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

Abstract

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.

Description

Method for producing decellularized tissue or organ using hydrostatic pressure

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

[0002] In organ transplants, when the living tissue of an organ donor is transplanted into the 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 known as hydrostatic pressure, a state quantity that can be applied uniformly to the entire tissue, to decellularize a uterus excised from a rat and transplant it into a uterine defect site, finding that the decellularized tissue produced 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 applying a hydrostatic pressure of 980 MPa for 10 minutes.

[0004] PLoS One 2014 July 24;9(7):e103201, doi: 10.1371 / journal.pone.0103201

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

[0006] Prolonged tissue washing after decellularization may increase the risk of matrix degeneration of the decellularized tissue itself. This has significant disadvantages in terms of cost and productivity of decellularized tissue production. Furthermore, chemical decellularization methods using surfactants such as SDS and Triton X-100 are highly toxic to tissue.

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

[0008] The problem to be solved by 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.

[0009] The present invention encompasses the following embodiments. Item 1. A method for producing decellularized tissue or organ, comprising the steps of incorporating a DNase into 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. Item 2. The method for producing 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. Item 3. The method for producing decellularized tissue or organ according to Item 1, wherein the medium does not contain a surfactant. Item 4. The method for producing decellularized tissue or organ according to Item 1, wherein the applying step is completed within 6 hours. Item 5. The method for producing decellularized tissue or organ according to Item 1, wherein the amount of DNA in the decellularized tissue or organ obtained after the applying step is 50% or less compared to the amount of DNA in a native tissue or organ that has not been subjected to decellularization treatment. Item 6. A decellularized tissue having an extracellular matrix, wherein 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 a decellularization treatment, and the decellularized tissue satisfies at least one of the following (i) to (vii):

[0010] (i) The rate of change of the amount of hydroxyproline in the decellularized tissue relative to the amount of hydroxyproline in natural tissue that has not been decellularized is within ±30%. (ii) The rate of change of the amount of elastin in the decellularized tissue relative to the amount of elastin in natural tissue that has not been decellularized is within ±30%. (iii) 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 of the storage modulus of the decellularized tissue relative to a strain in the range of 0.1 to 10% or less relative to the storage modulus of the natural tissue that has not been decellularized relative to a strain in the range of 0.1 to 10% or less is within ±30%. (v) The rate of change of the storage modulus of the decellularized tissue relative to a frequency in the range of 0.1 to 5 [1 / s] or less relative to the storage modulus of the natural tissue that has not been decellularized relative to a frequency 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 to the loss modulus of the decellularized tissue relative to a strain in the range of 0.1 to 10% or less is within ±30%. (vii) The rate of change in the loss modulus of the decellularized tissue relative to a frequency in the range of 0.1 to 5 [1 / s] or less compared to the loss modulus of the decellularized tissue relative to a frequency in the range of 0.1 to 5 [1 / s] or less is within ±30%. 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. Item 8. The decellularized tissue according to Item 6, wherein a biologically active substance is incorporated into the extracellular matrix. Item 9. A powdered tissue obtained by powdering the decellularized tissue according to Item 6. Item 10. A composition obtained by dissolving or suspending crushed material of the decellularized tissue according to Item 6 in a liquid medium. Item 11. Item 12. A solid organ comprising the decellularized tissue according to Item 6. Item 13. 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 growth, 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.

