WO2017175870A1

WO2017175870A1 - Graft material for reconstructing tissue of liver subjected to hepatectomy, method of manufacturing same, and method of reconstructing liver subjected to hepatectomy

Info

Publication number: WO2017175870A1
Application number: PCT/JP2017/014581
Authority: WO
Inventors: 八木　洋; 雄光北川; 一樹田島; 啓文下田
Original assignee: 学校法人慶應義塾
Priority date: 2016-04-08
Filing date: 2017-04-07
Publication date: 2017-10-12
Also published as: JPWO2017175870A1

Abstract

The present invention provides a graft material for reconstructing the tissue of a liver that has been subjected to a hepatectomy, wherein the graft material includes a decellularized scaffold comprising an extracellular matrix derived from the decellularized liver of a mammal, and a film which covers at least part of the extracellular matrix. Further, the present invention provides a method of manufacturing said graft material, including: a step of freezing a liver of a mammal; a step of thawing the frozen liver; a step of perfusing a cell disrupting medium including a surfactant into the thawed liver to disrupt the cells in the liver; and a step of washing the liver of which the cells have been disrupted. Further, the present invention provides a method of reconstructing a liver, including: a step of forming a liver adhering portion in the graft material in accordance with the shape of the excised section of the liver; and a step of bringing the excised section of the liver into contact with the liver adhering portion and suturing the liver thereto.

Description

Transplant for liver tissue reconstruction that has undergone hepatectomy, its production method, and method for reconstruction of liver that has undergone liver resection

The present invention relates to a transplant for tissue reconstruction of a liver that has undergone liver resection. The present invention also relates to a method for producing a transplant for liver tissue reconstruction that has undergone liver resection. The present invention also relates to a method for reconstructing a liver that has undergone hepatectomy.

Surgical treatment that removes a part of an organ for cancer treatment is important in aiming for radical cure, and more than tens of thousands of people undergo partial resection of various organs every year in Japan alone. However, organ failure after resection is sometimes fatal and is always the most important complication. There is no definitive treatment for organ dysfunction and relies on extracorporeal circulation. However, if organ failure occurs, for example, artificial dialysis or insulin treatment must be performed throughout life, and the quality of life of patients (QOL: (Quality of Life) gets worse. This also causes an increase in the number of cancer patients that cannot be treated.

Even in the liver, which is said to be reproducible, organ failure leads to death, and postoperative liver failure is a major problem, but at present there is no curative treatment other than liver transplantation. In order to perform liver transplantation, donors who provide the liver are necessary, but there are chronic donor shortages worldwide, including Japan, and more than a few percent of patients can receive transplants. It is not considered. Therefore, in order to eliminate such chronic donor shortage, development of a new treatment technique that replaces the conventional treatment is being demanded.

In recent years, various regenerative medical technologies have been developed to solve these problems, and in particular, a technology called tissue engineering, which is a fusion of medicine and engineering, has shown remarkable development. In addition, the development of stem cells using pluripotent stem cells (ES cells, iPS cells, etc.) or the differentiation induction of those cells has been developed in parallel.・ Attempts to build organs are being made all over the world.

As a method for constructing a regenerative organ using a tissue engineering technique, a so-called bottom-up type and top-down type technique have been developed. The former is a method of stacking cells, which are the smallest unit, for example, a method of stacking sheets of cells to form tissues / organs, or a method of stacking spheroid cells like blocks to store tissues / organs. The method of construction is mentioned. As the latter, for example, to use an organ derived from a mammal, leave an extracellular matrix (ECM) skeleton using an agent that crushes cell membranes such as a surfactant, and construct an organ there Examples include a technique of engrafting necessary cells again by seeding and culturing them, and creating an organ in vitro (Patent Document 1, Patent Document 2, Non-Patent Document 1, and Non-Patent Document 2). The former method is currently limited to the construction of thin tissues such as the retina and gastrointestinal mucosa. On the other hand, the latter method has not yet led to the development of an organ having a complete function as a substitute for an organ in a living body, and further research and development are required in the future.

Several decellularized scaffolds prepared from tissues collected from humans and non-human animals have already been commercialized, and are used in treatments that assist and fill tissue defect sites in the body (Non-patent Document 3). ). However, a decellularized skeleton that has been commercialized is applied to a part of an organ (heart valve, pericardium, etc.) or a part of a tissue (soft tissue, tendon, skin, etc.), and the organ itself No product has been developed.

International Publication No. 2007/025233 US Patent Application Publication No. 2005/0249816

As described above, a technique for treating an organ having a serious disease requiring transplantation, particularly a liver, has been developed, but a sufficient technique has not yet been provided. Then, this invention makes it a subject to provide the transplant material for the tissue reconstruction of the liver which received liver resection. Moreover, this invention makes it a subject to provide the method of manufacturing the transplant material for the tissue reconstruction of the liver which received liver resection. Moreover, this invention makes it a subject to provide the reconstruction method of the liver which received liver resection.

In order to solve the above-mentioned problems, the present inventors have conducted research and development by adding studies from various angles. As a result, surprisingly, when the transplant material for liver tissue reconstruction that has undergone hepatectomy according to the present invention is applied to a hepatectomy section, the vascular structure is quickly reconstructed from the hepatectomy section. The liver tissue was reconstructed from the liver excision section. It was also found that the reconstruction of the bile duct structure is also induced. That is, the present invention is as follows.

[1] A transplant for tissue reconstruction of a liver that has undergone liver resection,
A graft material, wherein the graft material includes a decellularized scaffold having an extracellular matrix derived from a decellularized mammalian liver and a coating covering at least a part of the extracellular matrix.
[2] The transplant according to [1], wherein the transplant is subjected to protein crosslinking treatment.
[3] The transplant according to [1] or [2], wherein the transplant is filled with hydrogel.
[4] The transplant according to any one of [1] to [3], wherein the transplant further has a liver adhesion part not having the coating and is applied to a cut section of the liver. .
[5] The transplant according to [[4], wherein the liver adhesion part is formed in conformity with the shape of the liver excision section immediately before application to the liver excision section.
[6] The transplant material according to any one of [1] to [5], wherein the mammal is a non-human mammal.
[7] The transplant material according to any one of [1] to [6], wherein angiogenesis and bile duct neoplasia are induced in the transplant material from a hepatectomy section.

[8] A method for producing an implant according to any one of [1] to [7],
(A) freezing the liver of the mammal;
(B) thawing the frozen liver;
(C) perfusing a cell destruction medium containing a surfactant into the thawed liver to destroy the cells;
(D) washing the liver from which the cells have been destroyed;
Including a method.
[9] (c) Step is
(C-1) perfusing a cell disruption medium containing an ionic surfactant to the thawed liver, and (c-2) a nonionic surfactant and a zwitterionic interface to the thawed liver Perfusing a cell disruption medium comprising an active agent;
The method according to [8], wherein the method comprises:
[10] The method according to [9], wherein the ionic surfactant is selected from the group consisting of sodium dodecyl sulfate (SDS), deoxycholate, cholate, sarkosyl, and combinations thereof.
[11] The method according to [9] or [10], wherein the nonionic surfactant is selected from the group consisting of Triton X-100, DDM, digitonin, Twin 20, Twin 80, and combinations thereof.
[12] The method according to any one of [9] to [11], wherein the zwitterionic surfactant is CHAPS.
[13] (e) treating the graft material with a protein crosslinking agent;
The method according to any one of [8] to [12], further comprising:
[14] The method according to [13], wherein the protein crosslinking agent is selected from aldehyde-based crosslinking agents, carboxyl group-amino group crosslinking agents, and combinations thereof.
[15] The method according to [14], wherein the carboxyl group-amino group cross-linking agent is water-soluble carbodiimide (WSC).
[16] The method according to [14] or [15], wherein the aldehyde-based crosslinking agent is glutaraldehyde.
[17] (f) a step of sterilizing the transplant material;
The method according to [8] to [16], further comprising:
[18] The step (f) is a step of sterilizing with gamma rays,
The method according to [17], comprising:
[19] The method according to any one of [8] to [18], further comprising (g) a step of filling the graft material with hydrogel.
[20] The method according to any one of [8] to [19], further comprising the step of (h) forming a liver adhesion part on the transplant material in accordance with the shape of the excised section of the liver.

