Method for producing decellularized tissue or organ by means of hydrostatic pressure

Abstract

To provide a method for producing a decellularized tissue or organ that enables short-term completion of decellularization of tissue or organ while ensuring superior structural retention in the produced decellularized tissue or organ.SOLUTION: Disclosed is a method for producing a decellularized tissue or organ, the method comprising: a step for incorporating a DNA-degrading enzyme into an animal-derived tissue or organ; and a step for applying a hydrostatic pressure of 100-500 MPa in a medium that contains the DNA-degrading enzyme to the animal-derived tissue or organ into which the DNA-degrading enzyme has been incorporated.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a method for producing a decellularized tissue or organ. [Background technology]

[0002] In organ transplants, when the living tissue of an organ donor is transplanted into an organ recipient, rejection and foreign body reactions occur. However, because decellularized organs do not contain cells, decellularized organ models are attracting attention as an alternative to organ transplants.

[0003] In a previous study, the inventors used a physical load called hydrostatic pressure as a state quantity that can be applied uniformly to the entire tissue to decellularize the uterus excised from a rat and transplant it into a defective site in the uterus, and found that the decellularized tissue created using hydrostatic pressure had good tissue regeneration ability (Non-Patent Document 1). In this document, a polyethylene bag filled with a salt solution containing a rat uterus sample was immersed in the transmission fluid in a chamber containing the transmission fluid, and decellularization was performed by holding it at a hydrostatic pressure of 980 MPa for 10 minutes. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] PLoS One 2014 July 24;9(7):e103201, doi: 10.1371 / journal.pone.0103201 Summary of the Invention [Problem to be solved by the invention]

[0005] In Non-Patent Document 1, hydrostatic pressure loading for 30 minutes or less was sufficient to kill cells in tissue, but there was a problem that DNA present in cell nuclei could not be easily removed. In addition, decellularization was performed using three methods using hydrostatic pressure (HP), SDS (sodium dodecyl sulfate), and Triton X-100, but in all cases, washing with a washing buffer containing DNA degrading enzyme for 7 days was required after decellularization.

[0006] Long-term tissue washing after decellularization may increase the risk of matrix degeneration of the decellularized tissue itself. There are also significant disadvantages in terms of the cost and productivity of decellularized tissue production. In addition, chemical decellularization methods using surfactants such as SDS and Triton X-100 are highly toxic to tissues.

[0007] There is a need for a method for producing decellularized tissue or organs that can significantly reduce the time required for the washing step of decellularized tissue while still using a physical means such as hydrostatic pressure and that is excellent in maintaining the structure of the tissue or organ.

[0008] An object of the present invention is to provide a method for producing decellularized tissue or organs that can complete decellularization of tissue or organs in a short period of time and that is excellent in maintaining the structure of the obtained decellularized tissue or organ. [Means for solving the problem]

[0009] The present invention encompasses the embodiments described below. Section 1. A step of incorporating a DNA enzyme into a tissue or organ of animal origin; and A method for producing decellularized tissue or organ, comprising the step of applying a hydrostatic pressure of 100 to 500 MPa to an animal-derived tissue or organ into which the DNase has been incorporated, in a medium containing the DNase. Section 2. Item 2. The method for producing a decellularized tissue or organ according to Item 1, wherein the step of incorporating a DNase into the animal-derived tissue or organ comprises immersing the animal-derived tissue or organ in a medium containing the DNase. Section 3. Item 2. The method for producing a decellularized tissue or organ according to Item 1, wherein the medium does not contain a surfactant. Section 4. Item 2. A method for producing a decellularized tissue or organ according to Item 1, wherein the voltage application step is completed within 6 hours. Section 5. Item 2. A method for producing a decellularized tissue or organ according to Item 1, wherein the amount of DNA in the decellularized tissue or organ obtained after the application step is 50% or less compared to the amount of DNA in a native tissue or organ not subjected to decellularization treatment. Section 6. A decellularized tissue having an extracellular matrix, The amount of DNA in the decellularized tissue is 10% or less compared to the amount of DNA in a natural tissue or organ that has not been decellularized, and A decellularized tissue that satisfies at least one of the following (i) to (viii).

[0010] (i) The rate of change in the amount of hydroxyproline in the decellularized tissue relative to the amount of hydroxyproline in native tissue not subjected to decellularization is within ±30%. (iii) The rate of change in the amount of elastin in the decellularized tissue relative to the amount of elastin in native tissue not subjected to decellularization is within ±30%. (iv) The exosomes in the decellularized tissue are present in amounts that can be observed under a fluorescent microscope after staining with a fluorescent dye. (v) The rate of change in the storage modulus of the decellularized tissue relative to ... (vi) The rate of change in the storage modulus of the decellularized tissue at frequencies in the range of 0.1 to 5 [1 / s] or less compared to the storage modulus of the undecellularized native tissue at frequencies in the range of 0.1 to 5 [1 / s] or less is within ±30%. (vii) The rate of change in the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue (viii) The rate of change in the loss modulus of the decellularized tissue at frequencies in the range of 0.1 to 5 [1 / s] or less relative to the loss modulus of the undecellularized native tissue at frequencies in the range of 0.1 to 5 [1 / s] or less is within ±30%. Section 7. Item 7. The decellularized tissue according to item 6, wherein the decellularized tissue contains a DNase at a concentration of 100 ng / ml or more. Section 8. Item 7. The decellularized tissue according to item 6, wherein a biologically active substance is incorporated into the extracellular matrix. Section 9. Item 7. A powdered tissue obtained by powdering the decellularized tissue according to Item 6. Section 10. Item 7. A composition comprising the disrupted decellularized tissue according to item 6 dissolved or suspended in a liquid medium. Section 11. Item 7. A solid organ comprising the decellularized tissue according to item 6. Section 12. Use of the decellularized tissue according to item 6, the powdered tissue according to item 9, the composition according to item 10, or the solid organ according to item 11 as a transplant for supporting cell proliferation, promoting cell regeneration, promoting angiogenesis, tissue replacement surgery, as a transplant for tissue repair or regeneration, or as a transplant for skin grafting and / or skin regeneration surgery. [Brief description of the drawings]

[0011] [Figure 1] From left to right, DIS microscopy of native small intestinal tissue, low pressure (LP), high pressure (HP), and SDS samples. Bars indicate 1 cm. [Diagram 2] A graph showing the relationship between the amount of residual DNA and hydrostatic pressure, expressed as mean ± standard error (n=4 per group). [Diagram 3]Hematoxylin and eosin (HE) staining of native small intestinal tissue, LP, HP, and DIS of SDS samples. Scale bar indicates 400 μm. [Figure 4] Quantification of DNA in LP, HP, and SDS-treated DIS compared to native tissue. Mean ± standard error (n=4 per group). *p < 0.05, **p < 0.005. [Diagram 5] Collagen content in each tissue sample. Masson's trichrome staining of native small intestinal tissue and DIS of LP, HP, and SDS samples. Scale bar indicates 400 μm. [Figure 6] Collagen levels in DIS of LP, HP, and SDS samples compared to native tissue. Mean ± standard deviation (n=4 per group), *p < 0.05, **p < 0.005. [Figure 7] Amount of elastin in each tissue sample. Masson's trichrome staining of native small intestinal tissue and DIS of LP, HP, and SDS samples. Scale bar indicates 400 μm. [Figure 8] The amount of elastin in the DIS of LP, HP, and SDS samples compared to native tissue, expressed as mean ± standard deviation (n=4 per group). [Figure 9] Expression of exosomes in each sample. [Figure 10] Scanning electron microscope images of small intestinal crypt structures in native tissue (top left), LP (top right), HP (bottom left), and SDS (bottom right). [Figure 11] Storage modulus of DIS versus strain for LP, HP, and SDS samples compared to native tissue. Expressed as mean ± standard deviation (n = 3–7 per group). [Figure 12] Storage modulus of DIS versus frequency for LP, HP, and SDS samples compared to native tissue. Expressed as mean ± standard deviation (n = 3–7 per group). [Figure 13]Loss modulus of DIS versus strain for LP, HP, and SDS samples compared to native tissue. Expressed as mean ± standard deviation (n = 3–5 per group). [Figure 14] Loss modulus of DIS versus frequency for LP, HP, and SDS samples compared to native tissue. Expressed as mean ± standard deviation (n=3–5 per group). [Figure 15] Photographs of intestinal tissues implanted with decellularized tissue samples from Sham, LP, HP, and SDS rats. [Figure 16] Immunohistological staining of each intestinal tissue. SMA: SMA staining, Enlarged SMA: Enlarged image of SMA staining, Vim: Vim staining, Enlarged Vim: Enlarged image of Vim staining, KRT18: KRT18 staining, Enlarged KRT18: Enlarged image of KRT18 staining, CD31: Enlarged image of CD31 staining. Black triangles indicate the sutured area. Red circle indicates the enlarged area in the center of the transplanted tissue fragment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In this specification, the term "comprise" is a concept that encompasses "consist essentially of" and "consist only of."

