Method for evaluating transplantation material

Abstract

To provide a method for preparing a transplantation model animal which makes it possible to realize a highly reliable and highly reproducible transplantation experimental system, in order to design a transplantation material which ensures performance and quality that are more suitable to clinical medical care.SOLUTION: A method for evaluating a transplantation material which is similar to that in autoplastic transplantation comprises: a step of administering, to a recipient newborn, a crude extract from a labeled marker expression donor to obtain a recipient which is immune tolerant to the donor; and a step of transplanting, to the recipient which is immune tolerant, the transplantation material which includes an artificial graft material that tissue or cells of the labeled marker expression donor cover, adhere to, infiltrate, or include. Thus, the method allows preparation of a transplantation model animal which is similar to that in autoplastic transplantation and which makes it possible to realize a highly reliable and highly reproducible transplantation experimental system.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for evaluating a graft material that can be used as a highly reliable and reproducible autologous transplantation experimental system and is prepared with high homology to clinical medicine.

Background Art

[0002] Regenerative medicine is a technique for repairing or compensating for the function and / or structure of biological tissues when the function and / or structure of biological tissues are damaged or missing in animals such as humans, livestock, and pets. In addition, regenerative medicine is also useful when growth is inhibited by some disease or trauma during the development or growth period of an animal, and the function and / or structure that should be obtained cannot be obtained.

[0003] As one of the most effective means in regenerative medicine, a method of transplanting a biological-derived medical material containing cells or tissues into the damaged part is used. In Japan, such medical materials are defined as "products for regenerative medicine, etc." by the three laws related to regenerative medicine (Regenerative Medicine Promotion Act, Regenerative Medicine Safety Assurance Act, and Amended Pharmaceutical Affairs Act).

[0004] From an ethical and immunological perspective, it is desirable to use "autoplastic transplantation" in which a product for regenerative medicine, etc. used for transplantation is manufactured by processing a material derived from the individual receiving the transplantation.

[0005] In addition, artificial materials are used to complement elements that are insufficient with only bio-derived medical materials. Such artificial materials need to be those that do not cause unwanted reactions in the body, including immune reactions. Conventionally, not only bio-derived materials including products such as regenerative medicine, but also various medical materials called biocompatible materials and bioaffinity materials have been used (for example, Patent Documents 1 to 4). However, it is difficult to verify whether those materials are optimal in terms of material, physical properties, and configuration before transplantation. In particular, it has been difficult to verify the performance of products such as regenerative medicine that contain living cells and tissues in advance.

[0006] In order to ensure the quality of products such as regenerative medicine, a clinical system that can perform animal experiments using mammals in advance and follow the course of immune reactions after transplantation is necessary. For such animal experiments, an animal experiment model that performs "autologous transplantation" in which cells and tissues collected from an individual scheduled for transplantation are returned to that individual is suitable.

[0007] However, animal experimental systems using autologous transplantation have hardly been used as research means so far. In particular, an autologous transplantation experiment model for small animals has hardly existed. The reason is that autologous transplantation is difficult in small animals such as mice and rats. Even if the amount of cells or tissues collected for transplantation is less than 1 g, it may be unbearable for small animals in terms of the collection procedure, or it may be a fatal amount depending on the tissue collected. In small animals, generally, after sacrificing the individual and bleeding it, tissues and cells are collected. Therefore, the individual is lost due to the collection procedure, and autologous transplantation cannot be performed.

[0008] In medium-sized and large-sized animals, autologous transplantation is possible, but it is difficult to prepare a sufficient number of experimental cases because breeding is costly. In addition, due to the spread of the current animal welfare spirit, there is a trend of strengthening the regulation of animal experiments themselves.

[0009] Therefore, many conventional studies are constructed based on experimental systems centered on allografts and heterografts / xenografts in small animals. Since allografts and heterografts are not autografts, in order to avoid immune reactions, it is necessary to use immunosuppressive agents or severely immunodeficient animals (nude mice, NOD / scid mice, NOG mice, etc.), or a combination of these. Therefore, it is necessary to use individuals that are highly costly to breed and maintain, and the problem is that the costs and labor increase. Furthermore, in such experimental systems, due to the high risk of infectious diseases due to the use of immunosuppressive agents or immunodeficiency, it was necessary to use a highly specialized facility at the aseptic level. There was also the problem that immunosuppressive agents had to be used for a long time and were more costly.

[0010] Furthermore, it was also a major problem that the immunodeficient experimental system was an experiment under circumstances that were greatly deviated from the clinical practice in medicine and veterinary medicine. Especially in humans, patients in an immunodeficient state and patients under long-term use of immunosuppressive agents are rarely the subjects of transplantation treatment. When a treatment method established in an immunodeficient experimental system is introduced into patients with normal immune function and livestock, etc., there is a risk of unexpected situations in terms of immune reactions. Therefore, in the present invention, an animal experimental system is provided that enables the design and development of a transplantation material with more appropriate performance and quality while avoiding these various problems. In particular, the present invention provides an animal experimental system capable of evaluating the engraftment of the target tissue, the reproducibility of the structure / function, etc. More specifically, the present invention provides an animal experimental system capable of evaluating not only the minimization of rejection reactions but also the presence / absence / appropriateness of reactions that support / support the transplanted tissue (regulation of the expression of blood flow induction / angiogenesis / neurotrophic factors, etc.).

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

[0012] There is a demand for constructing a reliable and reproducible system for verifying whether biomaterial-derived materials including products for regenerative medicine, biocompatible materials, and bioaffinity materials, which are materials used for transplantation, have an optimal composition and configuration for treatment. For this purpose, transplantation experiments using animals are indispensable, and the design of transplantation materials should be based on them.

