Evaluation methods for transplant materials

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 approximate autograft experimental system and is prepared with high homology to clinical medicine.

Background Art

[0002] Regenerative medicine is a technology for repairing or compensating for the function and / or structure of a living tissue when the function and / or structure of the living tissue is 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 a damaged part is used. Such medical materials are defined as "products for regenerative medicine, etc." in Japan by the three laws related to regenerative medicine (the Act on Promotion of Regenerative Medicine, the Act on Ensuring the Safety of Regenerative Medicine, and the 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] Furthermore, artificial materials are used to complement elements that are insufficient with bio-derived medical materials alone. Such artificial materials must not cause undesirable reactions in the body, including immune responses. Conventionally, various medical materials have been used, including not only bio-derived materials such as regenerative medicine products, but also biocompatible materials and biocompatible materials (for example, Patent Documents 1-4). However, it is difficult to verify whether these materials are optimal in terms of material, physical properties, and composition before transplantation. In particular, it has been difficult to verify the performance of regenerative medicine products that include living cells and tissues in advance.

[0006] To ensure the quality of regenerative medicine products, a clinically relevant system is needed that allows for prior animal testing using mammals and monitoring of the course of immune responses after transplantation. For such animal testing, an animal model that uses "autologous transplantation," in which cells and tissues collected from the organism to be transplanted are returned to that organism, is suitable.

[0007] However, animal experimental systems using autologous transplantation have been rarely used as a research tool until now. In particular, autologous transplantation experimental models for small animals did not even exist. The reason for this is that autologous transplantation is difficult in small animals, including mice and rats. Even if the amount of cells or tissue collected for transplantation is less than 1 gram, it may be too much for small animals to withstand the collection procedure, or it may be a lethal amount depending on the tissue collected. In small animals, tissues and cells are generally collected after the individual has died and blood has been removed. Therefore, the individual is lost during the collection procedure, making it impossible to perform autologous transplantation.

[0008] While autotransplantation is possible with medium and large animals, the cost of raising them makes it difficult to conduct a sufficient number of experiments. Furthermore, with the growing awareness of animal welfare, there is a trend towards stricter regulations on animal experimentation itself.

[0009] Therefore, much of the conventional research has been built around experimental systems centered on allograft and heterograft / xenograft in small animals. Since allograft and xenograft are not autotransplants, the use of immunosuppressants or severely immunodeficient animals (nude mice, NOD / scid mice, NOG mice, etc.), or a combination of both, is necessary to avoid immune responses. This necessitates the use of individuals that are expensive to breed and maintain, resulting in high costs and labor. Furthermore, these experimental systems require the use of highly specialized facilities, such as sterile rooms, due to the increased risk of infection from immunosuppressant use or immunodeficiency. There is also the problem of the need for long-term use of immunosuppressants, which adds to the cost.

[0010] Furthermore, a significant problem with immunodeficiency experimental systems is that experiments are conducted under conditions that are far removed from clinical practice in medicine and veterinary medicine. In particular, in humans, patients with immunodeficiency and those on long-term immunosuppressant use are rarely candidates for transplantation therapy. When treatment methods established in immunodeficiency experimental systems are introduced into patients with normal immune function or livestock, there is a risk of unforeseen complications in the immune response. Therefore, the present invention provides an animal experiment system that avoids these problems while enabling the design and development of transplant materials with more appropriate performance and quality. In particular, the present invention provides an animal experiment system that can evaluate the engraftment and reproducibility of structure / function of target tissue. More specifically, the present invention provides an animal experiment system that can not only minimize rejection but also evaluate the presence and suitability of responses that support / assist the transplanted tissue (e.g., blood flow induction, angiogenesis, regulation of trophic factor expression). [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] Japanese Patent Publication No. 2015-89433 [Patent Document 2] Japanese Patent Publication No. 2007-186831 [Patent Document 3] Japanese Patent Publication No. 2013-162796 [Patent Document 4] Special Publication No. 2008-523957 [Overview of the Initiative] [Problems that the invention aims to solve]

[0012] There is a need to establish a reliable and reproducible system for verifying whether bio-derived materials, including regenerative medicine products, as well as biocompatible and biocompatible materials, used as materials for transplantation, possess the optimal composition and structure for treatment. To this end, transplantation experiments using animals are indispensable, and the design of transplantation materials should be based on these experiments.

[0013] For animal experiments, small animals are suitable considering costs and other factors. However, because small animals are small, autologous transplantation is difficult because they often die during cell collection. On the other hand, allogeneic transplantation can lead to immune reactions (such as immune rejection) after transplantation, so it is performed in immunodeficiency systems or systems using immunosuppressants. However, these experimental systems deviate significantly from the reality of clinical treatment and lack reliability and reproducibility. Furthermore, immunodeficiency systems are problematic because they are costly in terms of experimental materials and require highly specialized equipment.