[0013] In the numerical ranges described in stages in this specification, the upper or lower limit of a certain numerical range can be arbitrarily combined with the upper or lower limit of another numerical range. In addition, in the numerical ranges described in this specification, the upper or lower limit of the numerical range may be replaced with a value shown in an example or a value that can be unambiguously derived from an example. Furthermore, in this specification, a numerical value connected with "~" means a numerical range that includes the numerical values ​​before and after "~" as the lower and upper limits.

[0014] According to a first aspect of the present invention, there is provided a method for producing a decellularized tissue or organ, comprising the steps of: incorporating a DNase into an animal-derived tissue or organ; and applying a hydrostatic pressure of 100 to 500 MPa to the animal-derived tissue or organ into which the DNase has been incorporated, in a medium containing the DNase.

[0015] According to such a method for producing decellularized tissue or organ, the animal-derived tissue or organ contains a DNA degrading enzyme, and hydrostatic pressure is applied to the tissue or organ cells in a state in which the DNA in the animal-derived tissue or organ is degraded, thereby facilitating the removal of cells from the animal-derived tissue or organ, and allowing cells to be removed from the animal-derived tissue or organ in a shorter period of time than with conventional methods in which hydrostatic pressure is applied in a medium that does not contain a DNA degrading enzyme.

[0016] The animal-derived tissue (also referred to as living tissue) is not particularly limited as long as it is a living tissue having cells derived from a vertebrate, but since there is less rejection reaction, living tissue derived from a mammal or bird is preferred, and mammals are more preferred. Examples of mammals include rats, mice, guinea pigs, hamsters, pigs, cows, horses, goats, sheep, camels, dogs, cats, rabbits, monkeys, and humans. The animal-derived tissue is preferably tissue derived from a rat, pig, cow, goat, sheep, rabbit, monkey, or human.

[0017] The body fluid includes interstitial fluid, blood, and lymphatic fluid. The body fluid preferably includes interstitial fluid.

[0018] Examples of animal-derived tissues include tissues of the heart, kidney, ureter, urethra, liver, spleen, pancreas, lung, brain, bone, uterus, fallopian tube, ovary, placenta, cartilage, tendon, stomach, small intestine, large intestine, bronchi, esophagus, bone marrow, teeth, blood vessels, nerves, or skin.

[0019] Examples of organs derived from animals include, but are not limited to, the heart, kidneys, ureters, urethra, liver, spleen, pancreas, lungs, brain, bones, uterus, oviducts, ovaries, placenta, cartilage, tendons, stomach, small intestine, large intestine, bronchi, esophagus, bone marrow, and teeth.

[0020] Any tissue or organ derived from an animal can be decellularized by introducing a DNase from the outside of the tissue or organ and then applying hydrostatic pressure, and therefore the method for producing a decellularized tissue or organ according to the first aspect of the present invention can be applied.

[0021] It is preferable that animal-derived tissues or organs are washed with physiological saline or various buffer solutions after being collected or removed from the animal.

[0022] By introducing a DNA enzyme into an animal-derived tissue or organ from the outside of the tissue or organ prior to the step of applying hydrostatic pressure, the concentration of the DNA enzyme in the animal-derived tissue or organ can be increased and DNA in the animal-derived tissue or organ can be decomposed prior to the step of applying hydrostatic pressure.

[0023] In some embodiments, the step of incorporating a DNase into an animal-derived tissue or organ comprises immersing the animal-derived tissue or organ in a medium containing the DNase. During the immersion, the animal-derived tissue or organ may be left stationary in the medium containing the DNase, or the medium containing the DNase may be shaken.

[0024] The medium containing the DNase used for the uptake of the DNase preferably contains water, an aqueous medium other than water, or a mixture thereof, and examples of the aqueous medium other than water include propylene glycol, glycerin, etc. The medium may be an aqueous solution or buffer solution containing at least one of an acid, a base, and a salt, and examples of such an aqueous solution or buffer solution include physiological saline, acetate buffer, phosphate buffer, citrate buffer, borate buffer, tartrate buffer, Tris buffer, HEPES buffer, MES buffer, etc. Alternatively, the medium may be an aqueous solution containing a sugar.

[0025] The medium containing the DNA degrading enzyme may contain a surfactant, but is preferably free of surfactant in terms of eliminating or reducing toxicity to tissues. Examples of the surfactant include anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants.

[0026] The amount of surfactant in the medium containing the DNA degrading enzyme is preferably 0.01% by mass or less, more preferably 0.001% by mass or less, and even more preferably 0% by mass, based on the total mass of the medium.

[0027] The step of incorporating the DNase into animal-derived tissues or organs is preferably carried out under a pressure lower than 100 MPa, particularly under atmospheric pressure.

[0028] The time for which the DNase is taken up by the animal-derived tissue or organ, or the time for which the animal-derived tissue or organ is immersed in the medium containing the DNase, can be selected depending on the site or size of the animal-derived tissue or organ and is not particularly limited, but is preferably 3 minutes to 1 hour, and more preferably 3 minutes to 30 minutes.

[0029] The step of applying hydrostatic pressure to an animal-derived tissue or organ into which a DNase has been incorporated includes the following series of steps: (i) increasing the pressure around the animal-derived tissue or organ into which the DNase has been incorporated (usually atmospheric pressure) to a higher pressure (usually the pressure applied in the next hydrostatic pressure step); (ii) after the pressure increasing step, applying a hydrostatic pressure within a certain range (i.e., 100 to 500 MPa) to the animal-derived tissue or organ in a medium containing the DNase; and (iii) after the hydrostatic pressure applying step, reducing the pressure around the animal-derived tissue or organ (usually the pressure applied in the hydrostatic pressure step) to a lower pressure (usually atmospheric pressure).

[0030] The medium containing the DNase used in the above series of steps preferably includes water, an aqueous medium other than water, or a mixture thereof, and examples of the aqueous medium other than water include propylene glycol, glycerin, etc. The medium may be an aqueous solution or buffer solution containing at least one of an acid, a base, and a salt, and examples of such an aqueous solution or buffer solution include physiological saline, acetate buffer, phosphate buffer, citrate buffer, borate buffer, tartrate buffer, Tris buffer, HEPES buffer, MES buffer, etc. Alternatively, the medium may be an aqueous solution containing a sugar.

[0031] The medium containing DNase used in the step of incorporating DNase into animal-derived tissue or organ and the medium containing DNase used in the step of applying hydrostatic pressure may be of the same composition or may be of different composition. The medium containing DNase used in the step of incorporating DNase may be used continuously as it is in the step of applying hydrostatic pressure, but it is preferable to replace the medium containing DNase used in the step of incorporating DNase with the medium containing DNase used in the step of applying hydrostatic pressure.