[0013] Considering costs and the like, it is suitable to use small animals in animal experiments. On the other hand, in small animals, since the body size is small, the individual sacrifices during cell collection, so autologous transplantation is difficult. On the other hand, when allogeneic transplantation is performed, an immune reaction (such as immune rejection) occurs after transplantation, so it is carried out in an immunodeficient experimental system and an experimental system using immunosuppressive agents. However, in such experimental systems, there is a large deviation from the actual situation of treatment in clinical practice, and they lack reliability and reproducibility. In addition, immunodeficient experimental systems also have problems such as high costs for experimental materials and the need for highly specialized facilities.

[0014] Therefore, an object of the present invention is to provide a method for producing a transplantation model animal capable of realizing a highly reliable and reproducible transplantation experimental system. [Means for Solving the Problems]

[0015] As medical materials for transplantation (Doner Graft), in addition to autografts, allografts or heterografts / xenografts are commonly used. All of them are Raw Grafts (unprocessed transplantation materials) that transplant the collected tissues "soon" without any special processing. "Soon" includes cases where transplantation is performed immediately after collection, as well as cases where there is a certain period required for refrigeration / freeze preservation, component separation and purification, etc., such as in component transfusion, blood products, and autologous blood transfusion. These Raw Grafts are premised on being transplanted for the purpose of directly using the traits / conditions of the cells and tissues at the time of collection. Since they are used as the tissue without processing and maintaining the traits / conditions, it has been considered that there is basically no need to evaluate their performance / quality.

[0016] On the other hand, the medical material "Auto-plast" (a coined term in the present invention) used for "Auto-plastic transplantation" as expressed in the present invention, which is produced as a medical material for transplantation with various processes such as differentiation and transformation according to the cells and tissues of the transplantation target, resulting in a trait / condition completely different from that at the time of collection, is a material that does not fall within the concept of conventional medical materials. Therefore, there has never been a system for appropriately evaluating its performance and the like.

[0017] It is clear that in the future, the number of medical materials produced as products in regenerative medicine and the like will increase. According to the Ministry of Health, Labour and Welfare's "Ensuring the Quality and Safety of Cell-Processed Pharmaceuticals, etc. (Pharmaceutical and Food Bureau Director General, No. 3, September 7, 2012)", animal experiments are recommended as "tests to verify efficacy or performance" for quality evaluation in animal experiments and the like.

[0018] To summarize the above, usually, autograft refers to Auto-graft. The transplantation material (graft) is transplanted into a tissue equivalent to the original tissue "soon" after collection, without going through processes such as processing, in the state in which it was collected, and is used while maintaining its original properties / traits. Transfusion is performed into blood vessels as blood, liver transplantation is performed on the liver, and skin transplantation is performed on the skin. The autotransplantation referred to in the present invention includes "Auto-plast" (a coined term in the present invention) which expands the concept of transplantation. It involves performing various processes on self-derived cells and tissues, and in some cases, using the manufactured product as a transplantation material in a state different from the original trait. Representative examples include products such as those defined in the three methods of regeneration in regenerative medicine. It proposes a method that enables the design method and quality evaluation of medical materials using biological-derived materials more widely.

[0019] The conventional concept of transplantation is "graft (including allografts)" ⊇ "Auto (autologous)-graft". In principle, the graft is used (transplanted) in the state as it is taken. The state where the taken transplantation material is processed is not included in the concept of "graft". Furthermore, there is still no clear indication of the concept of "plastic graft" created by adding some processing to biological-derived materials. "Auto-plast", the name of the transplantation material created by processing self-derived biological-derived materials as a material, is a coined term according to the present invention. Its concept also includes Auto-plast as a "composite material" combined with artificial materials, etc. At present, there has been no proposal for a specific verification method for using them in regenerative medicine.

[0020] In addition, not limited to tubular structures and membranous structures, solid organs are also included in Auto-plast. Or many composite medical materials are included, such as blood-related dosage forms (non-solid organs) that do not need to have a form as an organ, and endocrine system cells (atypical organs).

[0021] Furthermore, as a method for creating "Auto-plast", 1. It is assumed that in vitro (manufactured outside the body) is often used, but in part, 2. It is also assumed that ex vitro / in vivo (manufactured in the body) is possible. Regardless of the manufacturing method used, quality evaluation as a transplantation material is possible in the present invention.

[0022] In order to solve the above problems, the present inventors used an inbred mouse expressing EGFP as a donor, and administered a crude protein extracted from the donor's tissue to neonatal recipients of wild species several times subcutaneously to induce immune tolerance. Thereafter, an artificial graft material was transplanted into the donor. As a result, the donor's tissues and cells adhered to the transplanted artificial graft material. When a graft material containing the graft material prepared in the donor's body was transplanted into an immune-tolerant recipient, an immune response against donor-derived cells / tissues could be avoided. In addition, by fluorescence observation, engraftment of donor-derived cells and tissues together with the graft material could be easily observed in the recipient after transplantation. The present invention was completed based on these findings.

[0023] That is, the present invention provides a method for obtaining a recipient immune tolerant to a donor by administering a crude extract of a labeled marker-expressing donor to neonatal recipients of the donor, and a step of transplanting a graft material containing an artificial graft material coated, adhered to, infiltrated with, or containing the tissue or cells of the labeled marker-expressing donor into the immune-tolerant recipient and has a method for evaluating a graft material similar to autotransplantation, wherein the donor and the recipient are inbred animals.

[0024] The present invention also provides a method for evaluating a graft material, wherein in the above evaluation method, the graft material is a tubular structure, a membranous structure, or a solid artificial organ.

[0025] The present invention also provides a method for evaluating a graft material, which is recovered after transplanting a graft material into the living body of a donor and leaving it for 2 to 30 days in the above production method.