[0011] From left to right: Microscopic observation of DIS from native small intestinal tissue, low-pressure (LP), high-pressure (HP), and SDS samples. Bars represent 1 cm. Graph showing the relationship between DNA residue and hydrostatic pressure. Shown as mean ± standard error (SD). (n = 4 per group). Hematoxylin and eosin (HE) staining of DIS from native small intestinal tissue, LP, HP, and SDS samples. Scale bar represents 400 μm. Quantification of DNA in DIS from LP, HP, and SDS samples compared to native tissue. Shown as mean ± standard error (n = 4 per group). **p < 0.005. Collagen content in each tissue sample. Masson's trichrome staining of DIS from native small intestinal tissue, LP, HP, and SDS samples. Scale bar represents 400 μm. Collagen content in DIS from LP, HP, and SDS samples compared to native tissue. Shown as mean ± standard deviation (n = 4 per group). *p < 0.05. Elastin content in each tissue sample. Masson's trichrome staining of DIS from native small intestinal tissue and LP, HP, and SDS samples. Scale bar indicates 400 μm. Elastin content in DIS from LP, HP, and SDS samples compared to native tissue. Shown as mean ± standard deviation (n = 4 per group). Exosome expression in each sample. Scanning electron microscope images of small intestinal crypt structures from native tissue (top left), LP (top right), HP (bottom left), and SDS (bottom right). Storage modulus of DIS versus strain for LP, HP, and SDS samples compared to native tissue. Shown as mean ± standard deviation (n = 3-7 per group). Storage modulus of DIS versus frequency for LP, HP, and SDS samples compared to native tissue. Shown as mean ± standard deviation (n = 3-7 per group). Loss modulus of DIS versus strain for LP, HP, and SDS samples compared to native tissue. Shown as mean ± standard deviation (n = 3-7 per group). (n = 3-5 per group) 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) Photographs of intestinal tissues implanted with decellularized tissue samples from Sham-treated, LP, HP, and SDS rats. 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. The black triangle indicates the sutured area. The red circle indicates the enlarged area in the center of the transplanted tissue fragment.

[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. Furthermore, 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 "to" means a numerical range that includes the numbers before and after "to" as the upper and lower limits.

[0014] According to a first aspect of the present invention, there is provided a method for producing decellularized tissue or organ, comprising the steps of: incorporating a DNase into 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 this 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 within the animal-derived tissue or organ has been degraded. This promotes the removal of cells from the animal-derived tissue or organ, and allows cells to be removed from the animal-derived tissue or organ in a shorter period of time than 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 biological tissue) is not particularly limited as long as it is biological tissue containing cells derived from a vertebrate, but biological tissue derived from mammals or birds is preferred, and mammals are more preferred, as it is less likely to cause rejection. 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 rats, pigs, cows, goats, sheep, rabbits, monkeys, or humans.

[0017] Body fluids include interstitial fluid, blood, and lymphatic fluid, and preferably 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, and skin.

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

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

[0021] After being collected or removed from the animal, animal-derived tissues or organs are preferably washed with physiological saline or various buffer solutions.

[0022] By incorporating a DNase into an animal-derived tissue or organ from the outside of the tissue or organ before the step of applying hydrostatic pressure, the concentration of the DNase in the animal-derived tissue or organ can be increased and DNA in the animal-derived tissue or organ can be decomposed before 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 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 DNase-containing medium used for uptake of the DNase preferably comprises 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 DNase may contain a surfactant, but is preferably surfactant-free in order to eliminate or reduce toxicity to tissues. Examples of surfactants 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 and 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) a step of 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 subsequent hydrostatic pressure step); (ii) after the pressure increasing step, a step of 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, a step of decreasing 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 DNase-containing medium used in the step of incorporating the DNase into animal-derived tissues or organs and the DNase-containing medium used in the step of applying hydrostatic pressure may have the same composition or may have different compositions. Furthermore, the DNase-containing medium used in the step of incorporating the DNase may be used directly in the step of applying hydrostatic pressure, but it is preferable to replace the DNase-containing medium used in the step of incorporating the DNase with the DNase-containing medium 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, resulting in a large-scale facility. 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 decellularization treatment and minimizing the influence on the tissue.

[0035] The time for the pressure increasing step 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 viewpoint of maintaining the structure of the tissue or organ and speeding up the application of 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 pressure decreasing step 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 decellularized tissue or organ, it is preferable not to perform a process of degrading DNA in the decellularized tissue or organ with a DNase after the step of applying hydrostatic pressure, but optionally, to further promote the degradation 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 approximately 30 minutes to 6 hours. The DNase-containing medium used in this case may have the same composition as the DNase-containing medium used in the step of incorporating the DNase or the DNase-containing medium used in the step of applying hydrostatic pressure, or it may have a different composition.