[21] A method of reconstructing liver tissue that has undergone hepatectomy,
(I) a step of forming a liver adhesion part in the transplant material according to any one of [1] to [7] in accordance with the shape of the excised section of the liver;
(Ii) a step of bringing the excised section of the liver into contact with the liver adhesion part and attaching the liver;
Including methods.

The transplant material for liver tissue reconstruction that has undergone hepatectomy according to the present invention and the transplant material produced by the manufacturing method thereof can be easily molded into an appropriate size, and a large amount of liver for liver cancer with liver cirrhosis or metastatic liver cancer It is possible to cover and adhere to the size of the liver excision section that always occurs during resection. In the graft material attached to the cross section, the skeleton of the three-dimensional lumen structure left in the inside promotes early migration / engraftment of vascular endothelial cells, bile duct epithelial cells, etc. from the resected liver. It has a unique feature that allows the structure to be reproduced in advance. Through this regenerated vessel, hepatocytes can infiltrate extensively into the transplant made of extracellular matrix with high affinity, so that the structure of the liver itself is complemented early, reducing the risk of liver failure Therefore, early recovery after surgery is expected.

FIG. 1 is a conceptual diagram of the present invention. FIG. 2 shows a porcine decellularized skeleton. (A) It is a figure which shows the pig liver after washing | cleaning with PBS. (B) It is a figure which shows the pig liver currently processed with the SDS containing cell destruction medium. (C) Porcine decellularized skeleton after washing cells destroyed after treatment with cell disruption media. FIG. 3 is a diagram showing a decellularization system for porcine liver and decellularized porcine liver tissue. (A) It is a figure which shows the decellularization system (for pigs) for producing the transplant material containing the decellularization frame | skeleton of this invention. (B) HE-stained image in a tissue section of normal pig liver. (C) Fluorescence microscopic image showing nuclei (DAPI, blue) in tissue sections of normal pig liver. (D) HE-stained image in a tissue section of porcine liver that has been decellularized. (E) It is a fluorescence microscope image which shows the nucleus (DAPI, blue) in the tissue section | slice of the pig liver which performed the decellularization process. FIG. 4 is an enlarged view of the porcine decellularized skeleton with a scanning electron microscope. (A) Porcine decellularized skeleton decellularized using only SDS. (B) Porcine decellularized skeleton decellularized using SDS, Triton X-100 and CHAPS. FIG. 5 is a diagram showing a procedure for attaching a porcine decellularized skeleton to a resected liver stump. FIG. 6 shows the histological analysis results (10 days after transplantation) of the porcine decellularized skeleton after transplantation. (A) HE staining observation image of a tissue section in the vicinity of the skeleton boundary line between porcine decellularized skeleton and pig liver. (B) It is a fluorescence-microscope observation image of the tissue section near the skeleton boundary line of a pig decellularized skeleton and a pig liver. (C) Hepatocytes (albumin (ALB) positive cells, green) engrafted in a porcine decellularized skeleton. (D) It is a figure which shows the bile duct epithelial cell (CK19 positive, green) reconstructed to the porcine decellularization frame | skeleton. (E) It is a figure which shows the blood-vessel structure (CD31 positive, green) reconstructed to the porcine decellularization frame | skeleton. FIG. 7 shows the histological analysis results (28 days after transplantation) of the transplanted porcine liver decellularized skeleton. (A) It is a fluorescence-microscope observation image which shows the CK19 positive cell (green) of the porcine decellularization frame | skeleton and the skeleton boundary line vicinity of a pig liver. The bar indicates 1000 μm. (B, C) It is a fluorescence-microscope observation image which shows the CK19 positive cell (green) which expanded a part in the porcine decellularization frame | skeleton of (A). The bar represents 200 μm. (D) It is a fluorescence-microscope observation image which shows the vascular endothelial cell marker positive cell (CD31 positive cell, green) in the periphery of a porcine decellularization frame | skeleton. Blue (DAPI) indicates the position of the nucleus. (E) HE staining observation image of a tissue section in the vicinity of the skeleton boundary line between porcine decellularized skeleton and pig liver. A dotted line indicates a bounding boundary line. (F) It is a fluorescence-microscope observation image which shows the CK19 positive cell (green) of the post-resected stump of the pig liver as a control group which does not adhere to a decellularized skeleton. FIG. 8 is a view showing an HE staining observation image (28 days after transplantation) in the vicinity of the boundary line between the porcine decellularized skeleton and the porcine liver. Black arrows indicate vasculature including bile ducts and blood vessels. A white arrow indicates a vascular structure including bile ducts and blood vessels extending deep inside the skeleton. FIG. 9 is a diagram showing a rat liver decellularization system and a decellularized rat liver tissue. (A) It is a figure which shows the decellularization system (for rat) for producing the scaffold raw material containing the decellularization frame | skeleton of this invention. (B) It is a figure which shows a part of decellularization system (for rats) for producing the transplant material containing the decellularization frame | skeleton of this invention. (C) It is the figure which injected the 10 micrometer fluorescent bead and showed that the vascular network was maintained. (D) Heparinized blood collected from rats was injected to show that the vascular network was maintained and there was no leakage. (E) It is the figure which showed that the collagen fiber was maintained by the electron microscope analysis. FIG. 10 is a diagram showing a porcine liver decellularized skeleton after the crosslinking treatment. FIG. 11 is a diagram showing the boundary between the porcine liver decellularized skeleton and the pig liver on the 10th day after the pig liver decellularized skeleton after crosslinking treatment is attached to the pig liver. Right) The figure which HE-stained the section | slice of the whole pig liver in which the pig liver decellularization skeleton was attached is shown. (Left) An enlarged view of a part of the porcine liver decellularized skeleton (enlarged part of the left figure). Dotted line: hepatocytes, arrow: bile duct, arrowhead: blood vessel. FIG. 12 shows the rat liver decellularized skeleton after injection of collagen gel. (A) Rat liver decellularized skeleton after injection of type I collagen gel. (B) It is a figure which shows the pig liver 21 days after attaching the rat liver decellularization frame | skeleton obtained by (A). The rat liver decellularized skeletal part attached within the dotted line.

The terminology used herein is for the purpose of describing specific embodiments of the present invention and is not intended to limit the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In addition, the contents described in all documents and patent applications that are referred to before or after this specification are incorporated herein by reference in their entirety.