[0026] The present invention also provides a method for evaluating a graft material, which is a crude protein solution extracted from donor cells or tissues in the above production method.

[0027] The present invention also provides a method for evaluating a graft material, wherein the labeled marker is a fluorescent protein.

[0028] The present invention also provides a method for producing a transplanted model animal, comprising the steps of administering a crude extract of a donor expressing a labeling marker to a neonate recipient to obtain a recipient immunotolerant to the donor, and transplanting a graft material containing an artificial graft material coated, adhered or infiltrated with tissues or cells of the donor expressing the labeling marker into the immunotolerant recipient.

[0029] The present invention also provides the above-described transplanted model animal, which is a mouse.

Advantages of the Invention

[0030] By using the present invention, it is possible to provide a method for evaluating a graft material that can realize a highly reliable and reproducible transplantation experimental system as an alternative to autotransplantation.

Brief Description of the Drawings

[0031]

Figure 1

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Mode for Carrying Out the Invention

[0032] The present invention provides a method for evaluating a graft material and a graft model animal similar to autotransplantation for use in the method for evaluating a graft material, thereby enabling the design of a graft material and the verification of its performance and quality.

[0033] (Method for Evaluating Graft Material) The graft model animal of the present invention used in the method for evaluating a graft material is a graft model animal in which a graft material is transplanted and immune tolerance is induced at the time of transplantation. The graft model animal of the present invention can be suitably used for an experimental system for observing the reaction and course of a living body when a graft material is transplanted.

[0034] The animal used in the method for evaluating a graft material of the present invention is not particularly limited, but can be a non-human animal, for example, rodents such as mice, rats and guinea pigs, primates such as monkeys and marmosets, rabbits, ferrets, dogs, cats, pigs, sheep, goats, cows and horses, mammals, birds, nematodes such as nematodes, flatworms such as planarians, insects such as Drosophila, fish such as zebrafish, and amphibians such as newts and frogs, and reptiles such as lizards. The animal is preferably a rodent such as a mouse, a rat or a guinea pig, and more preferably a mouse.

[0035] The animals used in the present invention can be inbred animals. As used herein, the term "inbred animal" refers to a population of animals or plants that are genetically uniform, with little or no difference in genetic properties (traits) between generations and individuals. Inbred animals can be created by repeating brother-sister mating (full-sib mating) for about 20 generations or more to fix the genotype and eliminate individuals with different traits. The genetic trait homology at the level of monozygotic twins is ensured among individuals of inbred animals. Although not particularly limited, for example, Wister rats, DBA / 2 mice, C57BL / 6 mice, BALB / c mice, etc. can be used as inbred animals. When using inbred animals, animals of the same strain can be treated as the same individual.

[0036] The method for evaluating the transplantation material of the present invention includes a step of obtaining an immune-tolerant recipient by administering a crude extract of a donor expressing a labeling marker (hereinafter also simply referred to as a donor) to a neonatal recipient of the recipient (immune tolerance step), and a step of transplanting a transplantation material containing an artificial transplantation material coated, adhered, infiltrated or included with the tissue or cells of the donor expressing the labeling marker to the immune-tolerant recipient (transplantation step). As used herein, the term that a tissue or cell "coats" has its ordinary meaning in the art and includes that the tissue or cell covers part or all of the transplantation material. Also, as used herein, the term that a tissue or cell "adheres" has its ordinary meaning in the art and includes, for example, that the tissue or cell adheres to part or all of the transplantation material, for example, the tissue or cell sticks and does not separate. As used herein, the term that a tissue or cell "infiltrates" has its ordinary meaning in the art and includes, for example, that the tissue or cell enters and spreads inside part or all of the transplantation material, for example, adheres inside. As used herein, the term that a tissue or cell "includes" has its ordinary meaning in the art and includes, for example, covering all of the transplantation material.

[0037] (Donor and Recipient) In this specification, a "donor" is an animal that provides tissues or cells contained in the graft material. Also, in this specification, a "recipient" is an animal that receives the transplantation of the graft material.

[0038] (Recipient immune tolerance process) In the immune tolerance process, by administering a crude extract of a donor expressing a labeled marker to a neonatal recipient, immune tolerance to donor tissues is induced in the recipient to obtain an immune tolerant recipient. In this specification, "immune tolerance" refers to a state in which the immunological rejection reaction (immune response) against a specific antigen is suppressed.

[0039] The crude extract of the donor is not particularly limited, but can be a crude extract solution of a pulverized product derived from tissues or cells collected from the donor, for example, it can be a crude protein solution extracted from tissues or cells. The tissues or cells collected from the donor may include the tissues or cells planned to be transplanted. The crude extract can be prepared as a crude extract solution, for example, by crushing the tissues or cells collected from the donor with a homogenizer and then performing centrifugation or filtration, etc., and collecting the supernatant containing soluble proteins. The crude protein solution may be further concentrated, etc. Also, after adjustment and cryopreservation, it may be thawed and then used.

[0040] The crude extract to be administered is not particularly limited, but can contain a protein amount of 1 / 30 to 1 / 15 of the body weight of the neonatal recipient.

[0041] The step of administering the donor's crude extract to the recipient's newborn is performed prior to the transplantation step. The methods of administering the donor's crude extract to the recipient's newborn include subcutaneous administration, topical administration, transdermal administration, intradermal administration, transmucosal administration, oral administration, intranasal administration, intratracheal administration, sublingual administration, transnasal administration, buccal administration, rectal administration, vaginal administration, intravenous administration, intra-arterial administration, intramuscular administration, intracardiac administration, intraosseous administration, intraperitoneal administration, intraorbital administration, intravitreal administration, subconjunctival administration, suprachoroidal administration, subretinal administration, intra-articular administration, peri-articular administration, topical skin administration, and inhalation administration, etc., and preferably subcutaneous administration or intraperitoneal administration.

