JP6271298B2 - Method for producing decellularized tissue - Google Patents

Description

  The present invention relates to a method for producing a decellularized tissue that can be suitably used for regenerative medicine.

  When transplanting a graft derived from a living tissue of another person, rejection of the graft by the recipient side tissue becomes a problem. Development of artificial tissue is expected as a solution for such problems. Although various polymers have been tried to be used as materials, the compatibility between these materials and biological tissues is low, and there are cases where dropouts or infectious diseases occur at the joints between the graft and the biological tissues. Therefore, in order to improve the compatibility with the living tissue, a technique has been developed in which cells are removed from the living tissue and the decellularized living tissue that is the remaining supporting tissue is used as a graft. Since cells can be efficiently removed from a living tissue without using a special apparatus, a decellularization method using a surfactant (for example, see Patent Documents 1 to 3) is widely performed. However, the decellularization method using a surfactant causes a large damage to the tissue, and the mechanical strength of the decellularized tissue is greatly reduced. In particular, in the liver, kidney, lung, etc., organ damage may occur during transplantation. was there. On the other hand, a decellularization method using ultrahigh hydrostatic pressure treatment is known (for example, see Patent Documents 4 to 6). However, it takes a long time to wash cell pieces after treatment, or in the case of a large organ. Had a problem that it could not be cleaned sufficiently.

JP-A-60-501540 Japanese translation of PCT publication No. 2003-518981 Special table 2009-505752 gazette JP 2004-094552 A International Publication No. 2008/111530 Pamphlet Special table 2013-502275 gazette

  The problem to be solved by the present invention is to provide a method for producing a decellularized tissue that can clean a cell piece from a living tissue in a relatively short time and has little damage to the tissue.

  The present inventors have intensively studied and found that the above problems can be solved by applying a hydrostatic pressure to animal-derived tissue in a medium containing a surfactant, and have completed the present invention. That is, the present invention is a method for producing a decellularized tissue having a step of applying a hydrostatic pressure of 100 to 1500 MPa in a medium containing a surfactant.

  According to the present invention, a decellularized tissue with little damage can be produced in a relatively short time, and in particular, a decellularized tissue with little damage to large organs such as liver, kidney and lung, which has been difficult to produce conventionally, can be obtained. .

  Hereinafter, the present invention will be described in detail.

[Animal-derived tissue]
The animal-derived tissue (also referred to as a biological tissue) used in the method for producing a decellularized tissue of the present invention is not particularly limited as long as it is a biological tissue having vertebrate-derived cells. Alternatively, a biological tissue derived from birds is preferable, and a biological tissue derived from mammals, avian livestock, or humans is more preferable because it is easily available. Mammalian livestock includes cattle, horses, camels, llamas, donkeys, yaks, sheep, pigs, goats, deer, alpaca, dogs, raccoons, weasels, foxes, cats, rabbits, hamsters, guinea pigs, rats, mice, squirrels, Raccoon etc. are mentioned. In addition, examples of avian livestock include parakeets, parrots, chickens, ducks, turkeys, geese, guinea fowls, pheasants, ostriches, and quails. Among these, porcine, rabbit, and human biological tissues are preferable from the viewpoint of availability.

  As a part of a living tissue, a part having a matrix structure outside the cell can be used. Examples of such a part include a liver, kidney, ureter, bladder, urethra, tongue, tonsils, esophagus, stomach, small intestine, Examples include large intestine, anus, pancreas, heart, blood vessel, spleen, lung, brain, bone, spinal cord, cartilage, testis, uterus, fallopian tube, ovary, placenta, cornea, skeletal muscle, tendon, nerve, skin and the like. As the site of the living tissue, cartilage, bone, liver, kidney, heart, lung, brain, and spinal cord are preferable because of the high tissue regeneration effect. It is preferable that the biological tissue is subjected to a treatment for preventing spoilage and functional deterioration after collection. Examples of such treatment include sterilization treatment with a drug, freezing treatment by freezing, and the like, and freezing treatment is preferable because there is little damage to the tissue.