[0032] In the present invention, a hydrostatic pressure of 100 to 500 MPa is applied to the animal-derived tissue or organ into which the DNase has been incorporated in a medium containing the DNase.

[0033] If the applied hydrostatic pressure is lower than 100 MPa, the destruction of cells in the animal-derived tissue or organ will be insufficient. If the applied hydrostatic pressure exceeds 500 MPa, it will be difficult to maintain the structure of the animal-derived tissue or organ. In addition, a pressure vessel or device that can withstand the applied pressure will be required, and the equipment will be large-scale. The applied hydrostatic pressure is preferably 100 to 400 MPa, more preferably 200 to 400 MPa, and even more preferably 200 to 300 MPa.

[0034] The temperature in the step of increasing the pressure, the step of applying a hydrostatic pressure of 100 to 500 MPa, and the step of decreasing the pressure is preferably 0 to 45°C, more preferably 4 to 37°C, and most preferably 15 to 35°C, from the viewpoint of smoothly carrying out the decellularization process and having little effect on the tissue.

[0035] The time for the step of increasing the pressure is not particularly limited, but is preferably 30 seconds to 30 minutes, and more preferably 1 minute to 10 minutes. The time for applying a hydrostatic pressure of 100 to 500 MPa is more preferably 6 hours or less, more preferably 1 hour or less, and more preferably 30 minutes or less, from the viewpoints of maintaining the structure of the tissue or organ and accelerating the application of the pressure. The lower limit of the application time is not particularly limited, but is preferably 5 minutes or more, for example, 10 minutes or more. The time for the step of decreasing the pressure is not particularly limited, but is preferably 5 minutes or less, and more preferably 1 minute or less. The total time for the three steps (i) to (iii) is more preferably 6 hours or less, more preferably 1 hour or less, and more preferably 30 minutes or less.

[0036] In the above-mentioned method for producing a decellularized tissue or organ, it is preferable not to carry out a process of decomposing DNA in the decellularized tissue or organ with a DNase after the step of applying hydrostatic pressure, but optionally, in order to further promote the decomposition of DNA in the decellularized tissue or organ, the decellularized tissue or organ may be exposed to or incubated in a medium containing a DNase for about 30 minutes to 6 hours. The medium containing a DNase used in this case may be a medium of the same composition as the medium containing a DNase used in the step of incorporating the DNase or the medium containing a DNase used in the step of applying hydrostatic pressure, or may be a medium of a different composition.

[0037] The method for producing the decellularized tissue or organ may further include a step of washing the decellularized tissue or organ with a washing solution that does not contain DNase after the step of applying hydrostatic pressure. The washing solution may be an aqueous solution or buffer solution that preferably contains at least one of an acid, a base, and a salt, and examples of such an aqueous solution or buffer solution include physiological saline, acetate buffer, phosphate buffer, citrate buffer, borate buffer, tartrate buffer, Tris buffer, HEPES buffer, and MES buffer. Although a surfactant may be contained, it is preferable that the surfactant is not contained in terms of eliminating or reducing toxicity to the tissue. Although a surfactant may be contained, it is preferable that the surfactant is not contained in terms of eliminating or reducing toxicity to the tissue. The washing time is not particularly limited, but is preferably 30 minutes to 6 hours, more preferably 30 minutes to 1 hour.

[0038] According to the decellularized tissue or organ of the present invention, the time required for washing the decellularized tissue or organ after the step of applying hydrostatic pressure can be significantly reduced compared to conventional techniques. Also, by shortening the time required for the animal-derived tissue or organ to incorporate DNase, the time required from collection of the tissue or organ from the animal to the step of applying a hydrostatic pressure of 100 to 500 MPa can be significantly reduced.

[0039] The method for producing the decellularized tissue or organ may further include a step of binding a biologically active substance to the extracellular matrix of the decellularized tissue or organ after the step of applying hydrostatic pressure, or after the washing step if the method includes a washing step after the step of applying hydrostatic pressure.

[0040] Examples of the bioactive substance include a growth factor (or growth factor), an antibacterial agent, an analgesic, a hemostatic agent, an angiogenesis promoter, an angiogenesis inhibitor, or a combination thereof, or an exosome, etc. When the bioactive substance is an exosome, the exosome may be an exosome obtained from an animal that is a donor of an animal-derived tissue or organ, or may be another exosome, or a known or commercially available exosome may be used.

[0041] The bioactive agent may be incorporated into the extracellular matrix of the decellularized tissue or organ, for example as a suspension encapsulated as particles, microparticles or colloids, or the bioactive agent may be attached to the extracellular matrix of the decellularized tissue or organ by known means such as chemical treatment or the use of adhesion molecules.

[0042] The method for producing the decellularized tissue or organ may further comprise a step of powdering the decellularized tissue or organ after the step of applying hydrostatic pressure, or after the washing step if the method comprises a washing step after the step of applying hydrostatic pressure.

[0043] The decellularized tissue or organ can be powdered, for example, by drying the decellularized tissue or organ and pulverizing the dried decellularized tissue or organ. Methods for drying the decellularized tissue or organ include, but are not limited to, freeze-drying. The decellularized tissue or organ can be pulverized using a known pulverizing device such as a mill.

[0044] The resulting decellularized tissue or organ powder can be used, for example, by applying it to an affected area of ​​a patient to promote regeneration of the tissue or organ.

[0045] The above-mentioned method for producing a decellularized tissue or organ may further include, after the step of applying hydrostatic pressure, or after the washing step if a washing step is included after the step of applying hydrostatic pressure, a step of crushing the decellularized tissue or organ and dissolving or suspending the crushed decellularized tissue or organ in a liquid medium to obtain a composition.

[0046] The decellularized tissue or organ can be crushed, for example, by drying the decellularized tissue or organ and crushing the dried decellularized tissue or organ. Methods for drying the decellularized tissue or organ include, but are not limited to, freeze-drying. The crushing can be performed using a known crushing device such as a mill.

[0047] Examples of liquid solvents include solutions containing proteins. For example, a powder obtained by finely grinding decellularized tissue or organs is dissolved in a solution containing pepsin, and the resulting solution is appropriately adjusted in pH and diluted with a buffer solution to obtain a liquid composition. A base such as sodium hydroxide can be used to adjust the pH. A buffer solution such as a phosphate buffer can be used as the buffer solution.

[0048] The resulting composition can be used, for example, to fill a cartridge in a 3D printer to form a printed construct of a desired three-dimensional structure.

[0049] In some embodiments, the amount of DNA in the decellularized tissue or organ obtained after the step of applying hydrostatic pressure is 50% or less, preferably 40% or less, more preferably 30% or less, more preferably 20% or less, more preferably 10% or less, compared to the amount of DNA in a native tissue or organ that has not been subjected to decellularization treatment.

[0050] In certain embodiments, the amount of DNA in the decellularized tissue or organ obtained after the step of applying hydrostatic pressure is a value measured without further carrying out a step of washing the decellularized tissue or organ with a solution containing a DNAase after the step of applying hydrostatic pressure.

[0051] In the present specification, the amount of DNA in a decellularized tissue or organ can be measured by known DNA quantification methods such as the DAPI method, the Hoechst method, the picoGreen method, the MTT method, etc., and since all of these methods are based on standard DNA, the measured amount can be considered to be constant. In particular, it is preferable to measure the amount of DNA by the method described in the Examples.

[0052] According to this configuration, it is possible to largely remove cells in the decellularized tissue or organ after the step of applying hydrostatic pressure.

[0053] In some embodiments, the decellularized tissue obtained by the above-mentioned method for producing a decellularized tissue or organ, or the decellularized tissue in the obtained organ, satisfies at least one of the following (i) to (viii).