[0042] The donor's crude extract can be administered to the recipient's newborn once to several times. For example, the donor's crude extract may be administered to the recipient's newborn a total of 3 to 5 times at intervals of 1 to 2 days. The first administration to the recipient's newborn can be within 3 days after birth, preferably within 24 hours after birth. Also, all administrations to the recipient's newborn can be performed within 10 days after birth, preferably within 7 days after birth. Further, the donor's crude extract is administered to the recipient in an amount capable of inducing immune tolerance in the recipient. For example, if it is a crude extract containing a protein amount of 1 / 20 of the weight of the recipient newborn, within 24 hours after birth, the donor's crude extract with a weight of about 5% of the newborn's weight is administered for the first time, and within 7 days after birth, a total of 4 times every 2 days, the donor-derived crude extract with a weight of about 5% of the newborn's weight is subcutaneously administered to the abdomen or the like, whereby a recipient tolerant to the donor can be obtained.

[0043] (Transplantation step) In the transplantation step, a transplantation material containing an artificial transplantation material coated, adhered to, infiltrated with, or containing the donor's tissue or cells is transplanted into the recipient.

[0044] A graft material containing a grafting material that includes tissues or cells of a labeling marker-expressing donor includes a material derived from the donor's tissues or cells together with the grafting material. For example, the graft material can be a grafting material that includes the donor's tissues or cells. In the graft material, the donor's tissues or cells may cover the grafting material, or may adhere to, infiltrate, or be included in the grafting material.

[0045] The grafting material is not particularly limited and can be, for example, a material exhibiting bioabsorbability and a biocompatible material. Materials that can be used for the grafting material include, for example, PLLA: poly-L-lactic acid, PEG: polyethylene glycol, PGA: polyglycolic acid, a copolymer of PGA and PLA, polyhydroxybutyric acid, polycaprolactone, polyethylene succinate, polydioxanone, polybutylene succinate, etc. Also, materials based on soluble cellulose such as oxidized cellulose, proteins, peptides, polyamino acids, polysaccharides, polyesters, polyamides, their derivatives, crosslinked products and copolymers, metallic magnesium, calcium carbonate, calcium phosphate, hyaluronic acid and hydroxyapatite, acrylic resins, fluororesins, polyolefins, silicones, polystyrene, polyesters, polyurethanes, polycarbonates, polyimides, their derivatives, crosslinked products and copolymers, and titanium, silica, zirconia, etc. are included. Proteins, peptides, and polyamino acids include, for example, collagen, gelatin, α-polylysine, ε-polylysine, polyglutamic acid, polyaspartic acid, fibrin, and fibroin. Polysaccharides include, for example, chitin and chitosan. Polyesters include, for example, polylactic acid, polycaprolactam, polydioxanone, polyglycolic acid, and polyhydroxybutyric acid. The material of the grafting material may be one or more of the materials described above, or may be a composite of two or more materials.

[0046] In addition, the transplantation material may be coated with a coating material. Examples of the coating material include materials based on fibrin, materials based on collagen, materials based on hyaluronic acid, materials based on glycoprotein, and materials based on soluble cellulose such as oxidized cellulose.

[0047] The transplantation material is not particularly limited, and can be, for example, a tubular structure, a membranous structure, a structure of any other shape, and an artificial organ. The tubular structure can be used as a transplantation material for regenerative medicine for, for example, blood vessels, respiratory organs, digestive organs, and urinary tract organs. The membranous structure can be used as a transplantation material for regenerative medicine for, for example, dura mater, peritoneum, fascia, periosteum, and synovial membrane.

[0048] An artificial transplantation material coated, adhered to, or infiltrated with donor tissue or cells can be provided in any manner. For example, a transplantation material obtained by seeding and culturing donor cells on the transplantation material can be used.

[0049] In addition, the transplantation material can be one recovered after transplanting the transplantation material into the donor's body in advance and leaving it in place for 2 to 30 days. The transplantation material may be one obtained by performing transplantation and recovery to the donor a plurality of times. By transplanting the transplantation material into the donor and leaving it in place for a certain period of time, donor tissue or cells can be sufficiently adhered to or infiltrated into the transplantation material.

[0050] An artificial transplantation material coated, adhered to, or infiltrated with donor tissue or cells may be coated, adhered to, or infiltrated with donor tissue or cells throughout the entire transplantation material, or a part of the transplantation material may be coated, adhered to, or infiltrated with donor tissue or cells.

[0051] The tissue or cells of the donor expressing the labeled marker contained in the transplantation material may be the donor's tissue or cells themselves or those processed therefrom. The tissue or cells collected from the donor can be separated, cultured, differentiated, selected, proliferated, cryopreserved as needed for use as a transplantation material, and thawed later. Also, they may be tissue or cells that have been made into a cell line (cell straining) by subculture.

[0052] Cells and various bioactive substances (such as cytokines and cell growth factors) may be added to the transplantation material by co-culture, spraying, coating, infiltration, etc. Bioactive substances include, for example, vascular endothelial growth factor, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor, brain-derived neurotrophic factor, growth differentiation factor, erythropoietin (EPO), transforming growth factor, and bone morphogenetic protein.

[0053] The transplantation material is not particularly limited, and can be used, for example, as blood vessels, respiratory organs, digestive organs, urinary organs, dura mater, peritoneum, fascia, synovium, periosteum, and serosa.

[0054] The transplantation material is not particularly limited, and can be transplanted, for example, into internal organs such as the recipient's brain, liver, kidney, and heart, subcutaneous tissues, blood vessels, etc. The method of transplanting the transplantation material into the recipient is not particularly limited, and a conventionally known method can be used.

