-buffered saline. Then, after treating with G-Block (GENOSTAFF CO., LTD.) at room temperature for 10 minutes, they were treated with an Avidin / Biotin Blocking kit (VECTOR LABORATORIES) for blocking.

[0233] Then, using a rabbit monoclonal antibody against Lamin B1 (17416, Cell Signaling) as the primary antibody, an overnight antibody reaction was carried out at 4 °C. Then, after washing 2 times with Tris-buffered saline containing Tween 20, they were washed with Tris-buffered saline.

[0234] Then, an anti-rabbit Ig biotin (E0432, Dako), which serves as a secondary antibody, was used for a 30-minute antibody reaction at room temperature. Then, after washing twice with Tris-buffered saline containing Tween 20, it was washed with Tris-buffered saline.

[0235] Then, streptavidin peroxidase (NICHIREI CORPORATION.) was used to treat for 5 minutes at room temperature. Then, after washing twice with Tris-buffered saline containing Tween 20, it was washed with Tris-buffered saline.

[0236] Then, it was treated with DAB (3,3'-diaminobenzidine tetrahydrochloride) / hydrogen peroxide to develop color. Additionally, as a marker for observation, Mayer's Hematoxylin (MUTO PURECHEMICALS CO., LTD.) was used to counterstain the cell nuclei.

[0237] Then, dehydration and infiltration were performed, and it was encapsulated with an encapsulant (xylene-based encapsulating material: Malinol MUTO PURECHEMICALS CO., LTD.).

[0238] The results of immunohistochemical staining are shown in Figure 5 . In the left figure of each example (comparative example), the staining of lamin B1 is shown, and in the right figure, the negative control (stained with rabbit Ig) is shown. For the examples, it was confirmed that in the left figure, there were parts with darker staining compared to the right figure, and lamin B1 was detected. In contrast, for the comparative examples, there was no change in the staining between the left and right figures, and lamin B1 was not detected.

[0239] ≪Quantification of lamin B1 (enzyme immunoassay)≫

[0240] The content of lamin B1 in the decellularized ellipsoids, decellularized cell masses of the examples and comparative examples, or in comparative example 2 was quantified using an enzyme immunoassay (ELISA; Enzyme Linked Immunosorbent Assay).

[0241] To the decellularized ellipsoids, decellularized cell masses of the examples and comparative examples, or Comparative Example 2, add the Cell Extraction BufferPTR included in the ELISA kit (Human Lamin B1 ELISA Kit, ab252351, abcam), and homogenize using a disposable homogenizer (BioMasherII, Nippi, Incorporated). After incubating on ice for 20 minutes, centrifuge at 18,000 Xg for 20 minutes at 4 °C and recover the supernatant as the extract of the decellularized ellipsoids of the examples and comparative examples. Quantify lamin B1 in the extract according to the protocol of the ELISA kit manufacturer, and calculate the content of lamin B1 per unit mass of the sample.

[0242] The results obtained by quantitatively determining lamin B1 by enzyme immunoassay are shown in Figure 6 . It can be seen that the examples contain a large amount of lamin B1. The quantitative value of lamin B1 in Example 1 is particularly high. In addition, the quantitative values of lamin B1 in Example 3, Example 4, 5, and Example 8 are further relatively high. From Example 6, it can be seen that compared with the comparative examples, the decellularized cell aggregates of Example 1 can maintain the quantitative value of lamin B1 even when irradiated with gamma rays. The comparative examples do not contain lamin B1. Thus, it can be seen that the examples promote the proliferation of the seeded cells.

[0243] 《Calculation of Positive Area》

[0244] Microscopically observe the immunohistochemically stained sections obtained above, photograph the sections at a magnification of 40 times, and use image processing software (ImageJ) to calculate the positive areas of the examples and comparative examples. Obtain images of the stained sections, and count the total area of the stained sections and the area of the positively stained part of lamin B1 respectively.

[0245] First, calculate the positive area rates of the examples and comparative examples according to the following formula. As the measurement area, set a quadrilateral (30 pixel × 30 pixel) area in the depth direction and within the range of 100 μm from the cell seeding surface, and find the average value of the positive area rates at 3 positions.

[0246] Positive area rate (%) = Area of the positively stained part of lamin B1 / Total area of the stained section (measurement area) × 100

[0247] The results of calculating the positive areas of the immunohistochemical staining are shown in Figure 7It can be seen that the examples contain a large amount of lamin B1. In particular, Example 1 contains a relatively large amount of lamin B1. In addition, it can be seen that Examples 3, 4, 5, and 8 contain a further large amount of lamin B1. From Example 6, it can be seen that the decellularized cell aggregates of Example 1 can maintain lamin B1 even when irradiated with gamma rays, as compared with the comparative example. The comparative example does not contain lamin B1. Thus, it can be seen that the examples promote the proliferation of the seeded cells.

[0248] Industrial applicability

[0249] The decellularized cell aggregates of the present invention can be used as a substrate (scaffold material) for cell culture and a graft for organisms.

Claims

1. A decellularized cell aggregate, which is a decellularized cell aggregate obtained by decellularizing a cultured cell aggregate, and has 3 to 45 surface pores with a pore diameter of 5 μm or more per 11088 μm on the surface of the aggregate. 2 It has surface pores with a pore diameter of 5 μm or more.

2. A decellularized cell aggregate, which is a decellularized cell aggregate obtained by decellularizing a cultured cell aggregate, and contains 10 pg / mg or more of laminin with respect to the decellularized cell aggregate.

3. A decellularized cell aggregate, which is a decellularized cell aggregate obtained by decellularizing a cultured cell aggregate, having 3 to 45 surface pores with a pore diameter of 5 μm or more per 11088 μm on the surface of the aggregate, and containing 10 pg / mg or more of laminin with respect to the decellularized cell aggregate. 2 ​ 4. The decellularized cell aggregate according to any one of claims 1 to 3, wherein, The cells of the cultured cell aggregate are immortalized cells.

5. The decellularized cell aggregate according to any one of claims 1 to 3, wherein, The cells of the cultured cell aggregate are a mixture of two or more types of cells.

6. The decellularized cell aggregate according to any one of claims 1 to 3, wherein, The DNA content per unit dry mass of the decellularized cell aggregate is 1.000 mass% or less.

7. A substrate for cell culture, which contains the decellularized cell aggregate according to any one of claims 1 to 3.

8. A graft for an organism, which contains the decellularized cell aggregate according to any one of claims 1 to 3.

9. A composition, which contains the decellularized cell aggregate according to any one of claims 1 to 3 and cells.

10. A method for preparing a decellularized cell aggregate, which includes: A cultured cell aggregate formation step of forming a cultured cell aggregate by culturing cells; and A step of decellularizing the cultured cell aggregate. The decellularized cell condensate has 3 to 45 surface pores with a pore diameter of 5 μm or more per 11088 μm on the condensate surface. 2 ​ 11. A method for preparing a decellularized cell aggregate, which includes: A cultured cell aggregate formation step of forming a cultured cell aggregate by culturing cells; and A step of decellularizing the cultured cell aggregate. With respect to the decellularized cell aggregate, the decellularized cell aggregate contains 10 pg / mg or more of laminin.

12. The method for preparing a decellularized cell aggregate according to claim 10 or 11, wherein, The cells are immortalized cells.

13. The method for preparing a decellularized cell aggregate according to claim 10 or 11, wherein, The culturing of the cells is the culturing of two or more types of cells.

14. The method for preparing a decellularized cell aggregate according to claim 10 or 11, wherein, The decellularization is performed by high hydrostatic pressure treatment.

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