[0037] The method for producing decellularized tissue or organ may further include, after the step of applying hydrostatic pressure, a step of washing the decellularized tissue or organ with a washing solution that does not contain DNase. The washing solution may be an aqueous solution or buffer solution containing at least one of an acid, a base, and a salt. Examples of such aqueous solutions or buffer solutions include physiological saline, acetate buffer, phosphate buffer, citrate buffer, borate buffer, tartrate buffer, Tris buffer, HEPES buffer, and MES buffer. While the solution may contain a surfactant, it is preferable that it does not contain a surfactant in order to eliminate or reduce toxicity to the tissue. While the solution may contain a surfactant, it is preferable that it does not contain a surfactant in order to eliminate or reduce 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 this embodiment, 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. Furthermore, by shortening the time required for the uptake of DNase into the animal-derived tissue or organ, the time required from the collection of the tissue or organ from the animal to the step of applying 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 bioactive substances include growth factors, antibacterial agents, analgesics, hemostatic agents, angiogenesis promoters, angiogenesis inhibitors, or combinations thereof, or exosomes. When the bioactive substance is an exosome, the exosome may be exosomes obtained from an animal that is a donor of animal-derived tissue or organs, or may be other exosomes, and known or commercially available exosomes may also be used.

[0041] The bioactive substance 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 substance may be bound 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 above-mentioned method for producing decellularized tissue or organ may further include a step of powdering 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.

[0043] Decellularized tissues or organs can be powdered, for example, by drying the decellularized tissues or organs and pulverizing the dried decellularized tissues or organs. Methods for drying decellularized tissues or organs include, but are not limited to, lyophilization. Decellularized tissues or organs 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, to promote tissue or organ regeneration by applying it to the affected area of ​​a patient.

[0045] The method for producing the 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] Decellularized tissues or organs can be disrupted, for example, by drying the decellularized tissue or organ and disrupting the dried decellularized tissue or organ. Methods for drying decellularized tissues or organs include, but are not limited to, freeze-drying. This can be done using a known grinding device such as a mill.

[0047] Examples of liquid solvents include solutions containing proteins. For example, a powder of finely pulverized decellularized tissue or organs can be dissolved in a solution containing pepsin, and the resulting solution can be appropriately pH-adjusted 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.

[0048] The resulting composition can be used, for example, to fill a cartridge in a 3D printer and 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, of the amount of DNA in the native tissue or organ that has not been subjected to decellularization.

[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 performing a step of washing the decellularized tissue or organ with a solution containing a DNA degrading enzyme after the step of applying hydrostatic pressure.

[0051] In this specification, the amount of DNA in 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. 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] With this configuration, it is possible to significantly remove cells from the decellularized tissue or organ after the step of applying hydrostatic pressure.

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

[0054] (i) The rate of change in the amount of hydroxyproline in decellularized tissue relative to the amount of hydroxyproline in natural tissue not subjected to decellularization is within ±30%. (ii) The rate of change in the amount of elastin in decellularized tissue relative to the amount of elastin in natural tissue not subjected to decellularization is within ±30%. (iii) Exosomes in decellularized tissue are present in an amount observable 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 relative to the storage modulus of the natural tissue not subjected to decellularization 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 relative to a frequency in the range of 0.1 to 5 [1 / s] or less relative to the storage modulus of the natural tissue not subjected to decellularization relative to a frequency 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 to the loss modulus of the decellularized native tissue relative to a strain in the range of 0.1 to 10% or less is within ±30%. (vii) The rate of change in the loss modulus of the decellularized tissue relative to a frequency in the range of 0.1 to 5 [1 / s] or less compared to the loss modulus of the decellularized native tissue relative to a frequency in the range of 0.1 to 5 [1 / s] or less is within ±30%. Herein, the amount of hydroxyproline in the decellularized tissue and the amount of elastin in the decellularized tissue may be measured by known methods or using commercially available kits, and are particularly measured by the methods described in the Examples. The amount of hydroxyproline reflects the amount of collagen.

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

[0056] The amount of elastin in decellularized tissue relative to the amount of elastin in native tissue that has not been decellularized is calculated by (amount of elastin in decellularized tissue) / (amount of elastin in native tissue that has not been decellularized)*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 decellularized tissue and native tissue that has not been subjected to decellularization treatment can be measured using a commercially available rheometer, and in particular, are measured by the method described in the Examples.

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

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

[0060] The rate of change in the loss modulus of decellularized tissue relative to the loss modulus of decellularized natural tissue relative to the loss modulus of decellularized natural tissue is calculated by {(loss modulus of decellularized tissue relative to the strain) - (loss modulus of decellularized natural tissue relative to the strain)} / (loss modulus of decellularized natural tissue relative to the strain)*100 (%).