[0055] The tissue or cells of the donor contained in the transplantation material are not particularly limited, and can be collected from the donor's adult. When the donor is an inbred strain, not only can the individual itself from which a crude extract for administration to the recipient's neonate has been prepared be used as the donor, but another individual of the same strain can also be used as the donor, and a transplantation material derived from another individual of the same strain can be transplanted into the recipient.

[0056] Donor tissues or cells may be selected from the group consisting of, for example, fibroblasts, chondroblasts, osteoblasts, angioblasts, myoblasts, epithelial cells, smooth muscle cells, endothelial cells, vascular endothelial cells, fibrocytes, hepatocytes, chondrocytes, epithelial cells, urothelial cells, smooth muscle cells, keratinocytes, β cells, small intestinal epithelial cells, epidermal keratinocytes, bone marrow mesenchymal cells, cardiomyocytes, intervertebral disc cells, gastrointestinal mucosal epithelial cells, ureteral epithelial cells, skeletal joint synovial cells, periosteal cells, perichondrial cells, skeletal muscle cells, smooth muscle cells, cardiomyocytes, pericardial cells, dural cells, meningeal cells, pericytes, glial cells, neurons, amniotic cells, placental membrane cells, serous cells, epitheliocytes, periventricular region cells, oral mucosal epithelial cells, neurons and dendritic cells, genetically modified cells thereof, cells produced by genetic manipulation techniques such as genome editing, or transformation techniques such as by mRNA (messenger RNA), and cells obtained by culturing and / or differentiating these cells.

[0057] In addition, the donor tissue or cells may be selected from the group consisting of, for example, somatic tissue stem cells, somatic tissue progenitor cells, germ stem cells, umbilical cord blood stem cells and their differentiated cells, adipose stem cells, neural stem cells, neural progenitor cells, dental pulp stem cells, mesenchymal stem cells, blood stem cells, hematopoietic stem cells, oral mucosal stem cells, periodontal ligament stem cells, liver stem cells, bone marrow mesenchymal stem cells, undifferentiated stem cells, undifferentiated progenitor cells, pre-differentiated stem cells, pre-differentiated progenitor cells, keratinocyte progenitor cells, organ-specific stem cells, progenitor cells of organ-specific stem cells, genetically modified cells thereof, and genome-edited cells. Note that somatic tissue stem cells include, as human somatic stem cells, those defined in the Ministry of Health, Labour and Welfare Pharmaceutical and Food Safety Bureau Director's Notice No. 0907 of September 7, 2012, "Ensuring the Quality and Safety of Processed Pharmaceuticals and Other Products Using Human Allogeneic Somatic Stem Cells" (Yakushokuhatsu No. 0907). "Human somatic stem cells" refers to cells collected from a human or cells generated by the division of such cells, which have pluripotency and maintain self-renewal ability or are presumed to be similar thereto, and among the cells derived therefrom, the following: tissue stem cells (for example, hematopoietic stem cells, neural stem cells, mesenchymal stem cells (including bone marrow stromal stem cells and adipose tissue-derived stem cells), corneal epithelial stem cells, skin stem cells, hair follicle stem cells, intestinal stem cells, liver stem cells, and skeletal muscle stem cells) and cell populations rich in these (for example, total bone marrow cells including hematopoietic stem cells). Human somatic stem cells include vascular progenitor cells, umbilical cord blood, and bone marrow stromal cells. In addition, human somatic stem cells include cells obtained by culturing and / or differentiating these cells in vitro.

[0058] In addition, the donor tissue or cells may be "induced pluripotent stem cells" such as iPS cells and "induced pluripotent stem cell-like cells" produced by genetic manipulation techniques such as gene recombination and genome editing, and transformation techniques such as mRNA (messenger RNA), as well as cells obtained by culturing and / or differentiating these.

[0059] Donor tissues or cells can be, for example, adipose stem cells collected from subcutaneous fat, neural stem cells collected from the brain, bone marrow-derived stem cells, dental pulp stem cells collected from teeth, mesenchymal stem cells collected from submucosal oral tissues, immune cells contained in the thymus, bone marrow, lymph nodes, etc.

[0060] (Method for evaluating a graft using a donor expressing a labeling marker) The method for evaluating a graft using a donor expressing a labeling marker of the present invention includes a step of obtaining an immunotolerant recipient by administering a crude extract of the donor expressing the labeling marker to neonatal recipients, and a step of transplanting a graft containing an artificial graft material coated, adhered to, or infiltrated with tissues or cells of the donor expressing the labeling marker into the immunotolerant recipient.

[0061] As used herein, a "labeling marker" refers to something that enables the discrimination and observation of cells expressing it and cells not expressing it. The labeling marker may be any marker that can identify donor-derived cells after transplantation into the recipient. The labeling marker can be, for example, a fluorescent protein. Fluorescent proteins include, for example, green fluorescent protein (GFP) derived from Aequorea victoria; variants of GFP such as EGFP; mutants of GFP that emit different colors of fluorescence (for example, blue fluorescent protein (BFP), yellow fluorescent protein (YFP), and cyan fluorescent protein (CFP)); dsRed fluorescent protein (dsRed2FP); red fluorescent protein (eqFP611) isolated from Entacmaea quadricolor; cyan fluorescent protein (amFP486 or AmCyan1) isolated from Anemonia majano; fluorescent protein (Azami Green) isolated from the family Galaxeidae; fluorescent protein (ZSGREEN) isolated from the genus Zoanthus; and any other fluorescent protein, etc.

[0062] If the labeling marker is a fluorescent protein such as EGFP, it can be constantly and stably expressed in vivo, and donor-derived cells can be easily observed by fluorescence in the recipient's body after transplantation. In addition, if it is a fluorescent protein such as EGFP, since it is fluorescence by excitation light irradiation, there is no need to stain the tissue for observation, etc., and the observation tissue can be easily observed while alive.

