Decellularizated structure and methods for preparing the same

Abstract

The purpose of this invention is to provide a decellularized graft with any thickness or specific shape. The aforementioned problem can be solved by a decellularized cell structure, which is the decellularized cell structure of the present invention for decellularizing cultured cell structures, characterized by a porosity of 30% to 80%.

Description

Technical Field

[0001] This invention relates to a decellularized cell structure. Prior Technology

[0002] When transplanting tissue samples taken from other people or different animal species, there is often a problem of rejection by the recipient's tissue. Therefore, to improve biocompatibility, a technique using decellularized tissue as a transplant sample has been developed. This decellularized tissue consists of supporting tissue (extracellular matrix, ECM), which is the residue left after cells have been removed from the biological tissue. Decellularization refers to the removal of antigenic cellular components such as nucleic acids from the recipient, thereby avoiding immune rejection. [Previous Technical Documents] [Patent Literature]

[0003] [Patent Document 1] Japanese Patent Publication No. 2005-514971 [Patent Document 2] International Publication No. 2016 / 136633 [Patent Document 3] Japanese Patent Application Publication No. 2012-139541 [Patent Document 4] Japanese Re-appearance No. 2014-148321 [Patent Document 5] Japanese Re-registration No. 2018-051415 [Patent Document 6] Japanese Patent Application Publication No. 2004-357694 [Patent Document 7] Japanese Patent Application Publication No. 2020-198794 [Patent Document 8] Japanese Patent No. 4517125 Summary of the Invention

[0004] [The problem that the invention aims to solve]

[0005] The aforementioned decellularized tissue is useful in obtaining non-rejection grafts by decellularizing living tissue. However, to achieve the desired thickness, a lamination process is sometimes required. Furthermore, it is not easy to create specific shapes. Moreover, maintaining the strength of the graft itself is not easy. Therefore, the present invention aims to provide a decellularized graft with arbitrary thickness and the ability to maintain rigidity. [Methods for solving problems]

[0006] The inventors of this case, while conducting research on decellularized transplant sheets with arbitrary thickness or specific shape, unexpectedly discovered that by decellularizing cultured cell structures (Patent Documents 3-7) obtained from culture, decellularized transplant sheets with arbitrary thickness and maintaining rigidity can be obtained. This invention is based on this viewpoint. Therefore, the present invention relates to: [1] A decellularized cell structure, which is a decellularized cell structure obtained by decellularizing a cultured cell structure, characterized in that the porosity is 30%~80%; [2] As in [1], the decellularized cell structure has a hardness of 0.20~5.00 (N); [3] Decellularized cell structures as in [1] or [2], wherein the DNA content per unit dry mass of the decellularized cell structure is less than 0.020% by mass; [4] Decellularized cell structures, such as those in [1] or [2], contain proteins with (a) a molecular weight of 45,000 to 53,000 and (b) an isoelectric point of pI 4.00 to 4.90; [5] As in [4], the decellularized cell structure, in which the aforementioned protein is tubulin; [6] A method for manufacturing a decellularized cell structure, comprising: The cell structure formation step involves culturing cells to form cell structures; and The decellularization step involves decellularizing the aforementioned cultured cell structures to achieve a porosity of 30% to 80%. [7] The method for manufacturing decellularized cell structures as in [6], wherein in the step of forming cultured cell structures, the cells are cultured into arbitrary shapes before or simultaneously with the formation of cultured cell structures; [8] The method for manufacturing decellularized cell structures as described in [7], wherein in the aforementioned cell structure formation step, the cells are cultured into culture sheets of arbitrary shapes or cell aggregates before the formation of the cell structure; [9] The method for manufacturing decellularized cell structures as in [6], wherein after the cell structure formation step, the cells are cultured into arbitrary shapes;

[10] The method for manufacturing decellularized cell structures as described in any of [6] to [9], wherein the aforementioned decellularization is carried out by high hydrostatic pressure treatment;

[11] The method for manufacturing a decellularized cell structure as described in any of [6] to [9], wherein the thickness maintenance rate before and after decellularization in the aforementioned decellularization step is 90% or more. [Effects of the Invention]

[0007] According to the decellularized cell structure of the present invention, a decellularized graft with arbitrary thickness and maintained rigidity can be provided. Furthermore, the manufacturing method of the present invention can maintain the thickness after decellularization relative to the thickness before decellularization, thus maintaining the shape before and after decellularization. That is, since decellularized materials of arbitrary thickness can be provided, decellularized materials with specific shapes can be provided, and the decellularized materials can be used according to the target application such as grafts or cell therapy. Moreover, decellularized materials with strength and no rejection reaction can be produced. Simple Explanation of the Diagram

[0008] [Figure 1] is a graph showing the porosity of decellularized cell structures in Examples 1-4 and Comparative Example 1. [Figure 2] is a graph showing the thickness retention rate of decellularized cell structures in Examples 1-4 and Comparative Example 1. [Figure 3] is a microscopic photograph of decellularized cell structures stained with Sirius red. [Figure 4] is a graph showing the results of the hardness test of the decellularized cell structures of Examples 1-4 and Comparative Example 1. [Figure 5] is a photograph showing the color spots of proteins produced by two-dimensional electrophoresis in Example 1(A) and Comparative Example 1(B). Implementation

[0009] [Forms of Invention Implementation]

[0010] [1] Decellularized cell structures The decellularized cell structure system of the present invention decellularizes cultured cell structures, and the porosity of the decellularized cell structures is 30%~80%.

[0011] Cultured Cell Structures The cell culture system of this invention comprises cell structures obtained through in vitro culture. The cells in the culture system adhere to each other by generating extracellular matrix (ECM); the three-dimensional shape is not particularly limited as long as it can be maintained, but the minimum three-dimensional distance (thickness) is preferably 50 μm or more, more preferably 100 μm or more. Other two-dimensional dimensions are also not particularly limited, for example, 1 mm or more, preferably 6 mm or more. This allows for the effective fabrication of decellularized cell structures. The strength of the cultured cell structure is not particularly limited as long as it can maintain a certain three-dimensional shape, but it is preferable to have the strength to be handled with forceps or sutured in a moist state. The shape of the cultured cell structure is not particularly limited, and examples include sheet-like, tubular, or block-like forms. In this way, it is easy to prepare decellularized cell structures according to the target application such as transplantation sheets or cell therapy.

[0012] The decellularized cell structure of this invention can be in a moist or dry state. A decellularized cell structure provided in a moist state can be used immediately upon transplantation. On the other hand, a decellularized cell structure provided in a dry state can be stored for a long time, is easy to transport, and can be restored for use when needed.