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

[0062] In this specification, exosomes in decellularized tissue are 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 the exosomes can also be compared with that of exosomes in natural tissue that has not been subjected to decellularization.

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

[0064] The amount of hydroxyproline in the decellularized tissue is preferably 70% or more of the amount of hydroxyproline in natural 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 in response to a strain in the range of 0.1 to 10% or less is preferably 70% or more of the storage modulus of natural tissue not subjected to decellularization in response to a 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 treatment 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 of the loss modulus of natural tissue that has not been 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 in the amount of hydroxyproline in decellularized tissue relative to that in native tissue that has not been 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 the amount of elastin in native tissue that has not been 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 storage modulus of decellularized tissue relative to strain of 0.1 to 10% or less relative to that of undecellularized native tissue relative to strain 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 in the storage modulus of decellularized tissue at frequencies in the range of 0.1 to 5 [1 / s] or less relative to the storage modulus of undecellularized native tissue at frequencies in the range of 0.1 to 5 [1 / s] or less is preferably within ±20%, more preferably within ±10%. Alternatively, the rate of change in the storage modulus of decellularized tissue at frequencies in the 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 undecellularized native tissue at frequencies in the 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%. Furthermore, over the entire range of frequencies, preferably 0.1 to 2 [1 / s] or less, more preferably 0.1 to 3 [1 / s] or less, and more preferably 0.1 to 5 [1 / s] 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 of 0.1 to 10% or less relative to the loss modulus of undecellularized native tissue relative to a strain 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 in the loss modulus of decellularized tissue over a frequency range of 0.1 to 5 [1 / s] relative to the loss modulus of undecellularized native tissue over a frequency range of 0.1 to 5 [1 / s] is preferably within ±20%, more preferably within ±10%. Alternatively, the rate of change in the loss modulus of decellularized tissue over a frequency range of 0.1 to 3 [1 / s], more preferably 0.1 to 2 [1 / s], relative to the loss modulus of undecellularized native tissue over a frequency range of 0.1 to 3 [1 / s], more preferably 0.1 to 2 [1 / s], is preferably within ±20%, more preferably within ±10%. Furthermore, over the entire frequency range of 0.1 to 2 [1 / s], more preferably 0.1 to 3 [1 / s], and more preferably 0.1 to 5, 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 above-described method for producing 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 above-mentioned 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 decellularized tissue or organ, or the decellularized tissue in the obtained organ, satisfies one or both of (i) and (ii) above, and (iii) above.

[0080] In some embodiments, the decellularized tissue obtained by the above-described method for producing a decellularized tissue or organ, 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 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 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 of them.

[0083] In some embodiments, the decellularized tissue obtained by the above-mentioned method for producing 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-described 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, wherein 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 a decellularization treatment, and wherein the decellularized tissue satisfies at least one of the following (i) to (vii): (i) The rate of change in the amount of hydroxyproline in decellularized tissue relative to the amount of hydroxyproline in natural tissue not subjected to decellularization is within ±30%. (ii) The rate of change in the amount of elastin in decellularized tissue relative to the amount of elastin in natural tissue not subjected to decellularization is within ±30%. (iii) Exosomes in decellularized tissue are present in an amount observable 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 relative to the storage modulus of the natural tissue not subjected to decellularization 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 relative to a frequency in the range of 0.1 to 5 [1 / s] or less relative to the storage modulus of the natural tissue not subjected to decellularization relative to a frequency in the range of 0.1 to 5 [1 / s] or less is within ±30%. (vi) The rate of change of the loss modulus of the decellularized tissue relative to a strain in the range of 0.1 to 10% or less compared to 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 relative to a frequency in the range of 0.1 to 5 [1 / s] or less compared to the loss modulus of the natural tissue not subjected to decellularization treatment relative to a frequency 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 natural tissue that has not been subjected to decellularization treatment.

[0087] 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.

[0088] The storage modulus of decellularized tissue in response to a strain in the range of 0.1 to 10% or less is preferably 70% or more of the storage modulus of natural tissue not subjected to decellularization in response to a 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 treatment 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 of the loss modulus of natural tissue that has not been 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 in the amount of hydroxyproline in decellularized tissue relative to that in native tissue that has not been 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 the amount of elastin in native tissue that has not been subjected to decellularization treatment is preferably within ±20%, more preferably within ±10%.