[0063] A labeling marker-expressing donor is an animal in which a labeling marker gene has been introduced and the labeling marker is constantly and stably expressed (or capable of expression) in some or all cells in the body. A labeling marker-expressing donor can be produced by introducing a labeling marker gene by a conventionally known method.

[0064] In the method of the present invention, the recipient is not particularly limited, but can be an animal into which at least the labeling marker introduced into the donor has not been introduced, for example, a wild type (wild type mouse: WM) that has not been genetically engineered. Furthermore, inbred animals may be used to improve experimental accuracy.

[0065] Since the recipient does not have the labeling marker introduced, when transplanted by a normal transplantation method, there is a high possibility that an immunological rejection reaction will be induced by the labeling marker contained in the donor-derived cells or other donor-derived antigens. However, in the method of the present invention, by administering the crude extract of the labeling marker-expressing donor to the neonate of the recipient in advance, immunological tolerance can be induced against the donor-derived antigen containing the labeling marker, so that the immune reaction at the time of transplantation can be suppressed only against the donor-derived antigen. On the other hand, for antigens not derived from the donor, the induction of a normal immune reaction is maintained.

[0066] By using a labeling marker-expressing donor, donor-derived cells and tissues can be easily observed in the recipient after transplantation. In addition, it is possible to easily identify whether cells, tissues, etc. that have occurred in the recipient after transplantation are derived from the recipient or the donor. Therefore, for example, when a tumor or the like occurs in the recipient after transplantation, it is possible to easily distinguish whether it has originated from donor-derived cells or from the recipient.

[0067] (Transplantation model animal) The present invention also includes a step of administering a crude extract of a labeling marker-expressing donor to a neonate of a recipient to obtain a recipient that is immunotolerant to the donor, a step of transplanting a transplantation material containing an artificial transplantation material coated, adhered to, infiltrated with, or incorporated with tissues or cells of a labeling marker-expressing donor into the immunotolerant recipient, and provides a transplantation model animal similar to autotransplantation, produced by a method for producing a mouse having the above steps.

[0068] The transplantation model animal of the present invention can be produced by a method including the immunotolerance step and the transplantation step described in the evaluation method of the transplantation material using the above-described labeling marker-expressing donor. That is, the transplantation model animal of the present invention is obtained by administering a crude extract of a labeling marker-expressing donor to a neonate of a recipient to obtain an immunotolerant recipient, and then transplanting a transplantation material containing an artificial transplantation material coated, adhered to, infiltrated with, or incorporated with tissues or cells of a labeling marker-expressing donor into the immunotolerant recipient. Details of the immunotolerance step and the transplantation step are as described in the above evaluation method of the transplantation material.

[0069] The transplantation model animal produced as described above has the transplanted transplantation material labeled with a labeling marker. Therefore, the transplantation model animal of the present invention can easily confirm not only how the recipient transplantation model animal behaves, but also how the transplanted transplantation material behaves in the body of the recipient transplantation model animal.

[0070] In the transplantation model animal of the present invention, since the transplantation material is transplanted after inducing immune tolerance to the donor in the recipient in advance, the recipient transplantation model animal has a suppressed immune reaction to the transplantation material. Therefore, for example, it can be used in an experimental system for observing the reaction and course of a living body when a transplantation material is transplanted. On the other hand, the immune reaction to any antigen not derived from the donor is maintained, 1. Regarding the risk of interruption of the experiment due to infection during the course, 2. Regarding the risk of increased cancer risk, etc., experimental results close to clinical transplantation that have never been obtained can be expected.

[0071] By using inbred animals, the present invention can provide a so-called "quasi-autologous transplantation animal experimental model". By using inbred animals, the present invention can easily induce immune tolerance even in animals with normal immune functions, and can establish an animal model that can avoid the immune reaction to a specific individual. Therefore, by using the present invention, there is no need to use the conventional immunodeficient animal experimental system, and transplantation experiments can be carried out in a state close to the clinic using animals with normal immune functions. In addition, by using the present invention, it can be used in an experimental system for observing the reaction and course of a living body when a transplantation material is autotransplanted using small animals such as mice for which autotransplantation has been inherently difficult. Therefore, it is possible to substitute for autotransplantation experiments that could only be carried out in medium and large animals, and while following the trend of animal welfare, it is also possible to comply with the recommendations of the Ministry of Health, Labour and Welfare regarding the development of regenerative medicine products based on the three laws of regenerative medicine.

Example

[0072] Figure 1 shows an outline of the tests conducted in this example. In addition, the explanations of the abbreviations used in the following examples are shown below. Explanation of terms: GM: Green mouse (EGFP constitutively expressing genetically recombinant mouse), ICR mouse: A prolific and most popular inbred experimental mouse of the albino strain, P1 and P1A: These are experimental facilities that comply with the specific measures for preventing the spread of genetically modified organisms (measures for preventing spread) in accordance with the international treaty "Cartagena Protocol". Among them, P1 and P1A have the least restrictive conditions. P1 is mainly for handling cells, and P1A is mainly for handling animals. 1. P1A (Animal Experiment Facility) Collection of GM-derived tissues and preparation of crude proteins Process of collecting donor-derived antigen-presenting proteins to induce immune tolerance in recipients 2. P1A (Animal Experiment Facility) Immunotolerance treatment of ICR mice (wild type) Process of administering antigen proteins to neonatal recipients to induce immunotolerance 3. P1A (Animal Experiment Facility) Process of collecting cells or tissues to be used as transplantation candidates from donors 4. P1 (Cell Experiment Facility) Process of processing the collected donor-derived cells as needed or preparing them as transplantation materials 5. P1A (Animal Experiment Facility) Process of transplanting the materials collected from donors into immunotolerant recipients 6. P1A (Animal Experiment Facility) Process of observing the post-transplantation state at the appropriate time, evaluating the performance of the transplantation materials, and considering the design of the transplantation materials as needed.