The term “organ” generally refers to a living organ including cells that make up an organ, a skeleton based on an extracellular matrix, and a vascular system (arteries, veins, capillaries) that supply oxygen and nutrients to each cell. It is covered with a film. Examples of the organ include heart, liver, lung, kidney, pancreas, spleen, brain, uterus, bladder, brain and the like. Since the organ is covered with a coating, the fluid (for example, blood) introduced from the vascular system is basically an outlet if the organ has substantially no damage to the coating and the internal vascular system. It is not excreted from other than the vascular system. In other words, the organ has a continuous structure in which arteries that introduce blood branch off inside the organ to form fine capillary structures, and the capillary structures join again to form veins. Actually, liquid does not leak from other than a specific entrance.

In this specification, the term “film” means a film covering the surface of an organ. In general, the surface of an organ is covered with a single layer of squamous epithelium (mesothelial cells) called serosa. The lower layer of the serosa has connective tissue, and the serosa and connective tissue are also called the peritoneum. As used herein, “capsule” is taken as the broadest meaning including serosa, connective tissue, or peritoneum.

The terms “extracellular matrix” and “ECM” are used interchangeably in the present specification, and a substance rich in collagen and the like existing between cells of a mammalian tissue, and a substance derived therefrom are subjected to any treatment. Also includes other materials. “Extracellular matrix” and “ECM” are also called extracellular matrix, intercellular matrix. The component constituting the extracellular matrix is composed of, for example, collagen, proteoglycan, fibronectin, cadherin, laminin, tenascin, enctin, elastin and the like, and the composition varies depending on the tissue / organ. The extracellular matrix is mainly produced from cells that constitute the connective tissue, but a part thereof is also secreted from cells having a basement membrane such as epithelial cells and endothelial cells. In multicellular organisms, it plays various roles such as filling of extracellular space, skeletal structure to maintain the shape, scaffold for cell adhesion (scaffold) and promotion of cell differentiation induction. Due to the presence of the extracellular matrix, cells are arranged three-dimensionally, contributing to the formation of complex forms of organs and tissues.

In this specification, the term “decellularization” means that a structure (skeleton) composed of an extracellular matrix as a main constituent component is maintained from a part of a living body such as an organ or a tissue, and is adhered thereto. That the removed cells are removed by a desired method. A decellularized living organism-derived organ or tissue becomes a skeleton mainly constituted by an extracellular matrix, but the constituted protein is not limited to an extracellular matrix-related protein. When a decellularized living body-derived organ or tissue is observed with an electron microscope, it is observed as a skeleton having a network structure having voids whose main component is an extracellular matrix. The method of decellularization can be appropriately changed depending on the organ / tissue.

The term “decellularized scaffold” as used herein refers to a three-dimensional structure mainly having an extracellular matrix that remains after a cell is removed from a living organ or tissue or a part thereof by a decellularization treatment. Means the skeleton of the structure. In the present invention, the decellularized skeleton apparently maintains the same form as the organ or tissue before decellularization treatment or a part thereof. In particular, when the decellularized skeleton is derived from a living organ, at least a part of its surface has a coating. The decellularized skeleton contained in the transplant material of the present invention is a blood vessel that is continuous with an artery, a capillary network, and a vein held in an organ or tissue before decellularization skeleton decellularization or a part thereof. The structure skeleton is also substantially maintained. Thereby, when transplanted in the living body, the blood vessel structure is quickly reconstructed, and blood reperfusion is promoted. In addition, when the decellularized skeleton contained in the transplant material of the present invention is derived from the liver, the three-dimensional skeleton substantially formed by the bile duct is maintained. As a result, when transplanted into a living body, the bile duct structure is quickly reconstructed, promotes early migration / engraftment of vascular endothelial cells, bile duct epithelial cells, etc., and the vascular structure is regenerated in advance. It has unique characteristics. Furthermore, the decellularized skeleton contained in the transplant material of the present invention maintains a specific extracellular matrix that promotes the engraftment, proliferation, and differentiation of cells constituting the organ / tissue. Promotes the reconstruction of organs and tissues by the cells that make up

In the present invention, the “transplant” includes a decellularized skeleton obtained by decellularization of an organ or tissue derived from a living body or a part thereof, and optionally additionally, cell survival. Processing for adding, binding, etc., a protein, a drug or the like that promotes adhesion, growth or differentiation may be performed. Further, the transplant material according to the present invention may be a transplant material that has been engrafted (recellularized) by previously seeding and culturing desired cells. The cell type and the number of cells may be appropriately selected depending on the organ / tissue to be transplanted. Moreover, the cell to be used may be any cell as long as it constitutes an organ / tissue to be treated, and may be a commercially available cell or a cell collected from a living body. The method for collecting cells from a living body is not particularly limited as long as a known method is followed. Further, it may be a cell obtained by inducing differentiation from a pluripotent stem cell. In this specification, a pluripotent stem cell is a cell which has a self-replication ability and a pluripotency, and a cell provided with the ability (pluripotent) to form all the cells which comprise a body. Self-replicating ability refers to the ability to make two undifferentiated cells from one cell. The pluripotent stem cells used in the present invention include embryonic stem cells (embryonic stem cells: ES cells), embryonic carcinoma cells (embryonal carcinoma cells: EC cells), trophoblast stem cells (TS cells), shrimp. Blast stem cell (epiblast stem cell: EpiS cell), embryonic germ cell (embryonic germ cell: EG cell), pluripotent germline stem cell (mGS cell), artificial pluripotent stem cell (induced plumped stem cell) iPS cells), Muse cells (see: International Publication WO2011 / 007900) and the like. The method for inducing differentiation into an arbitrary somatic cell is not particularly limited as long as it is a known method. As a method of seeding cells on the transplant material of the present invention, for example, Yagi H. et al. Et al. (Yagi H., et al., Human-scale whol-organ bioengineering for live translation: a regenerative medicine app. Cell Transplant. However, the present invention is not limited to this.

The origin of the organ or tissue or part of the animal species used as the material for the transplant of the present invention is, for example, human, rat, mouse, guinea pig, marmoset, rabbit, dog, cat, sheep, pig, horse, cow, goat Mammals such as monkeys, chimpanzees or immunodeficient animals thereof. The organ or tissue collected or a part thereof may be collected from a living individual or may be collected from a dead body. Even when the animal species to which the transplant of the present invention is applied is human, it is very low in immunogenicity due to the decellularization treatment and hardly causes rejection. An organ or tissue or part thereof may be used.

As for the method of decellularizing an organ or tissue or a part thereof, a known method or a method obtained by partially modifying it can be carried out according to the organ or tissue as a material or the animal species thereof. Methods for decellularization include (1) cell membrane destruction by mechanical stimulation: high pressure, freezing, electroporation, osmotic pressure change, and (2) washing / cell destruction with cell destruction media such as drugs: surfactants, Acid / alkali, enzyme, and alcohol are roughly classified into two types, and these can be used in combination. These methods can destroy cells, remove immunogenic cell fragments, and leave extracellular matrix and other substances that promote engraftment, proliferation, migration or differentiation of cells.

Examples of the method for producing a transplant material for reconstructing liver tissue that has undergone liver resection according to the present invention include the following steps.
(A) freezing the liver of the mammal;
(B) thawing the frozen liver;
(C) perfusing a cell destruction medium containing a surfactant into the thawed liver to destroy the cells;
(D) A step of washing the liver in which cells are destroyed.