[0013] Cells can be broadly classified into planktonic cells and adhesive cells; the cells used for culturing cell structures are not limited, but adhesive cells are preferred based on the view that it is easier to prepare culturing cell structures. The types of adhesive cells are not particularly limited and can be selected appropriately based on the intended use of the cultured structure. Examples of differentiated adhesive cells include hepatocytes, astrocytes, Koff's cells, vascular endothelial cells, sinusoidal endothelial cells, endothelial cells, fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, epidermal cells, tracheal epithelial cells, digestive tract epithelial cells, cervical epithelial cells, epithelial cells, mammary gland cells, external cells, smooth muscle cells, cardiomyocytes, myocytes, kidney cells, pancreatic Langerhans cells, peripheral nerve cells, nerve cells, chondrocytes, or osteocytes. Examples of undifferentiated adhesive cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), germ cell lineage stem cells (GS cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, hematopoietic stem cells, neural stem cells, cardiac precursor cells, vascular endothelial precursor cells, neural precursor cells, adipose precursor cells, dermal fibroblasts, skeletal muscle myoblasts, osteoblasts, or dental cells. Based on the viewpoint that it is easy to produce cultured cell structures, skin fibroblasts, mesenchymal stem cells, and myocardial precursor cells are preferred. The source of the cells is not particularly limited and includes eukaryotic cells, prokaryotic cells, multicellular organism cells, or single-celled organism cells. Examples of eukaryotic cells include animal cells, insect cells, plant cells, fungi, algae, or protozoa. Animal cells are not limited and may include cells from humans, monkeys, dogs, cats, rabbits, ferrets, sheep, goats, cattle, pigs, horses, camels, mice, rats, hamsters, guinea pigs, or gerbils.

[0014] The cultured cell structures are not limited, but they are preferably adhered to the extracellular matrix. By adhering to the extracellular matrix, the cultured cell structures can acquire a certain strength. Examples of extracellular matrix include collagen, laminin, fibronectin, chondroitin sulfate, heparin sulfate, keratin sulfate, and hyaluronic acid, which vary depending on the cell type and are not particularly limited.

[0015] DNA Content The DNA content per unit 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 acquisition of appropriately decellularized cell structures with minimal rejection during transplantation. DNA content can be determined using the PicoGreen method. A dried sample of decellularized cellular components (hereinafter referred to as the sample) is dissolved in a proteolytic enzyme solution, treated with phenol / chloroform to remove proteins, and then the DNA is recovered by ethanol precipitation. The recovered DNA is then stained with PicoGreen (Life Technologies) and the fluorescence intensity is measured to quantify the DNA and calculate the sample's DNA content (mass). Quantification is performed using a calibration curve established using the standard DNA provided with PicoGreen. The DNA ratio is calculated from the dried mass of the sample and the DNA content using the following formula: (DNA content per unit dry mass (hereinafter referred to as decellularized DNA ratio) = (DNA content of the dried sample of decellularized cell structures) / (mass of the dried sample of decellularized cell structures)

[0016] 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%, still more preferably 42%, still more preferably 44%, still more preferably 47%, and most preferably 50%. The upper limit of the porosity is preferably 77%, more preferably 73%, still more preferably 70%, even more preferably 68%, even more preferably 66%, still more preferably 64%, and most preferably 55%. Within the aforementioned ranges, a transplant patch with sufficient strength and no rejection reaction can be obtained. Furthermore, decellularized transplant patches with any thickness or specific shape can be provided. In this specification, the porosity is calculated as follows: Sections of decellularized cellular structures are stained with Sirius red, a staining agent for the extracellular matrix. The tissue image is analyzed using image processing software and unstained portions by Sirius red are identified as voids, from which the porosity is calculated. Sirius Red staining was performed as follows: De-cellular cell sections were deparaffinized. Next, the sections were treated with iron hematoxylin. Then, they were treated with hydrochloric acid / ethanol solution and phosphomolybdic acid aqueous solution. Finally, they were treated with Sirius Red (Direct Red 80) for dehydration and mounting. Sirius red stained sections were observed under a microscope, and the porosity was calculated using image processing software (ImageJ). Sections were photographed at 10x magnification, and the area of ​​the entire decellularized cellular structure and the area of ​​the Sirius red stained portion 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 portion) / Area of ​​the entire decellularized cell structure × 100

[0017] [2] Method for manufacturing decellularized cell structures The method for manufacturing decellularized cell structures of the present invention includes: a cell structure formation step, which forms a cell structure by culturing cells; and a decellularization step, which decellularizes the aforementioned cell structure to achieve a porosity of 30% to 80%.

[0018] Steps for the formation of cell structure in culture (1) In the aforementioned step (1) of forming a cultured cell structure, a cultured cell structure is formed by culturing cells. The cells used are not particularly limited as long as they can be cultured in vitro. They can be, for example, primary cells taken directly from tissues or organs, or secondary cells obtained by passing primary cells taken directly from tissues or organs through several generations or sub-generations. They can also be secondary cells obtained by sub-generation over a long period of time. Specifically, the cells used are not limited to those described in the aforementioned "[1] Decellularized Cell Structure". Cultured cell structures are formed by the production of extracellular matrix (ECM) by cells outside the cell, allowing cells to adhere and bind together, thus maintaining a three-dimensional shape. The method for forming cultured cell structures is not particularly limited as long as it utilizes adhesion generated by the extracellular matrix; methods described in patent documents 3-8, for example, can be used. Specifically, examples include: a method for manufacturing sheet-like three-dimensional structures without using a scaffold (Patent Document 3); a method for stacking multiple cell sheets (Patent Document 4); a method for manufacturing structures by supplying a support body with a mesh-like space formed by multiple filaments or needle-like components to a cell aggregate (Patent Document 5); a method for manufacturing by culturing cell aggregates in a chamber (Patent Document 6); a method for manufacturing three-dimensional cell structures by culturing cell aggregates using a culture mold (Patent Document 7); or a method for manufacturing by culturing using a support body with filaments or needle-like structures penetrating the cell aggregates (Patent Document 8). The methods for manufacturing these structures all involve using the extracellular matrix secreted by cells to make cells adhere to each other, thereby forming cell structures. Examples of extracellular matrix include those described in the aforementioned "[1] Decellularized Cell Structures", but are not particularly limited to them.

[0019] For example, cultured cell structures can be formed using cell conglomerates (spheroids) as follows. The culture medium used can be selected appropriately based on the cells used. The cell conglomerates are placed into a cavity surrounded by a mesh or comb-like support with interstices finer than the size of the cell conglomerates. The cavity is formed into the desired shape by the mesh or comb-like support and the plate, and the support has multiple openings. The culture medium can be brought into contact with the cell conglomerates through these openings. Shaking the culture container while the cell conglomerates are held within the cavity allows the culture medium to effectively contact the cell conglomerates, resulting in successful cell conglomerate culture. The cell conglomerates can generate extracellular matrix, thus creating cultured cell structures with a three-dimensional structure in which the cell conglomerates adhere to each other and have the desired shape.

