Decellularized cell structure

Abstract

The purpose of the present invention is to provide a decellularized graft having an arbitrary thickness or a specific shape.　This problem can be solved by a decellularized cell structure, which is obtained by decellularizing a cultured cell structure of the present invention, characterized by having a porosity of 30-80%.

Description

Decellularized cell construct

[0001] The present invention relates to a decellularized cell structure.

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

[0003] Patent Document 1: JP-T-2005-514971 Publication No. WO 2016 / 136633 Patent Document 2: JP-A-2012-139541 Publication No. JP-A-2014-148321 Publication No. JP-A-2018-051415 Publication No. JP-A-2004-357694 Publication No. JP-A-2020-198794 Publication No. JP-A-4517125

[0004] The decellularized tissue is useful in that it can be used to obtain a transplant that does not induce rejection by decellularizing biological tissue. However, a lamination process may be required to obtain a desired thickness. It is also not easy to create a specific shape. Furthermore, it is not easy to maintain the strength of the transplant. Therefore, an object of the present invention is to provide a decellularized transplant that can have any thickness and maintain its hardness.

[0005] As a result of extensive research into decellularized grafts having any thickness or specific shape, the present inventors have surprisingly found that by decellularizing cultured cell structures obtained by culturing (Patent Documents 3 to 7), it is possible to obtain decellularized grafts having any thickness and maintaining hardness. The present invention is based on this finding. [1] A decellularized cell structure obtained by decellularizing a cultured cell structure, characterized in that the porosity is 30% to 80%; [2] The decellularized cell structure according to [1], wherein the hardness of the decellularized structure is 0.20 to 5.00 (N); [3] The decellularized cell structure according to [1] or [2], wherein the DNA content per dry mass of the decellularized cell structure is 0.020 mass% or less; [4] The decellularized cell structure according to [1] or [2], wherein the decellularized cell structure contains a protein with (a) a molecular weight of 45,000 to 53,000 and (b) an isoelectric point of pI 4.00 to 4.90; [5] The decellularized cell structure according to [4], wherein the protein is tubulin; [6] A method for producing a decellularized cell structure, comprising: a cultured cell structure formation step of forming a cultured cell structure by culturing cells; and a decellularization step of decellularizing the cultured cell structure to a porosity of 30% to 80%. [7] A method for producing a decellularized cell structure according to [6], wherein cells are cultured in any shape before or simultaneously with the formation of the cultured cell structure in the cultured cell structure formation step; [8] A method for producing a decellularized cell structure according to [7], wherein in the cultured cell structure formation step, the culturing of cells in any shape before the formation of the cultured cell structure is the culture of sheet-like cells or the culture of cell aggregates; [9] A method for producing a decellularized cell structure according to [6], wherein cells are cultured in any shape after the cultured cell structure formation step;

[10] A method for producing a decellularized cell structure according to any of [6] to [9], wherein the decellularization is performed by high hydrostatic pressure treatment; and

[11] A method for producing a decellularized cell structure according to any of [6] to [9], wherein the thickness retention rate before and after decellularization in the decellularization step is 90% or more.

[0006] The decellularized cell structure of the present invention can provide a decellularized graft that has a desired thickness and maintains its hardness. Furthermore, the manufacturing method of the present invention can maintain the thickness after decellularization relative to that before decellularization, and can maintain the shape before and after decellularization. In other words, since it is possible to provide a decellularized material of any desired thickness, it is possible to provide a decellularized material with a specific shape, and it is possible to create different decellularized materials depending on the intended use, such as a graft or cell therapy. Furthermore, it is possible to produce a decellularized material that is strong yet does not induce rejection.

[0007] 1 is a graph showing the porosity of the decellularized cell structures of Examples 1 to 4 and Comparative Example 1. 2 is a graph showing the thickness maintenance rate of the decellularized cell structures of Examples 1 to 4 and Comparative Example 1. 3 is a micrograph of a decellularized cell structure stained with Sirius Red. 4 is a graph showing the results of a hardness test of the decellularized cell structures of Examples 1 to 4 and Comparative Example 1. 5 is a photograph showing protein spots by two-dimensional electrophoresis of Example 1 (A) and Comparative Example 1 (B).

[0008] [1] Decellularized Cell Structure The decellularized cell structure of the present invention is a decellularized cell structure obtained by decellularizing a cultured cell structure, and has a porosity of 30% to 80%.

[0009] Cultured Cell Structure The cultured cell structure of the present invention is a cell structure obtained by in vitro culture. The cultured cell structure is not particularly limited as long as the cells adhere and maintain a three-dimensional shape by producing extracellular matrix (ECM) outside the cells. The smallest three-dimensional distance (thickness) is preferably 50 μm or more, more preferably 100 μm or more. The other two-dimensional dimensions are also not particularly limited, but are, for example, 1 mm or more, preferably 6 mm or more. This allows for efficient production of decellularized cell structures. The strength of the cultured cell structure is also not particularly limited as long as it maintains a certain three-dimensional shape. However, it is preferable that the strength be such that it can be handled with tweezers or sutured in a wet state. The shape of the cultured cell structure is not particularly limited, but examples include sheet, tube, and block shapes. This facilitates the production of decellularized cell structures tailored to specific applications, such as transplants and cell therapy.

[0010] The decellularized cell structure of the present invention may be in a wet state or a dry state. A decellularized cell structure provided in a wet state can be used immediately for transplantation. On the other hand, a decellularized cell structure provided in a dry state can be stored for a long period of time, is easy to transport, and can be restored and used at the time of use.

[0011] Cells can be broadly divided into suspension cells and adherent cells. The cells of the cultured cell construct are preferably, but not limited to, adherent cells from the viewpoint of ease of preparation of the cultured cell construct. The adherent cells are not particularly limited and can be appropriately selected depending on the purpose of use of the cultured cell construct. Examples of differentiated adherent cells include hepatocytes, stellate cells, Kupffer cells, vascular endothelial cells, meatal 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 gland cells, pericytes, smooth muscle cells, cardiac muscle cells, muscle cells, kidney cells, pancreatic islet cells of Langerhans, peripheral nerve cells, neurons, chondrocytes, and bone cells. Examples of undifferentiated adherent cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, hematopoietic stem cells, neural stem cells, cardiac progenitor cells, vascular endothelial progenitor cells, neural progenitor cells, preadipocytes, skin fibroblasts, skeletal muscle myoblasts, osteoblasts, and odontoblasts. From the viewpoint of ease of preparation of cultured cell structures, skin fibroblasts, mesenchymal stem cells, and cardiac progenitor cells are preferred. The origin of the cells is not particularly limited, and includes eukaryotic cells, prokaryotic cells, cells of multicellular organisms, and cells of unicellular organisms. Examples of eukaryotic cells include animal cells, insect cells, plant cells, fungi, algae, and protozoa. Animal cells include, but are not limited to, cells derived from a human, monkey, dog, cat, rabbit, ferret, sheep, goat, cow, pig, horse, camel, mouse, rat, hamster, guinea pig, or gerbil.

