JP7627920B1 - Methods for evaluating implant materials - Google Patents

Abstract

[Problem] To provide a method for producing a transplant model animal that enables the realization of a highly reliable and reproducible transplant experimental system in order to design transplant materials with performance and quality more suited to clinical medical care.
[Solution] The method of the present invention for evaluating transplantation materials similar to autotransplantation comprises 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 transplantation 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, thereby making it possible to produce a transplantation model animal similar to autotransplantation, which can realize a highly reliable and reproducible transplantation experimental system.
[Selected Figure] Figure 1

Description

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

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 an animal's growth is inhibited by some kind of disease or injury during its development or growth period, resulting in failure to obtain the desired functions and/or structures.

One of the most effective methods in regenerative medicine is to transplant medical materials derived from living organisms, including cells or tissues, into the missing parts of the body. In Japan, such medical materials are defined as "regenerative medicine products" under the three laws related to regenerative medicine (the Regenerative Medicine Promotion Act, the Regenerative Medicine Safety Assurance Act, and the revised Pharmaceutical Affairs Act).

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

In addition, artificial materials are used to complement the elements that bio-derived medical materials alone are insufficient for. Such artificial materials must not cause undesirable reactions in the body, including immune reactions. Conventionally, not only bio-derived materials, including regenerative medicine products, but also various medical materials called biocompatible materials and bioaffinity materials have been used (for example, Patent Documents 1 to 4). However, it is difficult to verify whether these materials have optimal materials, physical properties, and configurations before transplantation. In particular, it has been difficult to verify the performance of regenerative medicine products that contain living cells and tissues in advance.

To guarantee the quality of regenerative medicine products, a clinically appropriate system is required that allows for advance animal testing using mammals and for tracking the progress of immune responses after transplantation. For such animal testing, an animal testing model that performs "autologous transplantation," in which cells and tissues taken from an individual to be transplanted are returned to that individual, is suitable.

However, animal experimental systems using autotransplantation have rarely been used as a research tool up until now. In particular, there have been no autotransplantation experimental models for small animals. The reason for this is that autotransplantation is difficult in small animals, including mice and rats. Even if the amount of cells or tissue collected for transplantation is less than 1g, small animals cannot withstand the collection procedure, and depending on the tissue collected, the amount may be fatal. In small animals, tissues and cells are generally collected after the individual is killed and blood is removed. As a result, the individual is lost during the collection procedure, making it impossible to perform autotransplantation.

Although autotransplantation is possible in medium- and large-sized animals, it is difficult to arrange a sufficient number of experimental cases due to the expense of raising them. Furthermore, with the current spread of animal welfare sentiment, there is a trend toward stricter regulations on animal testing itself.

For this reason, most of the conventional research is based on experimental systems centered on allografts and xenografts in small animals. Since allografts and xenografts are not autotransplants, they require the use of immunosuppressants or severely immunodeficient animals (nude mice, NOD/scid mice, NOG mice, etc.), or a combination of these, in order to avoid immune reactions. This requires the use of animals that are expensive to produce and keep alive, which increases the cost and labor involved. Furthermore, such experimental systems require the use of immunosuppressants or the use of advanced dedicated facilities at the level of a clean room due to the high risk of infection caused by immunodeficiency. There is also the problem of the need to use immunosuppressants for a long period of time, which adds to the cost.

Furthermore, the immunodeficiency experimental system has a serious problem in that the experiment is conducted under conditions that are significantly different from clinical practice in medical and veterinary medicine. In particular, in humans, patients with immunodeficiency and patients who have been using immunosuppressants for a long time are rarely the subjects of transplantation therapy. When a treatment method established in an immunodeficiency experimental system is introduced to patients or livestock with normal immune functions, there is a risk of unexpected immune reaction occurring. Therefore, the present invention provides an animal experiment system that allows the design and development of transplant materials with more appropriate performance and quality while avoiding these problems. In particular, the present invention provides an animal experiment system that can evaluate the engraftment and reproducibility of structure/function of the target tissue. More specifically, the present invention provides an animal experiment system that can not only minimize rejection reactions, but also evaluate the presence/suitability of reactions that support/assist transplanted tissue (blood flow induction/angiogenesis/regulation of expression of nutritional factors, etc.).

JP 2015-89433 A JP 2007-186831 A JP 2013-162796 A Patent Publication No. 2008-523957

There is a need to develop a reliable and reproducible system for verifying whether the materials used in transplants, such as biologically derived materials, including regenerative medicine products, as well as biocompatible and biocompatible materials, have the optimal composition and structure for treatment. For this purpose, transplant experiments using animals are essential, and transplant materials should be designed based on these experiments.