[0020] In the aforementioned cell culture structure formation step (1), cells can be cultured into any shape, either before or simultaneously with the formation of the cell culture structure. Cultured into any shape means cultured to any thickness or top-view shape. For example, the area can be adjusted to culture cells into any top-view shape. Cells of any top-view shape can be stacked and cultured to any thickness and shape. Alternatively, the cultured cells can be cut into the desired shape. In the aforementioned cell culture structure formation step (1), cells can be cultured simultaneously with the formation of the cell culture structure to form any shape. For example, cells can be cultured into any shape simultaneously with the method for forming the aforementioned cell culture structure. For example, cells can also be cultured in three dimensions (3D) in a gel containing an extracellular matrix to form a cell culture structure.

[0021] Furthermore, cells can be cultured in any shape before the formation of the cultured cell structure. In the cell structure formation step, culturing cells into any shape before the formation of the cultured cell structure is preferably, for example, culturing sheet-like cells or culturing cell aggregates (spheres). Examples of culturing before the formation of the cultured cell structure include culturing cells into any top-view shape, or stacking multiple cell sheets (sheet-like cells) as described in Patent Document 4, and culturing these stacked cell sheets before the formation of the cultured cell structure. The culture of cell sheets can be carried out according to the methods commonly used in the art for preparing cell sheets using culture trays or the like. By stacking and culturing the obtained cell sheets, cells can be cultured to any thickness and shape. Subsequently, by secreting extracellular matrix from the cells, the stacked cell sheets adhere, and a cultured cell structure maintaining a three-dimensional shape can be obtained.

[0022] Furthermore, examples of culturing before the formation of cell structures include, for instance, the creation of cell aggregates (spheroids) through culturing before cell adhesion, as described in Patent Documents 5 or 7. Adhesive cells cannot survive for long periods while floating in 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 conglomerates (spheres) can be created by agglutinating multiple cells. After monolayer culture, cells are transferred to hydrophobic or cell-non-adhesive round-bottomed multi-well or U-shaped discs (concave discs) for further culture, whereby the cells will agglutinate and form cell conglomerates. From an efficiency point of view, the culture time before the formation of cell conglomerates is preferably 6–48 hours, more preferably 6–24 hours. However, the method for producing cell conglomerates is not limited to the aforementioned methods; methods such as: rotational culture in a rotating solution; centrifugation to precipitate the cell suspension in a test tube; alginate beading; or the methods described in Patent Documents 5, 6, or 8 can be used. From the viewpoint of being able to process large quantities of uniform cell conglomerates, the method of placing the cell suspension in hydrophobic or cell-non-adhesive multi-well discs is more efficient and preferred. By adjusting the culture time of the cell conglomerates (spheroids), the cell conglomerates can be cultured to any thickness. Furthermore, by connecting the cell conglomerates (spheroids) together, they can be cultured to any thickness and shape. Further culturing these cell conglomerates (spheroids) allows the cells to secrete extracellular matrix, causing them to adhere to each other, thus obtaining cultured cell structures that maintain a three-dimensional shape. Moreover, by culturing cell conglomerates (spheroids) using the method described above for inserting them into a cavity, cultured cell structures with the desired three-dimensional shape can be accurately fabricated. When cells are cultured into arbitrary shapes before the formation of cell structures, the amount of extracellular matrix (ECM) is preferably less than the amount of extracellular matrix when the cell structures are formed.

[0023] Alternatively, after the cell structure formation step, the cells can be cultured into any shape. For example, after performing the above-mentioned method for forming cell structures, the cells can be further cultured into any shape. Another example is coating the cells with an extracellular matrix and then accumulating them into any shape for culture. The amount of extracellular matrix (ECM) when cells are cultured into arbitrary shapes after the cell structure formation step is preferably the same as or greater than the amount of extracellular matrix when the cell structure is formed.

[0024] Decellularization Step (2) In the aforementioned decellularization step (2), the aforementioned cell structures are decellularized to achieve a porosity of 30% to 80%. That is, the porosity of the decellularized cell structures 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 falling within the aforementioned range, a transplant patch with sufficient strength and no rejection reaction can be obtained. Furthermore, decellularized transplant patches with any thickness or specific shape can be provided.

[0025] The decellularization step can be performed using methods already known to the public. The method of decellularization is not particularly limited as long as the effects of this invention are achieved. Examples include, for instance, 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 using proteolytic enzymes or nucleic acid degrading enzymes, and treatment with an alcohol solvent. Two or more of these methods can be combined. However, to effectively obtain decellularized cell structures and to achieve the effects of this invention, high hydrostatic pressure treatment is preferred.

[0026] When obtaining the aforementioned decellularized structure using 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 achieving sufficient decellularization, the applied hydrostatic pressure is preferably 50 MPa or higher; from the viewpoint of not requiring a pressure vessel capable of withstanding pressure, not requiring a large amount of energy, and preventing ice formation when the applied medium is aqueous, which could damage the cell structure, a pressure of 1500 MPa or lower is preferred. More preferably, the applied hydrostatic pressure is 80 to 1300 MPa, even more preferably 90 to 1200 MPa, even more preferably 95 to 1100 MPa, and even more preferably 95 to 700 MPa. From the viewpoint of achieving decellularization, sterilization, and virus deactivation effects while being easy to apply, the optimal pressure is 400 to 700 MPa.

[0027] Examples of media used to apply hydrostatic pressure include water, physiological saline, water for injection, propylene glycol or its aqueous solution, glycerol or its aqueous solution, and aqueous solutions of sugars. Examples of buffer solutions include acetate buffer, phosphate buffer, citrate buffer, borate buffer, tartrate buffer, tris(hydroxymethyl)aminomethane buffer, HEPES buffer, and MES buffer. These media may also contain surfactants.

[0028] The temperature for high hydrostatic pressure treatment is not particularly limited as long as it does not generate ice or damage cell structures due to heat. However, for optimal cell decellularization with minimal impact on cell structures, a range of 0–45°C is preferred, more preferably 4–37°C, and optimally 15–36°C. If the high hydrostatic pressure treatment time is too short, cell destruction will not be sufficient; if it is too long, energy will be wasted. Therefore, the optimal time to maintain the target pressure during high hydrostatic pressure treatment is 1–120 minutes, more preferably 5–60 minutes, and even more preferably 7–30 minutes.