[0073] (Donor) As the donor, a genetically modified inbred mouse (Green Mouse: C57BL / 6-(CAG-EGFP) C14-Y01-FM131Osb (RBRC00267: RIKEN BioResource Center)) that expresses enhanced green fluorescence protein (EGFP: green fluorescent protein derived from Aequorea victoria) throughout the body by genetic recombination was used (hereinafter also referred to as "Green Mouse (GM)"). An inbred mouse refers to a mouse that has repeated inbreeding and has homology of expressed traits comparable to that of monozygotic twins between generations and / or individuals in terms of genetic traits and their expression.

[0074] Figure 2 is a photograph showing the fluorescence of the green mouse as the donor. By irradiating excitation light (ultraviolet light in the case of GM), the green fluorescence due to EGFP in the cells can be confirmed. In neonates, fluorescence could be confirmed in the whole body epidermis (left side of Figure 2), and in adults, in the excised brain (center of Figure 2) and the exposed epidermis excluding body hair (right side of Figure 2).

[0075] (Preparation of Crude Extract from Donor) Tissues were collected from the green mouse serving as the donor, disrupted with a homogenizer (tissue disruption device), and then centrifuged or filtered to recover a crude extract containing soluble proteins, and crude proteins were prepared (Step 1 in Figure 1). It can be confirmed that there is fluorescence in the test tube containing the crude extract in the upper part of Figure 3. In addition, in order to efficiently induce immune tolerance, a crude extract containing transplantation candidate tissues can be prepared, or the crude extract can be further purified and used.

[0076] (Immune Tolerance Step) As the recipient ICR mice (wild-type inbred strain: Wild type), commercially available mice as experimental organisms were used. Immune tolerance treatment was performed on the ICR mice (Step 2 in Figure 1). Approximately 5% BW of the donor's crude extract was administered subcutaneously into the abdominal area of neonatal ICR mice within 24 hours after birth. Then, within 7 days after birth, the donor-derived crude extract was subcutaneously administered to the abdomen 4 times every 2 days. The subcutaneously administered crude extract contained a protein amount of 1 / 20 of the recipient's body weight. After that, they were normally bred for 4 weeks or more.

[0077] Figure 3 is a diagram showing the recipient mice subjected to immune tolerance treatment. The donor-derived crude extract in the test tube showing green fluorescence in the upper part of Figure 3 was subcutaneously administered to the midline of the abdomen of neonatal ICR mice (left in the lower part of Figure 3). Then, when excitation light was irradiated to the abdomen, the skin could be seen through and green fluorescence could be confirmed (right in the lower part of Figure 3).

[0078] (Preparation of Composite Material for Transplantation) As a graft material for transplantation, a tubular structure with a silicon tube as the core was fabricated. This tubular structure can be used as an artificial blood vessel. Also, when deployed, this tubular structure can be utilized as a membrane-like structure. As a prototype, in this example, a tube (product name: Safield Silicone Catheter: outer diameter 2.7 mm) made of biocompatible silicon or the like (hereinafter referred to as the silicon tube) was used. A sheet of collagen, which is a white self-assembling material (product name: Integra: sheet type 45×30 mm) (hereinafter referred to as the collagen sheet) was wound around this silicon tube in two or more layers. Next, in order to prevent unwinding or loosening, a blue-violet bioabsorbable PGA thread (PGA: polyglycolic acid) was further wound around and fixed (upper left figure in Fig. 4). At this point, the wound tubular collagen sheet is not watertight and thus easily leaks water.

[0079] Fig. 4 is a photograph showing the state of transplanting the material into a donor green mouse. The upper left of Fig. 4 shows the tubular structure that is the transplant material transplanted into the donor. The lower left of Fig. 4 shows the state of transplanting the transplant material into the green mouse. The upper right of Fig. 4 shows the state one week after transplanting the transplant material into the green mouse. It was observed that new blood vessels were induced one week after transplantation. The lower right of Fig. 4 shows the state of fluorescence observation of the transplant material including the transplant material recovered by excision from the green mouse.

[0080] (Transplantation process) The process of transplanting the above tubular structure (transplant material) into a donor green mouse is shown in Fig. 4 (lower left figure in Fig. 4). An approximately 1 cm skin incision was made on the back of an adult green mouse serving as the donor, and then the subcutaneous tissue was peeled in a tunnel shape for a length of approximately 3 cm to create an insertion cavity. A sheath was inserted into the insertion cavity, and through this, the tubular structure was inserted subcutaneously. With the entire inserted tubular structure being able to be buried smoothly under the skin, the sheath was withdrawn, and the insertion hole was sutured to close the wound.

[0081] One week after subcutaneous implantation, the skin of the implanted green mouse was incised and flipped to the extent that the entire tubular structure could be seen, and the entire tubular structure was confirmed (upper right in Fig. 4). The wrapped collagen sheet was moistened with interstitial fluid and covered with cells derived from the green mouse, becoming translucent. It is connected to the surrounding bed tissue by membranous connective tissue. Based on this connective tissue, abundant cross - bridges of new blood vessels are observed in the collagen tube, and it is confirmed that fine blood vessels are developing all around.

[0082] One week after transplantation, the tubular structure was removed and collected as a transplantation material (lower right in Fig. 4; corresponding to steps 3 and 4 in Fig. 1). It was incised at the part of the connective tissue connected to the bed tissue, and the collagen tube was removed without being damaged.

