Method for producing transplantation model animal for evaluating transplantation material

Abstract

[Problem] Provided is 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] This method for evaluating a transplantation material which is similar to that in autoplastic transplantation comprises: a step for 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 for 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 for 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.

Description

Method for producing a transplant model animal for evaluating transplant materials

[0001] The present invention relates to a method for evaluating transplant materials that can be used as a highly reliable and reproducible approximate autotransplantation experimental system and that are produced with high homology to clinical medicine.

[0002] Regenerative medicine is a technique for repairing or compensating for damaged or missing functions and / or structures of biological tissues in animals, including humans, livestock, and pets. Regenerative medicine is also useful when growth is inhibited by some disease or injury during the developmental or growth period of an animal, preventing the desired functions and / or structures from being obtained.

[0003] One of the most effective methods in regenerative medicine is the transplantation of bio-derived medical materials, including cells or tissues, into defective areas. In Japan, these medical materials are defined as "regenerative medicine products" under the three regenerative medicine-related laws (the Regenerative Medicine Promotion Act, the Regenerative Medicine Safety Assurance Act, and the revised Pharmaceutical Affairs Act).

[0004] From an ethical and immunological standpoint, it is desirable for regenerative medicine products used in transplants to be manufactured using processed materials derived from the individual receiving the transplant, a method known as "autoplastic transplantation."

[0005] Artificial materials are also used to complement the insufficient functions of biomedical materials alone. These artificial materials must not induce undesirable reactions in the body, including immune responses. Conventionally, various medical materials, including bio-derived materials, including regenerative medicine products, as well as biocompatible and bio-affinity materials, have been used (see, for example, Patent Documents 1 to 4). However, it is difficult to verify whether these materials have optimal properties and composition before transplantation. In particular, it has been difficult to verify the performance of regenerative medicine products containing living cells and tissues in advance.

[0006] To ensure the quality of regenerative medicine products, a clinically appropriate system is required, which allows for prior animal testing using mammals and for tracking the progress of immune responses after transplantation. For such animal testing, an animal testing model using "autotransplantation," in which cells and tissues taken from the recipient are returned to the recipient, is suitable.

[0007] However, animal experimental systems using autotransplantation have rarely been used as a research tool. In particular, there have been no autotransplantation experimental models for small animals. This is because autotransplantation 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, small animals cannot withstand the collection procedure, and depending on the tissue, the amount can be fatal. In small animals, tissues and cells are generally collected after the animal is killed and blood is drained. As a result, the animal is lost during the collection procedure, making autotransplantation impossible.

[0008] Although autotransplantation is possible in medium-sized and large animals, it is difficult to arrange a sufficient number of experimental cases due to the cost of raising them. Furthermore, with the current spread of animal welfare, there is a trend toward stricter regulations on animal experiments themselves.

[0009] Therefore, most conventional research has focused on allografts and xenografts in small animals. Because allografts and xenografts are not autografts, they require the use of immunosuppressants or severely immunodeficient animals (e.g., nude mice, NOD / scid mice, and NOG mice) to avoid immune reactions, or a combination of these. This necessitates the use of expensive animals, which are expensive to produce and maintain, resulting in significant costs and labor. Furthermore, these experimental systems require the use of immunosuppressants or the use of sophisticated, specialized facilities, often equivalent to a sterile room, due to the high risk of infection caused by immunodeficiency. Furthermore, the long-term use of immunosuppressants poses additional costs.

[0010] Furthermore, immunodeficiency experimental systems have a serious problem in that they are conducted under conditions significantly different from those observed in human and veterinary clinical settings. In particular, immunocompromised patients and those receiving long-term immunosuppressants are rarely candidates for transplantation therapy in humans. Treatments established in immunodeficiency experimental systems may result in unexpected immune reactions when administered to immunocompetent patients or livestock. Therefore, the present invention provides an animal experimental system that avoids these problems and enables the design and development of transplant materials with more appropriate performance and quality. In particular, the present invention provides an animal experimental system that can evaluate the engraftment and reproducibility of structure / function of target tissues. More specifically, the present invention provides an animal experimental system that can not only minimize rejection reactions but also evaluate the presence / adequacy of responses supporting / assisting transplanted tissues (blood flow induction, angiogenesis, and regulation of trophic factor expression, etc.).

[0011] JP 2015-89433 A JP 2007-186831 A JP 2013-162796 A JP 2008-523957 A

[0012] There is a need to establish a reliable and reproducible system to verify whether the materials used in transplants, such as bio-derived materials including regenerative medicine products, as well as biocompatible and bio-compatible materials, have the optimal composition and structure for treatment. To this end, transplant experiments using animals are essential, and transplant materials should be designed based on these experiments.

[0013] Considering costs and other factors, small animals are suitable for animal experiments. However, autotransplantation is difficult with small animals because their small size requires the individual to be sacrificed during cell collection. Allotransplantation, on the other hand, is often performed in immunodeficiency experimental systems or experimental systems using immunosuppressants, as post-transplant immune responses (such as immune rejection) occur. However, these experimental systems are significantly different from the actual clinical treatment and lack reliability and reproducibility. Furthermore, immunodeficiency experimental systems are problematic in that they require high costs for experimental materials and highly specialized equipment.

[0014] Therefore, an object of the present invention is to provide a method for producing a transplant model animal that can realize a highly reliable and reproducible transplantation experimental system.

[0015] Donor grafts commonly used for transplantation include autografts, allografts, and xenografts. These are raw grafts, meaning that the tissue is transplanted "shortly" after collection without any special processing. "Shortly" can refer to transplants performed immediately after collection, as well as those performed after a certain period of time, such as in component transfusions, blood products, and autologous blood transfusions, where refrigeration or freezing is required, or for component separation and purification. These raw grafts are intended to utilize the characteristics and state of the cells and tissues at the time of collection. Because the tissue is used without processing and retains its characteristics and state, it has generally been thought that there is no need to evaluate its performance or quality.

[0016] On the other hand, the medical material used in what we call "auto-plastic transplantation" is "auto-plast" (a term coined by us in this invention), which undergoes various processes such as differentiation and transformation to suit the cells and tissues to be transplanted, resulting in a transplant medical material with completely different characteristics and conditions from those at the time of collection.This material is not within the conventional concept of medical materials, and as such, there has been no system to date for appropriately evaluating its performance.

[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 is recommending that quality be evaluated through animal testing, etc. According to "Ensuring the Quality and Safety of Cell-Processed Pharmaceuticals, etc. (Yakushoku Notification No. 0907-3, September 7, 2012, Director-General of the Pharmaceutical and Food Safety Bureau)," animal testing is recommended as "testing to verify efficacy or performance."