[0054] (i) The rate of change in the amount of hydroxyproline in the decellularized tissue relative to the amount of hydroxyproline in native tissue not subjected to decellularization is within ±30%. (ii) The rate of change in the amount of elastin in the decellularized tissue relative to the amount of elastin in native tissue not subjected to decellularization is within ±30%. (iii) The exosomes in the decellularized tissue are present in amounts that can be observed under a fluorescent microscope after staining with a fluorescent dye. (iv) The rate of change in the storage modulus of the decellularized tissue relative to ... (v) The rate of change in the storage modulus of the decellularized tissue at frequencies in the range of 0.1 to 5 [1 / s] or less compared to the storage modulus of the undecellularized native tissue at frequencies in the range of 0.1 to 5 [1 / s] or less is within ±30%. (vi) The rate of change in the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue (vii) The rate of change in the loss modulus of the decellularized tissue at frequencies in the range of 0.1 to 5 [1 / s] or less compared to the loss modulus of the undecellularized native tissue at frequencies in the range of 0.1 to 5 [1 / s] or less is within ±30%. In the present specification, the amount of hydroxyproline in a decellularized tissue and the amount of elastin in a decellularized tissue may be measured by a known method or a commercially available kit, and are particularly measured by the method described in the Examples. The amount of hydroxyproline reflects the amount of collagen.

[0055] The rate of change of the amount of hydroxyproline in decellularized tissue relative to the amount of hydroxyproline in native tissue not subjected to decellularization is calculated by {(amount of hydroxyproline in decellularized tissue)-(amount of hydroxyproline in native tissue not subjected to decellularization)} / (amount of hydroxyproline in native tissue not subjected to decellularization)*100 (%).

[0056] The amount of elastin in decellularized tissue relative to the amount of elastin in native tissue not subjected to decellularization is calculated by (amount of elastin in decellularized tissue) / (amount of elastin in native tissue not subjected to decellularization)*100 (%).

[0057] In this specification, the storage modulus versus strain, storage modulus versus frequency, loss modulus versus strain, and loss modulus versus frequency of each of the decellularized tissue and the natural tissue not subjected to decellularization treatment can be measured using a commercially available rheometer, and are particularly measured by the method described in the examples.

[0058] The rate of change in the storage modulus against strain of decellularized tissue relative to the storage modulus against strain of natural tissue not subjected to decellularization is calculated by {(storage modulus against strain of decellularized tissue)-(storage modulus against strain of natural tissue not subjected to decellularization)} / (storage modulus against strain of natural tissue not subjected to decellularization)*100 (%).

[0059] The rate of change in the storage elastic modulus versus frequency of decellularized tissue relative to the storage elastic modulus versus frequency of natural tissue not subjected to decellularization is calculated by {(storage elastic modulus versus frequency of decellularized tissue)-(storage elastic modulus versus frequency of natural tissue not subjected to decellularization)} / (storage elastic modulus versus frequency of natural tissue not subjected to decellularization)*100 (%).

[0060] The rate of change in the loss modulus of strain of decellularized tissue relative to the loss modulus of strain of natural tissue not subjected to decellularization is calculated by {(loss modulus of strain of decellularized tissue)-(loss modulus of strain of natural tissue not subjected to decellularization)} / (loss modulus of strain of natural tissue not subjected to decellularization)*100 (%).

[0061] The rate of change in the loss elastic modulus versus frequency of decellularized tissue relative to the loss elastic modulus versus frequency of natural tissue not subjected to decellularization is calculated by {(loss elastic modulus versus frequency of decellularized tissue)-(loss elastic modulus versus frequency of natural tissue not subjected to decellularization)} / (loss elastic modulus versus frequency of natural tissue not subjected to decellularization)*100 (%).

[0062] In the present specification, the amount of exosomes in decellularized tissue is measured by the method described in the Examples. The amount of exosomes in decellularized tissue calculated from the fluorescence intensity of the fluorescent dye used to stain exosomes can also be compared with that in natural tissue that has not been subjected to decellularization treatment.

[0063] The rate of change in the amount of exosomes in decellularized tissue relative to that in native tissue not subjected to decellularization is calculated as {(amount of exosomes in decellularized tissue)-(amount of exosomes in native tissue not subjected to decellularization)} / (amount of exosomes in native tissue not subjected to decellularization)*100 (%).

[0064] The amount of hydroxyproline in the decellularized tissue is preferably 70% or more of the amount of hydroxyproline in native tissue that has not been subjected to decellularization treatment.

[0065] The amount of elastin in the decellularized tissue is preferably 70% or more of the amount of elastin in natural tissue that has not been subjected to decellularization treatment.

[0066] The storage modulus of decellularized tissue against strain in the range of 0.1 to 10% or less is preferably 70% or more relative to the storage modulus of natural tissue not subjected to decellularization against strain in the range of 0.1 to 10% or less.

[0067] The storage modulus of the decellularized tissue at frequencies in the range of 0.1 to 5 [1 / s] or less is preferably 70% or more of that of natural tissue that has not been subjected to decellularization processing at frequencies in the range of 0.1 to 5 [1 / s] or less.

[0068] The loss modulus of decellularized tissue for a strain in the range of 0.1 to 10% or less is preferably 70% or more relative to the loss modulus of natural tissue not subjected to decellularization treatment for a strain in the range of 0.1 to 10% or less.

[0069] The loss modulus of the decellularized tissue for frequencies in the range of 0.1 to 5 [1 / s] or less is preferably 70% or more of the loss modulus of the decellularized tissue for frequencies in the range of 0.1 to 5 [1 / s] or less.

[0070] The rate of change of the amount of hydroxyproline in decellularized tissue relative to that in native tissue not subjected to decellularization treatment is preferably within ±20%, more preferably within ±10%.

[0071] The rate of change in the amount of elastin in decellularized tissue relative to that in native tissue not subjected to decellularization treatment is preferably within ±20%, more preferably within ±10%.

[0072] The amount of exosomes in decellularized tissue is preferably 5% or more, more preferably 10% or more, and more preferably 20% or more, compared to the amount of exosomes in natural tissue that has not been subjected to decellularization treatment.

[0073] The rate of change in the storage modulus of decellularized tissue relative to a strain in the range of 0.1 to 10% or less compared to that of natural tissue not subjected to decellularization treatment relative to a strain in the range of 0.1 to 10% or less is preferably within ±20%, more preferably within ±10%. Moreover, over the entire range of strain, preferably 0.1 to 10%, the rate of change is preferably within ±30%, more preferably within ±20%, more preferably within ±10%.

[0074] The rate of change of the storage modulus of the decellularized tissue with respect to a frequency range of 0.1 to 5 [1 / s] or less relative to the storage modulus of the natural tissue not subjected to decellularization with respect to a frequency range of 0.1 to 5 [1 / s] or less is preferably within ±20%, more preferably within ±10%. Alternatively, the rate of change of the storage modulus of the decellularized tissue with respect to a frequency range of 0.1 to 3 [1 / s] or less, more preferably 0.1 to 2 [1 / s] or less relative to the storage modulus of the natural tissue not subjected to decellularization with respect to a frequency range of 0.1 to 3 [1 / s] or less, more preferably 0.1 to 2 [1 / s] or less is preferably within ±20%, more preferably within ±10%. Moreover, in the entire range of preferably 0.1 to 2 [1 / s] or less, more preferably 0.1 to 3 [1 / s] or less, more preferably 0.1 to 5 or less, the rate of change is preferably within ±30%, more preferably within ±20%, more preferably within ±10%.

[0075] The rate of change in the loss modulus of decellularized tissue relative to a strain in the range of 0.1 to 10% or less relative to the loss modulus of natural tissue not subjected to decellularization treatment relative to a strain in the range of 0.1 to 10% or less is preferably within ±20%, more preferably within ±10%. Moreover, over the entire range of strain, preferably 0.1 to 10%, the rate of change is preferably within ±30%, more preferably within ±20%, more preferably within ±10%.