[0029] Cellular structures subjected to the aforementioned high hydrostatic pressure treatment are preferably subjected to nucleic acid-degrading enzyme treatment. Nucleic acid-degrading enzymes are those used to remove nucleic acid components from cell structures subjected to hydrostatic pressure, and are not particularly limited, but can be exemplified by DNases (e.g., DNase I, DNase II) derived from the pancreas, spleen, or Escherichia coli. Nucleic acid-degrading enzymes can be added to the media used in the aforementioned high hydrostatic treatment (e.g., water, physiological saline, injection solution, or buffer solution) to activate their function. The amount of enzyme added varies depending on the type of enzyme or the definition of units (U), and can be appropriately set by the manufacturer. For example, if DNase I is used, a concentration of 50-1000 U / mL is sufficient. The treatment temperature also varies depending on the nucleic acid-degrading enzyme used, and can be set to, for example, 1°C to 40°C. The treatment time is not particularly limited and can be, for example, 1-120 hours (preferably 1-96 hours, more preferably 1-48 hours). Longer treatment times are possible at lower temperatures, while shorter treatment times are possible at higher temperatures.

[0030] The cell structure system treated with the aforementioned high hydrostatic pressure is then washed with a cleaning solution. The cleaning solution may be the same as or different from the medium used for high hydrostatic pressure treatment. Preferably, the cleaning solution contains an organic solvent or a chelating agent. The organic solvent can improve the lipid removal efficiency, while the chelating agent can prevent limening of the particle-based decellularized cell structure when applied to the affected area by deactivating calcium or magnesium ions in the decellularized cell structure. As an organic solvent, water-soluble organic solvents are preferred due to their high lipid removal efficiency; suitable options include ethanol, isopropanol, acetone, and dimethyl sulfoxide. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), nitrotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic 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), dicarboxymethyl glutamic acid (CMGA), 3-hydroxy-2,2'-iminodisuccinic acid (HIDA), dicarboxymethyl aspartic acid (ASDA), and other iminocarboxylic acid chelating agents or their salts; hydroxycarboxylic acid chelating agents or their salts, such as citric acid, tartaric acid, malic acid, and lactic acid; and sodium or potassium salts of these chelating agents. The washing temperature is not particularly limited as long as it will not damage the cell structure due to heat. However, for good cleaning performance and minimal impact on cell structure, the preferred temperature is 0~45℃, more preferably 1~40℃, and optimally 2~35℃. The washing solution may be shaken or stirred as needed during washing.

[0031] When obtaining the aforementioned decellularized cell structures using a freeze-thaw process, the following steps are repeated once or more (preferably 2 to 5 times): The cell structures are frozen at -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 20 to 40°C. Subsequently, nucleic acid-degrading enzyme treatment is performed, which can be done using the same method as the aforementioned high hydrostatic pressure treatment. After the freeze-thaw process, the cells in the cell structures are destroyed, and these cells are removed with a washing solution. The washing method can be the same as the washing method used in the high hydrostatic pressure treatment.

[0032] When obtaining the aforementioned decellularized cell structures by ultrasonic treatment, the cell structures are ultrasonically treated in, for example, physiological saline (e.g., intensity: 10 W / cm², frequency: 10 kHz, duration: 2 minutes). Subsequently, preferably, a surfactant solution (e.g., 1% by mass Triton X (polyoxyethylene octylphenyl ether) solution) is used, and the cells are shaken at 2-10°C (preferably 4°C) for 1-120 hours (preferably 12-120 hours). Next, nucleic acid-degrading enzyme treatment is preferably performed, which can be done using the same method as the aforementioned high hydrostatic pressure treatment. Afterward, preferably, washing is performed using the same method as the washing in the high hydrostatic pressure treatment.

[0033] The resulting decellularized cell structures are not particularly limited, but freeze-drying is preferred. Freeze-drying can be omitted depending on the location of the cell structure. Furthermore, the resulting decellularized cell structures can be sterilized by gamma irradiation, UV irradiation, or similar methods.

[0034] Thickness retention rate In the method for manufacturing the decellularized cell structure of the present invention, the thickness can be maintained before and after the decellularization step (2) described above. The thickness maintenance rate before and after decellularization is not limited, for example, 90% or more, more preferably 92% or more, and even more preferably 94% or more. Thickness retention rate can be calculated as follows: Thickness retention rate (%) = (Thickness of decellularized cell structures / Thickness of cultured cell structures before decellularization) × 100

[0035] Hardness Test The hardness of the decellularized cell structure of this invention is not particularly limited as long as the desired effect of this invention is achieved, for example, it is 0.20~5.00 (N). The lower limit of 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 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). Within the aforementioned ranges, a transplant patch with sufficient strength and no rejection reaction can be obtained. When this invention is used as a transplant patch, it is easy to perform operations such as grasping with forceps or puncturing with a surgical needle for suturing. Furthermore, decellularized grafts with any thickness or specific shape can be provided. In this invention, hardness is determined by texture profile analysis (TPA) as follows. The hardness is measured at room temperature using a texture analyzer TA.XT plus (Eihong Precision Machinery Co., Ltd.) equipped with a P / 4 cylindrical probe (4 mm in diameter). The point where a load of 0.01 N is applied to the probe is set as point 0, and the probe is compressed twice at a speed of 0.02 mm / s over a distance of 0.5 mm. The maximum force (N) during the first compression cycle is defined as the hardness.

[0036] "protein" The decellularized cell structure of this invention is not particularly limited as long as the effects of this invention can be obtained, and includes, for example, proteins with a molecular weight of 45,000 to 53,000 and an isoelectric point of 4.00 to 4.90. This further enhances the effects of this invention. The molecular weight and isoelectric point (pI) of the aforementioned proteins can be determined, for example, by two-dimensional electrophoresis, while the identification of proteins can be performed by mass spectrometry. In two-dimensional electrophoresis, either isoelectric point separation or molecular weight separation can be performed first (as the first dimension). However, from the perspective of improving the accuracy of the measurement, it is preferable to perform isoelectric point separation in the first dimension and molecular weight separation in the second dimension. Two-dimensional electrophoresis can be performed using common methods and commercially available kits and apparatus. For example, capillary gels or strip gels can be used as separation media for isoelectric point electrophoresis, and the electrophoresed gel can be separated into molecular weights by electrophoresis using a planar gel (e.g., SDS-polyacrylamide gel) in a direction perpendicular to the spreading direction of the isoelectric point electrophoresis. By staining the gel after two-dimensional electrophoresis using common methods, the presence of proteins, molecular weights, and isoelectric points can be confirmed.

[0037] The molecular weight of the aforementioned protein, from the viewpoint of maximizing the effects of the present invention, 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 aforementioned protein is preferably 4.00 to 4.90, more preferably 4.40 to 4.85, and even more preferably 4.60 to 4.80.