[0018] To summarize the above, autotransplantation usually refers to autograft, where the transplant material (graft) is 1. transplanted into tissue equivalent to the original tissue immediately after collection without any processing, 2. transplanted in the collected state, or 3. used with its original properties / characteristics. Blood transfusions are performed as blood into the blood vessels, liver transplants are performed on the liver, and skin is used as a skin transplant.

[0019] The term "autotransplantation" as used in this invention includes "auto-plast" (a term coined by this invention), which is an expanded concept of transplantation, and includes the use of tissues produced by subjecting self-derived cells and tissues to various processes, in some cases resulting in tissues with different characteristics from their original state, as transplant materials. Representative examples include regenerative medicine products defined in the Three Regeneration Methods, and this invention proposes a method that enables the wide-ranging design and quality evaluation of medical materials made from biological materials.

[0020] The conventional concept of transplantation is "transplant (including allogeneic) graft" ⊇ "autograft," and as a rule, grafts are used (transplanted) in the state in which they are harvested. The concept of "graft" does not include processed harvested transplant materials. Furthermore, the concept of "plastic grafts," which are made by processing biomaterials, has not yet been explicitly defined. The term "autoplast," a name for transplant materials made by processing autologous biomaterials, was coined by the present inventors. However, this concept also includes autoplast as a "composite material" combined with artificial materials, and no specific verification methods have yet been proposed for its use in regenerative medicine.

[0021] Auto-plast is not limited to tubular and membranous structures; it also includes solid organs, as well as many other composite medical materials, such as blood-related dosage forms (non-solid organs) and endocrine system cells (atypical organs), which do not necessarily have the shape of an organ.

[0022] Furthermore, it is anticipated that Auto-plast will be produced mostly by 1. in vitro (production outside the body), but some may also be produced by 2. ex vitro / in vivo (production inside the body). Regardless of the production method, the quality of the material as a transplant material can be evaluated in this invention.

[0023] To solve the above-mentioned problems, the present inventors used inbred mice expressing EGFP as donors and subcutaneously administered crude extracts containing crude proteins extracted from donor tissues to newborn wild-type recipients several times to induce immune tolerance. An artificial transplant material was then transplanted into the donors. As a result, donor tissues and cells adhered to the transplanted artificial transplant material. When transplantation materials containing the transplant material produced in the donor's body were transplanted into immunotolerant recipients, immune responses to donor-derived cells / tissues were avoided. Furthermore, by fluorescence observation, the engraftment of donor-derived cells and tissues along with the transplant material in the recipient after transplantation was easily observed. The present invention was completed based on these findings.

[0024] That is, the present invention provides a method for producing a transplant model animal for evaluating transplant materials similar to autotransplants, comprising the steps of: administering a crude extract of a labeled marker-expressing donor to a newborn recipient to obtain a recipient that is immunotolerant to the donor; and transplanting a transplant material containing an artificial transplant material coated, adhered, infiltrated, or encapsulated with tissues or cells of the labeled marker-expressing donor into the immunotolerant recipient, wherein the donor and the recipient are inbred animals.

[0025] The present invention also provides a method for producing a transplant model animal for evaluating a transplant material, wherein the transplant material in the above-mentioned production method is a tubular structure, a membranous structure or a solid artificial organ.

[0026] The present invention also provides a method for producing a transplant model animal for evaluating a transplant material, which is obtained by transplanting a transplant material into the body of a donor, leaving it in place for 2 to 30 days, and then recovering the transplant material, using the above-mentioned production method.

[0027] The present invention also provides a method for producing a transplant model animal for evaluating transplant materials, which is a crude protein solution extracted from cells or tissues of a donor, in the above-mentioned production method.

[0028] The present invention also provides a method for producing a transplant model animal for evaluating a transplant material, wherein the labeling marker is a fluorescent protein.

[0029] The present invention also provides a method for evaluating the behavior of a transplanted material, in which the crude extract is administered subcutaneously, intramuscularly, intravenously, intradermally, transdermally or orally to a newborn of the recipient in the step of obtaining a recipient that is immunotolerant to the donor.

[0030] The present invention also provides a method for evaluating the behavior of a transplant material, comprising the steps of: administering a crude extract of a labeled marker-expressing donor to a newborn recipient to obtain a recipient that is immunotolerant to the donor; and transplanting a transplant material containing an artificial transplant material coated, adhered, infiltrated, or encapsulated with tissues or cells of the labeled marker-expressing donor into the immunotolerant recipient, wherein the donor and the recipient are inbred animals; producing a transplant model animal by the production method; and observing the labeled marker in the transplant model animal, thereby providing a method for evaluating the behavior of a transplant material in the body of the transplant model animal by the labeled marker.

[0031] The present invention also provides the above-mentioned transplant model animal, wherein the animal is a mouse.

[0032] The present invention can provide a method for evaluating transplantation materials that can realize a highly reliable and reproducible transplantation experimental system as an alternative to autotransplantation.

[0033] 4 shows an outline of a test performed in one embodiment of the present invention. FIG. 4 shows fluorescent observation of a donor green mouse. FIG. 4 shows a recipient mouse that has undergone immune tolerance treatment. FIG. 4 shows the state of transplanting a material into a donor green mouse. The upper left of FIG. 4 shows the transplanted tubular structure, the lower left of FIG. 4 shows the state of transplanting the transplant material into a green mouse, the upper right of FIG. 4 shows the state of the transplant material one week after transplantation, and the lower right of FIG. 4 shows the state of a transplant material containing the transplant material recovered from a green mouse, observed under fluorescent light. FIG. 4 shows the state of transplanting a tubular structure, which is a transplant material, into a recipient mouse. FIG. 6 shows the transplant material one month after transplantation into a recipient. The left of FIG. 6 shows the tubular structure (transplant material) one month after transplantation, and the right of FIG. 6 shows the tubular structure (transplant material) one month after transplantation, observed under fluorescent light.

[0034] The present invention provides a transplant model animal that resembles autotransplantation for use in a method for evaluating transplant materials, and a method for evaluating transplant materials, thereby enabling the design of transplant materials and the verification of their performance and quality.

[0035] (Method for producing a transplant model animal for evaluating a transplant material) A transplant model animal produced by the method for producing a transplant model animal for evaluating a transplant material of the present invention is a transplant model animal into which a transplant material has been transplanted and in which immune tolerance has been induced at the time of transplantation. In other words, a transplant model animal produced by the method for producing a transplant model animal for evaluating a transplant material of the present invention is a recipient that has immune tolerance to a donor into which the transplant material has been transplanted. A transplant model animal produced by the method for producing a transplant model animal for evaluating a transplant material of the present invention can be suitably used in an experimental system for observing the biological response and progress, etc., when a transplant material is transplanted.

[0036] The animals used in the method for producing a transplant model animal for evaluating a transplant material of the present invention are not particularly limited, and may 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. The animals are preferably rodents such as mice, rats, and guinea pigs, and more preferably mice.