[0076] The rate of change of the loss modulus of the decellularized tissue with respect to a frequency range of 0.1 to 5 [1 / s] or less relative to the loss modulus of the natural tissue not subjected to decellularization with respect to a frequency range of 0.1 to 5 [1 / s] or less is preferably within ±20%, more preferably within ±10%. Alternatively, the rate of change of the loss modulus of the decellularized tissue with respect to a frequency range of 0.1 to 3 [1 / s] or less, more preferably 0.1 to 2 [1 / s] or less relative to the loss modulus of the natural tissue not subjected to decellularization with respect to a frequency range of 0.1 to 3 frequencies [1 / s] or less, more preferably 0.1 to 2 [1 / s] or less is preferably within ±20%, more preferably within ±10%. Moreover, in the entire range of preferably 0.1 to 2 [1 / s] or less, more preferably 0.1 to 3 [1 / s] or less, more preferably 0.1 to 5 or less, the rate of change is preferably within ±30%, more preferably within ±20%, more preferably within ±10%.

[0077] In some embodiments, the decellularized tissue obtained by the method for producing a decellularized tissue or organ, or the decellularized tissue in the obtained organ, satisfies one or two of (i) and (ii) above.

[0078] In some embodiments, the decellularized tissue obtained by the method for producing a decellularized tissue or organ, or the decellularized tissue in the obtained organ, satisfies (iii) above.

[0079] In some embodiments, the decellularized tissue obtained by the method for producing a decellularized tissue or organ, or the decellularized tissue in the obtained organ, satisfies one or two of (i) and (ii) above, and also satisfies (iii) above.

[0080] In some embodiments, the decellularized tissue obtained by the method for producing a decellularized tissue or organ described above, or the decellularized tissue in the obtained organ, satisfies (iv) and (v), (vi) and (vii), or both of the above.

[0081] In some embodiments, the decellularized tissue obtained by the method for producing a decellularized tissue or organ, or the decellularized tissue in the obtained organ, satisfies (iii) above, and (iv) and (v), (vi) and (vii) above, or both.

[0082] In some embodiments, the decellularized tissue obtained by the method for producing a decellularized tissue or organ, or the decellularized tissue in the obtained organ, satisfies one or two of (i) and (ii) above, (iii) above, and (iv) and (v), (vi) and (vii) above, or both.

[0083] In some embodiments, the decellularized tissue obtained by the above-mentioned method for producing a decellularized tissue or organ, or the decellularized tissue in the obtained organ, satisfies all of the above (i) to (vii).

[0084] The scope of the present invention also includes decellularized tissues or organs (particularly solid organs) produced by the above-mentioned method for producing decellularized tissues or organs.

[0085] According to a second aspect of the present invention, there is provided a decellularized tissue having an extracellular matrix, The amount of DNA in the decellularized tissue is 10% or less compared to the amount of DNA in a natural tissue or organ that has not been decellularized, and (i) The rate of change in the amount of hydroxyproline in the decellularized tissue relative to the amount of hydroxyproline in native tissue not subjected to decellularization is within ±30%. (ii) The rate of change in the amount of elastin in the decellularized tissue relative to the amount of elastin in native tissue not subjected to decellularization is within ±30%. (iii) The exosomes in the decellularized tissue are present in amounts that can be observed under a fluorescent microscope after staining with a fluorescent dye. (iv) The rate of change in the storage modulus of the decellularized tissue relative to ... (v) The rate of change in the storage modulus of the decellularized tissue at frequencies in the range of 0.1 to 5 [1 / s] or less compared to the storage modulus of the undecellularized native tissue at frequencies in the range of 0.1 to 5 [1 / s] or less is within ±30%. (vi) The rate of change in the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue relative to the loss modulus of the decellularized tissue (vii) The rate of change in the loss modulus of the decellularized tissue at frequencies in the range of 0.1 to 5 [1 / s] or less compared to the loss modulus of the undecellularized native tissue at frequencies in the range of 0.1 to 5 [1 / s] or less is within ±30%. Such decellularized tissue can be produced by the method for producing decellularized tissue or organ of the first aspect.

[0086] The amount of hydroxyproline in the decellularized tissue is preferably 70% or more of the amount of hydroxyproline in native tissue that has not been subjected to decellularization treatment.

[0087] The amount of elastin in decellularized tissue is preferably within 70% of the amount of elastin in native tissue that has not been subjected to decellularization treatment.

[0088] The storage modulus of decellularized tissue against strain in the range of 0.1 to 10% or less is preferably 70% or more relative to the storage modulus of natural tissue not subjected to decellularization against strain in the range of 0.1 to 10% or less.

[0089] The storage modulus of the decellularized tissue at frequencies in the range of 0.1 to 5 [1 / s] or less is preferably 70% or more of that of natural tissue that has not been subjected to decellularization processing at frequencies in the range of 0.1 to 5 [1 / s] or less.

[0090] The loss modulus of decellularized tissue for a strain in the range of 0.1 to 10% or less is preferably 70% or more relative to the loss modulus of natural tissue not subjected to decellularization treatment for a strain in the range of 0.1 to 10% or less.

[0091] The loss modulus of the decellularized tissue for frequencies in the range of 0.1 to 5 [1 / s] or less is preferably 70% or more of the loss modulus of the decellularized tissue for frequencies in the range of 0.1 to 5 [1 / s] or less.

[0092] The rate of change of the amount of hydroxyproline in decellularized tissue relative to that in native tissue not subjected to decellularization treatment is preferably within ±20%, more preferably within ±10%.

[0093] The rate of change in the amount of elastin in decellularized tissue relative to that in native tissue not subjected to decellularization treatment is preferably within ±20%, more preferably within ±10%.

[0094] The amount of exosomes in decellularized tissue is preferably 10% or more, and more preferably 20% or more, compared to the amount of exosomes in natural tissue that has not been subjected to decellularization treatment.

[0095] The rate of change in the storage modulus of decellularized tissue relative to a strain in the range of 0.1 to 10% or less compared to that of undecellularized natural tissue relative to a strain in the range of 0.1 to 10% or less is preferably within ±20%, more preferably within ±10%, and preferably satisfies such a rate of change over the entire range of strains of 10% or less.

[0096] The rate of change of the storage modulus of the decellularized tissue with respect to a frequency range of 0.1 to 5 [1 / s] or less relative to the storage modulus of the natural tissue not subjected to decellularization with respect to a frequency range of 0.1 to 5 [1 / s] or less is preferably within ±20%, more preferably within ±10%. Alternatively, the rate of change of the storage modulus of the decellularized tissue with respect to a frequency range of 0.1 to 3 [1 / s] or less, more preferably 0.1 to 2 [1 / s] or less relative to the storage modulus of the natural tissue not subjected to decellularization with respect to a frequency range of 0.1 to 3 [1 / s] or less, more preferably 0.1 to 2 [1 / s] or less is preferably within ±20%, more preferably within ±10%. Moreover, in the entire range of preferably 0.1 to 2 [1 / s] or less, more preferably 0.1 to 3 [1 / s] or less, more preferably 0.1 to 5 or less, the rate of change is preferably within ±30%, more preferably within ±20%, more preferably within ±10%.

[0097] The rate of change in the loss modulus of decellularized tissue relative to a strain in the range of 0.1 to 10% or less relative to the loss modulus of natural tissue not subjected to decellularization treatment relative to a strain in the range of 0.1 to 10% or less is preferably within ±20%, more preferably within ±10%. Moreover, over the entire range of strain, preferably 0.1 to 10%, the rate of change is preferably within ±30%, more preferably within ±20%, more preferably within ±10%.