[0038] The aforementioned proteins preferably include tubulin, more preferably α-tubulin or β-tubulin, and even more preferably β-tubulin. Human tubulin exists in five forms: α, β, γ, δ, and ε (spsilon). Tubulin is highly preserved across species, forming heterodimers, which then polymerize to form microfilaments. The heterodimer systems of α-tubulin and β-tubulin participate in the basic structural units of microtubules during cell division. α-tubulin and β-tubulin have similar 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 their C-terminal regions as type I (HM40), type II (Hβ9), type III (Hβ4), type IVa (H5β), type IVb (Hβ2), type V, and type VI (Hβ1) (Roman numerals indicate protein isotypes, and parentheses indicate human gene classifications). Type III (Hβ4), namely tubulin beta-4B chain (Homo sapiens), is a protein that forms microtubules, which are mainly composed of cytoskeletal proteins and is believed to play a role in maintaining cell shape and physiological activities. Tubulin beta-4B chain is a protein composed of 445 amino acids (sequence number 1), with a molecular weight of 50255 and a pI of 4.79.

[0039] The source of tubulin in decellularized cell structures is not particularly limited as long as it originates from eukaryotes, but it is preferred to obtain it from mammals or birds. Examples of mammals include cattle, horses, camels, alpacas, donkeys, yaks, sheep, pigs, goats, deer, dogs, raccoons, weasels, foxes, cats, rabbits, hamsters, guinea pigs, rats, mice, squirrels, or raccoons. Examples of birds include parrots, parrots, chickens, ducks, turkeys, geese, guinea fowl, pheasants, ostriches, quails, or emus.

[0040] Furthermore, the decellularized cell structure of this invention is not limited; as long as the effects of this invention can be achieved, it may include a microtubule mutant. Examples of microtubule mutants include: (1) A polypeptide or peptide having tubulin activity, comprising one or more amino acid sequences (e.g., amino acid sequences indicated by sequence number 1) at one or more sites in the amino acid sequence of tubulin (preferably 1 to 10, more preferably 1 to 7, and even more preferably 1 to 5), for example, comprising one or more amino acid sequences with deletions, substitutions, insertions, and / or additions, and having the activity of tubulin. (2) A polypeptide having an amino acid sequence that is more than 90% identical to the amino acid sequence of tubulin (e.g., the amino acid sequence represented by sequence number 1) and having the activity of tubulin. The activity of tubulin can be exemplified by the decellularized cell structure of this invention, which, compared with the non-mutant form, enhances the ability to maintain the thickness and rigidity after decellularization.

[0041] (An amino acid sequence with one or more amino acid deletions, substitutions, insertions, and / or additions) The aforementioned mutants may also be polypeptides composed of one or more amino acid sequences with deletions, substitutions, insertions, and / or additions in the amino acid sequence of tubulin (e.g., sequence number 1). Furthermore, the aforementioned mutant polypeptides can maintain the thickness and stiffness after decellularization. That is, polypeptides that do not demonstrate the ability to maintain the thickness and stiffness after decellularization are not included in the aforementioned mutant polypeptides. In this specification, "one or more amino acid sequences with deletions, substitutions, insertions, and / or additions" refers to mutations caused by amino acid substitutions, etc. The number of amino acid mutations can be, for example, 1 to 30, 1 to 20, 1 to 15, 1 to 10, preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 5, and most preferably 1 to 2. Examples of amino acid sequences that can be used in the mutants of the present invention are preferably amino acid sequences in which the amino acid has one or more (preferably one, two, three or four) preserved substitutions.

[0042] (An amino acid sequence with more than 90% identity to the amino acid sequence) The aforementioned mutant can also be a polypeptide composed of an amino acid sequence with at least 90% identity to the amino acid sequence of tubulin (e.g., sequence number 1). Furthermore, the aforementioned mutant polypeptide can maintain the thickness and stiffness after decellularization. That is, polypeptides that do not demonstrate the ability to maintain the thickness and stiffness after decellularization are not included in the aforementioned mutant polypeptide. More preferably, it is a polypeptide composed of an amino acid sequence with at least 95% identity, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% identity, and exhibits the ability to maintain the thickness and stiffness after decellularization.

[0043] The "amino acid sequences with one or more amino acid deletions, substitutions, insertions, and / or additions" or "amino acid sequences with more than 90% identity" in the aforementioned tubulin (e.g., sequence number 1) are substitutions of the amino acid sequence of tubulin (e.g., sequence number 1). This substitution of amino acid sequences is a preservative substitution that maintains the function of the tubulin used in this invention. In other words, "preservative substitution" refers to substitution that does not lose the excellent effects of tubulin. That is, even after the aforementioned insertions, substitutions, deletions, or additions, the thickness and rigidity after decellularization can still be maintained. Specifically, it refers to replacing amino acid residues with other chemically similar amino acid residues. Examples include replacing a hydrophobic residue with another hydrophobic residue, or replacing a polar residue with another polar residue carrying the same charge. Functionally similar amino acids that can be formed by such substitutions are those 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, glutamic acid, aspartic acid, 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.

[0044] The aforementioned proteins (such as tubulin) are proteins that form microtubules, which are mainly composed of cytoskeletal proteins, and are presumed to play a role in maintaining cell shape. That is, although not limited, the function of maintaining cell shape is believed to be related to maintaining the thickness and rigidity after decellularization. Therefore, it is believed that the aforementioned proteins (such as tubulin) can effectively play a role in the strength of decellularized cell structures.

[0045] In this case, the following examples can be cited. They relate to: [1] A decellularized cell structure, which is a decellularized cell structure obtained by decellularizing a cultured cell structure, characterized in that the porosity is 30%~80%; [2] As in [1], the decellularized cell structure has a hardness of 0.20~5.00 (N); [3] Decellularized cell structures as in [1] or [2], wherein the DNA content per unit dry mass of the decellularized cell structure is less than 0.020% by mass; [4] The decellularized cell structure of any one of [1] to [3] contains (a) a molecular weight of 45,000 to 53,000 and (b) an isoelectric point of pI 4.00 to 4.90; [5] As in [4], the decellularized cell structure, in which the aforementioned protein is tubulin; [6] A method for manufacturing a decellularized cell structure, comprising: The cell structure formation step involves culturing cells to form cell structures; and the cell removal step involves removing the aforementioned cell structures to achieve a porosity of 30% to 80%. [7] The method for manufacturing decellularized cell structures as in [6], wherein in the step of forming cultured cell structures, the cells are cultured into arbitrary shapes before or simultaneously with the formation of cultured cell structures; [8] The method for manufacturing decellularized cell structures as described in [7], wherein in the aforementioned cell structure formation step, the cells are cultured into culture sheets of arbitrary shapes or cell aggregates before the formation of the cell structure; [9] The method for manufacturing decellularized cell structures as in [6], wherein after the cell structure formation step, the cells are cultured into arbitrary shapes;

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

[11] The method for manufacturing a decellularized cell structure as described in any of [6] to

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

[0046] This invention is not limited to the embodiments described above. The embodiments described above are merely illustrative examples, and any invention having a substantially identical structure and function to the technical concept described in the claims of this invention is included within the scope of this invention. [Example]

[0047] The present invention is illustrated below by way of examples, but these are not intended to limit the scope of the invention.