[0037] The animals used in the present invention may be inbred animals. As used herein, the term "inbred animals" refers to a genetically homogeneous population of plants or animals with little or no difference in genetic properties (genetic traits) between generations or between individuals. Inbred animals can be produced by repeating inbreeding (full-sibling mating) for approximately 20 or more generations to fix the genotype and eliminating individuals with different traits. Inbred animals ensure the homology of genetic traits between individuals at the level of identical twins. Inbred animals are not particularly limited, but examples include the Wistar strain for rats and DBA / 2, C57BL / 6, and BALB / c strains for mice. When using inbred animals, animals of the same strain can be treated as the same individual.

[0038] The method for producing a transplant model animal for evaluating the transplant material of the present invention includes the steps of administering a crude extract from a labeled marker-expressing donor (hereinafter simply referred to as the donor) to a newborn recipient to obtain an immunotolerant recipient (immunotolerance step), and transplanting a transplant material containing an artificial transplant material coated, adhered, infiltrated, or encapsulated with tissues or cells from the labeled marker-expressing donor into the immunotolerant recipient (transplantation step). As used herein, the term "covering" by tissues or cells has its usual meaning in the art, including covering part or all of the transplant material with tissues or cells. As used herein, the term "adhesion" by tissues or cells has its usual meaning in the art, including, for example, attachment of tissues or cells to part or all of the transplant material, such as sticking to the tissues or cells and not detaching. As used herein, the term "infiltration" by tissues or cells has its usual meaning in the art, including, for example, penetration and spreading of tissues or cells into part or all of the transplant material, such as adhering to the interior. As used herein, the term "comprise" by tissue or cells has its usual meaning in the art, including, for example, covering the entire implant material.

[0039] (Donor and Recipient) As used herein, a "donor" refers to an animal that provides tissue or cells contained in a transplant material. Also, as used herein, a "recipient" refers to an animal that receives the transplant of the transplant material.

[0040] (Recipient Immune Tolerance Step) In the immune tolerance step, a crude extract from a donor expressing a labeled marker is administered to a newborn recipient to induce immune tolerance to the donor tissue in the recipient, thereby obtaining an immune-tolerant recipient. As used herein, "immune tolerance" refers to a state in which an immunological rejection reaction (immune response) against a specific antigen is suppressed.

[0041] The crude extract of the donor (also referred to as a crude extract) is not particularly limited, and can be a crude extract solution of pulverized material derived from tissues or cells collected from the donor, such as a crude protein solution extracted from tissues or cells. The tissues or cells collected from the donor may include tissues or cells to be transplanted. More specifically, donor-derived crude extracts include the whole body of a newborn immediately after birth, various organs, whether adult or newborn, including the brain, subcutaneous fat, limb muscles, internal organs such as the liver, lungs, heart, and kidneys, and supporting tissues including the connective tissue surrounding each organ. A transplant model animal for evaluating the transplant material of the present invention was produced, and the behavior of the transplant material in the body of the transplant model animal was evaluated. Among these, a crude extract obtained by crushing and extracting the whole body of a newborn, which covers antigenic proteins throughout the body, can be used for general purposes. When focusing more on nervous tissue, a crude extract derived from the brain can also be added to produce a transplant model animal for evaluating the transplant material of the present invention, and the behavior of the transplant material in the body of the transplant model animal was evaluated. The crude extract formulation can be varied for each target organ, depending on the target organ intended for transplantation.

[0042] Crude extracts can be prepared as a crude extract solution, for example, by disrupting tissues or cells collected from a donor using a homogenizer, followed by centrifugation or filtration, and collecting the supernatant containing soluble proteins. The crude protein solution may be further concentrated. Alternatively, the solution may be frozen and then thawed. Crude extracts can be prepared simply by the following procedure. For general-purpose use, whole bodies from 3 to 5 newborns are pre-minced into pieces several millimeters in diameter using scissors. An equal volume of ice-cold saline is then added to the tissue. The tissue is then placed on ice at 0-4°C and disrupted intermittently using a homogenizer (tissue disrupter) for approximately 3-5 minutes, while monitoring the disruption status, to prevent the temperature from rising until a sufficiently thick supernatant is extracted. The supernatant is then obtained by centrifugation. The disruption conditions, such as the centrifugation speed, vary depending on the equipment and tissue type, but can be between 200 and 1,000 revolutions per minute. To facilitate temperature control, tissue can be pre-frozen in the disruption container at -20°C or below, followed by the addition of ice-cold saline. When using neonatal tissue, pre-freezing the tissue in a tube or other container at -20°C or below can prevent degradation of antigen proteins associated with tissue disruption. For use, the tissue can be shredded immediately before extraction without thawing, and then disrupted by adding ice-cold saline. Both of these procedures are effective in minimizing denaturation and degradation of antigen proteins due to temperature rise. Since inbred animal species are used as donors (antigen-presenting animals), crude extracts can be prepared from tissues from multiple individuals, aliquoted, frozen, and then used for multiple antigen presentations. Furthermore, because there is no need to consider individual differences in newborns, unwanted newborns born irregularly and in uneven numbers during in-house breeding can be frozen and preserved as individuals for collecting presentation antigens. If sufficient supernatant cannot be collected, it can be collected by squeezing the sample in a filter with a pore size of 20 μm or less and squeezing the extract. Note that live cells should never be used as antigen-presenting proteins.This is because although they may present antigens, they may also engraft as viable cells, and if any surviving cells remain, after a transplant experiment, it will be impossible to distinguish between the cells that were already engrafted and the newly engrafted cells. The antigen-presenting protein must be completely degraded and absorbed after presentation, and disappear once. For this purpose, the process of freezing and crushing the cells is effective.

[0043] The crude extract to be administered is not particularly limited, but may contain a protein amount that is 1 / 30 to 1 / 15 of the body weight of the recipient newborn.

[0044] The step of administering the donor crude extract to the recipient neonate is carried out prior to the transplantation step. The method of administering the donor crude extract to the recipient neonate is not particularly limited, and for example, a method can be used in which the donor crude extract taken up by the recipient is recognized by antigen-presenting cells, activating T cells, and inducing regulatory T cells (Tregs) or an anergic state, thereby inducing immune tolerance. An anergic state is a state in which T cells become dysfunctional against donor antigens as well as against self-antigens, are not activated, and the immune response is suppressed. Such methods include, for example, subcutaneous administration, intramuscular administration, intravenous administration, intradermal administration, transdermal administration, and oral administration. Furthermore, in the present invention, the method of administering the donor crude extract to the recipient newborn is not limited to the methods described above, and examples that can be used include topical administration, transmucosal administration, intranasal administration, intratracheal administration, sublingual administration, nasal administration, buccal administration, rectal administration, intravaginal administration, intraarterial administration, intracardiac administration, intraosseous administration, intraperitoneal administration, intraorbital administration, intravitreal administration, subconjunctival administration, suprachoroidal administration, subretinal administration, intraarticular administration, periarticular administration, epicutaneous administration, and inhalation administration.