[0012] The cultured cell construct is preferably, but not limited to, adhered by an extracellular matrix. Adhesion to the extracellular matrix provides the cultured cell construct with a certain degree of strength. Examples of the extracellular matrix include collagen, laminin, fibronectin, chondroitin sulfate, heparin sulfate, keratan sulfate, and hyaluronic acid, but the type of extracellular matrix varies depending on the type of cell, and is not particularly limited.

[0013] <<DNA Content>> The DNA content per dry mass of the decellularized cell structure is 0.020% by mass or less, preferably 0.015% by mass or less, more preferably 0.010% by mass or less, even more preferably 0.008% by mass or less, even more preferably 0.006% by mass or less, even more preferably 0.004% by mass or less, even more preferably 0.002% by mass or less, even more preferably 0.0018% by mass or less, and even more preferably 0.0015% by mass or less. This allows for the production of a decellularized cell structure that is appropriately decellularized and has minimal rejection upon transplantation. DNA content can be measured using the Picogreen method. A dried test piece of the decellularized cell structure (hereinafter sometimes referred to as a sample) is immersed in a protease solution and dissolved, then treated with phenol / chloroform to remove proteins, and DNA is recovered by ethanol precipitation. The recovered DNA is fluorescently stained with Picogreen (Life Technologies) and the fluorescence intensity is measured to quantify the DNA, and the DNA content (mass) of the sample is calculated. Quantification is performed using a calibration curve created using the standard DNA provided with the Picogreen. The DNA ratio is calculated from the dry mass and DNA content of the sample according to the following formula: (DNA content per dry mass (hereinafter sometimes referred to as decellularized DNA ratio) = (DNA content of the dried test piece of the decellularized cell structure) / (mass of the dried test piece of the decellularized cell structure)

[0014] <<Porosity>> The porosity of the decellularized cell structure of the present invention is 30% to 80%. The lower limit of the porosity is preferably 33%, more preferably 37%, even more preferably 40%, even more preferably 42%, even more preferably 44%, even more preferably 47%, and most preferably 50%. The upper limit of the porosity is preferably 77%, more preferably 73%, even more preferably 70%, even more preferably 68%, even more preferably 66%, even more preferably 64%, and most preferably 55%. By keeping the porosity within this range, a graft with sufficient strength and no rejection reaction can be obtained. Furthermore, a decellularized graft with any thickness or specific shape can be provided. Herein, porosity is calculated as follows: A section of the decellularized cell structure is stained with Sirius Red, which stains the extracellular matrix. Microscopic tissue images are analyzed using image processing software, and areas not stained with Sirius Red are determined to be voids, and the porosity is calculated. Sirius Red staining was performed as follows. Section samples of the decellularized cell structure were deparaffinized. Next, the section samples were treated with iron hematoxylin. Next, they were treated with a hydrochloric acid / ethanol solution and a phosphomolybdic acid aqueous solution. Subsequently, they were treated with Sirius Red (Direct Red 80), dehydrated, and mounted. The obtained Sirius Red-stained sections were observed under a microscope, and the porosity was calculated using image processing software (ImageJ). The sections were photographed at 10x magnification, and the area of ​​the entire decellularized cell structure and the area of ​​the Sirius Red-stained area were measured (Figure 3). The porosity was calculated using the following formula: Porosity (%) = (area of ​​the entire decellularized cell structure - area of ​​the Sirius Red-stained area) / area of ​​the entire decellularized cell structure x 100

[0015] [2] Method for manufacturing a decellularized cell structure The method for manufacturing a decellularized cell structure of the present invention includes a cultured cell structure formation step of forming a cultured cell structure by culturing cells, and a decellularization step of decellularizing the cultured cell structure to a porosity of 30% to 80%.

[0016] <<Cultured Cell Structure Formation Step (1)>> In the cultured cell structure formation step (1), cells are cultured to form a cultured cell structure. The cells used are not particularly limited as long as they are culturable in vitro. For example, they may be primary cells collected directly from tissues or organs, or passaged cells obtained by passage of primary cells collected directly from tissues or organs for several generations. They may also be passaged cells that have been passaged for a long period of time. Specifically, any of the cells described in the above "[1] Decellularized Cell Structure" can be used without limitation. The cultured cell structure can be formed by cells producing extracellular matrix (ECM) outside the cells, adhering and bonding cells to each other, and maintains a three-dimensional shape. The method for forming the cultured cell structure is not particularly limited as long as it utilizes adhesion via the extracellular matrix. For example, the methods described in Patent Documents 3 to 8 can be used. Specifically, these methods include a method for producing a sheet-like three-dimensional structure without using a scaffold (Patent Document 3), a method for stacking multiple cell sheets (Patent Document 4), a method for producing a structure by supplying cell aggregates to a support having a mesh-like space formed by multiple threads or needle-shaped members (Patent Document 5), a method for producing a structure by culturing cell aggregates in a chamber (Patent Document 6), a method for producing a three-dimensional cell structure by culturing cell aggregates in a culture mold (Patent Document 7), or a method for producing a structure by culturing cell aggregates on a support having threads or needle-shaped members that penetrate the cell aggregates (Patent Document 8). All of these methods for producing structures utilize an extracellular matrix secreted by cells to adhere cells to each other and form a cell structure. Examples of extracellular matrices include, but are not limited to, those described in the above section "[1] Decellularized Cell Structure."

[0017] For example, a cultured cell structure can be formed by culturing cell aggregates (spheroids) as follows. A suitable medium can be selected for the culture depending on the cells used. The cell aggregates are placed in a cavity surrounded by a mesh-like or comb-like support with gaps smaller than the size of the cell aggregates. The cavity is formed into a desired shape by the mesh-like or comb-like support and plate, and the support has many openings. The medium can contact the cell aggregates through these openings. By shaking the shaking culture vessel while the cell aggregates are held in the cavity, the medium can effectively contact the cell aggregates, allowing the cell aggregates to be cultured well. The cell aggregates produce an extracellular matrix, and the cell aggregates begin to adhere to each other, allowing a cultured cell structure with a desired three-dimensional structure to be produced.

[0018] In the cultured cell structure formation step (1), cells may be cultured into any shape, for example, before or simultaneously with the formation of the cultured cell structure. Culturing into any shape refers to culturing cells to have any thickness or planar shape. For example, the area may be adjusted to culture cells into any planar shape. Cells of any planar shape may be layered and cultured to any thickness and shape. The cultured cells may also be cut into a desired shape. In the cultured cell structure formation step (1), cells may be cultured to form any shape simultaneously with the formation of the cultured cell structure. For example, cells may be cultured to form any shape simultaneously with the method for forming the cultured cell structure described above. For example, a cultured cell structure can also be formed by three-dimensional (3D) culture of cells in a gel containing an extracellular matrix.