[0098] The rate of change of the loss modulus of the decellularized tissue with respect to a frequency range of 0.1 to 5 [1 / s] or less relative to the loss modulus of the natural tissue not subjected to decellularization with respect to a frequency range of 0.1 to 5 [1 / s] or less is preferably within ±20%, more preferably within ±10%. Alternatively, the rate of change of the loss modulus of the decellularized tissue with respect to a frequency range of 0.1 to 3 [1 / s] or less, more preferably 0.1 to 2 [1 / s] or less relative to the loss modulus of the natural tissue not subjected to decellularization with respect to a frequency range of 0.1 to 3 frequencies [1 / s] or less, more preferably 0.1 to 2 [1 / s] or less is preferably within ±20%, more preferably within ±10%. Moreover, in the entire range of preferably 0.1 to 2 [1 / s] or less, more preferably 0.1 to 3 [1 / s] or less, more preferably 0.1 to 5 or less, the rate of change is preferably within ±30%, more preferably within ±20%, more preferably within ±10%.

[0099] In some embodiments, the decellularized tissue obtained by the method for producing a decellularized tissue or organ, or the decellularized tissue in the obtained organ, satisfies one or two of (i) and (ii) above.

[0100] In some embodiments, the decellularized tissue obtained by the method for producing a decellularized tissue or organ, or the decellularized tissue in the obtained organ, satisfies (iii) above.

[0101] In some embodiments, the decellularized tissue obtained by the method for producing a decellularized tissue or organ, or the decellularized tissue in the obtained organ, satisfies one or two of (i) and (ii) above, and also satisfies (iii) above.

[0102] In some embodiments, the decellularized tissue obtained by the method for producing a decellularized tissue or organ described above, or the decellularized tissue in the obtained organ, satisfies (iv) and (v), (vi) and (vii), or both of the above.

[0103] In some embodiments, the decellularized tissue obtained by the method for producing a decellularized tissue or organ, or the decellularized tissue in the obtained organ, satisfies (iii) above, and (iv) and (v), (vi) and (vii) above, or both.

[0104] In some embodiments, the decellularized tissue obtained by the method for producing a decellularized tissue or organ, or the decellularized tissue in the obtained organ, satisfies one or two of (i) and (ii) above, (iii) above, and (iv) and (v), (vi) and (vii) above, or both.

[0105] In some embodiments, the decellularized tissue obtained by the above-mentioned method for producing a decellularized tissue or organ, or the decellularized tissue in the obtained organ, satisfies all of the above (i) to (vii).

[0106] In some embodiments, the decellularized tissue contains DNase at a concentration of 100 ng / ml or more. The concentration of DNase in human serum is 3.2±1.4 ng / ml (Clin Exp Immunol.1998 Aug;113(2):289-96. doi: 10.1046 / j.1365-2249.1998.00647.x. PMID:9717980; PMCID: PMC1905046), but the concentration is higher than that. The concentration of DNase is preferably 1 μg / ml or more, more preferably 10 μg / ml or more.

[0107] In some embodiments the extracellular matrix has incorporated therein a bioactive substance, as described above in relation to the method for producing a decellularized tissue or organ of the first aspect.

[0108] According to a third aspect of the present invention, there is provided a powdered tissue obtained by powdering the decellularized tissue of the second aspect. The method for producing the powdered tissue is as described above for the method for producing the decellularized tissue or organ of the first aspect.

[0109] According to a fourth aspect of the present invention, there is provided a composition comprising the decellularized tissue of the second aspect suspended in a liquid medium. The method for producing the composition is as described above for the method for producing the decellularized tissue or organ of the first aspect.

[0110] According to a fifth aspect of the present invention, there is provided a solid organ comprising the decellularized tissue of the second aspect. The method for producing the solid organ is as described above for the method for producing the decellularized tissue or organ of the first aspect.

[0111] The decellularized tissue or organ produced by the method for producing a decellularized tissue or organ according to the first aspect of the present invention, the decellularized tissue according to the second aspect of the present invention, the powdered tissue according to the third aspect of the present invention, the composition according to the fourth aspect of the present invention, and the solid organ according to the fifth aspect of the present invention can be used as a transplant for supporting cell proliferation, promoting cell regeneration, promoting angiogenesis, for tissue replacement surgery, as a transplant for tissue repair or regeneration, or as a transplant for skin grafting and / or skin regeneration surgery.

[0112] In the present application, a technology has been developed that can remove cells contained in organs and tissues by applying a physical quantity called hydrostatic pressure. In conventional technology, decellularization by applying hydrostatic pressure required a washing process of the organ or tissue for about 1 to 2 weeks to remove cells after applying hydrostatic pressure. In addition, conventional decellularization by applying hydrostatic pressure had problems such as difficulty in decellularizing large organs and difficulty in controlling the mechanical properties of the organ because the organ structure could not be maintained.

[0113] The method for producing decellularized tissue or organ according to the first embodiment is a new technology that can complete sufficient decellularization in a short period of time despite low hydrostatic pressure, and is an alternative to organ transplantation with fewer immune rejections. This new decellularization method kills cells with a physical quantity called hydrostatic pressure in the presence of DNA enzyme, and therefore can significantly shorten the tissue washing step after hydrostatic pressure application compared to conventional techniques. In addition, since the hydrostatic pressure is low, a commercially available hydrostatic pressure device can be used, and a large-scale device required for applying 980 MPa is not required. Furthermore, the method for producing decellularized tissue or organ according to the first embodiment is advantageous in that it can decellularize even thick and complex organs, and there are many options for organs that can be decellularized. Furthermore, since the structure of the decellularized tissue or organ is well maintained, it also has excellent tissue regeneration ability.

[0114] There are high expectations for clinical application of decellularized organ models using such methods for producing decellularized tissues or organs. The methods for producing decellularized tissues or organs of the present invention and the decellularized tissues or organs obtained using the same can be widely applied in the medical field, beauty industry, pet industry, and horse racing industry.

[0115] For example, in the medical field, it is valuable as a decellularized organ / tissue that can replace organ transplantation. It is also possible to produce screw plates in the orthopedic field using decellularized bone. The powder or liquid tissue composition produced from the decellularized tissue or organ of the present invention can also be used as a material for fibrin gel used in basic research such as iPS. In the cosmetic field, the matrix of the decellularized organ / tissue is expected to have a cosmetic molding effect. Collagen, elastin, and placenta components developed by the method for producing the decellularized tissue or organ of the present invention can be applied to the shaping and / or formation of the face.

[0116] The disclosures of all patent applications and publications cited herein are hereby incorporated by reference in their entireties.

[0117] The following examples are intended for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Unless otherwise specified, reagents are commercially available or are obtained or prepared according to conventional techniques or procedures known in the art. EXAMPLES

[0118] Test Example 1. Preparation of decellularized tissue (Pretreatment) Step 1. The rat small intestine is removed of fatty tissue and opened longitudinally. Step 2. Prepare sterilized phosphate buffer solution (PBS) and Milli-Q water (optional) and wash the sample until it turns pure white (approximately 5 times). Step 3. The pretreated small intestine sample was treated by the following (1) EHHP method or (2) SDS method. (1) EHHP method (LP,HP) 1. In a dark place and using clean procedures, the small intestine sample was immersed in 10 ml of DNAse I solution contained in a plastic bag. 2. Heat-seal the opening of the plastic bag, being careful not to let any air in. 3. Until hydrostatic pressure was applied, the sealed small intestine sample was stored in a refrigerator or ice box at 4°C in a dark place. The small intestine sample was immersed in the DNAse I solution for approximately 30 minutes. 4. Using a hydrostatic pressure device, the samples were pressurized, pressurized at 30°C for 40 minutes, and then released. The sample with a hydrostatic pressure of 150 MPa was designated as LP, and the sample with a hydrostatic pressure of 960 MPa and pressurization time of 40 minutes was designated as HP. In both cases of hydrostatic pressure, the rate of pressure increase and decrease was 65.3 MPa / min. 5.Optional: If Milli-Q water was not used, the plastic bag containing the sample and DNAse I solution was incubated overnight on a shaker in a 4°C refrigerator. 6. The samples were removed and washed with PBS for 2 hours. (2)SDS method Prepare a 1.1% SDS solution, dissolve it sufficiently, and then filter it through a 0.22 μm filter. 2. Place the 1% SDS solution and the small intestine in a sealed container and wash for 2 hours using a shaker at room temperature. 3. Wash the samples with PBS solution for 2 hours, then transfer them to DNAse I buffer and wash them for seven days. Change the DNAse I buffer every day. (3)Native The sample after pretreatment step 2 was designated as Native.