Examples of the method of destroying the cell membrane by mechanical stimulation include freezing and thawing methods. By freezing and thawing, the moisture contained in the cells expands and the cell membrane can be destroyed. The freezing temperature is, for example, −10 ° C. or lower, −20 ° C. or lower, −30 ° C. or lower, −40 ° C. or lower, −50 ° C. or lower, −60 ° C. or lower, −70 ° C. or lower, −80 ° C. or lower, or −90 ° C. Hereinafter, it can be carried out at a temperature of −100 ° C. or lower or lower. The freezing temperature can be in the range of −150 ° C. to −10 ° C., −130 ° C. to −15 ° C., −100 ° C. to −20 ° C., for example. A lower temperature is preferable because it can be frozen in a short time and has a high cell membrane destruction effect. The thawing temperature may be, for example, 37 ° C., room temperature, room temperature, or 4 ° C. The thawing temperature may be, for example, 4 ° C. to 50 ° C., 4 ° C. to 45 ° C., 4 ° C. to 40 ° C. It is preferable to set -80 ° C. for freezing and room temperature for thawing as temperatures at which cells can be destroyed while maintaining the extracellular matrix as much as possible.

The surfactant contained in the cell disruption medium refers to an amphiphilic molecule having a hydrophobic group and a hydrophilic group in the molecule. Surfactant destroys the lipid bilayer that forms the cell membrane and nuclear membrane. Examples of the surfactant contained in the cell disruption medium include ionic surfactants, nonionic surfactants, and zwitterionic surfactants. Examples of the ionic surfactant used in the cell disruption medium include sodium dodecyl sulfate (SDS), deoxycholate, cholate, sarkosyl, Triton X-200, or a combination thereof. Nonionic surfactants used for cell disruption media include, for example, Triton X-100, n-dodecyl-β-D-maltoside (DDM), digitonin, twin 20 (Tween 20), twin 80 (Tween 80), or The combination is mentioned. Examples of the zwitterionic surfactant used in the cell disruption medium include 3-[; (3-cholamidopropyl) dimethylammonio];-1-propanesulfonic acid (CHAPS). These surfactants can be appropriately selected according to the organ / tissue to be decellularized. Taking the decellularization of the liver, particularly pig liver, as an example, it becomes possible to obtain a transplant that can reconstruct liver tissue by decellularization using an ionic surfactant, particularly SDS. Furthermore, by using a nonionic surfactant, particularly Triton X-100, it is possible to obtain a transplant that can reconstruct liver tissue. Furthermore, by using a zwitterionic surfactant, particularly CHAPS, it becomes possible to obtain a transplant that can reconstruct liver tissue with better cell engraftment. As the solvent contained in the cell disruption medium, water, physiological saline, buffer solution (for example, PBS), alcohols, acetic acid, tributyl phosphate (TBP), or the like can be used. The cell disruption medium may further contain nuclease, trypsin and / or dispase. Furthermore, a chelating material (EDTA, EGTA) may be included in order to prevent cells from adhering to the extracellular skeleton.

The time for cell destruction by the cell disruption medium may be appropriately adjusted according to the organ or tissue to be decellularized or the animal species.

For example, water, physiological saline, or a buffer solution (for example, PBS) can be used as a cleaning solution for cleaning the cell disruption medium. What is necessary is just to perform washing | cleaning time and the frequency | count of washing | cleaning to such an extent that the above-mentioned cell destruction medium does not remain | survive.

When deorganizing an organ or tissue or a part thereof, the above-described method for perfusing the cell disruption medium can be performed by a known method or a partially modified method. For example, cannulas (catheters) can be inserted into arteries and veins of organs, and the cell disruption medium and / or lavage fluid can be continuously perfused with a perfusion device. Depending on the type of organ or tissue, the cell destruction medium and / or the washing solution may be perfused from a tube other than an artery or vein, for example, in the case of the liver, from the portal vein or hepatic vein. In addition, in the case of having a plurality of arteries, veins, or tubes capable of flowing in / out the cell destruction medium and / or the washing solution, a part of them is ligated so that the inlet / drainage of the cell destruction medium and / or the washing solution is obtained. The exit may be restricted. Thereby, especially when performing decellularization processing of an organ, a cell destruction medium retains efficiently in an organ, and decellularization processing can be performed efficiently. The perfusion rate may be adjusted to such an extent that structures such as blood vessels in the decellularized skeleton are not destroyed, and may be appropriately adjusted according to the organ or tissue or animal species thereof or the degree of progression of decellularization. As a perfusion means, arbitrary pumps, such as a peristaltic pump, a centrifugal pump, a syringe pump, can be used, for example.

The transplant material of the present invention is preferably further subjected to protein crosslinking treatment. Protein cross-linking refers to linking two or more molecules by chemical covalent bonds. In this specification, different amino acid molecules in the same protein, for example, disulfide between cysteine residues in the same protein. A reaction for constructing a bond (sulfhydryl group) and the like is also included. The graft material of the present invention is subjected to protein cross-linking treatment, whereby the physical strength is increased and the skeletal form is more stable. This makes it possible to provide a space (scaffold) for regenerating the liver for a long time without being crushed in the abdominal cavity even after transplantation into a living body, and as a result, good liver regeneration is realized. .