[0094] The amount of exosomes in the decellularized tissue is preferably 10% or more, 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 storage modulus of decellularized tissue relative to strain in the range of 0.1 to 10% or less relative to that of undecellularized native tissue relative to strain in the range of 0.1 to 10% or less is preferably within ±20%, more preferably within ±10%, and preferably satisfies this rate of change over the entire range of strain up to 10%.

[0096] The rate of change in the storage modulus of decellularized tissue at frequencies in the range of 0.1 to 5 [1 / s] or less relative to the storage modulus of undecellularized native tissue at frequencies in the range of 0.1 to 5 [1 / s] or less is preferably within ±20%, more preferably within ±10%. Alternatively, the rate of change in the storage modulus of decellularized tissue at frequencies in the 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 undecellularized native tissue at frequencies in the 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%. Furthermore, over the entire range of frequencies, preferably 0.1 to 2 [1 / s] or less, more preferably 0.1 to 3 [1 / s] or less, and more preferably 0.1 to 5 [1 / s] 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 of 0.1 to 10% or less relative to the loss modulus of undecellularized native tissue relative to a strain 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 in the loss modulus of decellularized tissue over a frequency range of 0.1 to 5 [1 / s] relative to the loss modulus of undecellularized native tissue over a frequency range of 0.1 to 5 [1 / s] is preferably within ±20%, more preferably within ±10%. Alternatively, the rate of change in the loss modulus of decellularized tissue over a frequency range of 0.1 to 3 [1 / s], more preferably 0.1 to 2 [1 / s], relative to the loss modulus of undecellularized native tissue over a frequency range of 0.1 to 3 [1 / s], more preferably 0.1 to 2 [1 / s], is preferably within ±20%, more preferably within ±10%. Furthermore, over the entire frequency range of 0.1 to 2 [1 / s], more preferably 0.1 to 3 [1 / s], and more preferably 0.1 to 5, 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 above-described method for producing 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 above-mentioned 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 decellularized tissue or organ, or the decellularized tissue in the obtained organ, satisfies one or both of (i) and (ii) above, and (iii) above.

[0102] In some embodiments, the decellularized tissue obtained by the above-described method for producing a decellularized tissue or organ, 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 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 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 of them.

[0105] In some embodiments, the decellularized tissue obtained by the above-mentioned method for producing 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 greater, which is higher than the concentration of DNase in human serum, 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). The DNase concentration is preferably 1 μg / ml or greater, more preferably 10 μg / ml or greater.

[0107] In some embodiments, the extracellular matrix has incorporated therein a bioactive substance, as described above in relation to the method for producing the 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 to support cell proliferation, promote cell regeneration, promote angiogenesis, as a transplant 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] This application develops a technology that can remove cells contained in organs or tissues by applying a physical quantity called hydrostatic pressure. Conventional decellularization techniques using hydrostatic pressure require a 1-2 week washing process for the organ or tissue to remove cells after applying hydrostatic pressure. Furthermore, conventional decellularization techniques using hydrostatic pressure have had problems, such as difficulty in decellularizing large organs and difficulty in maintaining the organ's structure, making it difficult to control the organ's mechanical properties.

[0113] The method for producing decellularized tissue or organs according to the first aspect is a novel technique for producing decellularized tissue or organs with minimal immune rejection, replacing organ transplantation. This technique achieves sufficient decellularization in a short period of time despite the application of low hydrostatic pressure. This new decellularization method kills cells using the physical quantity known as hydrostatic pressure in the presence of a DNA-degrading enzyme, significantly shortening the tissue washing process after application of hydrostatic pressure compared to conventional techniques. Furthermore, the low hydrostatic pressure allows the use of commercially available hydrostatic pressure equipment, eliminating the need for the large-scale equipment required to apply 980 MPa. Furthermore, the method for producing decellularized tissue or organs according to the first aspect is advantageous in that it can decellularize even thick, complex organs, thereby broadening the range of organs that can be decellularized. Furthermore, the structure of the decellularized tissue or organ is well maintained, resulting in excellent tissue regeneration potential.