[0019] Furthermore, cells may be cultured in any shape before forming a cultured cell structure. In the cultured cell structure formation process, culturing cells in any shape before forming a cultured cell structure is preferably, for example, culturing cells in a sheet-like shape or culturing cell aggregates (spheroids). Examples of culturing cells before forming a cultured cell structure include culturing cells to form any shape in a planar view, or stacking multiple cell sheets (sheet-like cells) as described in Patent Document 4 and culturing these stacked cell sheets before forming a cultured cell structure. Cell sheet culture can be performed according to the method commonly used in this field for producing cell sheets using culture plates, etc. By stacking and culturing the obtained cell sheets, cells can be cultured to any thickness and shape. After this, the cells secrete extracellular matrix, allowing the stacked cell sheets to adhere, resulting in a cultured cell structure that maintains a three-dimensional shape.

[0020] Further examples of culturing prior to the formation of cultured cell structures include culturing to form cell aggregates (spheroids) before adhering cells, as described in Patent Documents 5 and 7. Adherent cells cannot survive for long periods of time if they are suspended in a solution. When adhesive cells are placed in a non-adhesive environment, they seek a foothold and adhere to each other, forming cell aggregates. Specifically, cell aggregates (spheroids) can be produced by aggregating multiple cells. After monolayer culture, cells are transferred to a water-repellent or non-cell-adhesive round-bottom multi-well or U-shaped plate (dimple plate) and incubated, resulting in cell aggregation. From the standpoint of efficiency, the incubation time required to form cell aggregates is preferably 6 to 48 hours, more preferably 6 to 24 hours. However, methods for producing cell aggregates are not limited to the above methods. Other methods that can be used include a rotational culture method in which a cell suspension is placed in a rotating solution, a method in which a cell suspension is placed in a test tube and precipitated using a centrifuge, or the alginate bead method, as well as the production methods described in Patent Documents 5, 6, and 8. From the viewpoint of processing a large amount of uniform cell aggregates, a method of placing a cell suspension in a water-repellent or cell-nonadhesive multiwell is efficient and preferable. Cell aggregates can be cultured to any thickness by adjusting the incubation time of the cell aggregates (spheroids). Furthermore, cell aggregates can be cultured to any thickness and shape by connecting the cell aggregates (spheroids) together. Further culturing the cell aggregates (spheroids) obtained in this manner allows the cells to secrete extracellular matrix, which allows the cells to adhere to each other, resulting in a cultured cell structure that maintains a three-dimensional shape. Furthermore, culturing the cell aggregates (spheroids) by the above-described method of placing them in a cavity allows accurate production of a cultured cell structure with a desired three-dimensional shape. It is preferable that the amount of extracellular matrix (ECM) used when culturing cells into a desired shape before the formation of the cultured cell structure is less than the amount of extracellular matrix used during the formation of the cultured cell structure.

[0021] Furthermore, after the cultured cell structure formation step, the cells may be cultured into any shape. For example, after the above-mentioned method for forming a cultured cell structure is performed, the cells may be further cultured to form any shape. Alternatively, the cells may be coated with an extracellular matrix and then cultured in layers to form any shape. It is preferable that the amount of extracellular matrix (ECM) when culturing the cells into any shape after the cultured cell structure formation step is the same as or greater than the amount of extracellular matrix when forming the cultured cell structure.

[0022] Decellularization Step (2) In the decellularization step (2), the cell structure is decellularized to a porosity of 30% to 80%. That is, the porosity of the cell structure after decellularization is 30% to 80%. The lower limit of the porosity is preferably 33%, more preferably 37%, even more preferably 40%, even more preferably 42%, even more preferably 44%, even more preferably 47%, and most preferably 50%. The upper limit of the porosity is preferably 77%, more preferably 73%, even more preferably 70%, even more preferably 68%, even more preferably 66%, even more preferably 64%, and most preferably 55%. By achieving a porosity within this range, a graft can be obtained that has sufficient strength and is free from rejection. Furthermore, a decellularized graft of any thickness or specific shape can be provided.

[0023] The decellularization step can be performed using conventionally known methods. The decellularization method is not particularly limited as long as it achieves the effects of the present invention, and examples include high hydrostatic pressure treatment, freeze-thaw treatment, ultrasonic treatment, enzyme treatment, treatment with a hypertonic electrolyte solution, physical stirring, hypertonic / hypotonic solution treatment, enzyme treatment with protease, nuclease, or the like, treatment with an alcohol solvent, and two or more of these may be combined. In order to efficiently obtain a decellularized cell structure and to achieve the effects of the present invention, a high hydrostatic pressure treatment is preferred.

[0024] When the decellularized structure is obtained by the high hydrostatic pressure treatment method, a hydrostatic pressure of 50 to 1500 MPa is applied to the obtained cell structure in a medium. From the viewpoint of sufficient decellularization, the applied hydrostatic pressure is preferably 50 MPa or more. A pressure vessel capable of withstanding the pressure is not required, a large amount of energy is not required, and, if the medium used for application is an aqueous medium, ice is generated. From the viewpoint of preventing damage to the cell structure by the generated ice, a hydrostatic pressure of 1500 MPa or less is preferred. The applied hydrostatic pressure is more preferably 80 to 1300 MPa, more preferably 90 to 1200 MPa, even more preferably 95 to 1100 MPa, even more preferably 95 to 700 MPa, and most preferably 400 to 700 MPa, from the viewpoint of exerting decellularization effects, sterilization effects, and viral inactivation effects, and from the viewpoint of ease of application.

[0025] Examples of media used for applying hydrostatic pressure include water, physiological saline, water for injection, propylene glycol or an aqueous solution thereof, glycerin or an aqueous solution thereof, and aqueous sugar solutions. Examples of buffer solutions include acetate buffer, phosphate buffer, citrate buffer, borate buffer, tartrate buffer, Tris buffer, HEPES buffer, and MES buffer. These media may contain a surfactant.

[0026] The temperature for the high hydrostatic pressure treatment is not particularly limited as long as it does not produce ice and does not cause thermal damage to the cell structure, but since decellularization is carried out smoothly and has little effect on the cell structure, it is preferably 0 to 45° C., more preferably 4 to 37° C., and most preferably 15 to 36° C. If the high hydrostatic pressure treatment time is too short, cells will not be sufficiently destroyed, and if it is too long, it will result in a waste of energy, so the time for which the desired applied pressure is maintained in the high hydrostatic pressure treatment is preferably 1 to 120 minutes, more preferably 5 to 60 minutes, and even more preferably 7 to 30 minutes.

[0027] The cell structure treated with high hydrostatic pressure is preferably treated with a nuclease. The nuclease removes nucleic acid components from the cell structure to which hydrostatic pressure has been applied, and is not particularly limited, and examples thereof include pancreatic-, spleen-, or Escherichia coli-derived DNases (e.g., DNase I and DNase II). The nuclease can be added to the medium used in the high hydrostatic pressure treatment (e.g., water, physiological saline, injection solution, buffer solution, etc.) and allowed to act. The amount of enzyme to be added varies depending on the type of enzyme and the definition of the number of units (U), but can be appropriately determined by those skilled in the art. For example, DNase I may be used at 50 to 1000 U / mL. The treatment temperature also varies depending on the nuclease used, but may be set to, for example, 1 to 40°C. The treatment time is also not particularly limited, and may be, for example, 1 to 120 hours (preferably 1 to 96 hours, more preferably 1 to 48 hours). Longer treatment times are required at lower temperatures, and shorter treatment times are required at higher temperatures.