[0048] Example 1 In this embodiment, fibroblasts are used to manufacture decellularized cell structures. (Preparation of cultured cell structures) NHDF-Neo-dermal fibroblasts (CC-2509, Lonza) were cultured in T-175 flasks (vent cap, 175 cm², VIOLAMO). When the flasks reached approximately 90-100% confluence, the cells were detached and a cell suspension (10 mL) (approximately 2.0 × 10⁷ cells) was prepared. A concave plate was placed on a 10 cm culture dish, and 10 mL of 100% ethanol was added, ensuring it completely covered the surface of the plate. After confirming the absence of air bubbles, the ethanol was removed. This process was repeated twice using phosphate-buffered saline (PBS, 0.01 M, pH 7.4). 10 mL of FKCM medium (serum-free fibroblast medium, FUKOKU) was added to the concave plate, along with the cell suspension, and the cells were incubated at 37°C in a 5% CO₂ incubator (IP400, Yamato Scientific). After culturing overnight, the spherical bodies (cell aggregation) within the concave plate were confirmed using an inverted microscope (CKX31, Olympus). The spherical bodies were removed from the concave plates using a pipette and transferred to a 10cm culture dish. The 10cm dish was rotated to concentrate the spherical bodies in the center, which were then collected and placed into a mesh mold (mesh mold kit V6, TISSUE By NET). The mesh mold was transferred to a 60mL safety container (WATSON) containing 80mL of FKCM medium and placed in a 37°C, 5% CO2 incubator with a shaker (OS-762, Optima) and cultured with shaking (45rpm). The medium was replaced with half the volume 1-2 times per week. After approximately 3 weeks of culture, the mesh mold was removed, yielding the cultured cell structures. The aforementioned method for forming cultured cell structures represents an example of: the method described in the claim where, in the step of forming cultured cell structures, cells are cultured into arbitrary shapes before the formation of the cultured cell structures; or an example of a method that, in the aforementioned step of forming cultured cell structures, cells are cultured into arbitrary shapes to form cell aggregates (spheroids) before the formation of the cultured cell structures. The aforementioned method for forming cultured cell structures represents an example of forming cultured cell structures by culturing the cell aggregates (spheroids) while they are held within a cavity, causing the cell aggregates to produce extracellular matrix (ECM).

[0049] (Decellularization of cultured cell structures) The cultured cell structures removed from the mesh mold and FKCM culture medium were placed in a nylon plastic vacuum bag (manufactured by Kashimi-jin or KURILON Chemicals), and sealed with a sealing machine to form a double layer on all four sides. Using FKCM culture medium, a high-pressure treatment device for research and development (manufactured by Dr. CHEF or Kobe Steel) was used to perform high hydrostatic treatment at 600 MPa for 10 minutes. The high hydrostatic treatment was carried out within a temperature range of minimum temperature: 23.3°C and maximum temperature: 35.9°C. After removing the cultured cell structures from the nylon plastic vacuum bag, they were transferred to an embedding cassette (manufactured by TISSUE-TEK) and immersed in physiological saline, then washed with shaking at 4°C for at least 5 minutes. Subsequently, the cultured cell structures were immersed in a DNase I solution (800 U / mL) and washed with shaking at 4°C for at least 18 hours. Subsequently, the cultured cell structures were immersed in physiological saline and washed three times by shaking at 4°C for more than 5 minutes to obtain decellularized cell structures.

[0050] Example 2 In this embodiment, fibroblasts were used to prepare decellularized cell structures by subjecting them to high hydrostatic pressure treatment at 200 MPa. Except for the high hydrostatic pressure treatment of 200MPa instead of 600MPa, the operation of Example 1 was repeated to obtain decellularized cell structures.

[0051] Example 3 In this embodiment, fibroblasts were used to prepare decellularized cell structures by subjecting them to high hydrostatic pressure treatment at 400 MPa. Except for the high hydrostatic pressure treatment of 400MPa instead of 600MPa, the operation of Example 1 was repeated to obtain decellularized cell structures.

[0052] Example 4 In this embodiment, decellularized cell structures are manufactured by freezing-thawing. The cultured cell structures removed from the mesh were frozen on dry ice or in a freezer at -80°C for at least 30 minutes, and then thawed in phosphate-buffered saline (PBS, 0.01M, pH 7.4) heated to 37°C. The cultured cell structures, after undergoing this freeze-thaw cycle three times, were immersed in a DNase I solution (800 U / mL) and washed with agitation at 4°C for 24 hours. Subsequently, they were washed with physiological saline at 4°C for at least 2 hours to obtain decellularized cell structures.

[0053] Comparative Example 1 In this comparative example, the decellularization process was performed using a surfactant. The cell structures obtained in Example 1 were decellularized by treatment with the following surfactant. The cultured cell structures removed from the mesh were washed with water for injection at 4°C for 1 hour. Subsequently, the cultured cell structures were treated with 0.25% sodium dodecyl sulfate solution (10 mM Tris, pH 8.0) at 4°C for 24 hours. Next, the cultured cell structures were treated with 0.5% Triton-X (polyoxyethylene octylphenyl ether) solution (10 mM Tris, pH 8.0) at 4°C for 24 hours. Afterward, the cultured cell structures were immersed in physiological saline and repeatedly washed with shaking for at least 5 minutes at 4°C three times to obtain decellularized cell structures.

[0054] Calculating the ratio of decellularized DNA The mass of decellularized cell structures from Examples 1-4 and Comparative Example 1 was determined. These structures were used as samples, dissolved in a proteolytic enzyme solution, treated with phenol / chloroform to remove proteins, and DNA was recovered by ethanol precipitation. The recovered DNA was stained with fluorescence using PicoGreen (Life Technologies) and the fluorescence intensity was measured. DNA quantification was performed, and the DNA content of the sample was calculated from the sample mass and the amount of DNA. Furthermore, DNA quantification was performed using a calibration curve established with the standard DNA provided with PicoGreen. Using other samples, 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 unit dried mass of the sample was then calculated from the sample DNA content and the drying loss ratio. The results are shown in Table 1. (Decellularized DNA ratio) = (DNA content of dried decellularized cell structure sample) / (mass of dried decellularized cell structure sample)

[0055]

[0056] Preparation of Paraffin-Embedded Tissue Specimens and Stained Sections As a pretreatment, the decellularized cell structures obtained in Examples 1-4 or Comparative Example 1 were immersed in 4% PFA (4% paraformaldehyde phosphate buffer, manufactured by FUJIFILM Wako Chemicals Co., Ltd. for tissue fixation) for at least 48 hours. Subsequently, the decellularized cell structures were immersed in phosphate buffer (PBS, 0.01M, pH 7.4) and washed with agitation at 4°C for at least 30 minutes. Next, the decellularized cell structures were immersed in 70% ethanol and washed with agitation at 4°C for at least 30 minutes to prepare samples for paraffin-embedded tissue specimens.