[Applying process]
In the production method of the present invention, a hydrostatic pressure of 100 to 1500 MPa is applied to animal-derived tissue in a medium containing a surfactant. The animal-derived tissue after application corresponds to the decellularized tissue in the present invention. When the applied hydrostatic pressure is lower than 100 MPa, the destruction of the cells in the animal-derived tissue is insufficient, and when it exceeds 1500 MPa, a pressure vessel that can withstand the application is required, which requires a lot of energy and is applied. When the medium used in the above is an aqueous medium, ice is likely to be generated. The applied hydrostatic pressure is preferably 300 to 1300 MPa, more preferably 500 to 1200 MPa, and most preferably 700 to 1100 MPa. According to the present invention, decellularization can be effectively performed even by application with a hydrostatic pressure having a lower value than conventional ones. Therefore, from the viewpoint of performing the application step at a lower pressure, the applied hydrostatic pressure may be 100 to 800 MPa, preferably 200 to 700 MPa, and more preferably 500 to 700 MPa.

  For applying the hydrostatic pressure, a medium containing one or more surfactants is used. Examples of the medium include water, physiological saline, propylene glycol or an aqueous solution thereof, glycerin or an aqueous solution thereof, and an aqueous saccharide solution. Examples of the buffer solution include acetate buffer solution, phosphate buffer solution, citrate buffer solution, borate buffer solution, tartaric acid buffer solution, Tris buffer solution, HEPES buffer solution, MES buffer solution and the like. Examples of the saccharide in the aqueous saccharide solution include erythrose, xylose, arabinose, allose, talose, glucose, mannose, galactose, erythritol, xylitol, mannitol, sorbitol, galactitol, sucrose, lactose, maltose, trehalose, dextran, alginic acid, hyaluronic acid, etc. Can be mentioned. Surfactants are classified into ionic to aqueous anionic surfactants, cationic surfactants, amphoteric surfactants and nonionic surfactants in the aqueous solution. In the present invention, the anionic surfactants and the cationic interfaces are classified. The active agent and the amphoteric surfactant may be collectively referred to as an ionic surfactant.

  Anionic surfactants include alkyl sulfonates (such as sodium dodecyl sulfonate), fatty acid soaps, alkoxy carboxylates, polyoxyethylene alkoxy carboxylates, alkyl benzene sulfonates, alkyl sulfates, polyoxy Examples include ethylene alkyl sulfate, alkyl phosphate, α-sulfo fatty acid ester, N-acyl glutamate, acyl-N-methyl taurate, N-alkyl sarcosine, cholic acid and its derivatives. .

  Examples of the cationic surfactant include cationic surfactants such as alkyldimethylamine, alkyldiethanolamine, alkyltrimethylammonium salt, dialkyldimethylammonium salt, alkylpyridium salt, and alkylbenzyldimethylammonium salt.

  Amphoteric surfactants include alkyldimethylamine oxide, alkylcarboxybetaine, alkylamidopropylbetaine, alkylamidopropylsulfobetaine, alkylimidazolium betaine, 3-[(3-cholamidopropyl) dimethylammonio] -2-hydroxy. Examples include -1-propanesulfonate salt, 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate salt, and the like.

  Nonionic surfactants include glycerin fatty acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene alkyl. Phenyl ether (polyoxyethylene octylphenyl ether, nonylphenol ethoxylate (polyoxyethylene nonylphenyl ether), etc.), polyoxyethylene polyoxypropylene ether, alkyl (poly) glycerin ether, fatty acid alkanolamide, polyoxyethylene fatty acid alkanolamide, Examples thereof include alkyl (poly) glycosides.

  Surfactants include alkyl sulfonate, alkyl sulfate ester salt, polyoxyethylene alkyl sulfate ester salt, α-sulfo fatty acid ester salt, polyoxyethylene alkyl ether, polyoxyethylene from the viewpoint of cell destruction and cell removal. Ethylene alkyl phenyl ethers and alkyl (poly) glycosides are preferred, and alkyl sulfonates, polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, and alkyl (poly) glycosides are more preferred.

  The content of the surfactant in the medium is preferably 0.05 to 5.00% by mass, more preferably 0.07 to 2.00% by mass with respect to the entire medium from the viewpoint of cell destruction and cell removal. . Note that only one surfactant may be used, or two or more surfactants may be used in combination. The content of the surfactant in the medium is specified by a known method such as liquid chromatography.