[0045] The donor crude extract can be administered to the newborn recipient once or several times. For example, the donor crude extract may be administered to the newborn recipient every 1-2 days for a total of 3-5 times. The first administration to the newborn recipient can be within 3 days after birth, preferably within 24 hours after birth. All administrations to the newborn recipient can be within 10 days after birth, preferably within 7 days after birth. The donor crude extract is administered to the recipient in an amount sufficient to induce immune tolerance in the recipient. For example, if the crude extract contains 1 / 20 the protein content of the recipient newborn's body weight, the first administration of the donor crude extract is administered within 24 hours after birth, at a weight of about 5% of the newborn's body weight, and then administered subcutaneously into the abdomen or other area every two days for a total of 4 times over the first 7 days after birth, at a weight of about 5% of the newborn's body weight. This will result in a recipient that is immune tolerant to the donor.

[0046] (Transplantation Step) In the transplantation step, a transplant material containing an artificial transplant material coated, adhered, infiltrated or encompassed with donor tissue or cells is transplanted into a recipient.

[0047] Transplant materials, including artificial transplant materials coated, adhered, infiltrated, or encompassed by donor tissue or cells expressing a marker, include materials derived from the donor tissue or cells along with the transplant material. For example, the transplant material can be a transplant material that encompasses donor tissue or cells. In the transplant material, the donor tissue or cells may coat, or may adhere to, infiltrate, or encompass the transplant material.

[0048] The implant material is not particularly limited and can be bioabsorbable or biocompatible. Materials that can be used for implants include, for example, PLLA (poly-L-lactic acid), PEG (polyethylene glycol), PGA (polyglycolic acid), copolymers of PGA and PLA, polyhydroxybutyric acid, polycaprolactone, polyethylene succinate, polydioxanone, and polybutylene succinate. Other materials include soluble cellulose-based materials such as oxidized cellulose, proteins, peptides, polyamino acids, polysaccharides, polyesters, polyamides, their derivatives, crosslinked materials, and copolymers, magnesium metal, calcium carbonate, calcium phosphate, hyaluronic acid, and hydroxyapatite, acrylic resins, fluororesins, polyolefins, silicones, polystyrene, polyesters, polyurethanes, polycarbonates, polyimides, their derivatives, crosslinked materials, and copolymers, as well as titanium, silica, and zirconia. Examples of proteins, peptides, and polyamino acids include collagen, gelatin, α-polylysine, ε-polylysine, polyglutamic acid, polyaspartic acid, fibrin, and fibroin. Examples of polysaccharides include chitin and chitosan. Examples of polyesters include polylactic acid, polycaprolactam, polydioxanone, polyglycolic acid, and polyhydroxybutyric acid. The material of the implant may be one or more of the above-mentioned materials, or may be a composite of two or more materials.

[0049] The implant material may also be coated with a coating material, including, for example, fibrin-based materials, collagen-based materials, hyaluronic acid-based materials, glycoprotein-based materials, and soluble cellulose-based materials such as oxidized cellulose.

[0050] The transplant material is not particularly limited, and can be, for example, a tubular structure, a membranous structure, any other shaped structure, an artificial organ, etc. Tubular structures can be used as transplant materials for regenerative medicine, such as blood vessels, respiratory system organs, digestive system organs, and urinary system organs. Membranous structures can be used as transplant materials for regenerative medicine, such as dura mater, peritoneum, fascia, periosteum, and synovium.

[0051] The artificial transplant material coated, adhered, infiltrated, or encompassed by donor tissue or cells can be provided in any manner. For example, the transplant material can be prepared by seeding donor cells onto the transplant material and culturing the cells.

[0052] The transplant material may be one that has been transplanted into the body of a donor, left in place for 2 to 30 days, and then retrieved. The transplant material may also be one that has been transplanted into the donor and retrieved multiple times. By transplanting the transplant material into the donor and leaving it in place for a certain period of time, the donor's tissue or cells can be sufficiently attached to or infiltrated into the transplant material.

[0053] The production method of the present invention may further include a step of transplanting the transplant material into the body of a donor, leaving it in place for 2 to 30 days, and then recovering the transplant material from the body of the donor, the transplant material containing the transplant material coated, adhered, infiltrated, or encapsulated with tissues or cells of the donor. This step may be carried out at any timing as long as it is carried out at least before the step of transplanting the transplant material into a recipient.

[0054] Transplant materials, including artificial transplant materials coated, adhered, infiltrated or encompassed by donor tissues or cells, may be coated, adhered, infiltrated or encompassed by donor tissues or cells throughout the entire transplant material, or only a portion of the transplant material may be coated, adhered, infiltrated or encompassed by donor tissues or cells.

[0055] The donor tissue or cells expressing the marker contained in the transplant material may be the donor tissue or cells themselves or processed versions thereof. The tissue or cells collected from the donor may be isolated, cultured, differentiated, selected, expanded, or cryopreserved as needed to prepare the transplant material, and then thawed. Alternatively, the tissue or cells may be established as a cell line by subculture.

[0056] Cells and various physiologically active substances (cytokines and cell growth factors) may be added to the transplant material by co-cultivation, spraying, application, infiltration, etc. Physiologically active 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.

[0057] The transplant material is not particularly limited, but can be used as, for example, blood vessels, respiratory system organs, digestive system organs, urinary system organs, dura mater, peritoneum, fascia, synovium, periosteum, serous membrane, and the like.

[0058] The transplant material is not particularly limited, but can be transplanted into, for example, an internal organ of the recipient, such as the brain, liver, kidney, or heart, or into the subcutaneous tissue, blood vessels, etc. The method for transplanting the transplant material into the recipient is not particularly limited, and any conventionally known method can be used.

[0059] The donor tissue or cells contained in the transplant material can be collected from, but are not limited to, an adult donor. When the donor is an inbred strain, not only can the donor be the individual from which the crude extract to be administered to the recipient's newborn be prepared, but also another individual of the same strain can be used as the donor, and the transplant material derived from that individual can be transplanted into the recipient.