[0083] When the extracted tubular structure was irradiated with excitation light, fluorescence was emitted from the transplantation material, indicating that cells derived from the green mouse were evenly seeded in the collagen tube. Also, at this stage, it was separately confirmed that water - tightness without water leakage had already been achieved. Furthermore, by culturing the extracted collagen tube, the cells seeded on the collagen sheet were viable cells that could also be cultured.

[0084] (Transplantation process) For wild - type ICR mice without a labeled marker that had been induced with immune tolerance in green mice, the same operation as above (preparation of the composite material for transplantation) was performed, and a transplantation material containing the tubular structure recovered from the green mouse was subcutaneously transplanted. Specifically, an approximately 1 - cm skin incision was made on the back of the ICR mouse, and the subcutaneous tissue was peeled in a tunnel shape for a length of 3 cm to create an insertion cavity. A sleeve was inserted into the insertion cavity, and through this, the tubular structure was inserted subcutaneously (Fig. 5). With the entire inserted tubular structure being able to be buried subcutaneously without difficulty, the sleeve was withdrawn, and the insertion hole was sutured to close the wound.

[0085] After subcutaneous implantation, the skin was incised and flipped one month later so that the entire appearance of the tubular structure could be observed, and the whole was confirmed (left side of Fig. 6). The wound collagen sheet was moistened with interstitial fluid or the like, and was covered with tissue cells derived from ICR mice and the like from above the cell layer derived from green mice, and the transparency to visible light was reduced. It is firmly connected by the surrounding bed tissue and connective tissue. Based on this connective tissue, abundant cross-linking of new blood vessels was observed in the tubular structure, and it was confirmed that the new blood vessels derived from green mice further developed and the blood vessels reached all around.

[0086] Furthermore, since fluorescence peculiar to green mice was confirmed by irradiation with excitation light (right side of Fig. 6), it was confirmed that cells derived from green mice engrafted without undergoing immune rejection even in the subcutaneous tissue where a particularly strong immune reaction is likely to be induced in the body of immunotolerant wild-type ICR mice. In addition, viable cells that could be cultured were confirmed from the tissue collected from the tubular structure transplanted into ICR mice, and coexistence of cells emitting green fluorescence and cells without fluorescence was confirmed.

[0087] Fig. 5 is a diagram showing a state in which a transplantation material containing a tubular structure was transplanted into a recipient mouse.

[0088] Fig. 6 is a diagram showing the transplantation material one month after transplantation into a recipient. The left side of Fig. 6 shows the tubular structure (transplantation material) one month after transplantation, and the right side of Fig. 6 shows a state in which the tubular structure (transplantation material) one month after transplantation was observed by fluorescence. One month after transplantation, by fluorescence observation, the distribution of donor-derived cells and tissues attached to the tubular structure and new cells and tissues generated therefrom was observed.

Industrial Applicability

[0089] The present invention can be used in a highly reliable and reproducible transplantation experimental system. In addition, compared with conventional animal experiments for the same purpose (such as experimental systems of immunodeficient animals), experiments can be conducted under conditions closer to actual clinical medicine. Based on this, in regenerative medicine, it is most important to ensure better performance and quality as a medical material for transplantation, including products for regenerative medicine. Furthermore, in the use of experimental materials (experimental animals such as mice and their breeding materials) and research facilities (clean rooms and facilities for preventing the spread of genetically modified animals), inexpensive and widely used ones can be selected, so it is also possible to significantly reduce the necessary expenses related to the implementation of experiments. In addition, since it is not necessary to use medium- and large-sized animals, which are indispensable in conventional autologous transplantation experimental systems, it does not conflict with social trends such as animal welfare, and it is easy to increase the number of experimental cases in terms of cost and labor. Because the Life Span is short, it is possible to respond to different biological conditions for each lifetime generation, and thus it is possible to design and evaluate the performance of various medical materials for transplantation with different conditions.

Claims

1. subcutaneously administering a crude protein solution extracted from cells or tissues of a donor expressing a labeled marker to a newborn of a recipient to obtain a recipient that is immunotolerant to the donor; Transplanting a transplant material comprising an artificial transplant material coated, adhered, infiltrated or encapsulated with tissue or cells of the labeled marker-expressing donor into the immunotolerant recipient; producing an immunotolerant recipient (excluding a human) produced by the method, wherein the donor and the recipient are inbred animals; and Observing the behavior of the labeled marker in the tolerized recipient; thereby identifying whether cells and tissues arising in the immunotolerant recipient after transplantation are derived from cells and tissues derived from the immunotolerant recipient or from the donor.

2. subcutaneously administering a crude protein solution extracted from cells or tissues of a donor expressing a labeled marker to a newborn of a recipient to obtain a recipient that is immunotolerant to the donor; Transplanting a transplant material comprising an artificial transplant material coated, adhered, infiltrated or encapsulated with tissue or cells of the labeled marker-expressing donor into the immunotolerant recipient; producing an immunotolerant recipient (excluding a human) produced by the method, wherein the donor and the recipient are inbred animals; and Observing the behavior of the labeled marker in the immune tolerant recipient and the response and progress of the transplant model animal; thereby ascertaining the immune-tolerant status of a recipient following transplantation of said transplant material.

3. 3. The evaluation method according to claim 1, wherein the transplant material is a tubular structure, a membrane structure, or an artificial organ.

4. 3. The evaluation method according to claim 1, wherein the transplant material is transplanted into the body of the donor, left in the body for 2 to 30 days, and then recovered.

5. The evaluation method according to claim 1 or 2, wherein the labeling marker is a fluorescent protein.

6. 3. The evaluation method according to claim 1, wherein the transplant material is a collagen sheet seeded with donor-derived cells.

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