[0014] Therefore, the present invention aims to provide a method for producing transplant model animals that can realize highly reliable and reproducible transplant experimental systems. [Means for solving the problem]

[0015] The medical materials commonly used for transplantation (donor graft) include autograft, allograft, and heterograft / xenograft. All of these are raw grafts (unprocessed transplant material) where the harvested tissue is transplanted "soon" without any special processing. "Soon" includes transplantation immediately after harvesting, as well as cases where there is a certain period of time required for refrigeration / freezing storage and separation / purification of components, such as in component transfusions, blood products, and autologous blood transfusions. These raw grafts are intended to be transplanted with the premise that the characteristics / condition of the cells and tissues at the time of harvesting are used as they are. Because they are used as the tissue in question without processing, it has generally been thought that there is no need to evaluate their performance / quality.

[0016] On the other hand, "Auto-plast" (a term coined in this invention), a medical material used in what is called "Auto-plastic transplantation" in this invention, is produced as a transplantable medical material with completely different characteristics / conditions from those at the time of collection, through various processing such as differentiation and transformation to suit the cells or tissues to be transplanted. This material does not exist in the conventional concept of medical materials, and therefore, there has been no system to properly evaluate its performance and other properties until now.

[0017] It is clear that the number of medical materials produced as regenerative medicine products will increase in the future, and the Ministry of Health, Labour and Welfare also recommends animal testing as a way to evaluate quality, as stated in "Regarding the Securing of Quality and Safety of Cell-Processed Pharmaceuticals, etc. (Pharmaceuticals and Food Safety Bureau Director's Notice No. 0907-3, September 7, 2012)" as a "test to support efficacy or performance."

[0018] In summary, autografting typically refers to an auto-graft, where the transplant material (graft tissue) is 1. transplanted immediately after harvesting, without any processing, to tissue equivalent to the original tissue, 2. transplanted in the same state as when it was harvested, and 3. used while retaining its original properties / characteristics. Blood transfusions are performed as blood into the blood vessels, liver transplants are performed in the liver, and skin transfusions are performed as skin grafts. The autologous transplantation referred to in the present invention includes "Auto-plast" (a coined term in the present invention) which expands the concept of transplantation, and involves performing various processes on cells or tissues derived from oneself, and in some cases, using the manufactured product as a transplantation material in the form of a tissue in a state different from the original trait. Representative examples include products such as regenerative medicine defined in the three regenerative methods, and it proposes a method that enables the design method and quality evaluation of medical materials using biological-derived materials in a broad sense.

[0019] The conventional concept of transplantation is "graft (including allogeneic)" ⊇ "Auto (autologous)-graft", and 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 explicit concept of "plastic graft" created by adding some processing to biological-derived materials. The term "Auto-plast" for the transplantation material created by processing biological-derived materials derived from oneself is a coined term according to the present invention, and its concept also includes Auto-plast as a "composite material" combined with artificial materials, etc., and there has been no specific verification method proposed for using them in regenerative medicine at present.

[0020] Also, not limited to tubular structures or membranous structures, solid organs are also included in Auto-plast, or many composite medical materials such as blood-related dosage forms (non-solid organs) that do not need to have the form of an organ, or endocrine cells (non-typical organs), etc. are included.

[0021] Furthermore, as a method for creating "Auto-plast", 1. It is assumed that in vitro (produced outside the body) is often used, but in part, 2. It is also assumed that ex vitro / in vivo (produced inside the body) is possible, Regardless of the production method, 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 wild-type recipients subcutaneously several times 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 the 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 has been 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, 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 the cells or tissues of a donor 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 includes the step of administering a crude extract of a labeled marker-expressing donor to a newborn recipient to obtain a recipient immune to the donor, The present invention provides a transplant model animal produced by a method for producing mice, comprising the step of transplanting a transplant material, which includes an artificial transplant material in which tissue or cells of a donor expressing the labeling marker are covered, adhered to, or infiltrated, into an immunotolerant recipient.

[0029] The present invention also provides the above-mentioned transplantation model animal, which is a mouse. [Effects of the Invention]

[0030] By using the present invention, it is possible to provide a method for evaluating transplant materials that enables the realization of a highly reliable and reproducible transplantation experimental system as an alternative to autologous transplantation. [Brief explanation of the drawing]

[0031] [Figure 1] A diagram illustrating the outline of a test conducted according to one embodiment of the present invention. [Figure 2] This diagram shows the appearance of a donor green mouse under fluorescent observation. [Figure 3] A diagram showing recipient mice that have undergone immunotherapy. [Figure 4] The diagram shows the transplantation of the material into a donor green mouse. The upper left of Figure 4 shows the transplanted tubular structure, the lower left shows the transplantation of the material into the green mouse, the upper right shows the condition of the material one week after transplantation, and the lower right shows the fluorescence observation of the transplant material, including the transplant material recovered from the green mouse. [Figure 5] This diagram shows the transplantation of a tubular structure, which is the transplant material, into a recipient mouse. [Figure 6] This figure shows the transplant material one month after transplantation into the recipient. The left side of Figure 6 shows the tubular structure (transplant material) one month after transplantation, and the right side of Figure 6 shows the tubular structure (transplant material) one month after transplantation as observed by fluorescence. [Modes for carrying out the invention]

[0032] This invention provides a method for evaluating transplant materials and a transplant model animal that approximates autologous transplantation for use in the method for evaluating transplant materials, thereby enabling the design of transplant materials and the verification of their performance and quality.