[0060] The donor tissue or cells may be selected from the group consisting of, for example, fibroblasts, chondroblasts, osteoblasts, hemangioblasts, myoblasts, epithelial cells, smooth muscle cells, endothelial cells, vascular endothelial cells, fibrocytes, hepatocytes, chondrocytes, epithelial cells, urothelial cells, smooth muscle cells, keratinocytes, beta cells, small intestinal epithelial cells, epidermal keratinocytes, bone marrow mesenchymal cells, cardiac muscle cells, intervertebral disc cells, gastrointestinal mucosal epithelial cells, ureteral epithelial cells, skeletal joint synoviocytes, periosteal cells, perichondrocytes, skeletal muscle cells, smooth muscle cells, cardiac muscle cells, pericardial cells, dura mater cells, meningeal cells, pericytes, glial cells, neurons, amniotic cells, placental membrane cells, serous cells, ependymal cells, periventricular zone cells, oral mucosal epithelial cells, neurons and dendritic cells, genetically modified cells thereof, cells produced by gene manipulation techniques such as genome editing or transformation techniques using mRNA (messenger RNA) or the like, and cells obtained by culturing and / or differentiating these.

[0061] The donor tissue or cells may be selected from 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, blood 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, keratinocyte progenitor cells, organ-specific stem cells, progenitor cells of organ-specific stem cells, genetically modified cells thereof, and genome-edited cells. Somatic tissue stem cells include human somatic stem cells as defined in the Notification of the Director-General of the Pharmaceutical and Food Safety Bureau of the Ministry of Health, Labour and Welfare, dated September 7, 2012, No. 0907-3, entitled "Ensuring the Quality and Safety of Pharmaceuticals and Medical Devices Processed from Human Allogeneic Somatic Stem Cells." "Human somatic stem cells" refer to cells collected from a human or cells resulting from the division of such cells that are presumed to be pluripotent and maintain the ability to self-renew, or similar, 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 enriched therein (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. Human somatic stem cells also include cells obtained by culturing and / or differentiating these cells in vitro.

[0062] In addition, the donor tissues or cells may be "induced pluripotent stem cells" and "induced pluripotent stem cell-like cells" such as iPS cells produced by genetic engineering techniques such as gene recombination and genome editing, and transformation techniques using mRNA (messenger RNA), as well as cells cultured and / or differentiated from these.

[0063] The donor tissue or cells can be, for example, adipose 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 immunocompetent cells contained in the thymus, bone marrow, lymph nodes, etc.

[0064] (Method for producing a transplant model animal for evaluating transplant materials using a donor that expresses a labeled marker) A method for producing a transplant model animal for evaluating transplant materials using a donor that expresses a labeled marker of the present invention includes the steps of administering a crude extract of the donor that expresses the labeled marker to a newborn recipient to obtain an immunotolerant recipient, and transplanting into the immunotolerant recipient a transplant material containing an artificial transplant material coated, adhered, infiltrated or encompassed by tissues or cells of the donor that expresses the labeled marker.

[0065] As used herein, the term "labeled marker" refers to a marker that allows cells expressing the marker to be distinguished from cells not expressing the marker and observed. The labeled marker may be any marker that allows cells derived from a donor to be identified after transplantation into a recipient. The labeled marker may be, for example, a fluorescent protein. Fluorescent proteins include, for example, green fluorescent protein (GFP) from Aequorea victoria; modified versions of GFP such as EGFP; mutant versions of GFP that emit different colors of fluorescence (e.g., blue fluorescent protein (BFP), yellow fluorescent protein (YFP), and cyan fluorescent protein (CFP)); dsRed fluorescent protein (dsRed2FP); red fluorescent protein isolated from the coral sea anemone Entacmaea quadricolor (eqFP611); cyan fluorescent protein isolated from Anemonia majano (amFP486 or AmCyan1); fluorescent protein isolated from Galaxeidae (Azami Green); fluorescent protein isolated from Zoanthus (ZSGREEN); and any other fluorescent protein.

[0066] If the labeling marker is a fluorescent protein such as EGFP, it can be stably expressed in vivo, allowing for easy fluorescent observation of donor-derived cells in the recipient's body after transplantation. Furthermore, fluorescent proteins such as EGFP emit fluorescence upon irradiation with excitation light, eliminating the need for tissue staining and allowing for easy observation of live tissue. When the labeling marker is EGFP, for example, green fluorescence with a maximum fluorescence wavelength of 507 nm can be obtained by using excitation light between 470 nm and 530 nm, particularly the EGFP-specific excitation light of 488 nm.

[0067] A labeled marker-expressing donor is an animal into which a labeled marker gene has been introduced and which constitutively and stably expresses (or is capable of expressing) the labeled marker in some or all of its cells. A labeled marker-expressing donor can be prepared by introducing the labeled marker gene by a conventionally known method.

[0068] In the method of the present invention, the recipient may be, but is not limited to, an animal that does not have at least the marker introduced into the donor, such as a wild-type mouse (WM) that has not been genetically engineered. Furthermore, to increase the accuracy of the experiment, the recipient may be an inbred animal.

[0069] Because the recipient does not have a marker introduced, there is a high possibility that the marker marker contained in the donor-derived cells or other donor-derived antigens will induce an immunological rejection reaction if transplanted using a conventional transplantation method. However, with the method of the present invention, by administering a crude extract from a donor expressing a marker to the newborn recipient in advance, immune tolerance can be induced against donor-derived antigens, including the marker marker, thereby suppressing immune responses during transplantation limited to only donor-derived antigens. Meanwhile, normal immune responses to antigens not derived from the donor are maintained.

[0070] By using a labeled marker-expressing donor, donor-derived cells and tissues can be easily observed in the recipient after transplantation. Furthermore, it is easy to distinguish whether cells and tissues, etc., developed in the recipient after transplantation are recipient-derived or donor-derived. Therefore, for example, if a tumor or the like develops in the recipient after transplantation, it can be easily determined whether it arose from donor-derived cells or recipient-derived.

[0071] (Method for evaluating the behavior of a transplant material) The method for evaluating the behavior of a transplant material of the present invention comprises the steps of first administering a crude extract from a donor expressing a labeled marker to a newborn recipient to obtain a recipient that is immunotolerant to the donor, and then transplanting into the immunotolerant recipient a transplant material comprising an artificial transplant material coated, adhered, infiltrated, or encapsulated with tissues or cells of the donor expressing the labeled marker. The method for producing a transplant model animal as a transplant model animal is as described above in (Method for producing a transplant model animal for evaluating a transplant material). The donor and recipient are inbred animals. The transplant model animal produced by the method for producing a transplant model animal for evaluating a transplant material of the present invention is a transplant model animal into which a transplant material has been transplanted and in which immunotolerance has been induced at the time of transplantation.

[0072] The method for evaluating the behavior of a transplant material of the present invention further includes a step of observing the transplant material in a recipient into which the transplant material has been transplanted. This allows the behavior of the transplant material in the body of a transplant model animal to be evaluated. As used herein, "evaluating" includes observing. Furthermore, evaluating includes assessing whether the transplant material used in the transplant material has qualities suitable for transplantation. Qualities suitable for transplantation include, for example, biocompatibility, functionality at the transplant site, and the presence or absence of toxicity. Evaluating includes, for example, assessing the biocompatibility of the transplant material by observing the immune response and local reaction of an immunotolerant recipient after transplantation. Evaluating also includes assessing functionality by confirming the function of the transplant site. Evaluating also includes assessing the presence or absence of toxicity by observing the recipient's overall condition, observing changes in weight, appetite, and behavior, and conducting blood tests and urine tests.