[0028] The cell structure treated with high hydrostatic pressure is washed with a washing solution. The washing solution may be the same as or different from the medium used in the high hydrostatic pressure treatment. The washing solution preferably contains an organic solvent or a chelating agent. The organic solvent can improve the efficiency of lipid removal, and the chelating agent can inactivate calcium ions and magnesium ions in the decellularized cell structure, thereby preventing calcification when the particulate decellularized cell structure of the present invention is applied to a diseased area. As the organic solvent, a water-soluble organic solvent is preferred because of its high lipid removal effect, and ethanol, isopropanol, acetone, and dimethyl sulfoxide are preferred. Examples of the chelating agent include iminocarboxylic acid chelating agents such as ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriminepentaacetic acid (DTPA), hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraminehexaacetic acid (TTHA), 1,3-propanediaminetetraacetic acid (PDTA), 1,3-diamino-2-hydroxypropanetetraacetic acid (DPTA-OH), hydroxyethyliminodiacetic acid (HIDA), dihydroxyethylglycine (DHEG), glycol ether diaminetetraacetic acid (GEDTA), dicarboxymethylglutamic acid (CMGA), 3-hydroxy-2,2'-iminodisuccinic acid (HIDA), and dicarboxymethylaspartic acid (ASDA), or salts thereof; and hydroxycarboxylic acid chelating agents such as citric acid, tartaric acid, malic acid, and lactic acid, or salts thereof. Examples of the salts of these chelating agents include sodium salts and potassium salts. The washing temperature is not particularly limited as long as it does not cause thermal damage to the cell structure, but in order to provide good washing properties and little effect on the cell structure, it is preferably 0 to 45° C., more preferably 1 to 40° C., and most preferably 2 to 35° C. When washing, the washing solution may be shaken or stirred as necessary.

[0029] When the decellularized cell structure is obtained by the freeze-thaw treatment method, the cell structure is frozen by holding it 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), and then thawed at a temperature of 20 to 40°C. This process is repeated once or twice or more times (preferably 2 to 5 times). This is then preferably followed by a nuclease treatment, which may be the same as the treatment in the high hydrostatic pressure treatment. The cells in the cell structure that have been subjected to the freeze-thaw treatment are destroyed, and these cells are removed with a washing solution. The washing method may be the same as the washing method in the high hydrostatic pressure treatment.

[0030] When the decellularized cell structure is obtained by a method of treatment by ultrasonic treatment, the cell structure is subjected to ultrasonic treatment (for example, intensity: 10 W / cm) in, for example, physiological saline. ２ , frequency: 10 kHz, exposure time: 2 minutes). Thereafter, preferably, the sample is shaken in a surfactant solution (e.g., a 1% by mass Triton X (polyoxyethylene octylphenyl ether) solution) for 1 to 120 hours (preferably 12 to 120 hours) at 2 to 10°C (preferably 4°C). Furthermore, it is preferable to perform a nuclease treatment, which may be performed by the same method as in the high hydrostatic pressure treatment. Thereafter, it is preferable to perform washing by the same method as in the high hydrostatic pressure treatment.

[0031] The obtained decellularized cell structure is preferably, but not limited to, subjected to a freeze-drying process. The freeze-drying process may be omitted depending on the part of the cell structure. The obtained decellularized cell structure may also be sterilized by gamma irradiation, UV irradiation, or the like.

[0032] <<Thickness Maintenance Rate>> In the method for producing a decellularized cell structure of the present invention, the thickness can be maintained before and after decellularization in the decellularization step (2). The thickness maintenance rate before and after decellularization is not limited, but is, for example, 90% or more, more preferably 92% or more, and even more preferably 94% or more. The thickness maintenance rate can be calculated as follows: Thickness maintenance rate (%) = (thickness of decellularized cell structure / thickness of cultured cell structure before decellularization) × 100

[0033] <Hardness Test> The hardness of the decellularized cell structure of the present invention is not particularly limited as long as the effects of the present invention are obtained, but is, for example, 0.20 to 5.00 (N). The lower limit of the hardness is preferably 0.30 (N), more preferably 0.40 (N), even more preferably 0.50 (N), even more preferably 0.60 (N), even more preferably 0.65 (N), even more preferably 0.70 (N), and most preferably 0.73 (N). The upper limit of the hardness is preferably 3.00 (N), more preferably 1.00 (N), even more preferably 0.95 (N), even more preferably 0.90 (N), even more preferably 0.87 (N), even more preferably 0.83 (N), and most preferably 0.80 (N). By keeping the hardness within the above range, a graft can be obtained that has sufficient strength and is free from rejection reactions. This facilitates handling when implanting the present invention as a graft, such as by pinching with tweezers or piercing with a surgical needle for suturing. Furthermore, decellularized grafts with any thickness or specific shape can be provided. In the present invention, hardness is measured by Texture Profile Analysis (TPA) as follows: Measurements are made at room temperature using a texture analyzer TA.XT plus (Eiko Seiki Co., Ltd.) equipped with a P / 4 cylindrical probe (diameter 4 mm). The point at which a load of 0.01 (N) is applied to the probe is set as the zero point, and the sample is compressed twice over a distance of 0.5 mm at a speed of 0.02 mm / sec. The maximum force (N) during the first compression cycle is taken as the hardness.

[0034] <<Protein>> The decellularized cell structure of the present invention is not particularly limited as long as the effects of the present invention are obtained. For example, it may contain a protein having (a) a molecular weight of 45,000 to 53,000 and (b) an isoelectric point of 4.00 to 4.90. This enhances the effects of the present invention. The molecular weight and isoelectric point (pI) of the protein can be determined, for example, by subjecting the protein to two-dimensional electrophoresis, followed by identification of the protein by mass spectrometry. In two-dimensional electrophoresis, either isoelectric focusing or molecular weight separation can be performed first (as the first dimension). However, from the perspective of improving measurement accuracy, it is preferable to perform isoelectric focusing in the first dimension and molecular weight separation in the second dimension. Two-dimensional electrophoresis can be performed according to conventional methods, and commercially available kits and devices can be used. For example, isoelectric focusing can be performed using a capillary gel or strip gel as a separation medium, and the gel after electrophoresis can be electrophoresed perpendicular to the direction of development of the isoelectric focusing using a planar gel (e.g., SDS-polyacrylamide gel) to separate the molecular weights. By staining the gel after two-dimensional electrophoresis in a conventional manner, the presence or absence of protein, its molecular weight, and its isoelectric point can be confirmed.

[0035] From the viewpoint of exerting the effects of the present invention, the molecular weight of the protein is preferably 45,000 to 53,000, more preferably 47,000 to 51,000, and even more preferably 50,000 to 50,500. The isoelectric point (pI) of the protein is preferably 4.00 to 4.90, more preferably 4.40 to 4.85, and even more preferably 4.60 to 4.80.