[0114] The clinical application of decellularized organ models using such methods for producing decellularized tissues or organs is highly anticipated. 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 racehorse industry.

[0115] For example, in the medical field, decellularized organs / tissues have value as an alternative to organ transplants. It is also conceivable that decellularized bones could be used to create screw plates in orthopedic surgery. Powder or liquid tissue compositions produced from the decellularized tissue or organ of the present invention can also be used as materials for fibrin gels used in basic research on iPS cells and other tissues. In the cosmetic field, the matrix of decellularized organs / tissues is expected to have cosmetic molding effects. Collagen, elastin, and placenta components developed using the method for producing decellularized tissue or organs of the present invention can be applied to facial shaping and / or formation.

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

[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 literature procedures in the art.

[0118] Test Example 1. Preparation of decellularized tissue (pretreatment) Step 1. Fat tissue was removed from the rat small intestine, and it was opened longitudinally. Step 2. Sterilized phosphate buffer solution (PBS) and Milli-Q water (optional) were prepared, and the sample was washed until it turned pure white (approximately 5 times). Step 3. The pretreated small intestine sample was treated using either (1) the EHHP method or (2) the SDS method as follows. (1) EHHP method (LP, HP) 1. In a dark place, using clean procedures, the small intestine sample was immersed in 10 ml of DNAse I solution placed in a plastic bag. 2. The opening of the plastic bag was heat-sealed. Care was taken to prevent air from entering during this process. 3. Until hydrostatic pressure was applied, the sealed small intestine sample was stored in a refrigerator or icebox at 4°C, protected from light. The small intestine sample was immersed in the DNAse I solution for approximately 30 minutes. 4. The sample was pressurized using a hydrostatic pressure device, maintained at 30°C for 40 minutes, and then released. The sample was designated LP when subjected to a hydrostatic pressure of 150 MPa, while the sample was designated HP when subjected to a hydrostatic pressure of 960 MPa for 40 minutes. For both hydrostatic pressures, the pressure increase and decrease rate 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 refrigerator at 4°C. 6. The sample was removed and washed with PBS for 2 hours. (2) SDS Method: 1. Prepare a 1% SDS solution, dissolve it thoroughly, and 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 on a shaker at room temperature. 3. Wash the sample with PBS for 2 hours, then transfer it to DNAse I buffer and wash for seven days. Change the DNAse I buffer daily. (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 briefly washed 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-2Na and incubated at 65°C for at least 15 hours. The samples were further pulverized using a homogenizer and ultrasonic cell disrupter. These samples were 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 using 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 4 M NaOH and heated at 120°C for 30 minutes. A 1.4 N citric acid and chloramine T solution was then added to the sample. After 20 minutes, the aldehyde-perchlorate reagent was added to the solution and incubated at 70°C for 20 minutes. Each sample was analyzed by fluorescence spectrophotometer at a wavelength of λ = 450 nm.

[0122] Test Example 5: Elastin Assay. Elastin content was assessed using a commercially available elastin assay kit (Fastin elastin assay kit; Biocolor, UK). 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. 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 using 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 fluorescence 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. The samples were then dehydrated through gradient ethanol, embedded in paraffin blocks, and sectioned at 7 μm thickness. Staining was performed with hematoxylin and eosin (HE, nuclei 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 optical microscope (Zeiss, Germany). The submucosa of the implant was stained with vimentin (Abcam; 1:400 dilution), the smooth muscle layer with smooth muscle α-actin (α-SMA) (Abcam; 1:400 dilution), the mucosa with KRT18 (Abcam; 1:100 dilution), and the endothelial cells with CD31 (Santa Cruz; 1:50 dilution). Heat-induced epitope retrieval (10 mM citrate buffer, pH 6.0) was performed prior to protein blocking and inactivation of endogenous peroxidase. Primary antibody incubation was performed overnight at 4°C, followed by secondary antibody (anti-rabbit IgG; Dako) incubation for 2 hours at room temperature. Samples were then stained with 3,3'-diaminobenzidine (DAB) and counterstained with hematoxylin.