[0033] (Method for evaluating transplant materials) The transplant model animal of the present invention, used in the method for evaluating transplant materials, is a transplant model animal in which the transplant material has been transplanted and immune tolerance has been induced at the time of transplantation. The transplant model animal of the present invention can be suitably used in experimental systems for observing the biological response and progress when a transplant material is transplanted.

[0034] The animals used in the method for evaluating transplant materials of the present invention are not particularly limited, but can be non-human animals, such as rodents such as mice, rats, and guinea pigs; primates such as monkeys and marmosets; mammals such as rabbits, ferrets, dogs, cats, pigs, sheep, goats, cows, and horses; birds; nematodes such as nematodes; flatworms such as planarians; insects such as fruit flies; fish such as zebrafish; amphibians such as newts and frogs; and reptiles such as lizards. Preferably, the animals are rodents such as mice, rats, and guinea pigs, and more preferably mice.

[0035] The animals used in this invention may be inbred animals. In this specification, "inbred animals" refers to a population of animals or plants that is genetically homogeneous, with little or no difference in genetic characteristics (hereditary traits) between generations and between individuals. Inbred animals can be created by repeatedly performing inbreeding (full-sibling mating) for about 20 generations or more to fix the genotype and eliminate individuals with different traits. Inbred animals ensure homology of genetic traits at the level of identical twins between individuals. Inbred animals are not particularly limited, but for example, Wister rats, DBA / 2, C57BL / 6, and BALB / c mice can be used. When using inbred animals, animals of the same strain can be treated as the same individual.

[0036] The present invention provides a method for evaluating transplant materials, comprising the steps of: administering a crude extract of a labeled marker-expressing donor (hereinafter also simply referred to as "donor") to a newborn recipient to obtain an immune-tolerant recipient (immune tolerance step); and transplanting a transplant material, which includes an artificial transplant material in which tissue or cells of a labeled marker-expressing donor have covered, adhered to, infiltrated, or incorporated, into the immune-tolerant recipient (transplantation step). In this specification, "covering" of tissue or cells has the common sense in the art and includes the tissue or cells covering part or all of the transplant material. Also, in this specification, "adhering" of tissue or cells has the common sense in the art and includes, for example, the tissue or cells adhering to part or all of the transplant material, for example, sticking to the tissue or cells and not letting go. In this specification, "infiltrating" of tissue or cells has the common sense in the art and includes, for example, the tissue or cells penetrating and spreading inside part or all of the transplant material, for example, adhering to the inside. In this specification, "contains" a tissue or cell in the sense commonly used in the art, including, for example, covering the entirety of the transplant material.

[0037] (Donor and recipient) In this specification, "donor" refers to an animal that provides the tissue or cells to be included in the transplant material. In this specification, "recipient" refers to an animal that receives the transplant material.

[0038] (Recipient's immune tolerance process) In the immunotolerance step, a crude extract of a labeled marker-expressing donor is administered to the recipient's newborn offspring to induce immune tolerance to the donor tissue in the recipient, thereby obtaining an immunotolerant recipient. In this specification, "immune tolerance" refers to a state in which the immunological rejection reaction (immune response) to a specific antigen is suppressed.

[0039] The donor crude extract is not particularly limited, but can be a crude extract solution of pulverized material derived from tissue or cells collected from the donor, for example, a crude protein solution extracted from tissue or cells. The tissue or cells collected from the donor may include the tissue or cells intended for transplantation. The crude extract can be prepared, for example, by homogenizing the tissue or cells collected from the donor, then centrifuging or filtering, and collecting the supernatant containing soluble protein. The crude protein solution may be further concentrated. Alternatively, after preparation, it may be frozen and then thawed before use.

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

[0041] The step of administering the crude extract of the donor to the recipient's newborn is performed prior to the transplantation process. Methods for administering the crude extract of the donor to the recipient's newborn include subcutaneous administration, local administration, transdermal administration, intradermal administration, transmucosal administration, oral administration, intranasal administration, intratracheal administration, sublingual administration, transnasal administration, buccal administration, rectal administration, intravaginal administration, intravenous administration, intra-arterial administration, intramuscular administration, intracardiac administration, intraosseous administration, intraperitoneal administration, intraorbital administration, intravitreous administration, subconjunctival administration, suprachoroidal administration, subretinal administration, intra-articular administration, periarticular administration, cutaneous administration, and inhalation administration, with subcutaneous or intraperitoneal administration being preferred.

[0042] The donor crude extract can be administered to the recipient's newborn in a single to several doses. For example, the donor crude extract may be administered to the recipient's newborn every 1 to 2 days for a total of 3 to 5 doses. The first dose to the recipient's newborn can be given within 3 days of birth, preferably within 24 hours of birth. All doses to the recipient's newborn can be given within 10 days of birth, preferably within 7 days of birth. Furthermore, the amount of donor crude extract administered to the recipient is such that it can induce immune tolerance to the recipient. For example, if the crude extract contains 1 / 20th of the recipient's newborn body weight in protein, an initial dose of the donor crude extract weighing approximately 5% of the newborn's body weight can be administered within 24 hours of birth, and then approximately 5% of the newborn's body weight of the donor-derived crude extract can be administered subcutaneously to the abdomen or other areas four times, every two days for a total of four doses, over the first seven days of life, to obtain a recipient immune to the donor.