[0036] The protein is preferably tubulin, more preferably α-tubulin or β-tubulin, and even more preferably β-tubulin. In humans, there are five types of tubulin: α (alpha), β (beta), γ (gamma), δ (delta), and ε (epsilon). Tubulin is highly conserved across species, forming heterodimers that further multimerize to form microfilaments. Heterodimers of α-tubulin and β-tubulin are the basic structural units of microtubules involved in cell division. α-tubulin and β-tubulin are similar, with molecular weights of approximately 55,000. At least seven β-tubulin isotypes have been identified in humans. These isotypes are classified according to the sequence of the carboxy-terminal domain: class I (HM40), class II (Hβ9), class III (Hβ4), class IVa (H5β), class IVb (Hβ2), class V, and class VI (Hβ1). (Roman numerals indicate protein isotypes, and the notation in parentheses indicates human gene classification.) Class III (Hβ4), i.e., tubulin beta-4B chain (Homo sapiens), is a protein that forms microtubules composed mainly of cytoskeletal proteins and is believed to play a role in maintaining cell shape and physiological activity. The tubulin beta-4B chain is a protein consisting of 445 amino acids (SEQ ID NO: 1), with a molecular weight of 50255 and a pI of 4.79.

[0037] The origin of the tubulin contained in the decellularized cell structure is not particularly limited as long as it is derived from a eukaryote, but tubulin derived from a mammal or bird is preferred. Examples of mammals include cows, horses, camels, llamas, donkeys, yaks, sheep, pigs, goats, deer, alpacas, dogs, raccoon dogs, weasels, foxes, cats, rabbits, hamsters, guinea pigs, rats, mice, squirrels, and raccoons. Examples of birds include parakeets, parrots, chickens, ducks, turkeys, geese, guinea fowl, pheasants, ostriches, quails, and emus.

[0038] Furthermore, the decellularized cell structure of the present invention may include, but is not limited to, a modified tubulin, as long as the effects of the present invention can be obtained. Examples of modified tubulin include: (1) a polypeptide comprising an amino acid sequence in which one or more amino acids (preferably 1 to 10, more preferably 1 to 7, and even more preferably 1 to 5) have been deleted, substituted, inserted, and / or added, for example, one to several amino acids, at one or more positions in the amino acid sequence of tubulin (e.g., the amino acid sequence represented by SEQ ID NO: 1), and which has tubulin activity; or (2) a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence of tubulin (e.g., the amino acid sequence represented by SEQ ID NO: 1) and which has tubulin activity. Examples of tubulin activity include improved ability of the decellularized cell structure of the present invention to maintain thickness and hardness after decellularization, compared to a decellularized cell structure not containing the modified tubulin.

[0039] (Amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, and / or added) The variant may be a polypeptide consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, and / or added in the amino acid sequence of tubulin (e.g., SEQ ID NO: 1). The variant polypeptide is capable of maintaining thickness and stiffness after decellularization. In other words, polypeptides that do not exhibit the ability to maintain thickness and stiffness after decellularization are not included in the variant polypeptide. As used herein, "an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, and / or added" means that the amino acid sequence has been modified by amino acid substitution or the like. The number of amino acid modifications can be, for example, 1 to 30, 1 to 20, 1 to 15, or 1 to 10, preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 5, and most preferably 1 to 2. An example of a variant amino acid sequence that can be used in the present invention is preferably an amino acid sequence having one or several (preferably 1, 2, 3, or 4) conservative substitutions in the amino acids.

[0040] (Amino acid sequence having 90% or more identity to an amino acid sequence) The variant may be a polypeptide consisting of an amino acid sequence having 90% or more identity to the amino acid sequence of tubulin (e.g., SEQ ID NO: 1). The variant polypeptide is capable of maintaining thickness and stiffness after decellularization. In other words, polypeptides that do not exhibit the ability to maintain thickness and stiffness after decellularization are not included in the variant polypeptide. More preferably, the variant is a polypeptide consisting of an amino acid sequence having an identity of 95% or more, more preferably an amino acid sequence having an identity of 96% or more, more preferably an amino acid sequence having an identity of 97% or more, more preferably an amino acid sequence having an identity of 98% or more, or more preferably an amino acid sequence having an identity of 99% or more, and which exhibits the ability to maintain thickness and stiffness after decellularization.

[0041] The "amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, and / or added" or "amino acid sequence with 90% or greater identity" to the amino acid sequence of tubulin (e.g., SEQ ID NO: 1) refers to a substitution in the amino acid sequence of tubulin (e.g., SEQ ID NO: 1), and this substitution is a conservative substitution that maintains the function of the tubulin used in the present invention. In other words, a "conservative substitution" refers to a substitution that does not impair the excellent effects of tubulin. That is, a substitution that allows the thickness and hardness of the tubulin to be maintained after decellularization, even when the insertion, substitution, deletion, or addition occurs. Specifically, this refers to the replacement of an amino acid residue with another chemically similar amino acid residue. Examples include replacing a hydrophobic residue with another hydrophobic residue, or replacing a polar residue with another polar residue having the same charge. Functionally similar amino acids that can be obtained by such substitutions are known in the art for each amino acid. Examples of nonpolar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0042] The protein (e.g., tubulin) is a protein that forms microtubules composed mainly of cytoskeletal proteins and is presumed to play a role in maintaining the shape of cells. That is, although not limited to, the function of maintaining cell shape is thought to be related to maintaining thickness and hardness after decellularization. Therefore, the protein (e.g., tubulin) is thought to effectively act on the strength of decellularized cell structures.

[0043] The present disclosure includes the following aspects. [1] A decellularized cell structure obtained by decellularizing a cultured cell structure, characterized in that the decellularized cell structure has a porosity of 30% to 80%; [2] The decellularized cell structure according to [1], wherein the hardness of the decellularized structure is 0.20 to 5.00 (N); [3] The decellularized cell structure according to [1] or [2], wherein the DNA content per dry mass of the decellularized cell structure is 0.020 mass% or less; [4] The decellularized cell structure according to any of [1] to [3], wherein the decellularized cell structure contains a protein with (a) a molecular weight of 45,000 to 53,000 and (b) an isoelectric point of pI 4.00 to 4.90; [5] The decellularized cell structure according to [4], wherein the protein is tubulin; [6] A method for producing a decellularized cell structure, comprising: a cultured cell structure formation step of forming a cultured cell structure by culturing cells; and a decellularization step of decellularizing the cultured cell structure to a porosity of 30% to 80%. [7] The method for producing a decellularized cell structure according to [6], wherein cells are cultured in any shape before or simultaneously with the formation of the cultured cell structure in the cultured cell structure formation step; [8] The method for producing a decellularized cell structure according to [7], wherein in the cultured cell structure formation step, the culturing of cells in any shape before the formation of the cultured cell structure is the culturing of sheet-like cells or the culturing of cell aggregates; [9] The method for producing a decellularized cell structure according to [6], wherein cells are cultured in any shape after the cultured cell structure formation step;

[10] The method for producing a decellularized cell structure according to any of [6] to [9], wherein the decellularization is performed by high hydrostatic pressure treatment; and

[11] The method for producing a decellularized cell structure according to any of [6] to

[10] , wherein the thickness retention rate before and after decellularization in the decellularization step is 90% or more.