[0057] Prepare paraffin-embedded tissue specimens from the above samples according to the following procedure, and prepare staining sections (described below). Rinse the tissue with tap water for 30-40 minutes to remove formaldehyde. Dehydrate the samples in an ethanol bath using a paraffin embedding device (CT-Pro20, Genostaff) in the following order. 70% ethanol (by mass) for 20 minutes (×1) → 95% ethanol for 20 minutes (×2) → 100% ethanol, 20 minutes (×2) Next, the sample was washed twice in xylene for 20 minutes each time. The sample was then incubated twice in a paraffin bath at 65°C for 30 minutes each time. The dissolved paraffin was poured into a mold, and the sample was placed in the mold and allowed to cool for 15-20 minutes to prepare a paraffin-embedded tissue specimen. Then, using the paraffin-embedded tissue specimen, sections were continuously prepared on a Microtome at a thickness of 5 μm, and the sections were floated in a 37°C water bath containing deionized water. Each section was placed on a glass slide on distilled water, stretched on a stretching table at 30-40°C, and then dried in a thermostat (37°C) for 1-2 nights to prepare a stained section specimen.

[0058] (Sirius red dye) The following procedure was followed to stain the sectioned specimens with Sirius red, thereby staining the extracellular matrix (ECM). First, the stained section samples were deparaffinized, hydrated with distilled water, and rinsed with distilled water. Next, the stained section samples were treated with iron hematoxylin (iron hematoxylin I (1% hematoxylin (SIGMA-ALDRICH) / 95% ethanol) and iron hematoxylin II (Muto Chemical) in equal volumes) for 2 minutes, and rinsed with distilled water. Then, they were treated with 0.5% hydrochloric acid / 70% ethanol solution for 3 minutes. Next, they were treated with 0.2% phosphomolybdic acid aqueous solution for 5 minutes. Next, they were treated with 0.1% Direct Red 80 (Sirius Red, SIGMA-ALDRICH) / saturated picric acid solution for 90 minutes. Next, they were impregnated three times with 0.01N hydrochloric acid. Afterwards, the stained section samples were dehydrated by permeation with xylene and then sealed with an imprinting agent (MOUNT-QUICK: Daido Sangyo Co., Ltd.).

[0059] Calculation of porosity The Sirius red stained sections were observed under a microscope, and the sections were photographed at 10x magnification. The porosity of the decellularized cell structures in Examples 1-4 and Comparative Example 1 was calculated using image processing software (ImageJ). The images of the stained samples were read, and the area of ​​the entire decellularized cell structure and the area of ​​the Sirius red stained portion (ECM) were measured respectively. The porosity of decellularized cell structures was calculated using the following formula. A quadrilateral region (310 μm × 310 μm) was defined as the measurement area, and the average porosity at three locations was calculated. To remove large voids, a region containing no voids larger than 34,000 (approximately 35%) μm² was defined. Porosity (%) = (Area of ​​the entire decellularized cell structure (measurement area) - Area of ​​the Sirius red stained portion of the example or comparative example) / Area of ​​the entire decellularized cell structure (measurement area) × 100

[0060] Calculation of Thickness Retention Rate The thickness maintenance rate was calculated by comparing the thickness of the cultured cell structures with the thickness of the decellularized cell structures in Examples 1-4 and Comparative Example 1. Furthermore, the thickness of the cultured cell structures, as shown in Figure 2 below, refers to the untreated structure. Thickness maintenance rate (%) = (Thickness of decellularized cell structures / Thickness of cultured cell structures) × 100

[0061] The porosity results are shown in Figure 1, and the thickness retention results are shown in Figure 2. Regarding the porosity, Examples 1-4 have a porosity of 30-80%, while Comparative Example 1 has a larger value. Furthermore, regarding the thickness retention, Examples 1-4, compared to Comparative Example 1, can maintain the thickness after decellularization relative to the thickness before decellularization, indicating that the shape before and after decellularization can be maintained. Therefore, it can be seen that by setting the porosity of the decellularized cell structure to a fixed value, the shape of the cell structure before and after decellularization can be maintained, and the decellularized cell structure can be made into any shape.

[0062] Hardness Test To determine the stiffness of the decellularized cell structures obtained in the examples and comparative examples, texture profile analysis (TPA) was performed as follows. TPA was performed at room temperature using a texture analyzer TA.XT plus (Eng Hong Precision Machinery Co., Ltd.) equipped with a P / 4 cylindrical probe (4 mm in diameter). The point where a load of 0.01 N was applied to the probe was set as point 0, and the probe was compressed twice at a speed of 0.02 mm / s to a distance of 0.5 mm. The maximum force (N) during the first compression cycle is used to represent hardness. The same measurements were also performed on the following manufacturing examples 1 and 2.

[0063] (Manufacturing Example 1) The bovine heart membrane was cut into slices, and all the fat was removed (hereafter referred to as "heart membrane slices"). These slices were used to manufacture the heart membrane slices in Example 1.

[0064] (Manufacturing Example 2) The cardiac membrane patch from Manufacturing Example 1 was placed in a nylon plastic vacuum bag. Using phosphate-buffered saline (PBS, 0.01M, pH 7.4) added to water for injection as the medium, it was subjected to high hydrostatic treatment at 600 MPa for 10 minutes using a research and development high-pressure treatment device. The high-hydrostatic treated cardiac membrane patch was then immersed in DNase I (125 U / mL), a nucleic acid-degrading enzyme, 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 freeze-drying, it was irradiated with gamma rays (25 kGy). This was used as the decellularized cardiac membrane patch (decellularized tissue) for Manufacturing Example 2.

[0065] The test results are shown in Figure 4. The hardness of the cardiac patch of Manufacturing Example 1 was 10.13 (N), and the hardness of the decellularized cardiac patch (decellularized tissue) of Manufacturing Example 2 was 13.62 (N). It is evident that, compared to the comparative examples, the embodiments maintain rigidity and can be stably used as transplant pieces. In particular, it is evident that Embodiment 1 achieves good results in terms of stability as a transplant piece. Furthermore, it is evident that the embodiments are softer than the cardiac membrane sheet of Manufacturing Example 1 and the decellularized tissue of Manufacturing Example 2, exhibiting a different level of rigidity.