[0043] (Transplanting process) The transplantation process involves transplanting a transplant material, which includes an artificial transplant material that covers, adheres to, infiltrates, or incorporates donor tissue or cells, into the recipient.

[0044] A transplant material containing tissue or cells of a labeled marker-expressing donor includes material derived from the donor's tissue or cells together with the transplant material. For example, the transplant material may be a transplant material containing donor tissue or cells. In the transplant material, the donor's tissue or cells may cover the transplant material, or adhere to, infiltrate, or be incorporated into the transplant material.

[0045] The transplant material is not particularly limited and can be a bioabsorbable material or a biocompatible material. Materials that can be used as transplant materials include, for example, PLLA (poly-L-lactic acid), PEG (polyethylene glycol), PGA (polyglycolic acid), copolymers of PGA and PLA, polyhydroxybutyric acid, polycaptolactone, polyethylene succinate, polydioxanone, polybutylene succinate, etc. Also included are materials based on soluble cellulose such as oxidized cellulose, proteins, peptides, polyamino acids, polysaccharides, polyesters, polyamides, derivatives thereof, crosslinked and copolymerized materials, metallic magnesium, calcium carbonate, calcium phosphate, hyaluronic acid and hydroxyapatite, acrylic resins, fluororesins, polyolefins, silicones, polystyrene, polyesters, polyurethanes, polycarbonates, polyimides, derivatives thereof, crosslinked and copolymerized materials, as well as titanium, silica and zirconia, etc. 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 implantation material may be one or more of the above-mentioned materials, or a composite of two or more materials.

[0046] Furthermore, the transplant material may be covered with a covering material. Covering materials include, for example, fibrin-based materials, collagen-based materials, hyaluronic acid-based materials, glycoprotein-based materials, and soluble cellulose-based materials such as oxidized cellulose.

[0047] The transplant material is not particularly limited, but can be, for example, tubular structures, membranous structures, other structures of any shape, and artificial organs. Tubular structures can be used as transplant materials for regenerative medicine, such as blood vessels, respiratory organs, digestive organs, and urinary tract organs. Membranous structures can be used as transplant materials for regenerative medicine, such as dura mater, peritoneum, fascia, periosteum, and synovial membrane.

[0048] Artificial transplant materials, in which donor tissue or cells are covered, adhered to, or infiltrated, can be provided in any form. For example, transplant material can be prepared by seeding and culturing donor cells onto the transplant material.

[0049] Furthermore, the transplant material may be obtained by pre-implanting the transplant material into the donor's body, leaving it in place for 2 to 30 days, and then retrieving it. The transplant material may also be obtained by repeatedly implanting and retrieving it from the donor. By implanting the transplant material into the donor and leaving it in place for a certain period, the donor's tissue or cells can be sufficiently adhered to or infiltrated into the transplant material.

[0050] An artificial graft material covered, adhered to, or infiltrated by donor tissue or cells may be covered, adhered to, or infiltrated by donor tissue or cells throughout the entire graft material, or only a portion of the graft material may be covered, adhered to, or infiltrated by donor tissue or cells.

[0051] The donor tissue or cells expressing the labeled marker included in the transplant material may be the donor tissue or cells themselves or processed thereof. The tissue or cells collected from the donor can be isolated, cultured, differentiated, selected, amplified, and cryopreserved as needed for use as transplant material, and then thawed later. Alternatively, the tissue or cells may be those that have been subcultured to form a cell line.

[0052] The transplant material may be supplemented with cells and various bioactive substances (such as cytokines and cell growth factors) by co-culture, spraying, coating, and infiltration. Examples of bioactive substances include 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 and differentiation factors, erythropoiesis-promoting factor (EPO), transformation growth factor, and bone morphogenetic protein.

[0053] The transplant material is not particularly limited, but can be used as, for example, blood vessels, respiratory organs, digestive organs, urinary tract organs, dura mater, peritoneum, fascia, synovial membrane, periosteum, and serosal membrane.

[0054] The transplant material is not particularly limited, but can be transplanted into internal organs such as the brain, liver, kidneys, and heart of the recipient, as well as subcutaneous tissue and blood vessels. The method of transplanting the transplant material into the recipient is not particularly limited, and conventionally known methods can be used.

[0055] The donor tissue or cells included in the transplant material are not particularly limited, but can be collected from an adult donor. If the donor is an inbred line, the individual from which the crude extract for administration to the recipient's newborn offspring is prepared can be used as the donor, or another individual from the same line can be used as the donor, and the transplant material derived from that other individual from the same line can be transplanted into the recipient.

[0056] The donor tissue or cells may be selected from, for example, fibroblasts, chondrocytes, osteoblasts, angioblasts, myoblasts, epithelial cells, smooth muscle cells, endothelial cells, vascular endothelial cells, fibrous cells, hepatocytes, chondrocytes, epithelial cells, urothelial cells, smooth muscle cells, keratinizing cells, 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, chondrocytes, skeletal muscle cells, smooth muscle cells, cardiomyocytes, pericardial cells, dura mater cells, meningeal cells, pericytes, glial cells, nerve cells, amnion cells, placental membrane cells, serosal cells, ependymal cells, periventricular cells, oral mucosal epithelial cells, nerve cells and dendritic cells, genetically modified cells of these, cells produced by gene manipulation techniques such as genome editing or transformation techniques using mRNA (messenger RNA), and cells cultured and / or differentiated from these.