  The temperature of the high hydrostatic pressure treatment is not particularly limited as long as ice is not generated and the tissue is not damaged by heat. However, the decellularization treatment is performed smoothly and the influence on the tissue is small. 45 ° C is preferable, 4-37 ° C is more preferable, and 15-35 ° C is most preferable. When the time for the high hydrostatic pressure treatment is too short, cell destruction is not sufficiently performed, and when it is long, energy is wasted, so 5 to 60 minutes is preferable, and 7 to 30 minutes is more preferable.

[Washing process]
A biological tissue to which a high hydrostatic pressure is applied may be washed with a washing solution in order to remove broken cells in the tissue. The cleaning liquid may have the same composition as the medium used in the high hydrostatic pressure treatment or a different composition. The washing liquid used in the present invention preferably contains a nucleolytic enzyme (DNase, RNase, etc.), an organic solvent or a chelating agent, but may not contain it. From biological tissues to which high hydrostatic pressure is applied, nucleolytic enzymes can improve the removal efficiency of nucleic acid components, organic solvents can remove lipids, and chelating agents can eliminate calcium ions and magnesium ions in decellularized tissues. By activating, calcification when the particulate decellularized tissue of the present invention is applied to a diseased part can be prevented. As the organic solvent, a water-soluble organic solvent is preferable because it has a high effect of removing lipids, and ethanol, isopropanol, acetone, and dimethyl sulfoxide are preferable. As chelating agents, ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriminepentaacetic acid (DTPA), hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraminehexaacetic acid (TTHA), 1,3-propane Diamine tetraacetic acid (PDTA), 1,3-diamino-2-hydroxypropanetetraacetic acid (DPTA-OH), hydroxyethyliminodiacetic acid (HIDA), dihydroxyethylglycine (DHEG), glycol ether diamine tetraacetic acid (GEDTA), Iminocarboxylic acid-based chelating agents such as dicarboxymethylglutamic acid (CMGA), 3-hydroxy-2,2′-iminodisuccinic acid (HIDA), dicarboxymethylaspartic acid (ASDA) or salts thereof; citric acid, tartaric acid, apple Examples thereof include hydroxycarboxylic acid-based chelating agents such as acids and lactic acid or salts thereof. Examples of salts of these chelating agents include sodium salts and potassium salts.

  In the case of washing the tissue subjected to the high hydrostatic pressure treatment, the tissue is immersed in the washing solution or perfused with the washing solution. When the tissue is immersed in the cleaning liquid, the cleaning liquid may be shaken or stirred as necessary. The washing time may be selected according to the site and size of the biological tissue and the type and content of the surfactant in the medium, and is not particularly limited. For example, the washing time is 12 to 144 hours, preferably 12 to 72 hours, and more preferably. May be 12 to 48 hours. According to the present invention, decellularization can be performed in a shorter time than conventional techniques.

[Decellularized tissue in the present invention]
In a decellularized tissue decellularized with a treatment solution containing a surfactant, the physical strength is usually greatly reduced, whereas in the method for producing a decellularized tissue of the present invention, a surfactant is used for high hydrostatic pressure treatment. Despite the use of a medium containing, the decrease in physical strength is small. Although the cause of this is not known, it is considered that the protein has been altered by the application of high hydrostatic pressure and is less likely to be damaged by the surfactant.

  By the production method of the present invention, decellularized tissue with little damage to the tissue can be produced in a relatively short time, and particularly large organs that have been difficult to obtain by the method using high hydrostatic pressure, such as the heart, liver, kidney, Decellularization of pancreas and the like can be easily performed.

  The decellularized tissue produced by the production method of the present invention can be suitably applied to transplantation into a living body and repair of a diseased site or damaged site. When the decellularized tissue of the present invention is applied to a living body, it may be applied alone, or may be applied together with other components having a regeneration / healing effect on a disease site. Examples of such other components include growth factors, proteoglycans or glycosaminoglycans, cells, β-1,3-glucan, mevalonic acid and the like. In addition, the decellularized tissue of the present invention may be applied to a living body as it is, or may be applied after cells are seeded, cultured and fixed.