[0073] Furthermore, the evaluation includes confirming whether new blood vessels derived from the donor are formed around the transplant material or whether donor-derived cells have engrafted, and assessing the transplant material as biocompatible if these are observed. The method for evaluating the behavior of a transplant material of the present invention uses a donor that expresses a labeled marker, and therefore the use of the labeled marker makes it possible to easily distinguish between donor-derived cells and recipient-derived cells. This makes it possible to evaluate the behavior of the transplant material in the body of a transplant model animal. Therefore, as used herein, "observing" includes observing the behavior of donor-derived cells, i.e., the labeled marker, in the recipient transplant model animal.

[0074] (Transplantation model animal and method for producing same) The present invention also provides a transplantation model animal that resembles an autologous transplant, produced by a method for producing a transplantation model animal, the method comprising: administering a crude extract of a donor expressing a labeled marker to a newborn recipient to obtain a recipient that is immunotolerant to the donor; and transplanting a transplantation material containing an artificial transplant material coated, adhered, infiltrated, or encapsulated with tissues or cells of the donor expressing the labeled marker into the immunotolerant recipient.

[0075] The present invention also provides a method for producing a transplantation model animal that resembles an autotransplant, comprising the steps of administering a crude extract from a donor that expresses a labeled marker to a newborn recipient to obtain a recipient that is immunotolerant to the donor, and transplanting a transplantation material containing an artificial transplant material coated, adhered, infiltrated, or encompassed by tissues or cells of the donor that expresses the labeled marker into the immunotolerant recipient. The transplantation model animal of the present invention can be used to evaluate transplantation materials that resemble autotransplantation.

[0076] The transplantation model animal of the present invention can be produced by a method comprising an immune tolerance step and a transplantation step, as explained above in (Method for producing a transplantation model animal for evaluating transplantation materials using a labeled marker-expressing donor). That is, the transplantation model animal of the present invention is produced by administering a crude extract from a labeled marker-expressing donor to a newborn recipient to obtain an immune-tolerant recipient, and then transplanting into the immune-tolerant recipient a transplantation material comprising an artificial transplant material coated, adhered, infiltrated, or encapsulated with tissues or cells of the labeled marker-expressing donor. Details of the immune tolerance step and the transplantation step are as explained above in (Method for producing a transplantation model animal for evaluating transplantation materials).

[0077] In the transplant model animal prepared as described above, the transplanted transplant material is labeled with a labeling marker. Therefore, the transplant model animal of the present invention can easily confirm not only how the recipient transplant model animal behaves, but also how the transplanted transplant material behaves inside the recipient transplant model animal. The location of donor-derived cells and the like in the recipient can be easily observed by fluorescence.

[0078] The transplant model animal of the present invention is a recipient that is transplanted with a transplant material after immune tolerance to the donor has been induced in advance, so the recipient transplant model animal has a suppressed immune response to the transplant material. Therefore, for example, it can be used as an experimental system for observing the biological response and progression of transplantation of a transplant material. Meanwhile, immune responses to all antigens not derived from the donor are maintained, and experimental results closer to those of clinical transplantation than ever before can be expected, with regard to 1. the risk of interrupting the experiment due to infection during the course of the experiment and 2. the risk of increased risk of cancer.

[0079] The present invention provides a so-called "approximate autotransplantation animal experimental model" by using inbred animals. By using inbred animals, the present invention can easily induce immune tolerance even in immunocompetent animals, thereby establishing an animal model that can avoid immune reactions against specific individuals. Therefore, the present invention eliminates the need for conventional immunodeficient animal experimental systems and allows transplantation experiments to be performed under conditions similar to clinical conditions using immunocompetent animals. Furthermore, the present invention can be used as an experimental system to observe the biological responses and progress of autotransplantation of transplant materials using small animals, such as mice, for which autotransplantation is traditionally difficult. Therefore, it can serve as an alternative to autotransplantation experiments that were previously only possible using medium- and large-sized animals, and it is in line with animal welfare trends while also complying with the Ministry of Health, Labor, and Welfare's recommendations regarding the development of regenerative medicine products based on the three regenerative medicine laws.

[0080] In the future, various regenerative medicine products will be researched, developed, and commercialized, but most of these will likely be artificial organs that humanity has never had before. By using the transplant model animal of the present invention, various conditions can be examined through animal experiments and other methods before artificial organs for which no prior performance information is available are provided for clinical use in humans, and the design, performance, and surgical procedures that are more suitable for clinical use can be thoroughly examined in advance.

[0081] Furthermore, by using the transplant model animal of the present invention, it is possible to conduct experiments under conditions that are closer to actual clinical care than conventional animal experiments with similar purposes (such as experimental systems using immunodeficient animals) due to immune tolerance.

[0082] Until now, transplant treatments have relied on the "homologous use" of derived organs. Because the function (performance) of transplanted organs / tissues was confirmed and guaranteed in advance within the donor's body, performance evaluation of the organ alone prior to transplantation was unnecessary and should not have been performed. In contrast, while performance evaluation of regenerative medicine products is possible in vitro, in vivo performance is not guaranteed in advance. Therefore, in vivo post-transplant function evaluation, such as through animal testing, is essential. Furthermore, in conventional transplants, the success of post-transplant function depends solely on the transplantation procedure and the success of suppressing immune rejection. In this regard, the conditions suitable for transplantation procedures for regenerative medicine products should be considered in advance, and efforts should be made to manufacture products that avoid immune rejection as much as possible.

[0083] The use of the transplant model animal of the present invention enables experiments similar to autologous transplants, which were previously impossible in small animals. This allows for the design and development of regenerative medicine products using "autologous biological tissue" as organs that are semi-permanently implanted within an individual as living tissue. This allows for the production of transplant materials that do not require immune response. This allows for the development of artificial organs that do not pose the risk of prolonged immunosuppression (infections, carcinogenicity) due to the permanent use of immunosuppressants, and also reduces permanent treatment costs (immunosuppressants, etc.). Given these factors, the most important aspect of regenerative medicine is ensuring the performance and quality of transplant medical materials, including regenerative medicine products. Furthermore, the use of the transplant model animal of the present invention allows for the selection of widely available, inexpensive experimental materials (laboratory animals such as mice and their breeding materials) and research facilities (clean rooms and facilities for preventing the spread of genetically modified animals), thereby significantly reducing the necessary expenses (initial and ongoing costs) associated with conducting experiments. Immunocompromised experimental animals are expensive, require specialized facilities and feed, are expensive to maintain, and are prone to infection. The transplant model animal of the present invention has normal immune function and immune responses other than those to presented antigens are not suppressed at all, so the initial and maintenance costs are low, it is resistant to infection, and there are few inconveniences in raising it.