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

[0126] Test Example 9. Viscoelasticity Testing. A 5 mm biopsy punch was used to cut round samples from each tissue prepared in Test Example 1. The thickness of the samples was first measured using a micromaterials testing machine (Shimadzu Corporation). Viscoelasticity testing was performed using a rheometer (Physica MCR301) according to the accompanying manual. The samples were humidified at 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] Experimental Example 10: Transplantation of Decellularized Tissues. SD rats (9 weeks old, male) were used as subjects for decellularized tissue transplantation. These rats were divided into four groups (n = 3; sham, LP, HP, SDS). From 12 hours before surgery to 48 hours after surgery, the subjects' solid diet was replaced with a 5% glucose solution. In the sham surgery, half of the intestinal tissue was incised and sutured without intervention. In the experimental group, half of the intestinal wall was resected and replaced with decellularized tissue (15 mm x 10 mm). Each decellularized tissue prepared in Experimental Example 1 was sutured to the native tissue at least 10 times, including the four corners, with non-degradable polypropylene thread. After suturing, the transplanted area was wrapped in Seprafilm (KAKEN, Japan) to prevent adhesions. The rats were maintained for 1 month and then sacrificed. Prior to histological examination, the grafts were excised and removed of connective tissue and fat.

[0128] Results (1) Preparation of decellularized tissue Samples after pretreatment in Test Example 1 are shown in Figure 1. In Test Example 1, for the EHHP samples (LP, HP), the time for immersing the tissue in a solution containing DNase was shorter than for the SDS method, and the washing time after application of hydrostatic pressure, which required 7 days or more in prior art (PLoS One 2014 July 24;9(7):e103201), was unnecessary or only required in a short time. As a result, the decellularization process was able to be performed in a short time of just a few tens of minutes from organ procurement to complete decellularization.

[0129] (2) Residual DNA in Tissues. As shown in Figure 2, the DNA assay revealed low levels of residual DNA in all samples prepared using the EHHP method at hydrostatic pressures of 100 MPa, 150 MPa, and 200 MPa, with the 150 MPa LP sample showing the lowest levels. HE staining revealed blue cell nuclei and pink tissues. As shown in Figure 3, the native sample showed intensely concentrated nuclei and non-nuclear tissues, whereas the LP, HP, and SDS samples showed reduced or no blue dots, confirming cell removal. As shown in Figure 4, the LP sample showed significantly reduced DNA content in tissues below 10% compared to the native sample. Furthermore, the DNA content of the LP sample was even lower than that of the HP sample. Based on the results of Figure 2, a hydrostatic pressure of 150 Pa and an application time of 60 minutes (excluding pressure rise and fall) were selected as the optimal pressure and time conditions for reducing residual DNA in tissues to 5%. These were used to create new LP samples. The samples used in the inventors' previous research, subjected to a hydrostatic pressure of 980 MPa and a pressure time of 10 minutes (not including the time for pressure increase and decrease), were designated as HP samples. In both cases, the pressure increase and decrease rate was 65.3 MPa / min. The SDS samples were those prepared in Test Example 1. Figure 4 shows that the amount of residual DNA was significantly reduced in the LP, HP, and SDS samples compared to the native sample. The higher amount of residual DNA in the HP samples is likely due to the short pressure application time 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 the absence of black and red staining confirmed the removal of cells. Furthermore, as shown in Figure 6, the LP sample had significantly higher hydroxyproline content than the HP and SDS samples, suggesting that a greater amount of collagen remained after decellularization, suggesting that the structural proteins of the tissue were better maintained 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 in the native, LP, and HP samples confirmed the preservation of elastin. 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 better maintained in the hydrostatic pressure sample than in the SDS sample.

[0132] (5) Expression of exosomes in decellularized tissue As shown in Figure 9, the LP sample appeared to be the darkest in the immunostaining of the small intestine. This suggests that exosomes are best retained or retained in the LP decellularized tissue compared to the HP and SDS decellularized tissues.

[0133] (6) Scanning Electron Microscope (SEM) Measurements As shown in Figure 10, the villi-crypt structure on the inner surface of the small intestine was observed. The white dots of cells present in the native sample were absent in the LP sample, but the villi-crypt structure was confirmed to be preserved. Some structural collapse was observed in the HP sample (top right of the image). The SDS sample was completely destroyed, with no visible holes.