[0057] Furthermore, the donor's tissue or cells may be selected from, for example, the group consisting of somatic tissue stem cells, somatic tissue progenitor cells, germline 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, hematopoietic stem cells, oral mucosal stem cells, periodontal ligament stem cells, hepatic stem cells, bone marrow mesenchymal stem cells, undifferentiated stem cells, undifferentiated progenitor cells, predifferentiated stem cells, predifferentiated progenitor cells, keratin-producing cell 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 those defined as human somatic stem cells in the Ministry of Health, Labour and Welfare's Pharmaceutical and Food Safety Bureau Notification No. 0907-3 dated September 7, 2012, "Regarding the assurance of quality and safety of human allogeneic somatic stem cell-processed pharmaceuticals, etc." "Human somatic stem cells" refers to cells taken from humans or cells produced by the division of such cells that possess multipotency and maintain self-renewal ability, or are presumed to do so, and cells derived therefrom, including the following: tissue stem cells (e.g., 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, hepatic stem cells, and skeletal muscle stem cells) and cell populations rich in these (e.g., whole bone marrow cells including hematopoietic stem cells). Human somatic stem cells include vascular progenitor cells, umbilical cord blood, and bone marrow stromal cells. Furthermore, human somatic stem cells include cells obtained by culturing and / or differentiating these cells in vitro.

[0058] Furthermore, the donor tissue or cells may be "induced pluripotent stem cells" and "induced pluripotent stem cell-like cells," such as iPS cells, produced by genetic engineering technologies such as genetic recombination and genome editing, as well as transformation technologies using mRNA (messenger RNA), and cells cultured and / or differentiated from these.

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

[0060] (Method for evaluating transplant materials using donors expressing labeled markers) The present invention provides a method for evaluating transplant materials using a labeled marker-expressing donor, comprising the steps of: administering a crude extract of the labeled marker-expressing donor to a newborn recipient to obtain an immune-tolerant recipient; and transplanting a transplant material containing an artificial transplant material coated, adhered to, or infiltrated by the tissue or cells of the labeled marker-expressing donor into the immune-tolerant recipient.

[0061] In this specification, "labeling marker" refers to a marker that enables the identification and observation of cells that express it and cells that do not express it. A labeling marker may be any marker capable of identifying donor-derived cells after transplantation into a recipient. A labeling marker may be, for example, a fluorescent protein. Fluorescent proteins include, for example, green fluorescent protein (GFP) from the jellyfish Aequorea victoria; variants of GFP such as EGFP; mutants of GFP that emit fluorescence of different colors (e.g., blue fluorescent protein (BFP), yellow fluorescent protein (YFP), and cyan fluorescent protein (CFP)); dsRed fluorescent protein (dsRed2FP); red fluorescent protein (eqFP611) isolated from the sea anemone 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 proteins.

[0062] If the labeling marker is a fluorescent protein such as EGFP, it can be stably expressed in vivo at all times, allowing for easy fluorescence observation of donor-derived cells in the recipient's body after transplantation. Furthermore, since fluorescence in fluorescent proteins such as EGFP is triggered by excitation light irradiation, there is no need to stain the tissue for observation, allowing for convenient observation of living tissue.

[0063] A labeled marker expression donor is an animal into which a labeled marker gene has been introduced, resulting in the constitutive and stable expression (or ability to express) of the labeled marker in some or all of its cells. Labelled marker expression donors can be created by introducing the labeled marker gene using conventionally known methods.

[0064] In the method of the present invention, the recipient is not particularly limited, but can be an animal that has not been introduced with the labeling markers introduced in the donor, and can be, for example, a wild-type mouse (WM) that has not been genetically modified. Furthermore, it may be an inbred animal to improve experimental accuracy.

[0065] Since the recipient has not been introduced with a labeling marker, if transplanted using a conventional 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, with the method of the present invention, by administering a crude extract of a donor expressing the labeling marker to the newborn recipient in advance, immune tolerance to donor-derived antigens including the labeling marker can be induced, thereby suppressing the immune response at the time of transplantation, limited to donor-derived antigens only. On the other hand, the induction of a normal immune response to antigens not derived from the donor is maintained.

[0066] By using donors expressing labeled markers, donor-derived cells and tissues can be easily observed in the recipient after transplantation. Furthermore, it is easy to distinguish whether cells and tissues generated in the recipient after transplantation originate from the recipient or the donor. Therefore, for example, if a tumor develops in the recipient after transplantation, it can be easily determined whether it originated from donor-derived cells or from the recipient.

[0067] (Transplant model animals) The present invention also includes the step of administering a crude extract of a labeled marker-expressing donor to a newborn recipient to obtain a recipient that is immune to the donor, A step of transplanting a transplant material, which includes an artificial transplant material in which tissue or cells from a labeled marker-expressing donor are covered, adhered to, infiltrated, or incorporated, into an immunotolerant recipient, This invention provides a transplant model animal that approximates autotransplantation, produced by a method for producing mice possessing [specific characteristic].