Considering costs, etc., it is suitable to use small animals for animal experiments. However, autotransplantation is difficult with small animals because the animals are small in size and must be sacrificed when cells are harvested. On the other hand, allotransplantation can cause immune reactions after transplantation (such as immune rejection), so it is performed in immunodeficiency experimental systems and experimental systems using immunosuppressants. However, such experimental systems are significantly different from the actual situation in clinical treatment, and lack reliability and reproducibility. Another problem with immunodeficiency experimental systems is that they are expensive for experimental materials and require highly specialized equipment.

The present invention aims to provide a method for producing a transplant model animal that can realize a highly reliable and reproducible transplantation experimental system.

Donor grafts are commonly used for transplantation, including autografts, allografts, and xenografts. All of these are raw grafts (unprocessed transplant materials) in which the tissue is harvested and transplanted "shortly" without any special processing. "Shortly" can mean transplantation immediately after harvesting, or a certain amount of time has passed since refrigeration or freezing, or for component transfusions, blood preparations, and autologous blood transfusions, which require separation and purification of components. The premise of these raw grafts is that they are transplanted with the aim of utilizing the characteristics/state of the cells and tissues at the time of harvesting. Because they are used without processing and with their characteristics/state intact as the tissue in question, it has generally been thought that there is no need to evaluate their performance/quality.

On the other hand, the medical material "auto-plast" (a term coined in this invention) used in what is referred to as "auto-plastic transplantation" in this invention is a material that does not fit the conventional concept of medical materials, and as such, there has been no system to properly evaluate its performance. Auto-plast is a medical material that is used in what is referred to in this invention as "auto-plastic transplantation" in which various processes such as differentiation and transformation are carried out to suit the cells and tissues to be transplanted, resulting in a material that is completely different in character and condition from the time of collection.

It is clear that the amount of medical materials produced as regenerative medicine products will increase in the future, and according to the Ministry of Health, Labour and Welfare's "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 a "test to verify efficacy or performance" to evaluate quality.

To summarize the above, autotransplantation usually refers to autograft, and transplant materials (grafts) are 1. transplanted immediately after collection into tissue equivalent to the original tissue without processing, 2. transplanted in the collected state, and 3. used with their original properties/characteristics. Blood transfusions are performed as blood into blood vessels, liver transplants are performed on the liver, and skin is used as a skin transplant.
In this invention, autotransplantation includes "auto-plast" (a term coined by this invention), which is an expanded concept of transplantation, and includes the use of tissues produced by processing cells and tissues derived from the patient in various ways, and in some cases producing tissues in a state different from the original state, as transplant materials. A representative example is regenerative medicine products defined in the three regeneration methods, which propose a method that enables the design and quality evaluation of medical materials made from biological materials.

The conventional concept of transplantation is "transplant (including allogeneic) graft" ⊇ "auto-graft," and in principle, grafts are used (transplanted) in the state in which they are harvested. The concept of "graft" does not include the state in which harvested transplant materials have been processed. Furthermore, the concept of "plastic grafts" created by processing biomaterials has not yet been clearly stated. "Auto-plast," the name of transplant materials created by processing autologous biomaterials, is a coined word by the present invention, and the concept also includes auto-plast as a "composite material" combined with artificial materials, and no specific verification method has been proposed for their use in regenerative medicine.

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.

Furthermore, as a method for creating "Auto-plast",
1. It is expected that most will be produced in vitro, but some may be
2. It is also assumed that ex vitro/in vivo (intracellular production) is possible.
Regardless of the method used for preparation, the quality of the material as a transplant material can be evaluated in the present invention.

In order to solve the above problems, the present inventors used inbred mice expressing EGFP as donors, and administered crude protein extracted from the donor's tissue subcutaneously several times to newborn wild-type recipients to induce immune tolerance. An artificial transplant material was then transplanted into the donor. As a result, the donor's tissues and cells adhered to the transplanted artificial transplant material. When a transplant material containing the transplant material produced in the donor's body was transplanted into an immune-tolerant recipient, an immune response to the donor-derived cells/tissues could be avoided. Furthermore, by fluorescent observation, the engraftment of the donor-derived cells and tissues together with the transplant material in the recipient after transplantation could be easily observed. The present invention was completed based on these findings.