[0084] Furthermore, the use of the transplantation animal model of the present invention eliminates the need for medium- or large-sized animals, which are essential for conventional autologous transplantation experimental systems. This eliminates the need for animal welfare and other social trends, and offers cost and labor advantages. As a result, the ease of conducting experiments and the short lifespan of these animals allow for the design and performance evaluation of various transplantation medical materials under different conditions. For example, if the lifespan is divided into childhood (pre-reproductive), reproductive, and post-reproductive (senile) stages, small animals can reach the appropriate age for senile experiments after only a few months of care. Even if processing donor-derived transplant candidate cells (such as iPS cell generation and various differentiation processes, or the preparation of composite transplant materials with autologous materials or bioabsorbable materials) requires time, as long as the donor is an inbred animal species and has undergone immune tolerance treatment, multiple individuals from each lifespan can be used simultaneously for experiments. As a result, the ability to conduct multiple experiments simultaneously not only enables evaluation of the performance of the artificial organ itself, but also the effects of variations in surgical technique, concomitant medications, and equipment. Furthermore, it will also be possible to consider the design of more customized artificial organs (i.e. regenerative medicine products) that are tailored to the differences in surgical conditions and individual recipient conditions.

[0085] Figure 1 shows an overview of the tests performed in this example. The following explains the abbreviations used in the following examples. Explanation of terms: GM: Green mouse (a genetically modified inbred mouse strain that constitutively expresses EGFP); ICR mouse: an inbred laboratory mouse strain with the most prolific albino lineage; P1, P1A: Experimental facilities that comply with specific measures (containment measures) to prevent the spread of genetically modified organisms in accordance with the Cartagena Protocol, an international treaty. Among these, P1 and P1A have the least restrictive conditions. P1 is equipment required primarily for handling cells, and P1A is equipment required primarily for handling animals. 1. P1A (Animal Experimental Facility) Collection of GM-derived tissues and production of crude proteins The process of collecting donor-derived antigen-presenting proteins to induce immune tolerance in the recipient. 2. P1A (Animal Experimental Facility) Immune tolerance treatment of ICR mice (wild species) The process of administering antigen proteins to newborn recipients to induce immune tolerance. 3. P1A (Animal Experimental Facility) The process of collecting cells and tissues as transplant candidates from donors. 4. P1 (Cell Experimental Facility) The process of processing the collected donor-derived cells, etc. as needed, or preparing them as transplant material. 5. P1A (Animal Experimental Facility) The process of transplanting material collected from donors into immune-tolerant recipients. 6. P1A (Animal Experimental Facility) The process of observing the condition after transplantation at the appropriate time, evaluating the performance of the transplant material, and, if necessary, considering the design of the transplant material.

[0086] (Donor) The donor used was a genetically engineered inbred mouse strain (Green Mouse: C57BL / 6-(CAG-EGFP) C14-Y01-FM131Osb (RBRC00267: RIKEN BioResearch Center)) that expresses EGFP (Enhanced green fluorescence protein) throughout its body through genetic modification (hereinafter referred to as "Green Mouse (GM)"). Note that an inbred mouse strain is a mouse strain that has undergone repeated inbred mating and exhibits phenotypic homology between generations and / or between individuals equivalent to that of identical twins in terms of genetic traits and their expression.

[0087] Figure 2 shows photographs of the fluorescence of a donor green mouse. By irradiating it with excitation light (ultraviolet light in the case of GM), green fluorescence due to intracellular EGFP can be confirmed. Fluorescence was observed in the whole-body epidermis of a newborn mouse (left side of Figure 2), and in the excised brain (center of Figure 2) and exposed epidermis (excluding body hair) of an adult mouse (right side of Figure 2).

[0088] (Preparation of Donor-Derived Crude Extract) Tissues were collected from donor green mice and disrupted using a homogenizer (tissue disruption device), followed by centrifugation or filtration to recover the solution. Specifically, the whole bodies of 3–5 newborn pups were pre-minced into pieces several millimeters in diameter using scissors or other tools. An amount of ice-cold saline equal to the weight of the tissue was then added. The tissue was then homogenized for approximately 3–5 minutes, maintaining the temperature at 0–4°C, while being intermittently disrupted using a homogenizer (tissue disruption device) until a sufficiently thick supernatant was extracted. The supernatant was then centrifuged (200–1000 rpm). This solution containing soluble proteins was designated the crude extract. The crude extract is also referred to as the crude protein solution (Step 1 in Figure 1). The top panel of Figure 3 shows the recovered crude protein solution. As shown in the figure, fluorescence from the crude extract in the test tube was confirmed when irradiated with EGFP-specific excitation light. In order to efficiently induce immune tolerance, a crude extract containing the transplant candidate tissue can be prepared, or the crude extract can be further purified before use.

[0089] (Immune Tolerance Step) ICR mice (wild type inbred strain) were used as recipients and were commercially available as experimental animals. The ICR mice were subjected to immune tolerance treatment (Step 2 in Figure 1). Approximately 5% of the donor's body weight (BW) of the crude extract was initially administered subcutaneously into the abdomen of newborn ICR mice within 24 hours of birth. Over the next seven days, the donor-derived crude extract was administered subcutaneously into the abdomen four times every two days. The subcutaneously administered crude extract contained a protein amount equivalent to 1 / 20 of the recipient's body weight. The mice were then housed normally for at least four weeks.

[0090] Figure 3 shows recipient mice that underwent immune tolerance treatment. A donor-derived crude extract in a test tube, which exhibited green fluorescence when irradiated with the EGFP-specific excitation light shown in the upper panel of Figure 3, was administered subcutaneously to the midline of the abdomen of an ICR neonate (lower left panel of Figure 3). When the same excitation light was then irradiated onto the abdomen, the presence of green fluorescent protein could be confirmed through the skin (lower right panel of Figure 3). The donor-derived crude extract taken up by the recipient is thought to be recognized by antigen-presenting cells in the recipient's body, activating T cells and inducing regulatory T cells (Tregs) or an anergic state, thereby inducing immune tolerance.

[0091] (Preparation of Composite Transplant Materials) A tubular structure with a silicone tube as a 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. In this example, a tube made of biocompatible materials such as silicone (product name: Saffied Silicone Catheter, outer diameter 2.7 mm) (hereinafter referred to as silicone tube) was used as a prototype. A white self-assembling collagen sheet (product name: Integran, sheet type 45 × 30 mm) (hereinafter referred to as collagen sheet) was wrapped around this silicone tube in two or more layers. Next, to prevent unwinding or unraveling, a blue-purple bioabsorbable PGA thread (PGA: polyglycolic acid) was further wrapped around it to secure it in place (Figure 4, upper left). At this stage, the wrapped tubular collagen sheet was not watertight and thus easily leaked.