The protein cross-linking agent used in the present invention is known in the art, and includes, for example, an inter-amino group cross-linking agent: for example, DSG (disuccinimidyl glutarate); DSS (disuccinimidyl suberate); BS3 ( Bis (sulfosuccinimidyl) suberate); TSAT (tris- (succinimidyl) aminotriacetate); BS (PEG) 5 (PEGylated bis (sulfosuccinimidyl) suberate); BS (PEG) 9; DSP (dithiobis) (Succinimidyl propionate)); DTSSP (3,3′-dithiobis (sulfosuccinimidyl propionate)); DST (disuccinimidyl tartrate); BSOCOES (bis [2- (succinimidooxycarbonyl) Oxy) ethyl] sulfone); EGS (ethylene glycol) Bis (sulfosuccinimidyl succinate)); sulfo-EGS; DMA (dimethyl adipimidate hydrochloride); DMP (dimethylpimelimidate hydrochloride); DMS (dimethyl suberimidate hydrochloride), sulfhydryl group Intercrosslinking agents: for example, BMOE (bismaleimide ethane); BMB (1,4-bismaleimide butane); BMH (bismaleimide hexane); TMEA (tris (2-maleimidoethyl) amine); BM (PEG) 2 (1 , 8-bismaleimide-diethylene glycol); BM (PEG) 3; DTME (dithiobismaleimide ethane), amino group-sulfhydryl group cross-linking agent: for example, AMAS (N-α-maleimidoaceto-oxysuccinimide ester); BMPS ( N-β-maleimide Pill-oxysuccinimide ester); GMBS (N-γ-maleimidobutyryl-oxysuccinimide ester) and sulfo-GMBS; MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester) and sulfo-MBS; SMCC (succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate) and sulfo-SMCC; EMCS (N-ε-maleimidocaproyl-oxysulfosuccinimide ester) and sulfo-EMCS; SMPB (succinimidyl-4- (p-maleimidophenyl) Butyrate) and sulfo-SMPB; SMPH (succinimidyl-6- (β-maleimidopropionamido) hexanoate); LC-SMCC (succinimidyl-4- (N-maleimidometh) ) -Cyclohexane-1-carboxy (6-amidocaproate)); sulfo-KMUS (N-κ-maleimidoundecanoyl-oxysulfosuccinimide ester); SPDP (succinimidyl-3- (2-pyridyldithio) propionate) LC-SPDP (sulfosuccinimidyl-6- [3 (2-pyridyldithio) propionamido] hexanoate) and sulfo LC-SPDP; SMPT (4-succinimidyloxycarbonyl-α-methyl-α (2 -Pyridyldithio) toluene); SIA (succinimidyl iodoacetate); SBAP (succinimidyl 3- (bromoacetamido) propionate); SIAB (succinimidyl (4-iodoacetyl) aminobenzoate) and sulfo-SIAB, carbo Sil group-amino group cross-linking agent: for example, DCC (N, N′-dicyclohexylcarbodiimide); EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) (also known as water-soluble carbodiimide (WSC)) NHS (N-hydroxysuccinimide) and sulfo NHS, sulfhydryl group-sugar chain cross-linking agent; for example, BMPH (N-β-maleimidopropionic acid hydrazide); EMCH (N-ε-maleimidocaproic acid hydrazide); MPBH (4 -(4-N-maleimidophenyl) butyric acid hydrazide); KMUH (N- (κ-maleimidoundecanoic acid) hydrazide); PDPH (3- (2-pyridyldithio) propionylhydrazide), photoreactive cross-linking agent: for example, ANB -NOS (N-5-azido-nitrobenzo Sulfo-SANPAH (sulfosuccinimidyl 6- (4′-azido-2′-nitrophenylamino) hexanoate); SDA (succinimidyl 4,4′-adipanoate) and sulfo-SDA; LC-SDA (succinimidyl 6- (4,4′-adipanoamido) hexanoate) and sulfo-LC-SDA; SDAD (succinimidyl 2-[(4,4′-adiptanamido) ethyl) -1,3 ′ -Dithiopropionate) and sulfo-SDAD. Moreover, as a protein crosslinking agent, you may use aldehyde type crosslinking agents: For example, formaldehyde, paraformaldehyde, glutaraldehyde, etc .; Polyfunctional epoxides: For example, polyethyleneglycol diglycidyl ether. In one embodiment of the present invention, as the protein crosslinking agent, the above-mentioned protein crosslinking agent may be used alone, or a combination of two or more protein crosslinking agents may be used. In the present specification, the use of a “combination” of two or more types of protein cross-linking agents is used in the meaning including both cases where two or more types of protein cross-linking agents are used simultaneously and separately. For example, two or more types of protein cross-linking agents may be processed using a mixed solution dissolved in the same solvent, and multiple transplants may be prepared using different solutions containing different protein cross-linking agents. It may be processed, or may be processed simultaneously using different solutions containing different protein cross-linking agents. These usage modes are appropriately changed depending on the type of protein crosslinking agent to be used. In another embodiment of the present invention, when two or more kinds of protein cross-linking agents are used separately, the order of the protein cross-linking agents to be used is not particularly limited.

The protein cross-linking agent for treating the graft material of the present invention is preferably selected from aldehyde-based cross-linking agents, carboxyl group-amino group cross-linking agents, and combinations thereof. More preferably, it is selected from glutaraldehyde, EDC (water-soluble carbodiimide (WSC)) and combinations thereof. Most preferred is a combination of glutaraldehyde and EDC (water-soluble carbodiimide (WSC)).

The transplant material of the present invention may be transplanted to a subject as it is after being treated with a protein cross-linking agent, or may be transplanted after a washing treatment. Preferably, the graft material is washed after the protein crosslinking treatment. As a washing solution used for washing the transplant of the present invention, a known solution can be used. For example, phosphate buffered saline (PBS), saline, Tris buffered saline, HEPES buffer Physiological saline, Ringer's solution, 5% aqueous glucose solution, liquid medium for mammalian culture, isotonic agents (eg, glucose, D-sorbitol, D-mannitol, lactose, sodium chloride) can be used.

The transplant material of the present invention is also preferable because the form can be stably maintained when the hydrogel is filled therein. The transplant material of the present invention filled with hydrogel can provide a space (scaffold) for regenerating the liver for a long period of time without being crushed in the abdominal cavity even after transplantation into a living body. Thereby, good liver regeneration is realized. The hydrogel used in the present invention is composed of a material that can be transplanted into a living body without causing toxicity to tissues, cells, and the like constituting the living body. As used herein, “hydrogel” is a substance that can contain a large amount of water, and it is useful for cell survival, such as oxygen, water, water-soluble nutrients, polypeptides such as enzymes and cytokines. It has a material or form capable of easily diffusing and transferring necessary substances and wastes. It usually means something that is biocompatible. As the shape or form of the hydrogel applied to the present invention, an aqueous solution containing suspended or colloidal particles is preferable. If it is a flowable hydrogel, it becomes possible to fill the inside of the transplant material of the present invention. Examples of the hydrogel that can be used in the present invention include water-soluble, water-affinity, or water-absorbing synthesis such as polyacrylamide, polyacrylic acid, polyhydroxyethyl methacrylate, polyvinyl alcohol, polylactic acid, and polyglycolic acid. It is a particle made of a hydrogel obtained by chemically cross-linking polymers, polysaccharides, proteins, nucleic acids and the like. Examples of the polysaccharide include, but are not limited to, glycosaminoglycans such as hyaluronic acid and chondroitin sulfate, starch, glycogen, agarose, pectin, and cellulose. Examples of proteins include collagen and hydrolysates thereof such as gelatin, proteoglycan, fibronectin, vitronectin, laminin, entactin, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, Matrigel (registered trademark) and the like. However, it is not limited to these. Preferably, a hydrogel made of a material that is biocompatible and can be degraded by cells in vivo is suitable for the present invention. If the hydrogel has such properties, good liver regeneration can be realized without inhibiting the effect of cells infiltrating, proliferating and engrafting in the transplant material of the present invention. More preferably, it is a hydrogel that enhances the effect of infiltration, proliferation, and engraftment of cells, such as collagen, gelatin, proteoglycan, fibronectin, vitronectin, laminin, entactin, tenascin, thrombospondin, von Willebrand factor, osteopontin , Fibrinogen, Matrigel (registered trademark) and other protein hydrogels.

The method of filling the graft material of the present invention with hydrogel is not particularly limited. For example, using a syringe, the method of filling from the portal vein and / or hepatic vein of the graft material, the solution containing the hydrogel is perfused. The method etc. are mentioned. When the hydrogel form applied to the present invention is an aqueous solution containing suspended or colloidal particles, the concentration is not limited because it varies depending on the properties of the main component of the hydrogel selected, It is preferable that the concentration is such that it can be filled in the container and does not flow out after filling.

The transplant material of the present invention must be used by being implanted in a living body by transplantation and highly sterilized from the viewpoint of preventing pathogen infection by transplantation. Known methods can be used to sterilize the transplant material of the present invention. For example, sterilization with antibiotics (penicillin, ampicillin, tetracycline, streptomycin, gentamicin, amteforicin B, etc.), autoclave sterilization, UV irradiation sterilization, gamma ray Irradiation sterilization, ozone sterilization, ethylene oxide gas (EOG) sterilization, or a combination thereof may be mentioned. The treatment by these sterilization methods is preferably carried out to such an extent that the structure and properties of the extracellular matrix contained in the transplant material are not impaired. In the present invention, gamma irradiation is particularly preferable because of its high sterilization effect.