[0044] The present invention is not limited to the above-described embodiments. The above-described embodiments are merely examples, and any configuration that is substantially identical to the technical idea described in the claims of the present invention and that provides similar effects is included within the technical scope of the present invention.

[0045] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0046] Example 1 In this example, a decellularized cell structure was produced using fibroblasts. (Preparation of cultured cell structure) A T-175 flask (vent cap, 175 cm ２ NHDF-Neo-dermal fibroblasts (CC-2509, manufactured by Lonza) were cultured on a 100-well plate (Biolamo). When the cells reached approximately 90-100% confluence, they were detached and a cell suspension (10 mL) was prepared (approximately 2.0 × 10 ７ The dimple plate was placed in a 10 cm dish, and 100% ethanol (10 mL) was added and spread over the entire surface of the dimple plate. After confirming that there were no air bubbles, the ethanol was removed. This procedure was repeated twice using phosphate buffer (PBS, 0.01 M, pH 7.4). 10 mL of FKCM medium (serum-free medium for fibroblasts, manufactured by Fukoku Co., Ltd.) was added to the dimple plate, and the above cell suspension was poured evenly over the plate. The plate was then incubated at 37°C and 5% CO ２ The cells were cultured in an incubator (IP400, Yamato Scientific). After overnight culture, spheroids (aggregations of cells) in the dimple plate were confirmed using an inverted microscope (CKX31, Olympus). The spheroids were removed from the depressions of the dimple plate by pipetting and transferred to a 10 cm dish. The 10 cm dish was rotated to collect and recover the spheroids in the center, and then placed in a net mold (Net Mold Starter Kit V6, Tissue by Net). The net mold was transferred to a 60 mL safe container (WATSON) containing FKCM medium (80 mL) and incubated at 37°C and 5% CO ２The cells were placed in a shaker (OS-762, Optima) in an incubator and culture was initiated under shaking (45 rpm). Half of the medium was replaced once or twice a week, and culture was continued. After approximately three weeks of culture, the net mold was removed to obtain a cultured cell structure. The above-mentioned method for forming a cultured cell structure is an example of a method in which, in the cultured cell structure formation step, cells are cultured into a desired shape before the formation of the cultured cell structure, and in the cultured cell structure formation step, the culture of cells into a desired shape before the formation of the cultured cell structure is the culture of cell aggregates (spheroids). The above-mentioned method for forming a cultured cell structure is an example in which cell aggregates (spheroids) are held in a cavity and cultured, causing the cell aggregates to produce extracellular matrix (ECM), thereby forming a cultured cell structure.

[0047] (Decellularization of Cultured Cell Constructs) The cultured cell constructs removed from the net mold and FKCM medium were placed in a nylon poly vacuum bag (Kashibijin, manufactured by Kurilon Chemical Co., Ltd.) and double-sealed on all four sides with a sealer. Using FKCM medium as 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 performed at a minimum temperature of 23.3°C and a maximum temperature of 35.9°C. The cultured cell constructs were removed from the nylon poly vacuum bag and transferred to an embedding cassette (manufactured by TISSUE-TEK Co., Ltd.), then immersed in physiological saline and washed with shaking at 4°C for at least 5 minutes. The cultured cell constructs were then immersed in a nuclease DNase I solution (800 U / mL) and washed with shaking at 4°C for at least 18 hours. Thereafter, the cultured cell structure was immersed in physiological saline and washed with shaking at 4°C for 5 minutes or more, repeated three times, to obtain a decellularized cell structure.

[0048] Example 2 In this example, a decellularized cell structure was produced using fibroblasts by high hydrostatic pressure treatment at 200 MPa. The procedure of Example 1 was repeated except that a high hydrostatic pressure treatment of 200 MPa was performed instead of a high hydrostatic pressure treatment of 600 MPa, to obtain a decellularized cell structure.

[0049] Example 3 In this example, a decellularized cell structure was produced using fibroblasts by high hydrostatic pressure treatment at 400 MPa. The procedure of Example 1 was repeated, except that a high hydrostatic pressure treatment of 400 MPa was performed instead of a high hydrostatic pressure treatment of 600 MPa, to obtain a decellularized cell structure.

[0050] Example 4 In this example, a decellularized cell structure was produced by freeze-thawing. The cultured cell structure removed from the net mold was frozen on dry ice or in a freezer, stored at -80°C for at least 30 minutes, and then thawed in phosphate buffer solution (PBS, 0.01 M, pH 7.4) warmed to 37°C. After this freeze-thawing process was repeated three times, the cultured cell structure was immersed in a solution of the nuclease DNase I (800 U / mL) and washed with shaking at 4°C for 24 hours. The cultured cell structure was then washed with shaking in physiological saline at 4°C for at least 2 hours to obtain a decellularized cell structure.

[0051] Comparative Example 1 In this comparative example, decellularization was performed using a surfactant treatment. The cell structure obtained in Example 1 was decellularized using the following surfactant treatment. The cultured cell structure removed from the net mold was washed with water for injection at 4°C for 1 hour. The cultured cell structure was then treated with 0.25% by mass sodium dodecyl sulfate solution (10 mM Tris, pH 8.0) at 4°C for 24 hours. The cultured cell structure was then treated with 0.5% by mass Triton-X (polyoxyethylene octylphenyl ether) solution (10 mM Tris, pH 8.0) at 4°C for 24 hours. The cultured cell structure was then immersed in physiological saline and washed with shaking for at least 5 minutes at 4°C three times to obtain a decellularized cell structure.

[0052] Calculation of Decellularized DNA Ratio: The masses of the decellularized cell structures from Examples 1 to 4 and Comparative Example 1 were measured. These samples were then immersed in a protease solution and dissolved. Proteins were then removed by treatment with phenol / chloroform, and DNA was recovered by ethanol precipitation. The recovered DNA was fluorescently stained with Picogreen (Life Technologies) and the fluorescence intensity was measured to quantify the DNA. The DNA content of the sample was calculated from the sample mass and DNA quantity. A calibration curve prepared using the standard DNA provided with the Picogreen was used to quantify the DNA. Using a separate sample, the drying loss ratio was calculated from the sample mass (70% moisture content) and the mass of the dried sample (stored in a 60°C incubator for 12 hours). The DNA content per dry mass of the sample was calculated from the sample DNA content and drying loss ratio. The results are shown in Table 1. (Decellularized DNA ratio) = (DNA content of the dried test piece of the decellularized cell structure) / (mass of the dried test piece of the decellularized cell structure)

[0053]

[0054] Preparation of Paraffin-Embedded Tissue Specimens and Stained Section Samples As a pretreatment, the decellularized cell structures obtained in Examples 1 to 4 or Comparative Example 1 were immersed in 4% by mass PFA (4% by mass paraformaldehyde phosphate buffer, for tissue fixation, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) for 48 hours or more. The decellularized cell structures were then immersed in phosphate buffer (PBS, 0.01 M, pH 7.4) and washed with shaking at 4°C for 30 minutes or more. The decellularized cell structures were then immersed in 70% by mass ethanol and washed with shaking at 4°C for 30 minutes or more to prepare samples for paraffin-embedded tissue specimens.