  As growth factors, insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), transforming growth factor-α (TGF-α), transforming growth factor (TGF-β), bone morphogenetic protein (BMP), platelet-derived growth factor (PDGF), keratinocyte growth factor (KGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), hematopoietic promoter ( EPO), granule macrophage growth factor (GM-CSF), granule cell growth factor (G-CSF), nerve cell growth factor (NGF), heparin binding (EGF) and the like. Examples of proteoglycan or glycosaminoglycan include chondroitin sulfate, heparan sulfate, keratan sulfate, dermatan sulfate, hyaluronic acid, heparin and the like.

  The decellularized tissue of the present invention can be applied to a substrate of a culture medium for cell culture, for example, stem cell (ES cell, iPS cell, etc.), in addition to application to a living body.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited by these Examples. Unless otherwise specified, “%” and “ppm” in the examples are based on mass.

[Sample preparation]
The kidney was removed from the rat, and the artery and vein connected to the kidney were cannulated and fixed with an instantaneous adhesive. A sample obtained by immersing the kidney in physiological saline, injecting physiological saline from the artery side, and perfusing for 12 hours was used as a sample.

[Medium / Cleaning liquid]
The following solutions were used as media or cleaning solutions in the high hydrostatic pressure treatment step and the cleaning step.
PBS: phosphate buffered saline 0.10% SDS: phosphate buffered physiology containing 0.1% sodium dodecylsulfonate
Saline 0.50% SDS: phosphate buffered physiology containing 0.5% sodium dodecyl sulfonate
Saline 0.50% NPE: 0.5% nonylphenol ethoxylate (EO10)
Phosphate buffered saline 20 ppm DN: phosphate buffered saline containing 20 pppm DNase

[Applying process]
A sample and a medium for high hydrostatic pressure treatment are put into a bag with polyethylene chuck, and a hydrostatic pressure of 500 MPa or 1000 MPa is applied for 15 minutes using a high-pressure treatment apparatus for research and development (product name: Dr. CHEF). Applied.

[Cleaning method]
The sample was immersed in a washing solution, and the washing solution was injected from the artery side and perfused for 24 to 48 hours to obtain a decellularized sample. The numbers in parentheses in Table 1 represent the time of perfusion, and the arrow “→” represents the order of washing. The process described before “→” is the process performed previously.

<Evaluation of decellularity>
The decellularized sample was visually observed, and decellularity was evaluated according to the following criteria. The results are shown in Table 2.
○: No redness is seen, clear to clean white and decellularization is good Δ: Some redness is seen, and the decellularity is slightly poor ×: Clear redness remains, and the decellularity is poor Is

<Evaluation of shape retention>
The decellularized sample was placed in a waist-high petri dish and the height of the sample was measured. The waist-high petri dish was stored in a thermostatic bath at 25 ° C. for 24 hours, and then the sample height was measured again. From the height reduction rate, shape retention was judged according to the following criteria. The results are shown in Table 2. In addition, it shows that there is so little damage of a decellularized tissue that shape retainability is high.
○: Height reduction rate is 0-20% and shape retention is high Δ: Height reduction rate is 20-50% and shape retention is slightly low ×: Height reduction rate is 50% or more And shape retention is low

  As is clear from the results in Table 2, it can be seen that the decellularized tissue obtained by the production method of the present invention has been decellularized in a relatively short time and has little damage to the tissue.

Claims (2)

An application step of applying a hydrostatic pressure of 100 to 1500 MPa in a medium containing a surfactant to the animal-derived tissue;
The content of the surfactant in the medium is 0.05 to 5.00% by mass with respect to the whole medium,
The surfactant includes alkyl sulfonate, alkyl sulfate ester salt, polyoxyethylene alkyl sulfate ester salt, α-sulfo fatty acid ester salt, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether and alkyl (poly) glycoside. One or more selected surfactants ,
A method for producing a decellularized tissue. The method for producing a decellularized tissue according to claim 1, further comprising a step of washing the animal-derived tissue for 12 to 144 hours after the applying step.