[0092] Figure 4 is a photograph showing the state of transplantation of a material into a donor green mouse. The upper left of Figure 4 shows the tubular structure that is the transplant material transplanted into the donor. The lower left of Figure 4 shows the state of transplanting the transplant material into a green mouse. The upper right of Figure 4 shows the state one week after transplantation of the transplant material into the green mouse. One week after transplantation, it was observed that new blood vessels had been induced. The lower right of Figure 4 shows the state of fluorescent observation of the transplant material containing the transplant material that was extracted and recovered from the green mouse.

[0093] (Step of transplanting the transplant material into a donor) Figure 4 shows the step of transplanting the above-mentioned tubular structure (transplant material) into a donor green mouse (lower left diagram of Figure 4). A skin incision of approximately 1 cm was made on the back of an adult green mouse that will serve as the donor, and then a tunnel-like subcutaneous tissue of approximately 3 cm in length was excised to create an insertion cavity. An outer tube was inserted into the insertion cavity, and the tubular structure was inserted subcutaneously through this. When the entire inserted tubular structure could be comfortably embedded subcutaneously, the outer tube was removed, and the insertion hole was sutured closed.

[0094] One week after subcutaneous implantation, the skin of the transplanted green mouse was incised and turned over enough to allow the entire tubular structure to be seen, and the entire tubular structure was confirmed (Figure 4, upper right). The wrapped collagen sheet was moistened with interstitial fluid and covered with green mouse-derived cells, making it translucent. It was connected to the surrounding bed tissue by a membranous connective tissue. Abundant bridging of new blood vessels was observed in the collagen tube, based on this connective tissue, and it was confirmed that fine blood vessels had grown around the entire circumference.

[0095] One week after transplantation, the tubular structure was extracted and collected as a transplant material (lower right of Figure 4; corresponding to steps 3 and 4 in Figure 1). The connective tissue connected to the host tissue was incised, and the collagen tube was extracted without damaging it.

[0096] When the extracted tubular structure was irradiated with excitation light, the transplant material emitted fluorescence, revealing that green mouse-derived cells had been evenly seeded into the collagen tube. It was also confirmed separately that at this stage, the structure had already achieved watertightness with no water leakage. Furthermore, the cells seeded on the collagen sheet from the extracted collagen tube were viable cells that could be cultured.

[0097] (Step of Transplanting the Transplant Material into an Immunotolerant Recipient) The transplant material containing the tubular structure recovered from the green mouse was subcutaneously transplanted into wild-type ICR mice lacking a marker and induced tolerogenic to the green mouse, using the same procedure as described above (Preparation of Composite Material for Transplantation). Specifically, a 1 cm skin incision was made on the back of the ICR mouse, and a 3 cm tunnel was excised from the subcutaneous tissue to create an insertion cavity. A mantle tube was inserted into the insertion cavity, and the tubular structure was inserted subcutaneously through this tube (Figure 5). When the entire inserted tubular structure could be comfortably embedded subcutaneously, the mantle tube was removed, and the insertion hole was sutured closed.

[0098] One month after subcutaneous implantation, the skin was incised enough to allow a full view of the tubular structure, and the structure was turned over to confirm its overall appearance (Figure 6, left side). The wrapped collagen sheet was moistened with interstitial fluid and was covered with tissue cells derived from ICR mice, starting from the green mouse-derived cell layer, reducing its transmittance to visible light. It was firmly connected to the surrounding bed tissue by connective tissue. Abundant bridging of new blood vessels was observed in the tubular structure, based on this connective tissue, and it was confirmed that the green mouse-derived new blood vessels had further developed, providing a vascular network around the entire circumference.

[0099] Furthermore, the fluorescence characteristic of green mice was confirmed when irradiated with excitation light (right side of Figure 6), confirming that green mouse-derived cells were able to engraft without immune rejection, even in the subcutaneous tissue of immune-tolerized wild-type ICR mice, where a particularly strong immune response is likely to be elicited. Furthermore, culturable living cells were confirmed in tissues extracted from the tubular structures transplanted into ICR mice, and the coexistence of green fluorescent cells and non-fluorescent cells was confirmed.

[0100] FIG. 5 shows the state of a transplant material containing a tubular structure transplanted into a recipient mouse.

[0101] Figure 6 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, observed under fluorescent light. One month after transplantation, fluorescent light observation revealed the distribution of donor-derived cells and tissues attached to the tubular structure, as well as new cells and tissues that developed from them.

[0102] This invention can be used as a highly reliable and reproducible transplantation experiment system, and will contribute to the evaluation of the design, performance, and surgical procedures of unknown artificial organs that may be developed in the future.

Claims

1. A method for producing a transplant model animal for evaluating a transplant material, comprising the steps of: administering a crude extract of a labeled marker-expressing donor to a newborn recipient to obtain a recipient that is immunotolerant to the donor; and transplanting a transplant material containing an artificial transplant material coated, adhered, infiltrated or contained with tissues or cells of the labeled marker-expressing donor into the immunotolerant recipient, wherein the donor and the recipient are inbred animals.

2. A method for producing a transplant model animal for evaluating the transplant material according to claim 1, wherein the transplant material is a tubular structure, a membrane structure or an artificial organ.

3. A method for producing a transplant model animal for evaluating a transplant material according to claim 1 or 2, wherein the transplant material is transplanted into the body of the donor, left therein for 2 to 30 days, and then recovered.

4. A method for producing a transplant model animal for evaluating a transplant material according to claim 1 or 2, wherein the crude extract is a crude protein solution extracted from cells or tissues of the donor.

5. A method for producing a transplant model animal for evaluating a transplant material according to claim 4, wherein the labeling marker is a fluorescent protein.

6. A method for producing a transplant model animal for evaluating the transplant material described in claim 1 or 2, wherein in the step of obtaining a recipient immunotolerant to the donor, the crude extract is administered subcutaneously, intramuscularly, intravenously, intradermally, transdermally or orally to a newborn of the recipient.

7. A method for evaluating the behavior of a transplant material, comprising: administering a crude extract of a donor expressing a labeled marker to a newborn recipient to obtain a recipient that is immunotolerant to the donor; and transplanting a transplant material containing an artificial transplant material coated, adhered, infiltrated or contained with tissues or cells of the donor expressing the labeled marker into the immunotolerant recipient, the method comprising: producing a transplant model animal similar to an autologous transplant by a production method, the donor and the recipient being inbred animals; and observing the labeled marker in the transplant model animal, thereby evaluating the behavior of the transplant material in the body of the transplant model animal by the labeled marker.

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