[0134] (7) Viscoelasticity Testing The viscoelasticity of the tissue slices was measured using a rheometer. Figure 11 shows the relationship between the storage modulus (a physical quantity representing elasticity) and the strain applied to the sample, Figure 12 shows the relationship between the storage modulus (a physical quantity representing elasticity) and the frequency applied to the sample, Figure 13 shows the relationship between the loss modulus (a physical quantity representing viscosity) and the strain applied to the sample, and Figure 14 shows the relationship between the loss modulus (a physical quantity representing viscosity) and the frequency applied to the sample. All of these figures show the same trend. That is, among the LP, HP, and SDS samples, the LP sample exhibited viscosity and elasticity closest to that of the native tissue. On the other hand, HP and SDS had viscoelastic properties different from that of the native tissue. This is thought to be related to the damage to the tissue caused by the decellularization process. In other words, the LP sample exhibited the least damage to the tissue's mechanical properties.

[0135] (8) Transplantation of Decellularized Tissue Figure 15 shows photographs of samples extracted one month after implantation of each tissue fragment prepared in Test Example 1 into the rat intestine. The arrows indicate the location of the tissue fragment, and it was confirmed that all tissue fragments had integrated with the native tissue. The black triangle in Figure 16 indicates the sutured area. The red circle is a magnified area in the center of the transplanted tissue fragment. SMA is a smooth muscle marker that labels smooth muscle in the outermost part of the intestinal tract and in the villi. Vim is an abbreviation for vimentin and labels the submucosa and connective tissue of the small intestine. KRT18 is a mucosal marker. CD31 is a marker for vascular endothelial cells. By staining the tissue fragment brown with these four markers, the regeneration status of the tissue fragment can be confirmed. A magnified image of SMA reveals that the brown-stained smooth muscle is connected. Smooth muscle appears discontinuous in HP and SDS. In other words, smooth muscle regeneration was best in LP. There was no significant difference in Vim regeneration between LP, HP, and SDS. Numerous fibroblasts were found in each tissue fragment, and the overall appearance resembled scar tissue. KRT18 staining revealed a partial loss of villous structure and mucosa in SDS (upper left magnified image). A single ring-shaped intact mucosa layer was confirmed in LP and HP samples. Staining of vascular endothelial cells confirmed the regeneration of capillaries in the mucosa and submucosa in LP and HP. Capillaries in the mucosa were not detected in SDS.

Claims

1. A method for producing decellularized tissue or organ, comprising the steps of: incorporating a DNA enzyme 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 DNA enzyme has been incorporated, in a medium containing the DNA enzyme.

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

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

4. A method for producing decellularized tissue or organs as described in claim 1, wherein the step of applying the voltage to the animal-derived tissue or organ into which the DNA degrading enzyme has been incorporated is completed within 6 hours.

5. A method for producing a decellularized tissue or organ as described in 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, wherein the amount of DNA in the decellularized tissue is 10% or less compared to the amount of DNA in a native tissue or organ that has not been subjected to a decellularization process, and which satisfies at least one of the following (i) to (vii): (i) The rate of change of the amount of hydroxyproline in the decellularized tissue relative to the amount of hydroxyproline in natural tissue not subjected to decellularization is within ±30%. (ii) The rate of change of the amount of elastin in the decellularized tissue relative to the amount of elastin in natural tissue not subjected to decellularization is within ±30%. (iii) Exosomes in the decellularized tissue are present in an amount observable under a fluorescent microscope after staining with a fluorescent dye. (iv) The rate of change of the storage modulus of the decellularized tissue relative to a strain in the range of 0.1 to 10% or less relative to the storage modulus of the natural tissue not subjected to decellularization relative to a strain in the range of 0.1 to 10% or less is within ±30%. (v) The rate of change of the storage modulus of the decellularized tissue relative to a frequency in the range of 0.1 to 5 [1 / s] or less relative to the storage modulus of the natural tissue not subjected to decellularization relative to a frequency in the range of 0.1 to 5 [1 / s] or less is within ±30%. (vi) The rate of change of the loss modulus of the decellularized tissue relative to a strain in the range of 0.1 to 10% or less compared to the loss modulus of the decellularized natural tissue 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 relative to a frequency in the range of 0.1 to 5 [1 / s] or less compared to the loss modulus of the decellularized natural tissue relative to 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 of claim 6 suspended in a liquid medium.

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

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

Images