That is, the present invention relates to a method for producing a recipient having immune tolerance to said donor, comprising administering a crude extract of a donor expressing a labeled marker to a newborn of the recipient;
and transplanting the transplant material, which comprises an artificial transplant material coated, adhered, infiltrated or encapsulated with tissue or cells of the labeled marker-expressing donor, into the immunotolerant recipient;
The donor and recipient are inbred animals, providing a method for evaluating transplant materials that approximates autologous transplants.

The present invention also provides a method for evaluating a transplant material, in which the transplant material is a tubular structure, a membrane structure, or a solid artificial organ.

The present invention also provides a method for evaluating a transplant material, which is prepared by the above-mentioned production method, and is then transplanted into the body of a donor, allowed to remain there for 2 to 30 days, and then recovered.

The present invention also provides a method for evaluating a transplant material, which is a crude protein solution extracted from donor cells or tissues, using the above-mentioned production method.

The present invention also provides a method for evaluating transplant materials, in which the labeling marker is a fluorescent protein.

The present invention also provides a method for the preparation of a recombinant human ovarian tumor comprising administering to a newborn recipient a crude extract of a donor expressing a target marker, thereby obtaining a recipient that is immunotolerant to said donor;
and a step of transplanting a transplant material comprising an artificial transplant material coated, adhered or infiltrated with tissue or cells of the labeled marker-expressing donor into the immunotolerant recipient.

The present invention also provides the above-mentioned transplant model animal being a mouse.

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.

FIG. 1 is a diagram showing an outline of a test carried out in an embodiment of the present invention. FIG. 1 shows the fluorescence observation of a donor green mouse. FIG. 1 shows a recipient mouse that underwent immune tolerance treatment. FIG. 4 shows the appearance of the material transplanted into a donor green mouse. The upper left of FIG. 4 shows the transplanted tubular structure, the lower left of FIG. 4 shows the appearance of the transplant material in a green mouse, the upper right of FIG. 4 shows the appearance of the transplant material one week after transplantation, and the lower right of FIG. 4 shows fluorescent observation of the transplant material including the transplant material recovered from the green mouse. FIG. 1 shows the state of a tubular structure, which is a transplant material, transplanted into a recipient mouse. This is a diagram showing the transplant material one month after transplantation into the recipient. The left side of FIG. 6 shows the tubular structure (transplant material) one month after transplantation, and the right side of FIG. 6 shows the tubular structure (transplant material) one month after transplantation, observed under fluorescent light.

The present invention provides a method for evaluating transplant materials and an animal transplant model similar to an autologous transplant for use in the method for evaluating transplant materials, thereby enabling the design of transplant materials and the verification of their performance and quality.

(Method of evaluating transplantation materials)
The transplant model animal of the present invention used in the method for evaluating a transplant material 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. The transplant model animal of the present invention can be suitably used in an experimental system for observing the reaction and progress of the living body when a transplant material is transplanted.

The animals used in the method for evaluating transplant materials of the present invention are not particularly limited, but 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, flatworms such as planaria, 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.

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

The method for evaluating transplantation materials of the present invention includes a step of administering a crude extract of a labeled marker-expressing donor (hereinafter also simply referred to as a donor) to a newborn recipient to obtain an immunotolerant recipient (immunotolerance step), and a step of transplanting a transplantation material containing an artificial transplantation material coated, adhered, infiltrated or contained with tissues or cells of the labeled marker-expressing donor into the immunotolerant recipient (transplantation step). In this specification, the term "covering" by tissues or cells has the usual meaning in the art, and includes tissues or cells covering a part or all of the transplantation material. In addition, in this specification, the term "adhesion" by tissues or cells has the usual meaning in the art, and includes, for example, tissues or cells attaching to a part or all of the transplantation material, for example, sticking to the tissues or cells and not releasing them. In this specification, the term "infiltrating" by tissues or cells has the usual meaning in the art, and includes, for example, tissues or cells penetrating and spreading into a part or all of the transplantation material, for example, adhering to the inside. As used herein, "containing" a tissue or cell has its usual meaning in the art, including, for example, covering the entire implant material.

(Donor and Recipient)
As used herein, a "donor" is an animal that provides tissue or cells contained in the transplant material, and a "recipient" is an animal that receives the transplantation of the transplant material.

(Recipient immune tolerance process)
In the immune tolerance step, a crude extract from a donor expressing a labeled marker is administered to a newborn of a 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.

The crude extract of the donor 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 tissue or cells to be transplanted. The crude extract can be prepared as a crude extract solution, for example, by disrupting tissue or cells collected from the donor with a homogenizer, centrifuging or filtering, etc., and collecting a supernatant containing soluble proteins. The crude protein solution may be further concentrated, etc. Also, after preparation, it may be frozen and stored, and then thawed.