[0055] Paraffin-embedded tissue specimens were prepared from the above samples using the following procedure to prepare section-stained samples for staining (described below). The tissues were rinsed with tap water for 30 to 40 minutes to remove formaldehyde. Using a paraffin embedding device (CT-Pro20, manufactured by Genostaff), the samples were dehydrated in an ethanol bath in the following order: 70% ethanol by weight for 20 minutes (×1) → 95% ethanol by weight for 20 minutes (×2) → 100% ethanol by weight for 20 minutes (×2). The samples were then washed twice in xylene for 20 minutes each. The samples were then incubated twice in a 65°C paraffin bath for 30 minutes each. The melted paraffin was poured into a mold, the sample was placed in the mold, and allowed to cool for 15-20 minutes to prepare paraffin-embedded tissue specimens. The paraffin-embedded tissue specimens were then used to prepare serial sections at a thickness of 5 μm on a microtome, which were then floated in a water bath containing deionized water at 37°C. Each section was floated on distilled water placed on a slide glass, heated (30-40°C) on an extension table, and dried in an incubator (37°C) for 1-2 nights to prepare stained section samples.

[0056] (Sirius Red Staining) The section-stained sample was stained with Sirius Red to stain the extracellular matrix (ECM) according to the following procedure. First, the section-stained sample was deparaffinized, hydrated in distilled water, and rinsed with distilled water. Next, the section-stained sample was treated with iron hematoxylin (a mixture of equal parts of iron hematoxylin I (1% by mass hematoxylin (Sigma-Aldrich) / 95% by mass ethanol) and iron hematoxylin II (Muto Chemicals)) for 2 minutes and rinsed with distilled water. The section-stained sample was then treated with a 0.5% by mass hydrochloric acid / 70% by mass ethanol solution for 3 minutes. Subsequently, the sample was treated with a 0.2% by mass aqueous phosphomolybdic acid solution for 5 minutes. Subsequently, the sample was treated with a 0.1% by mass Direct Red 80 (Sirius Red, Sigma-Aldrich) / saturated picric acid solution for 90 minutes. Subsequently, the sections were dipped three times in 0.01 N hydrochloric acid, and then the stained section samples were dehydrated, dehydrated in xylene through a filter, and mounted in a mounting medium (Mount Quick, manufactured by Daido Sangyo Co., Ltd.).

[0057] <Calculation of Porosity> The obtained Sirius Red stained sections were observed under a microscope, and the sections were photographed at 10x magnification. Image processing software (ImageJ) was used to calculate the porosity of the decellularized cell structures of Examples 1 to 4 and Comparative Example 1. Images of the stained section samples were imported, and the area of ​​the entire decellularized cell structure and the area of ​​the Sirius Red stained portion (ECM) were measured. The porosity of the decellularized cell structure was calculated according to the following formula. A rectangular area (310 μm x 310 μm) was set as the measurement area, and the average porosity of three locations was calculated. In order to exclude large cavities, a 34,000 (approximately 35%) μm ２ The above regions that did not contain cavities were set. Porosity (%) = (area of ​​entire decellularized cell structure (measurement region) - area of ​​Sirius Red stained portion of Example or Comparative Example) / area of ​​entire decellularized cell structure (measurement region) x 100

[0058] <Calculation of thickness maintenance rate> The thickness maintenance rate was calculated from the thickness of the cultured cell structure and the thickness of the decellularized cell structures of Examples 1 to 4 and Comparative Example 1. Note that the thickness of the cultured cell structure refers to the untreated state in Figure 2 described below. Thickness maintenance rate (%) = (thickness of decellularized cell structure / thickness of cultured cell structure) x 100

[0059] The results for porosity are shown in Figure 1, and the results for thickness retention are shown in Figure 2. With regard to porosity, Examples 1 to 4 had values ​​of 30 to 80%, while Comparative Example 1 had a higher value. Furthermore, with regard to thickness retention, Examples 1 to 4 maintained the thickness after decellularization relative to that before decellularization compared to Comparative Example 1, and it was found that the shape before and after decellularization was maintained. This shows that by setting the porosity of the decellularized cell structure to a predetermined value, the shape of the cell structure before and after decellularization is maintained, and the decellularized cell structure can be formed into any shape.

[0060] <Hardness Test> Texture profile analysis (TPA) was performed as follows to measure the hardness of the decellularized cell structures obtained in the Examples and Comparative Examples. TPA was performed at room temperature using a texture analyzer TA.XT plus (Eiko Seiki Co., Ltd.) equipped with a P / 4 cylindrical probe (diameter 4 mm). The point at which a load of 0.01 (N) was applied to the probe was set as point 0, and the probe was compressed twice over a distance of 0.5 mm at a speed of 0.02 mm / sec. The maximum force (N) during the first compression cycle indicates the hardness. Similar measurements were performed for Production Examples 1 and 2 below.

[0061] (Production Example 1) Bovine pericardium was cut open to form a sheet, and fat was removed entirely (hereinafter, this sheet-like bovine pericardium is referred to as a "pericardium sheet"). This was used as the pericardium sheet of Production Example 1.

[0062] (Production Example 2) The pericardium sheet from Production Example 1 was placed in a nylon poly vacuum bag and subjected to high hydrostatic pressure treatment at 600 MPa for 10 minutes using a medium prepared by adding phosphate buffered saline (PBS, 0.01 M, pH 7.4) to water for injection. The high-hydrostatic-pressure-treated pericardium sheet was immersed in the nuclease DNase I (125 U / mL) and washed with shaking at 4°C for at least 18 hours. It was then treated in 80% ethanol at 4°C for at least 1 hour, and finally washed with 4 L of water for injection at 4°C. After lyophilization, the sheet was subjected to gamma irradiation (25 kGy). This was designated the decellularized pericardium sheet (decellularized tissue) of Production Example 2.

[0063] The test results are shown in Figure 4. The hardness of the pericardial sheet of Production Example 1 was 10.13 (N), and the hardness of the decellularized pericardial sheet (decellularized tissue) of Production Example 2 was 13.62 (N). It was found that the Examples maintained their hardness compared to the Comparative Examples and could be used stably as grafts. In particular, it was found that Example 1 produced good results in terms of stability as a graft. It was also found that the Examples were softer than the pericardial sheet of Production Example 1 and the decellularized tissue of Production Example 2, and had a different hardness.