[0066] Protein Analysis The differences in composition between the decellularized cell structures of Example 1 and Comparative Example 1 were analyzed by two-dimensional electrophoresis. 0.3 mL of protein lysis solution (containing urea and surfactant) was added to the decellularized cell structures of the examples and comparative examples, and the mixture was shaken at 1,400 rpm for 2 hours at room temperature. After homogenization using a Polytron homogenizer, the mixture was centrifuged at 20,000 x g at room temperature for 30 minutes, and the supernatant was collected. The sample solution was purified using a 2-D Clean-Up Kit (Cytiiva, Global Life Sciences Technologies Japan). Subsequently, protein quantification and conductivity measurements were performed to confirm that the measured values ​​were within the permissible range for two-dimensional electrophoresis, thus preparing the sample for two-dimensional electrophoresis. After adding buffer to the sample for 2D electrophoresis, a protein dissolving solution (containing urea and surfactant) was added to adjust the total volume to 0.34 mL, thus preparing the sample solution. The sample solution was poured into a swelling tray, and a pre-prepared gel for 1D electrophoresis (Immobiline DryStrip, Cytiiva, Global Life Sciences Technologies Japan) was placed on top of the solution. Then, a dry-peeling solution (PlusOne DryStrip Cover Fluid, Cytiiva, Global Life Sciences Technologies Japan) was added and allowed to stand overnight. The swollen pre-prepared gel was then placed in an electrophoresis apparatus (Multiphor II Electrophoresis Unit, Cytiiva, Global Life Sciences Technologies Japan) for electrophoresis (500V for 1 minute, 3500V for 7.5 hours, 20°C). Use acrylamide gels with a concentration gradient of 10-20%. After preparation, allow the gels to stand for 24 hours to allow complete polymerization of acrylamide. Place the equilibrated first-dimensional electrophoresis pre-prepared gel onto the acrylamide gel and fix it with a 1% agarose solution containing 0.125% bromophenol blue. Mark the left end of the gel with a molecular weight label as a standard. Perform electrophoresis at 80V for 17 hours until a bromophenol blue band is visible at the bottom of the gel.

[0067] Protein Staining After electrophoresis, the gel was stained with SYPRO Ruby protein gel stain (S21900, Thermo Fisher Scientific Inc.) for total protein detection, and the images were saved using a fluorescence scanner. Image readings were performed using an excitation wavelength of 488 nm, a fluorescence filter of 640 nm (Bandpass), and a resolution of 100 micrometers. The obtained TIFF images of the fluorescent staining were imported into ImageMaster Platinum (Cytiiva, Global Life Sciences Technologies Japan) for numerical analysis. For numerical analysis, common spots of each gel were first marked according to the manual. After marking up to 340 spots for each gel, gel matching was performed. Based on this process, common spot IDs were assigned to the spots spread on the two gels (the gel from Example 1 and the gel from Comparative Example 1). Subsequently, spot detection was performed on the gels, and the spot signal concentration (%Volume) was calculated. The concentration of the color dot signal is calculated as the proportion of the color dot signal relative to the sum of all color dot signals in the gel. The %Volume value is expressed as a percentage, with the minimum value set at 0.001%. Color spots that meet the following two conditions will be used as criteria for protein identification. • The total concentration of the chromophore signal in the SYPRO Ruby staining image (the sum of the gels when two pieces are combined) is 0.1 or higher. • The concentration change of the color point (the change between gels when combining 2 pieces each) is less than 0.5 times or more than 2 times. The results of two-dimensional electrophoresis are shown in Figure 5. In Example 1, one spot was detected at an isoelectric point pI of 4.00-4.90 and a molecular weight of 45,000-53,000 (1). In contrast, no spot was detected in Comparative Example 1. It can be seen that Example 1 contains a specific protein not present in Comparative Example 1.

[0068] (Identification of color spots) After reading the fluorescence staining image, silver staining was performed using silver nitrate (195-09382, FUJIFILM Wako Chemicals Co., Ltd.). Select identifiable spots by silver staining. Add 100 μL each of 15 mM potassium ferricyanide and 50 mM sodium thiosulfate to gel slides cut into 1 mm squares and shake for 10 minutes. Remove the solution, add Milli-Q water and shake, washing until the stain fades from the gel slide. Add acetonitrile to the gel slide and dehydrate. Add 10 μL each of 100 mM ammonium bicarbonate and 0.02 μg / μL trypsin enzyme solution to swell the gel slide and incubate at 37°C for 16 hours. Add 50 μL of 0.1% TFA and 50% acetonitrile and shake for 20 minutes to extract the peptides. Perform extraction twice. Concentrate the peptide extract to approximately 10 μL using a vacuum centrifuge. Adsorb the sample onto a ZipTip C18 (Millipore, ZTC18S960) and dissolve the peptides with 2.5 μL of 60% acetonitrile and 0.1% TFA solution. Mix 1 μL of the sample solution with 1 μL of the CHCA matrix solution, add the mixture dropwise to the target disk, and allow it to dry before measuring using a mass spectrometer (ultrafleXtreme). Identify the proteins listed in the NCBI RefSeq database based on the obtained mass values. Analysis device: UltrafleXtreme (Bruker Daltonics) Target drive: 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 Criteria Identification software Mascot (Matrix Science) NCBI RefSeq database Searching for the biological species human (Genome assembly GRCh38.p13) Enzyme trypsin Fixed modification urea methylation

[0069] The results of mass spectrometry identification of the spots from Example 1 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.

[0070] [Industry-level availability]

[0071] The decellularized cell structure of this invention can be used as a non-rejection graft for the treatment of various diseases.

[0072]

Claims

1. A decellularized cell structure, which is a decellularized cell structure formed by decellularizing a cultured cell structure, characterized in that the aforementioned cultured cell structure is formed solely from cultured cells, and the aforementioned decellularized cell structure system is obtained by high hydrostatic pressure treatment of 80~700 MPa, with a porosity of 30%~80%.

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

3. The decellularized cell structure as requested in item 1 or 2, wherein the DNA content per unit dry mass of the decellularized cell structure is less than 0.020% by mass.

4. The decellularized cell structure as claimed in claim 1 or 2, which contains (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 as requested in item 4, wherein the aforementioned protein is tubulin.

6. A method for manufacturing a decellularized cell structure, comprising: a cell structure formation step, wherein the cell structure is formed by culturing cells; and a cell removal step, wherein the aforementioned cell structure is decellularized by high hydrostatic pressure treatment at 80-700 MPa to reduce the porosity to 30%-80%, and the aforementioned cell structure is formed solely from cultured cells.

7. The method for manufacturing decellularized cell structures as claimed in claim 6, wherein in the step of forming cultured cell structures, the cells are cultured into arbitrary shapes before or simultaneously with the formation of the cultured cell structures.

8. The method for manufacturing decellularized cell structures as claimed in claim 7, wherein in the aforementioned cell structure formation step, the cells are cultured into culture sheets of arbitrary shapes or cell aggregates before the cell structure is formed.

9. The method for manufacturing a decellularized cell structure as claimed in claim 6, wherein after the cell structure formation step, the cells are cultured into an arbitrary shape.

10. A method for manufacturing a decellularized cell structure as claimed in any of claims 6 to 9, wherein the thickness retention rate before and after decellularization in the aforementioned decellularization step is 90% or more.

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