The amount of protein in the crude extract to be administered is not particularly limited, but may be 1/30 to 1/15 of the body weight of the recipient newborn.

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 may be subcutaneous administration, topical administration, transdermal administration, intradermal administration, transmucosal administration, oral administration, intranasal administration, intratracheal administration, sublingual administration, nasal administration, buccal administration, rectal administration, intravaginal administration, intravenous administration, intraarterial administration, intramuscular administration, intracardiac administration, intraosseous administration, intraperitoneal administration, intraorbital administration, intravitreal administration, subconjunctival administration, suprachoroidal administration, subretinal administration, intraarticular administration, periarticular administration, epicutaneous administration, inhalation administration, or the like, with subcutaneous administration or intraperitoneal administration being preferred.

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

(Transplantation process)
In the transplantation process, the transplant material, which includes an artificial transplant material that is coated, attached, infiltrated or encapsulated with donor tissue or cells, is implanted into a recipient.

Transplant materials, including transplant materials containing donor tissue or cells expressing a labeled 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 includes the donor tissue or cells. In the transplant material, the donor tissue or cells may cover the transplant material or may adhere to, infiltrate, or be included in the transplant material.

The graft material is not particularly limited and may be a bioabsorbable material or a biocompatible material. Materials that can be used for the graft material 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, polybutylene succinate, etc. Also included are materials based on soluble cellulose such as oxidized cellulose, proteins, peptides, polyamino acids, polysaccharides, polyesters, polyamides, derivatives, crosslinked bodies and copolymers thereof, magnesium metal, calcium carbonate, calcium phosphate, hyaluronic acid and hydroxyapatite, acrylic resins, fluororesins, polyolefins, silicones, polystyrene, polyesters, polyurethanes, polycarbonates, polyimides, derivatives, crosslinked bodies and copolymers thereof, 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 graft material may be one or more of the above-mentioned materials, or may be a composite of two or more of these materials.

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.

The transplant material is not particularly limited, but can be, for example, a tubular structure, a membranous structure, any other structure of any shape, and an artificial organ. 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.

Artificial transplant materials coated, attached, or infiltrated with donor tissue or cells can be provided in any manner. For example, donor cells can be seeded on the transplant material and cultured to provide the transplant material.

The transplant material may be one that has been transplanted into the body of a donor in advance, left in place for 2 to 30 days, and then retrieved. The transplant material may 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.

An artificial graft material coated, adhered, or infiltrated with donor tissue or cells may be coated, adhered, or infiltrated with donor tissue or cells throughout the entire graft material, or only a portion of the graft material may be coated, adhered, or infiltrated with donor tissue or cells.

The donor tissue or cells expressing the labeled marker contained in the transplant material may be the donor tissue or cells themselves or processed versions of these. The tissue or cells collected from the donor can be separated, cultured, differentiated, selected, expanded, and frozen and stored as necessary, and then thawed, in order to be used as a transplant material. The tissue or cells may also be tissue or cells that have been subcultured to create a cell line.

Cells and various physiologically active substances (cytokines and cell growth factors) may be added to the transplant material by co-cultivation, spraying, coating, 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.

The transplant material can be, but is not limited to, blood vessels, respiratory system organs, digestive system organs, urinary system organs, dura mater, peritoneum, fascia, synovium, periosteum, serous membrane, etc.

The transplant material is not particularly limited, but can be transplanted into the recipient's internal organs, such as the brain, liver, kidney, or heart, or subcutaneously and into blood vessels. The method for transplanting the transplant material into the recipient is not particularly limited, and any conventionally known method can be used.

The donor tissue or cells contained in the transplant material can be taken from an adult donor, but are not particularly limited thereto. When the donor is an inbred strain, not only can the donor be the individual from which the crude extract for administration to the recipient's newborn is 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.

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, β 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 synovial cells, periosteal cells, perichondrium cells, skeletal muscle cells, smooth muscle cells, cardiac muscle cells, pericardial cells, dura cells, meningeal cells, pericytes, glial cells, nerve cells, amniotic cells, placental membrane cells, serous cells, ependymal cells, periventricular zone cells, oral mucosal epithelial cells, nerve cells and dendritic cells, genetically modified cells thereof, cells produced by gene manipulation techniques such as genome editing or transformation techniques using mRNA (messenger RNA) and the like, and cells cultured and/or differentiated from these.