[0064] Protein Analysis: The differences in the composition of the decellularized cell constructs of Example 1 and Comparative Example 1 were analyzed by two-dimensional electrophoresis. 0.3 mL of protein dissolution solution (containing urea and surfactant) was added to the decellularized cell constructs of Example and Comparative Example, and the mixture was shaken at 1,400 rpm at room temperature for 2 hours. After homogenization using a Polytron homogenizer, the mixture was centrifuged at 20,000 xg for 30 minutes at room temperature, and the supernatant was collected. The sample solution was purified using a 2-D Clean-Up Kit (Cytiiva, Global Life Science Technologies Japan). Protein quantification and electrical conductivity measurements were then performed to confirm that the measured values ​​were within the acceptable range for two-dimensional electrophoresis, and the sample was used for two-dimensional electrophoresis. A buffer solution was added to the two-dimensional electrophoresis sample, and then the protein dissolution solution (containing urea and surfactant) was added to adjust the total volume to 0.34 mL, creating a sample solution. The sample solution was placed in a swelling tray, and a first-dimensional electrophoresis precast gel (Immobiline DryStrip, Cytiiva, Global Life Science Technologies Japan) was placed on top of the solution. DryStrip cover fluid (PlusOne DryStrip Cover Fluid, Cytiiva, Global Life Science Technologies Japan) was then layered on top and allowed to stand overnight. The swollen precast gel was placed in an electrophoresis apparatus (Multiphor II Electrophoresis Unit, Cytiiva, Global Life Science Technologies Japan) and run (500 V for 1 minute, 3500 V for 7.5 hours, 20°C). An acrylamide gel with a 10-20% gradient was used. The gel was allowed to stand for 24 hours after preparation to allow complete polymerization of acrylamide. The equilibrated first-dimensional electrophoresis precast gel was placed on an acrylamide gel and fixed with 1% agarose solution containing 0.125% bromophenol blue. A molecular weight marker was applied to the left end of the gel as a molecular weight standard. Electrophoresis was performed at 80 V for 17 hours until the bromophenol blue band was visible at the bottom of the gel.

[0065] Protein Staining After electrophoresis, the gel was stained with a fluorescent stain for detecting total proteins (SYPRO Ruby protein gel stain, S21900, Thermo Fisher Scientific Inc.), and the image was saved using a fluorescent scanner. Images were captured using the scanner at an excitation wavelength of 488 nm, a fluorescence filter of 640 nm Bandpass, and a resolution of 100 micrometers. The resulting tiff image file of the fluorescently stained image was imported into ImageMaster Platinum (cytiiva, Global Life Science Technologies Japan, Inc.) and subjected to digitization analysis. In the digitization analysis, landmarks were first manually added to common spots on the gel. Landmarking was performed up to 340 spots per gel, and then gel matching was performed. This procedure assigned a common spot ID to the spots developed on the two gels (the gel to which Example 1 was applied and the gel to which Comparative Example 1 was applied). Spot detection was then performed on the gels, and the spot signal concentration (% Volume) was calculated. The spot signal concentration was calculated from the ratio of the spot signal to the total signal of all spots in the gel. The % Volume value was expressed as a percentage, with a minimum display of 0.001%. Spots that met the following two conditions were used as conditions for protein identification: ・The total spot signal concentration of the SYPRO Ruby stained image (the sum of the two gels when combined in pairs) is 0.1 or more. ・The change in spot concentration (the change between the two gels when combined in pairs) is 0.5-fold or less or 2-fold or more. The results of two-dimensional electrophoresis are shown in Figure 5. In Example 1, one spot (1) was detected at an isoelectric point pI of 4.00 to 4.90 and a molecular weight of 45,000 to 53,000. In contrast, in Comparative Example 1, the spot detected in Example 1 was not detected. It was found that Example 1 contains a specific protein not present in Comparative Example 1.

[0066] <Identification of Spots> After capturing the fluorescent stained image, silver staining was performed using silver nitrate (195-09382, Fujifilm Wako Pure Chemical Industries, Ltd.). Spots visualized by silver staining were excised and cut into 1 mm cubes. 100 μL of 15 mM potassium ferricyanide and 50 mM sodium thiosulfate were added to the gel pieces, which were then shaken for 10 minutes. The solution was discarded, and Milli-Q water was added and the pieces were washed with shaking until the color disappeared. The gel pieces were dehydrated with acetonitrile. 10 μL of an enzyme solution containing 100 mM ammonium bicarbonate and 0.02 μg / μL trypsin was added to swell the gel pieces, which were then left at 37°C for 16 hours. 50 μL of 0.1% TFA and 50% acetonitrile was added and the pieces were shaken for 20 minutes to extract peptides. This extraction was performed twice. The peptide extract was concentrated to approximately 10 μL using a vacuum centrifuge. The sample was adsorbed onto a ZipTip C18 (Millipore, ZTC18S960), and the peptides were eluted with 2.5 μL of 60% acetonitrile, 0.1% TFA solution. 1 μL of the sample solution was mixed with 1 μL of CHCA matrix solution, dropped onto a target plate, dried, and then measured using a mass spectrometer (Ultraflex Xtreme). Proteins registered in the database (NCBI RefSeq) were identified based on the obtained mass values. Analysis device: Ultraflex Xtreme (Bruker Daltonics) Target plate: MTP Anchorchip 600 / 384 (209513, Bruker Daltonics) Polarity: Positive mode Detection mode: Reflector mode (300-6,000 m / z) CHCA matrix solution: 0.3 g / L CHCA, 33% acetone, 66% ethanol MS / MS Ion Search Search conditions: Identification software: Mascot (Matrix Science) Database: NCBI RefSeq Search species: Human (Genome assembly GRCh38.p13) Enzyme: Trypsin Fixed modification: Carbamidomethylation

[0067] The spots in Example 1 were identified using a mass spectrometer and the results are shown in Table 2. The spot numbers in Table 2 correspond to the spot numbers in Figure 5. The spots were identified as tubulin.

[0068]

[0069] The decellularized cell structure of the present invention can be used as a transplant that does not cause rejection reactions in the treatment of various diseases.

Claims

1. A decellularized cell structure obtained by decellularizing a cultured cell structure, characterized in that the decellularized cell structure has a porosity of 30% to 80%.

2. The decellularized cell structure according to claim 1, wherein the hardness of the decellularized structure is 0.20 to 5.00 (N).

3. A decellularized cell structure as described in claim 1 or 2, wherein the DNA content per dry mass of the decellularized cell structure is 0.020 mass% or less.

4. A decellularized cell structure according to claim 1 or 2, comprising a protein having (a) a molecular weight of 45,000 to 53,000 and (b) an isoelectric point of pI 4.00 to 4.

90.

5. The decellularized cell structure described in claim 4, wherein the protein is tubulin.

6. A method for producing a decellularized cell structure, comprising: a cultured cell structure formation step of forming a cultured cell structure by culturing cells; and a decellularization step of decellularizing the cultured cell structure to a porosity of 30% to 80%.

7. A method for producing a decellularized cell structure as described in claim 6, wherein, in the cultured cell structure formation process, cells are cultured into any shape before or simultaneously with the formation of the cultured cell structure.

8. A method for producing a decellularized cell structure as described in claim 7, wherein in the cultured cell structure formation process, the culture of cells in any shape prior to the formation of the cultured cell structure is the culture of sheet-shaped cells or the culture of cell aggregates.

9. A method for producing a decellularized cell structure according to claim 6, wherein the cells are cultured into any shape after the cultured cell structure formation step.

10. A method for producing a decellularized cell structure described in any one of claims 6 to 9, wherein the decellularization is carried out by high hydrostatic pressure treatment.

11. A method for producing a decellularized cell structure described in any one of claims 6 to 9, wherein the thickness maintenance rate between before and after decellularization in the decellularization process is 90% or more.