The temperature at which the graft material of the present invention is stored may be, for example, 4 ° C. to 30 ° C., 4 ° C. to 28 ° C., 4 ° C. to 26 ° C., room temperature, or room temperature until use. It may be frozen and stored. The freezing temperature is, for example, −10 ° C., −20 ° C., −30 ° C., −40 ° C., −50 ° C., −60 ° C., −70 ° C., −80 ° C., −90 ° C., −100 ° C., or The following temperatures may be used. In addition, the freezing temperature may be in the range of −150 ° C. to −10 ° C., −130 ° C. to −15 ° C., −100 ° C. to −20 ° C., for example. Examples of the solution for storing the transplant of the present invention include water, physiological saline, PBS and the like. Further, the temperature for thawing the frozen transplant material of the present invention may be, for example, 37 ° C., room temperature, room temperature, or 4 ° C. The thawing temperature may be, for example, 4 ° C. to 50 ° C., 4 ° C. to 45 ° C., 4 ° C. to 40 ° C. As the optimum temperature for holding the extracellular matrix as much as possible and maximizing the effect of the transplant material of the present invention, it is preferable to set the freezing at −80 ° C. and the thawing at room temperature.

The transplant material of the present invention can reconstruct an organ or tissue by transplanting into a living body. In particular, when the organ to which the transplant of the present invention is applied is the liver, it can be applied to the excised section of the liver to reconstruct liver tissue, which has conventionally been difficult to regenerate from the excised section of the liver. . In particular, when the transplant material of the present invention contains a liver-derived decellularized skeleton, migration and engraftment of endothelial cells and bile duct epithelial cells are promoted inside the transplant material, and a functional structure with a capillary structure and a bile duct structure is promoted. The vasculature is reconstructed and regeneration of liver tissue is promoted (FIG. 1). This is a transplant material that has not been realized by conventional transplant materials, and was realized for the first time by the present invention.

The liver is recognized as a regenerating organ, but the mechanism of the regeneration is not the extension and enlargement of the liver after excision as it was, but the remaining liver enlarges compensably (compensatory) Overall capacity is secured by “hypertrophy”. In order for this “compensatory hypertrophy” to occur sufficiently, the balance between the amount of hepatic resection and the reserve capacity (health state) of the liver greatly contributes, and if the preoperative evaluation is mistaken, there is a risk of death from liver failure. Therefore, especially when a patient suffering from cirrhosis undergoes hepatectomy, a strict upper limit of resection is determined by the patient's own liver function. This method of determining the upper resection limit is called “intra-curtain classification”. The liver is said to regenerate even if about 70% is excised, but as specified in this classification, depending on the degree of cirrhosis, there is a risk that hepatic failure may occur even if nearly 70% remains. This can be said to be because the liver is fibrotic due to cirrhosis, in other words, in an environment where the internal hepatocytes cannot proliferate in a state of extracellular matrix failure. On the other hand, the transplant material of the present invention provides a pericellular environment / stereostructure composed of a normal extracellular matrix, and can promote regeneration on the excision stump side of the liver which is not originally regenerated. By using the transplant material of the present invention, it is possible to avoid a dangerous state in which compensatory hypertrophy is insufficient, to enable safe hepatectomy, and thus to perform radical treatment for more cancer patients.

A known surgical means may be used as a method of excising an organ or tissue or a part thereof to which the transplant material of the present invention is applied, and examples thereof include a scalpel, an electric knife, a scissors, a scissors, a forceps and the like. There is no particular limitation. A known surgical technique can be used for the method of attaching the transplant of the present invention to a cut section of an organ or tissue or a part thereof, for example, a coating of an organ or tissue remaining after excision and the present invention. And a method of suturing and ligating the coating of the graft (attachment). At this time, the graft of the present invention is formed with an adhesive portion having no coating at least in part according to the shape of the cut section of the organ or tissue to be applied or a part thereof. For example, a scalpel, an electric knife, a scissors, a scissors, a forceps, or the like is used as the adhesive portion. For example, the decellularized skeleton is cut to form the adhesive portion having no coating on the cut surface. For example, when the transplant material includes a decellularized skeleton derived from the liver, the graft material can be cut with a scalpel to form a liver adhesion portion that matches the shape of the excised section of the liver to be applied. The graft material can be transplanted by attaching the liver adhesion portion in contact with the excised section of the liver. As a result, cells that form liver tissue such as vascular endothelial cells, bile duct epithelial cells, hepatic parenchymal cells, hepatic progenitor cells, Kupffer cells, homologous endothelial cells, and stellate cells migrate from the hepatectomized section and live. It induces angiogenesis and bile duct neoplasia in the transplant material, and promotes remodeling of liver tissue.

The transplant of the present invention may be provided in a state in which an adhesive portion is formed in advance, or may be formed at the surgical site according to the cut section of an organ or tissue or a part thereof immediately before attachment. In addition, the transplant material of the present invention can be prepared in various size variations according to the volume to be excised.

When the transplant material of the present invention is used for reconstruction of liver tissue, it can be used for the treatment of primary or metastatic liver cancer, fatty liver, cirrhosis, hepatitis, autoimmune hepatitis and the like. Due to the above-mentioned diseases, the graft material of the present invention is sutured and adhered to the excised surface obtained by performing hepatectomy, so that the vascular structure / bile duct structure of the excised part different from the conventional hypertrophic regeneration mechanism It is possible to promote hepatocyte infiltration by the regenerative mechanism, dramatically increase the post-operative liver capacity at an early stage, and prevent liver function from being compensated and liver failure. This realizes early structure regeneration and cell infiltration in cm units, which are clearly different in scale from currently available fibrin glue and sheet-like structures.

In the following, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way. The experimental protocol using rats and pigs in this example was approved by the Ethics Committee on Animal Experiments at Keio University. “Basic Guidelines for Implementation of Animal Experiments at Research Institutions” (Ministry of Science).

Example 1
<Animals>
Male LWD pigs (All-Agricultural Feed and Livestock Research Center, Ibaraki, Japan) were used for the production of porcine decellularized scaffolds and for transplantation experiments. Pigs were anesthetized with 0.2 mg midazolam (kg Astellas, Tokyo, Japan) and 0.08 mg medetomidine (Nippon Zenyaku Kogyo, Fukushima, Japan) per kg, and then connected to a standard respiratory system. During the procedure, isoflurane was inhaled to maintain anesthesia.

<Collection of liver and pre-treatment (collected from live pig)>
Yagi H. The liver was partially modified by using the method (Human-scale whol-organ bioengineering for live translation: a regenerative medicine approach. Cell Transplant. 2013; 22 (2): 231-242). went. Specifically, immediately before collecting the pig liver, heparin 5000 IU was introduced from the vein, and then the liver was collected by vertical midline incision. The gallbladder was removed from the collected liver. The bile duct, hepatic artery, hepatic vein and lower hepatic inferior vena cava were ligated. The portal vein and upper hepatic inferior vena cava were cannulated. Heparin-containing (5000 U / L) 0.9 w / v% physiological saline was perfused from the portal vein until no blood came out. After injecting physiological saline, it was frozen and stored at -80 ° C.