[0068] The transplant model animal of the present invention can be prepared by a method including an immune tolerance step and a transplant step as described in the method for evaluating transplant materials using the labeled marker-expressing donor described above. That is, the transplant model animal of the present invention is prepared by administering a crude extract of the labeled marker-expressing donor to a newborn recipient to obtain an immune-tolerant recipient, and then transplanting a transplant material containing an artificial transplant material coated, adhered to, infiltrated, or encapsulated by the tissue or cells of the labeled marker-expressing donor into the immune-tolerant recipient. Details of the immune tolerance step and the transplant step are as described in the method for evaluating transplant materials described above.

[0069] As described above, the transplant model animals prepared have the transplanted material labeled with a labeling marker. Therefore, with the transplant model animals of the present invention, it is possible to easily confirm not only how the recipient transplant model animal behaves, but also how the transplanted material behaves within the body of the recipient transplant model animal.

[0070] In the transplant model animals of the present invention, immune tolerance to the donor is induced in the recipient before transplantation of the transplant material. Therefore, the recipient transplant model animals have a suppressed immune response to the transplant material. Consequently, they can be used, for example, in experimental systems to observe the biological response and progress after transplantation of the transplant material. On the other hand, the immune response to all antigens that are not derived from the donor is preserved. 1. The risk of interrupting the experiment due to infection during the process, 2. Regarding the risk of increased cancer risk, etc. We can expect experimental results that are closer to clinical transplantation than ever before.

[0071] This invention provides a so-called "approximate autotransplantation animal experimental model" by using inbred animals. By using inbred animals, this invention makes it possible to easily induce immune tolerance even in animals with normal immune function and establish an animal model that can avoid immune responses against specific individuals. Therefore, by using this invention, it is not necessary to use conventional immunodeficient animal experimental systems, and transplantation experiments can be performed in a near-clinical state using animals with normal immune function. Furthermore, this invention can be used as an experimental system to observe the biological response and progress when autotransplantation material is performed on small animals such as mice, which are normally difficult to autotransplant. Thus, it can serve as an alternative to autotransplantation experiments that could only be performed on medium-sized and large animals, and it is in line with the trend of animal welfare and can comply with the Ministry of Health, Labour and Welfare's recommendations regarding the development of regenerative medicine products based on the three laws on regenerative medicine. [Examples]

[0072] Figure 1 shows an overview of the tests conducted in this embodiment. An explanation of the abbreviations used in the following embodiments is provided below. Glossary: GM: Green mouse (EGFP constitutively expressing genetically modified mouse) ICR mouse: A highly prolific and most popular inbred albino strain of experimental mouse. P1 and P1A: These are experimental facilities that comply with the Cartagena Protocol, an international treaty, and adhere to specific measures (containment measures) to prevent the spread of genetically modified organisms. Among these, P1 and P1A have the least stringent regulations. P1 is mainly required when handling cells, while P1A is mainly required when handling animals. 1. P1A (Animal Experimentation Facility): Collection of GM-derived tissue and preparation of crude protein. The process of collecting donor-derived antigen-presenting proteins to induce immune tolerance in the recipient. 2. P1A (Animal Experiment Facility) Immune Tolerance Treatment of ICR Mice (Wild Species) The process of administering an antigen protein to a recipient's newborn to induce immune tolerance. 3. P1A (Animal Experimentation Facility) The process of collecting cells or tissues from a donor to be used as transplant candidates. 4. P1 (Cell Experiment Facility) The process of processing collected donor-derived cells, etc., as needed, or preparing them as transplant material. 5. P1A (Animal Experimentation Facility) The process of transplanting material collected from a donor into an immune-tolerant recipient. 6. P1A (Animal Experimentation Facility) This process involves observing the post-transplant condition at the appropriate time, evaluating the performance of the transplant material, and, if necessary, designing the transplant material.

[0073] (donor) As a donor, a genetically modified inbred mouse (Green Mouse: C57BL / 6-(CAG-EGFP) C14-Y01-FM131Osb (RBRC00267: RIKEN BioResearch Center)) that expresses EGFP (Enhanced green fluorescence protein: green fluorescent protein derived from the jellyfish Aequorea victoria) throughout its body through genetic engineering was used (hereinafter also referred to as "Green Mouse (GM)"). An inbred mouse is a mouse that has undergone repeated inbreeding and exhibits homology of expression traits in its genetic traits and their expression between generations and / or between individuals, similar to that of identical twins.

[0074] Figure 2 shows a photograph of fluorescence observed in a donor green mouse. By irradiating with excitation light (ultraviolet light in the case of GM), green fluorescence due to EGFP in cells can be confirmed. Fluorescence was observed in the entire epidermis of newborns (left side of Figure 2), and in adults, it was observed in the excised brain (center of Figure 2) and exposed epidermis excluding body hair (right side of Figure 2).

[0075] (Preparation of crude extract derived from donor) Tissue was collected from donor green mice, homogenized, and then centrifuged or filtered to recover a crude extract containing soluble proteins, thereby preparing crude protein (Step 1 in Figure 1). The fluorescence of the crude extract in the test tube can be confirmed in the upper part of Figure 3. To efficiently induce immune tolerance, a crude extract containing candidate transplant tissue can also be prepared, or the crude extract can be further purified before use.