[0119] Test Example 2. Sample preparation The decellularized tissue samples obtained in Test Example 1 were washed briefly with PBS, and then freeze-dried for at least 16 hours using a vacuum freeze dryer (Eyela, Japan). The dried samples were immersed in a lysis buffer containing 446 μg / ml papain, 5 mM cysteine-HCl, and 5 mM EDTA-2 Na, and incubated at 65° C. for at least 15 hours. The samples were further broken down into smaller pieces using a homogenizer and an ultrasonic cell disrupter. This sample was used in the following "Test Example 3: DNA assay", "Test Example 4: Hydroxyproline assay", and "Test Example 5: Elastin assay".

[0120] Test Example 3. DNA Assay The DNA content was measured using the samples prepared in Test Example 2. A commercially available DNA assay kit (Quant-iT PicoGreen dsDNA assay kit; Invitrogen, USA) was used to quantify the DNA content. Each sample was treated according to the standard protocol and analyzed with a fluorescence spectrophotometer at a wavelength of λ = 522 nm.

[0121] Test Example 4. Hydroxyproline assay The collagen protein content of each sample prepared in Test Example 2 was measured for hydroxyproline content using standard procedures. The samples were mixed with 4M NaOH and heated at 120°C for 30 minutes. Then, 1.4N citric acid and chloramine T solution were added to the samples. After 20 minutes, aldehyde-perchloric acid reagent was added to the solution and incubated at 70°C for 20 minutes. Each sample was analyzed by fluorescence spectrophotometer at wavelength λ = 450 nm.

[0122] Test Example 5. Elastin Assay A commercially available elastin assay kit (Fastin elastin assay kit; Biocolor, UK) was used to evaluate the elastin content. The samples were mixed with elastin precipitation reagent and incubated for 15 minutes. The samples were centrifuged at 10,000 g for 10 minutes, and the precipitate was collected. Reagent (TPPS: 5, 10, 15, 20-tetraphenyl-21, 23-porphine sulphonate) was added and gently mixed for 90 minutes. The dye-bound elastin was collected by centrifugation at 12,000 g for 10 minutes and re-diluted with dye dissociation reagent. Each sample prepared in Test Example 1 was analyzed with a fluorescence spectrophotometer at a wavelength of λ = 513 nm.

[0123] Test Example 6. Identification of exosomes in decellularized tissue Each small intestine sample prepared in Test Example 1 was immunostained with TSG101 antibody (Abcam, 300-fold dilution), which is an exosome marker, and observed under a fluorescent microscope.

[0124] Test Example 7. Tissue staining Each tissue prepared in Test Example 1 was briefly rinsed in PBS and fixed in 10% neutral buffered formalin solution. Samples were then dehydrated in gradient ethanol, embedded in paraffin blocks, and sectioned at a thickness of 7 μm. They were stained with hematoxylin and eosin (HE, nucleus and cytoplasm), Masson's trichrome stain (MT, collagen), and Verhoeff's Van Gieson (VVG, elastin) according to standard protocols. Samples were observed under a camera-equipped light microscope (Zeiss, Germany). Implants were stained for vimentin (Abcam; 1:400 dilution) for the submucosa, smooth muscle α-actin (α-SMA) (Abcam; 1:400 dilution) for the smooth muscle layer, KRT18 (Abcam; 1:100 dilution) for the mucosa, and CD31 (Santa Cruz; 1:50 dilution) for the endothelial cells. Heat-induced epitope retrieval (10 mM citrate buffer, pH 6.0) was performed before protein blocking and inactivation of endogenous peroxidases. Incubation with primary antibodies was performed overnight at 4°C, followed by incubation with secondary antibodies (anti-rabbit IgG; Dako) for 2 h at room temperature. Samples were then stained with 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin.

[0125] Test Example 8. Scanning Electron Microscope (SEM) Measurement Each sample prepared in Test Example 1 was first fixed overnight using 50% cacodylic acid and 42% glutaraldehyde aqueous solution. After dehydration with graded ethanol, CO2 critical point drying (Hitachi, HCP 2) was performed. The samples were observed using a scanning electron microscope (JOEL) according to the attached manual.

[0126] Test Example 9. Viscoelasticity test For each tissue prepared in Test Example 1, a 5 mm biopsy punch was used to cut a round sample from the vertically opened tissue. The thickness of the sample was first measured using a micromaterial testing machine (Shimadzu Corporation). Viscoelasticity testing was performed using a rheometer (Physica MCR301) according to the attached manual. The samples were humidified under conditions of 37°C and tested using a rotating parallel plate with a diameter of 8 mm. The storage modulus and loss modulus were automatically calculated and recorded by the testing machine.

[0127] Test Example 10. Transplantation of decellularized tissue SD rats (9 weeks old, male) were used as subjects for decellularized tissue transplantation. These rats were divided into 4 groups (n=3, sham, LP, HP, SDS). The subjects' solid food was replaced with 5% glucose solution from 12 hours before surgery to 48 hours after surgery. In the sham surgery, half of the intestinal tissue was incised and sutured without any intervention. In the experimental group, half of the intestinal wall was excised and replaced with decellularized tissue (15mm*10mm). Each decellularized tissue prepared in Test Example 1 was sutured to the native tissue at least 10 places, including the four corners, with non-degradable polypropylene thread. After suturing, the transplanted area was wrapped in Seprafilm (KAKEN, Japan) to prevent adhesion. The rats were raised for 1 month and then sacrificed. Prior to histological examination, the grafts were excised and the connective tissue and fat were removed.

[0128] result (1) Preparation of decellularized tissue The sample after pretreatment in Test Example 1 is as shown in FIG. In Test Example 1, for the EHHP samples (LP, HP), the time for which the tissue was immersed in the solution containing DNA degrading enzyme was shorter than for the SDS method, and the washing time after application of hydrostatic pressure, which required 7 days or more in the prior art (PLoS One 2014 July 24;9(7):e103201), was either unnecessary or only required a short time, making it possible to perform the decellularization method in a short period of just a few tens of minutes from obtaining the organ to completing decellularization.

[0129] (2) Amount of DNA remaining in tissue In the DNA assay, as shown in Figure 2, all of the samples produced by the EHHP method with hydrostatic pressures of 100 MPa, 150 MPa, and 200 MPa had low amounts of residual DNA, with the 150 MPa LP sample having the lowest amount. HE staining stains cell nuclei blue and non-nuclear tissues pink. As shown in Figure 3, cell nuclei and non-nuclear tissues were observed to be dark in the native sample, whereas the blue spots decreased or disappeared in the LP, HP, and SDS samples, confirming the removal of cells. As shown in Figure 4, the DNA content in the tissues of the LP samples was significantly reduced compared to the native samples, being less than 10%. Furthermore, the DNA content of the LP samples was even lower than that of the HP samples. Next, based on the results of FIG. 2, a hydrostatic pressure of 150 Pa and an application time of 60 minutes (not including the time for pressure increase and high pressure) were selected as the optimal pressure and time conditions for reducing the amount of DNA remaining in the tissue to 5%, and these were used as new LP samples. A sample with a hydrostatic pressure of 980 MPa and a pressurization time of 10 minutes (not including the time for pressure increase and high pressure) used in the inventors' previous research was used as an HP sample. In both cases, the rate of pressure increase and decrease was 65.3 MPa / min. The SDS sample was the sample prepared in Test Example 1. In FIG. 4, the amount of DNA remaining in the LP sample, HP sample, and SDS sample was significantly reduced compared to the native sample. The reason why the amount of DNA remaining in the HP sample was high is probably because the pressurization time was short in this experiment.