<Decellularization treatment of liver (SDS + TritonX-100 + CHAPS)>
A frozen liver is thawed at room temperature in a clean bench, and a solution containing 500 U / L heparin sodium (AY Pharma, Tokyo) is drained through the portal vein at 100 ml / min to remove blood. Perfused until clear. The liver was then perfused with deionized water containing 0.5 w / v% sodium dodecyl sulfate (SDS; SERVA Electrophoresis, Germany) for the first 24 hours. Subsequently, the liver was washed with deionized water for 15 minutes, 12 hours, 1 v / v% Triton X-100 (Sigma), 0.05 w / v% ethylene glycol bis (2-aminoethyl ether) -N, N, Washed with a PBS solution containing N ′, N′-tetraacetic acid (EGTA; Tokyo Chemical Industry Co., Ltd.), 0.05 w / v% sodium azide (Sigma), 4 mM CHAPS (Dojindo Laboratories, Kumamoto) (FIG. 3 (A)). Decellularized liver was washed with PBS for 1 hour. PBS containing antibiotics (1 v / v% penicillin / streptomycin, 1 v / v% gentamicin, 1 v / v% amphotericin B) was perfused at 20 mL / min for 30 to 1 hour. Thereafter, the decellularized liver was further sterilized using gamma rays (25 kGy). It preserve | saved at 4 degreeC until use (refer FIG. 2, FIG. 3 (A)). It was confirmed that the porcine liver was decellularized by the above treatment (FIGS. 3C and 3D).

<Decellularization of liver (SDS + TritonX-100)>
The frozen liver was thawed at room temperature and perfused with PBS overnight at 30 ml / min through the portal vein to remove blood. The liver was then perfused with deionized water containing 0.01 w / v% sodium dodecyl sulfate (SDS; Sigma, St. Louis, MO, USA) for the first 24 hours, followed by 0.1 w / v for 24 hours. Perfusion was performed with deionized water containing% SDS, followed by perfusion with deionized water containing 1 w / v% SDS for at least 48 hours. Subsequently, the liver was washed with deionized water for 15 minutes, and washed with deionized water containing 1 v / v% Triton X-100 (Sigma) for 30 minutes. Decellularized liver was washed with PBS for 1 hour. Decellularized livers were washed with PBS containing 0.1 v / v% peracetic acid (Sigma, St. Louis, MO, USA) for 1 hour. The decellularized liver was thoroughly washed with sterile PBS. Until use, livers decellularized in PBS supplemented with antibiotics (1 v / v% penicillin / streptomycin, 1 v / v% gentamicin, 1 v / v% amphotericin B) were stored at 4 ° C.

<Result>
The decellularized liver was observed using a scanning electron microscope. As a result, as compared with the decellularization method mainly using SDS (FIG. 4 (A)), the liver decellularized with the perfusate further containing CHAPS has a good residual state of the internal vasculature. (FIG. 4B). Compared with the decellularized skeleton (Fig. 4 (A)) that has been decellularized without using CHAPS has a hard matrix shape, the decellularized skeleton that has been decellularized with CHAPS has undergone decellularization. It has been clarified that the cellular skeleton has a delicate matrix remaining in detail, and extremely few microparticles that are considered to have cytotoxicity considered to be particles in the SDS washing solution (FIG. 4 ( B)).

Example 2
<Evaluation of cell engraftment on decellularized liver (transplant)>
The porcine liver was decellularized by the same method as in Example 1 liver decellularization (SDS + TritonX-100, SDS + TritonX-100 + CHAPS). Yagi H. Et al. (Yagi H., et al., Human-scale whol-organ bioengineering for liver translation: a regenerative medicaine approach. Cell Transplant. Pig liver parenchymal cells and vascular endothelial cells were seeded and cultured, and the cells were observed to be engrafted.

<Result>
When perfusion is performed in a culture solution at 37 ° C., the decellularized liver treated with CHAPS has higher tissue stability compared to the decellularized liver not used with CHAPS. The engraftment of vascular endothelial cells to the vascular wall after conversion was good.

Example 3
Porcine liver was decellularized by the same method as in Example 1 liver decellularization (SDS + TritonX-100 + CHAPS).

<Transplantation of decellularized liver (transplant)>
The decellularized transplant was molded into a form similar to that of the liver to be excised. A 15-20 kg female LWD SPF pig (Shiroishi Animal Co., Ltd., Saitama, Japan) was laparotomized, and part of the liver (left and middle lobe left) was excised. The liver excision surface and the cut surface of the graft material were brought into contact with each other, and the respective coatings were sutured to close the abdomen (FIG. 5). After the operation, normal breeding was performed.

<Result>
The decellularized skeleton in the vicinity of the adhesion boundary region 10 days after transplantation and 28 days after transplantation includes hepatocytes (albumin positive cells, FIG. 6 (C)) and bile duct epithelial cells (CK19 positive cells, FIG. 6 (D), It was confirmed that FIGS. 7A to 7C were engrafted. It was also confirmed that vascular endothelial cells were engrafted to form a luminal structure in the peripheral part of the transplanted decellularized skeleton (FIG. 7D). Further, also from FIGS. 7E and 8, by transplanting the hepatocyte skeleton to the cut surface of the liver, vascular endothelial cells are engrafted from the anchoring region to the peripheral part using the decellularized skeleton as a scaffold, and the luminal structure It was clarified that the capillary network accompanied with the blood flow was reconstructed and blood flow was restored. It was also revealed that the hepatic lobule structure was reconstructed while gradually reconstructing the bile duct structure from the vicinity of the attachment area. On the other hand, in the control group that did not wear the graft material of the present invention, a phenomenon in which the vascular structure and the vascular structure accompanied with the bile duct structure were regenerated was not observed (FIG. 7F).

In addition, the decellularized liver decellularized with CHAPS shows a clearly lower infection rate compared with the case without gamma sterilization when combined with sterilization with gamma rays, and is good in the liver after adhesion. Cell engraftment was observed.

In the regeneration of the liver using the scaffold, the phenomenon of regenerating the vascular structure accompanied with the vascular structure and the bile duct structure as seen in the present invention is not known. The transplant material of the present invention makes it possible for the first time to regenerate liver tissue having a vascular structure with blood vessels and bile ducts.

Example 4
The purchased pig liver was frozen at −80 ° C. and thawed at room temperature. The portal vein and upper hepatic inferior vena cava were cannulated. Thereafter, the gallbladder was removed, and the bile duct, artery and lower inferior vena cava were ligated. From the portal vein side, heparinized physiological saline was perfused until no blood came out. The following decellularization treatment, sterilization treatment and transplantation to the hepatectomy surface were performed in the same procedure as in Examples 1 and 2. As a result, the same results as in Examples 1 and 2 were obtained.

Example 5
<Animals>
Female Lewis rats (200-250 g, Sankyo Institute) and male Lewis rats (450-500 g, Sankyo Institute) were used.

<Isolation and decellularization of liver>
Rats were maintained under anesthesia with inhalation of 1.5-3.0% isoflurane (Mylan). After abdominal incision, heparin (450 units) was injected into the space in the heart. A 20G cannula was inserted into the portal vein and 10-15 mL of PBS containing heparin (50 units) was injected. IHVC (lower hepatic vena cava) was ligated and the entire liver was excised. SHVC (superhepatic vena cava) was excised without ligation. The resulting liver was frozen at −80 ° C. for at least 24 hours. Soto-Gutierrez et al. (Soto-Gutierrez A, et al., A whol-organ regenerative medical for live replacement. Tissue Eng. Decellularization treatment was performed by a modified method. Specifically, after thawing at 37 ° C., PBS containing trypsin (0.2 v / v%), Triton X-100 (0.1 v / v%), EGTA (0.05 w / v%) from SHVC. The cells were perfused for 24 hours at 3 mL / hour for decellularization treatment (FIG. 9). The resulting decellularized liver was sterilized by gamma radiation and the small caudate, left and right lobule were removed.