[0076] (Immune tolerance process) The recipient mice used were commercially available ICR mice (wild-type inbred mice). The ICR mice underwent immunotolerance treatment (Step 2 in Figure 1). Within 24 hours of birth, newborn ICR mice received an initial subcutaneous injection of a donor crude extract containing approximately 5% body weight. Subsequently, over the first seven days of life, the donor-derived crude extract was administered subcutaneously to the abdomen four times, every two days. The subcutaneously administered crude extract contained a protein amount equivalent to 1 / 20th of the recipient's body weight. The mice were then reared under normal conditions for at least four weeks.

[0077] Figure 3 shows recipient mice that underwent immunotherapy. The crude extract from the donor, which shows green fluorescence in a test tube (upper panel of Figure 3), was subcutaneously administered into the midline of the abdomen of ICR newborns (lower left panel of Figure 3). Subsequently, when the abdomen was irradiated with excitation light, green fluorescence could be confirmed through the skin (lower right panel of Figure 3).

[0078] (Preparation of composite materials for transplantation) A tubular structure with a silicone tube as the core was fabricated as a transplant material. This tubular structure can be used as a pseudo-blood vessel. Furthermore, when unfolded, this tubular structure can also be used as a membrane-like structure. As a prototype, in this embodiment, a tube made of biocompatible silicone (product name: Safeseed Silicone Catheter: outer diameter 2.7 mm) (hereinafter referred to as silicone tube) was used. Two or more layers of a white self-assembling collagen sheet (product name: Integrain: sheet type 45 x 30 mm) (hereinafter referred to as collagen sheet) were wrapped around this silicone tube. Next, to prevent unwinding or unraveling, a blue-violet bioabsorbable PGA thread (PGA: polyglycolic acid) was further wrapped around and fixed (Figure 4, upper left). At this point, the wrapped tubular collagen sheet is not watertight and easily leaks water.

[0079] Figure 4 is a photograph showing the transplantation of the material into a donor green mouse. The upper left of Figure 4 shows the tubular structure, which is the transplant material, transplanted into the donor. The lower left of Figure 4 shows the transplantation of the material into the green mouse. The upper right of Figure 4 shows the condition of the green mouse one week after transplantation. One week after transplantation, neovascularization was observed. The lower right of Figure 4 shows the fluorescence observation of the transplant material, including the transplant material, excised and recovered from the green mouse.

[0080] (Transplanting process) Figure 4 shows the process of transplanting the above-mentioned tubular structure (transplant material) into a donor green mouse (Figure 4, lower left). A 1 cm skin incision was made on the back of the adult donor green mouse, and then the subcutaneous tissue was peeled back in a tunnel shape for about 3 cm to create an insertion cavity. The mantle tube was inserted into the insertion cavity, and the tubular structure was implanted subcutaneously through it. Once the entire implanted tubular structure was comfortably embedded subcutaneously, the mantle tube was withdrawn, and the insertion hole was sutured closed.

[0081] One week after subcutaneous implantation, the skin of the transplanted green mouse was incised and turned over to allow a full view of the tubular structure, and its entire structure was confirmed (Figure 4, upper right). The collagen sheet that had been wrapped around it was moistened with interstitial fluid and covered with cells derived from the green mouse, making it semi-transparent. It was connected to the surrounding host tissue by membranous connective tissue. Based on this connective tissue, abundant cross-linking of neovascularization was observed in the collagen tube, and it was confirmed that fine blood vessels were extending around its entire circumference.

[0082] One week after transplantation, the tubular structure was excised and collected as transplant material (Figure 4, lower right; corresponding to steps 3 and 4 in Figure 1). An incision was made at the connective tissue portion connected to the host tissue, and the collagen tube was excised carefully to avoid damaging it.

[0083] When the excised tubular structure was irradiated with excitation light, fluorescence was emitted from the transplant material, indicating that green mouse-derived cells had been evenly seeded in the collagen tube. Separately, it was also confirmed that the structure had already achieved watertightness at this stage, preventing water leakage. Furthermore, culturing the cells from the excised collagen tube revealed that the cells seeded on the collagen sheet were viable cells suitable for culture.

[0084] (Transplanting process) In wild-type ICR mice lacking a labeling marker and in which immune tolerance had been induced by green mice, a transplant material containing a tubular structure recovered from green mice was subcutaneously transplanted using the same procedure as described above (preparation of the transplant composite material). Specifically, a 1 cm skin incision was made on the back of the ICR mouse, and the subcutaneous tissue was dissected in a tunnel shape for a length of 3 cm to create an insertion cavity. A mantle tube was inserted into the insertion cavity, and the tubular structure was subcutaneously implanted through it (Figure 5). With the entire implanted tubular structure comfortably embedded subcutaneously, the mantle tube was withdrawn, and the insertion hole was sutured closed.

[0085] One month after subcutaneous implantation, the skin was incised to allow a full view of the tubular structure, which was then inverted and examined (Figure 6, left). The collagen sheet that had been wrapped around it was moistened with interstitial fluid and covered with tissue cells derived from ICR mice on top of the cell layer derived from green mice, reducing the transmittance of visible light. It was firmly connected to the surrounding host tissue by connective tissue. Based on this connective tissue, abundant cross-linking of neovascularization was observed in the tubular structure, and it was confirmed that neovascularization derived from green mice had further developed, resulting in blood vessels extending all the way around.