[0130] (3) Collagen content in tissue Masson's trichrome staining stains collagen fibers blue, nuclei black, and actin cytoskeleton red. As shown in Figure 5, the preservation of blue collagen fibers was confirmed in all samples, and black and red were not observed, confirming the removal of cells. Furthermore, as shown in Figure 6, the LP sample had a significantly higher amount of hydroxyproline than the HP and SDS samples, suggesting that a larger amount of collagen remained even after decellularization treatment and that the structural proteins of the tissue were maintained better than those in the HP and SDS samples.

[0131] (4) Amount of elastin in tissue Verhoeff's Van Gieson staining stains elastin fibers black and collagen fibers red or orange. As shown in Figure 7, the presence of black lines confirmed the preservation of elastin in the Native, LP, and HP samples. Furthermore, as shown in Figure 8, the amount of elastin remaining in the LP sample was not significantly different from that in the native sample. This suggests that a large amount of elastin remained even after decellularization, and that it was maintained in the sample treated using hydrostatic pressure compared to the SDS sample.

[0132] (5)Exosome expression in decellularized tissue As shown in Figure 9, the LP sample appeared to be the darkest in the immunostaining of the small intestine. In other words, the LP decellularized tissue was considered to have the best retention or retention of exosomes compared to the HP and SDS decellularized tissues.

[0133] (6) Scanning electron microscope (SEM) measurement As shown in Figure 10, the villi-crypt structure on the inner surface of the small intestine was observed. The cells, which are white dots present in the native sample, were no longer present in the LP sample, but it was confirmed that the villi-crypt structure was preserved. Some collapse of the structure was observed in the HP sample (top right of the image). In the SDS sample, the structure was completely destroyed, with no visible holes.

[0134] (7) Viscoelasticity test The viscoelasticity of the tissue slices was measured using a rheometer. Figure 11 shows the relationship between the storage modulus (physical quantity representing elasticity) and the strain applied to the sample, Figure 12 shows the relationship between the storage modulus (physical quantity representing elasticity) and the frequency applied to the sample, Figure 13 shows the relationship between the loss modulus (physical quantity representing viscosity) and the strain applied to the sample, and Figure 14 shows the relationship between the loss modulus (physical quantity representing viscosity) and the strain applied to the sample. The same trend was observed in all the figures. That is, among the LP, HP, and SDS samples, the LP sample showed the closest viscosity and elasticity to the native sample. On the other hand, HP and SDS had different viscoelasticity from the native sample. The reason for this is thought to be related to the damage to the tissue caused by the decellularization process. In other words, it can be said that LP caused the least damage to the mechanical properties of the tissue.

[0135] (8) Transplantation of decellularized tissue Figure 15 is a photograph of the samples taken one month after implantation of each tissue fragment produced in Test Example 1 into the intestinal tract of a rat. The arrow indicates the location of the tissue fragment, and it was confirmed that all tissue fragments had healed with the native tissue. The black triangle in Figure 16 indicates the sutured area, and the red circle is a magnified area in the center of the transplanted tissue piece. SMA is a marker for smooth muscle, and marks the smooth muscle in the outermost part of the intestinal tract and in the villi. Vim is an abbreviation for Vimentin, and marks the submucosa and connective tissue of the small intestine. KRT18 is a marker for the mucosa. CD31 is a marker for vascular endothelial cells. By staining the tissue pieces brown with these four markers, the regeneration status of the tissue pieces can be confirmed. The enlarged image of the SMA showed that the smooth muscle stained brown was connected. The smooth muscle appeared intermittent in the HP and SDS samples. In other words, smooth muscle regeneration was the best in the LP samples. There was no particular difference in Vim regeneration among the LP, HP, and SDS samples. Many fibroblasts were found in each tissue fragment, and the overall appearance was like scar tissue. KRT18 staining showed a villus structure and partial loss of the mucosa in the SDS samples (upper left of the enlarged image). A single (ring-shaped) intact mucosa layer was confirmed in the LP and HP samples. Staining of vascular endothelial cells confirmed regeneration of capillaries in the mucosa and submucosa in the LP and HP samples. Capillaries in the mucosa were not detected in the SDS samples.

Claims

1. A step of incorporating a DNA degrading enzyme into a tissue or organ of animal origin; and A method for producing decellularized tissue or organ, comprising the step of applying a hydrostatic pressure of 100 to 500 MPa to an animal-derived tissue or organ into which the DNase has been incorporated, in a medium containing the DNase.

2. 2. The method for producing a decellularized tissue or organ according to claim 1, wherein the step of incorporating a DNA enzyme into the animal-derived tissue or organ comprises immersing the animal-derived tissue or organ in a medium containing the DNA enzyme.

3. The method for producing a decellularized tissue or organ according to claim 1 , wherein the medium does not contain a surfactant.

4. The method for producing a decellularized tissue or organ according to claim 1 , wherein the step of applying voltage to the animal-derived tissue or organ into which the DNase has been incorporated is completed within 6 hours.

5. 2. The method for producing a decellularized tissue or organ according to claim 1, wherein the amount of DNA in the decellularized tissue or organ obtained after the application step is 50% or less compared to the amount of DNA in a native tissue or organ not subjected to decellularization treatment.

6. A decellularized tissue having an extracellular matrix, The amount of DNA in the decellularized tissue is 10% or less compared to the amount of DNA in a natural tissue or organ that has not been subjected to decellularization treatment; and A decellularized tissue that satisfies at least one of the following (i) to (vii): (i) The rate of change in the amount of hydroxyproline in the decellularized tissue relative to the amount of hydroxyproline in native tissue not subjected to decellularization treatment is within ±30%. (ii) The rate of change of the amount of elastin in the decellularized tissue relative to the amount of elastin in native tissue not subjected to decellularization treatment is within ±30%. (iii) The exosomes in the decellularized tissue are present in an amount that can be observed under a fluorescent microscope after staining with a fluorescent dye. (iv) The rate of change in the storage modulus of the decellularized tissue relative to a strain in the range of 0.1 to 10% or less compared with the storage modulus of a natural tissue not subjected to decellularization treatment relative to a strain in the range of 0.1 to 10% or less is within ±30%. (v) The rate of change in the storage modulus of the decellularized tissue at frequencies in the range of 0.1 to 5 [1 / s] or less compared with the storage modulus of a natural tissue not subjected to decellularization treatment at frequencies in the range of 0.1 to 5 [1 / s] or less is within ±30%. (vi) The rate of change in the loss modulus of the decellularized tissue relative to a strain in the range of 0.1 to 10% or less compared with the loss modulus of the natural tissue not subjected to decellularization treatment relative to a strain in the range of 0.1 to 10% or less is within ±30%. (vii) The rate of change of the loss modulus of the decellularized tissue at a frequency in the range of 0.1 to 5 [1 / s] or less relative to the loss modulus of the natural tissue not subjected to decellularization treatment at a frequency in the range of 0.1 to 5 [1 / s] or less is within ±30%.

7. The decellularized tissue according to claim 6 , wherein the decellularized tissue contains a DNA degrading enzyme at a concentration of 100 ng / ml or more.

8. The decellularized tissue of claim 6 , wherein a biologically active substance is incorporated into the extracellular matrix.

9. A powdered tissue obtained by powdering the decellularized tissue according to claim 6.

10. A composition comprising the disrupted decellularized tissue according to claim 6 suspended in a liquid medium.

11. A solid organ comprising the decellularized tissue according to claim 6.

12. 13. Use of a decellularized tissue according to claim 6, a powdered tissue according to claim 9, a composition according to claim 10, or a solid organ according to claim 11 for supporting cell proliferation, promoting cell regeneration, promoting angiogenesis, as an implant for tissue replacement surgery, as an implant for tissue repair or regeneration, or as an implant for skin grafting and / or skin regeneration surgery.

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