<Transplantation of decellularized liver (transplant) and results>
The obtained decellularized skeleton was molded according to the size of the hepatectomy surface. The molded decellularized skeleton was brought into contact with the liver excision surface of another rat from which a part of the liver (left lobe and left side of the middle lobe) was excised, and each coat was sutured and attached, and the abdomen was closed. After the operation, normal breeding was performed. As a result, as in Examples 1 to 3, a phenomenon was observed in which the vascular structure accompanied by the vascular structure and the bile duct structure was regenerated from the liver cut surface.

Example 6
Porcine liver was decellularized by the same method as in Example 1 liver decellularization (SDS + TritonX-100 + CHAPS). The obtained porcine liver decellularized skeleton was subjected to a treatment for cross-linking proteins. The crosslinking treatment was performed using the following reagents and procedures.

<Reagent>
25% glutaraldehyde (GA) solution (Nacalai Tesque, Cat; 17003-05)
・ PBS
Water-soluble carbodiimide (WSC) (Dojindo, Cat; 346-03632)

<Protein crosslinking method>
1) Circulate PBS through the porcine liver decellularized scaffold at a rate of 100 ml / min.
2) The decellularized liver is immersed for 5 minutes in a GA solution diluted to 5% with PBS while being circulated.
3) After changing to a PBS solution, wash by immersing for 1 minute × 3 times.
4) Sterilize with gamma rays.
5) After filter sterilization of WSC adjusted to 120 mM with PBS, 100 ml is injected from the portal vein of the decellularized liver with external fixation.

The cross-linked porcine liver decellularized skeleton was obtained by the method described above (FIG. 10). By performing the cross-linking treatment, it became possible for the porcine liver decellularized skeleton to maintain a more stable form as compared to the skeleton not subjected to the cross-linking treatment.

The porcine liver decellularized skeleton subjected to crosslinking treatment was attached to a part of the pig liver according to the procedure of Example 3. A tissue section of porcine liver 10 days after the attachment was prepared and observed by HE staining, and as a result, hepatic parenchymal cell group (dotted line on the right in FIG. 11), bile duct structure (right on FIG. 11, arrow), blood vessel structure (see FIG. 11 right, arrow) was observed, and it became clear that internal bile duct cell infiltration was enhanced. These structures were also observed at the site farthest from the attachment surface in the transplanted porcine liver decellularized skeleton (left side of FIG. 11).

The cross-linking treatment increased the physical strength of the porcine liver decellularized skeleton, and it became possible to maintain a stable morphology even after the transplantation. As a result, a space (scaffold) for regenerating the liver for a long period of time without being crushed in the abdominal cavity even after transplantation can be provided, and good liver regeneration can be realized.

Example 7
As in Example 5, the rat liver was decellularized. The obtained rat liver decellularized skeleton was injected with collagen gel according to the following reagents and procedures.

<Reagent>
Type I collagen Rat tail high concentration (9.61 mg / ml) (Corning, Cat; 354249)
・ 10 × PBS
・ 1N NaOH
・ MilliQ water

<Filling method of collagen gel>
• Make a 6 mg / ml collagen solution.
• Mix 1 ml of 10 × PBS, 2.62 ml of milliQ, and 144 μL of 1N NaOH.
-Add 6.24 ml of collagen solution to the above solution and mix.
Inject 5 ml from the hepatic vein of the decellularized rat liver and 5 ml from the portal vein.

The rat liver decellularized skeleton filled with collagen gel was obtained by the above-described method (FIG. 12A). The obtained rat liver decellularized skeleton was attached to a part of the rat liver according to the procedure of Example 5. Examination of the rat liver 21 days after implantation confirmed that the transplanted decellularized skeleton did not collapse and remained engrafted (FIG. 12 (B), dotted line portion).

Claims

A transplant for tissue reconstruction of the liver that has undergone hepatectomy,
A graft material, wherein the graft material includes a decellularized scaffold having an extracellular matrix derived from a decellularized mammalian liver and a coating covering at least a part of the extracellular matrix.
The graft material according to claim 1, wherein the graft material has been subjected to protein crosslinking treatment.
The transplant material according to claim 1 or 2, wherein the transplant material is filled with hydrogel.
The transplant material according to any one of claims 1 to 3, wherein the transplant material further has a liver adhesion part not having the coating, and is applied to a resected section of the liver.
The transplant according to claim 4, wherein the liver adhesion part is formed in accordance with the shape of the liver excision section immediately before application to the liver excision section.
The transplant material according to any one of claims 1 to 5, wherein the mammal is a non-human mammal.
The transplant material according to any one of claims 1 to 6, which induces angiogenesis and bile duct neoplasia in the transplant material from a hepatectomy section.
A method for producing an implant according to any one of claims 1 to 7,
(A) freezing the liver of a mammal;
(B) thawing the frozen liver;
(C) perfusing a cell disruption medium containing a surfactant into the thawed liver to destroy the cells;
(D) washing the liver from which the cells have been destroyed;
Including a method.
(C) The process is
(C-1) perfusing a cell disruption medium containing an ionic surfactant to the thawed liver, and (c-2) a nonionic surfactant and a zwitterionic interface to the thawed liver Perfusing a cell disruption medium comprising an active agent;
The method according to claim 8, wherein the method comprises:
10. The method of claim 9, wherein the ionic surfactant is selected from the group consisting of sodium dodecyl sulfate (SDS), deoxycholate, cholate, sarkosyl, Triton X-200 and combinations thereof.
The method according to claim 9 or 10, wherein the nonionic surfactant is selected from the group consisting of Triton X-100, DDM, digitonin, Twin 20, Twin 80, and combinations thereof.
The method according to any one of claims 9 to 11, wherein the zwitterionic surfactant is CHAPS.
(E) treating the graft with a protein cross-linking agent;
The method according to any one of claims 8 to 12, further comprising:
The method according to claim 13, wherein the protein crosslinking agent is selected from an aldehyde-based crosslinking agent, a carboxyl group-amino group crosslinking agent, and a combination thereof.
The method according to claim 14, wherein the carboxyl group-amino group cross-linking agent is water-soluble carbodiimide (WSC).
The method according to claim 14 or 15, wherein the aldehyde-based crosslinking agent is glutaraldehyde.
(F) sterilizing the graft material;
The method according to any one of claims 8 to 16, further comprising:
(F) the step of sterilizing with gamma rays;
The method of claim 17, comprising:
(G) filling the graft material with hydrogel;
The method according to any one of claims 8 to 18, further comprising:
(H) a step of forming a liver adhesion part in the transplant material in accordance with the shape of the excised section of the liver;
The method according to any one of claims 8 to 19, further comprising:
A method of reconstructing liver tissue that has undergone hepatectomy,
(I) a step of forming a liver adhesion part in the transplant material according to any one of claims 1 to 7 in accordance with the shape of the excised section of the liver;
(Ii) a step of bringing the excised section of the liver into contact with the liver adhesion part and attaching the liver;
Including methods.