[0086] Furthermore, since fluorescence characteristic of green mice was confirmed upon irradiation with excitation light (Figure 6, right side), it was confirmed that cells derived from green mice engrafted without immune rejection even in subcutaneous tissue, which is particularly prone to eliciting a strong immune response, within the body of immune-tolerated wild-type ICR mice. In addition, cultureable viable cells were confirmed in tissue collected from tubular structures transplanted into ICR mice, and the coexistence of cells emitting green fluorescence and cells without fluorescence was confirmed.

[0087] Figure 5 shows the transplantation of a recipient mouse with a tubular structure-containing transplant material.

[0088] Figure 6 shows the transplant material one month after transplantation to the recipient. The left side of Figure 6 shows the tubular structure (transplant material) one month after transplantation, and the right side of Figure 6 shows the tubular structure (transplant material) one month after transplantation as observed by fluorescence. One month after transplantation, fluorescence observation revealed the distribution of donor-derived cells and tissues attached to the tubular structure, as well as new cells and tissues that arose from them. [Industrial applicability]

[0089] This invention can be used in highly reliable and reproducible transplantation experimental systems. Furthermore, compared to conventional animal experiments for similar purposes (such as experimental systems using immunodeficient animals), it allows for experiments under conditions closer to actual clinical medicine. Based on this, in regenerative medicine, it is of utmost importance to ensure better performance and quality as transplantable medical materials, including regenerative medicine products. In addition, because widely available and inexpensive experimental materials (experimental animals such as mice and their rearing materials) and research equipment (clean rooms and facilities for preventing the spread of genetically modified animals) can be selected, it is possible to significantly reduce the necessary expenses for conducting experiments. Moreover, since it does not require the use of medium-sized or large animals, which are essential for conventional autologous transplantation experimental systems, it does not conflict with social trends such as animal welfare, and it is easier to increase the number of experimental cases in terms of cost and labor. Furthermore, because of the short life span, it can accommodate different biological conditions at different stages of life, making it possible to design and evaluate the performance of various transplantable medical materials under different conditions.

Claims

1. A step of obtaining a recipient immune to the donor by subcutaneously administering a crude protein solution extracted from the cells or tissues of a labeled marker-expressing donor to a newborn recipient, The process involves transplanting a transplant material, which includes an artificial transplant material in which tissue or cells of a donor expressing the labeling marker are covered, adhered to, infiltrated, or incorporated, into the immunotolerant recipient. An immune tolerance recipient (excluding humans) produced by, wherein the donor and the recipient are inbred animals, and Observe the behavior of the labeling marker in the immune-tolerant recipient. A method comprising, thereby identifying whether cells and tissues generated in the immune-tolerant recipient after transplantation originate from the immune-tolerant recipient or from the donor.

2. A step of obtaining a recipient immune to the donor by subcutaneously administering a crude protein solution extracted from the cells or tissues of a labeled marker-expressing donor to a newborn recipient, The process involves transplanting a transplant material, which includes an artificial transplant material in which tissue or cells of a donor expressing the labeling marker are covered, adhered to, infiltrated, or incorporated, into the immunotolerant recipient. An immune tolerance recipient (excluding humans) produced by, wherein the donor and the recipient are inbred animals, and Observe the behavior of the labeling marker in the immune-tolerant recipient, and the response and progress of the transplant model animals. A method comprising, thereby confirming the state of immune tolerance in a recipient of the transplanted material after transplantation.

3. A method for identifying whether cells and tissues generated in an immune-tolerant recipient after transplantation are derived from the immune-tolerant recipient or from the donor, according to claim 1, wherein the transplant material is a tubular structure, a membranous structure, or an artificial organ.

4. A method for identifying whether cells and tissues generated in an immune-tolerant recipient after transplantation are derived from the immune-tolerant recipient or the donor, as described in claim 1, wherein the transplant material is retrieved after transplanting the transplant material into the donor's body and leaving it in place for 2 to 30 days.

5. A method for identifying whether cells and tissues generated in an immune-tolerant recipient after transplantation are derived from the immune-tolerant recipient or from the donor, according to claim 1, wherein the labeling marker is a fluorescent protein.

6. The method for identifying whether cells and tissues generated in an immune-tolerant recipient after transplantation are derived from the immune-tolerant recipient or from the donor, wherein the transplant material is a collagen sheet on which donor-derived cells have been seeded.

7. A method for confirming the state of immune tolerance in a recipient of a transplanted material according to claim 2, wherein the transplanted material is a tubular structure, a membranous structure, or an artificial organ.

8. A method for confirming the state of immune tolerance in a recipient of a transplanted material according to claim 2, wherein the transplanted material is obtained by transplanting the transplanted material into the living body of the donor and leaving it in place for 2 to 30 days before retrieval.

9. A method for confirming the state of immune tolerance in a transplant recipient after transplantation of a transplant material, according to claim 2, wherein the labeling marker is a fluorescent protein.

10. The method for confirming the state of immune tolerance in a recipient after transplantation of a transplant material according to claim 2, wherein the transplant material is a collagen sheet on which donor-derived cells have been seeded.

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