In addition, the donor's 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 mucosa 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, organ-specific stem cell precursors, genetically modified cells thereof, and genome-edited cells. Note that 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, "Ensuring the quality and safety of pharmaceuticals and other products processed from human allogeneic somatic stem cells." "Human somatic stem cells" refers to cells taken from a human or cells resulting from the division of such cells that are presumed to have pluripotency and maintain the ability to self-replicate, or similar, and cells derived from these, 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 that are enriched 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. Human somatic stem cells also include cells obtained by culturing and/or differentiating these cells in vitro.

In addition, the donor's 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), etc., as well as cells that have been cultured and/or differentiated from these.

The donor's 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, and immune cells contained in the thymus, bone marrow, lymph nodes, etc.

(Method of evaluating transplantation materials using labeled marker-expressing donors)
The method for evaluating a transplant material using a donor expressing a labeled marker of the present invention comprises the steps of administering a crude extract of the donor expressing a labeled marker to a newborn recipient to obtain an immunotolerant recipient, and transplanting a transplant material containing an artificial transplant material coated, adhered or infiltrated with tissues or cells of the donor expressing the labeled marker into the immunotolerant recipient.

As used herein, a "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; mutants 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 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.

If the labeling marker is a fluorescent protein such as EGFP, it can be constantly and stably expressed in the body, and the donor-derived cells can be easily observed fluorescently in the recipient's body after transplantation. Furthermore, since fluorescent proteins such as EGFP fluoresce when exposed to excitation light, there is no need to stain the tissue for observation, and the tissue can be easily observed while still alive.

A marker-expressing donor is an animal into which a marker gene has been introduced and into which the marker gene is constitutively and stably expressed (or capable of being expressed) in some or all of the cells in the body. A marker-expressing donor can be produced by introducing the marker gene using a conventional method.

In the method of the present invention, the recipient is not particularly limited, but may be an animal to which at least the marker introduced into the donor has not been introduced, and may be, for example, a wild type mouse (WM) that has not been genetically manipulated. Furthermore, to increase the accuracy of the experiment, the recipient may be an inbred animal.

Because the recipient does not have a marker introduced, there is a high possibility that the marker marker contained in the donor-derived cells and other donor-derived antigens will induce an immunological rejection reaction if transplanted using a normal transplantation method. However, with the method of the present invention, a crude extract of the marker-expressing donor can be administered to the newborn recipient in advance to induce immune tolerance to donor-derived antigens including the marker marker, so that immune reactions at the time of transplantation can be suppressed only against donor-derived antigens. Meanwhile, normal immune reactions are maintained against antigens not derived from the donor.

By using a donor expressing a labeled marker, donor-derived cells and tissues can be easily observed in the recipient after transplantation. In addition, it is easy to distinguish whether cells and tissues, etc. that arise 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 distinguished whether it arose from donor-derived cells or from the recipient.

(Animals with transplantation models)
The present invention also provides a method for the preparation of a recombinant human ovarian tumor graft comprising administering a crude extract of a donor expressing a target marker to a newborn recipient to obtain a recipient that is immunotolerant to the donor;
Transplanting the graft material, which comprises an artificial graft material coated, adhered, infiltrated or encapsulated with tissue or cells of a donor expressing a target marker, into an immunotolerant recipient;
The present invention provides a transplantation model animal similar to an autotransplantation, which is produced by the method for producing a mouse having the above-mentioned gene.

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

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 makes it easy to confirm not only how the recipient transplant model animal behaves, but also how the transplanted transplant material behaves inside the body of the recipient transplant model animal.

In the transplantation model animal of the present invention, the recipient is first induced to have immune tolerance to the donor before transplanting the transplant material, and therefore the recipient transplantation model animal has a suppressed immune response to the transplant material. Therefore, for example, the transplantation model animal can be used as an experimental system for observing the reaction and progress of the living body when transplanted with a transplant material. On the other hand, the immune response to all antigens not derived from the donor is maintained,
1. The risk of interrupting the experiment due to ongoing infection,
2. Regarding the risk of increased cancer risk,
We can expect experimental results that are closer to clinical transplantation than ever before.

The present invention can provide a so-called "approximate autotransplant animal experimental model" by using inbred animals. The present invention can easily induce immune tolerance in animals with normal immune functions by using inbred animals, and can establish an animal model that can avoid immune reactions to specific individuals. Therefore, by using the present invention, it is not necessary to use a conventional immunodeficient animal experimental system, and transplant experiments can be performed under conditions close to clinical conditions using animals with normal immune functions. In addition, by using the present invention, it can be used as an experimental system for observing the reaction and progress of the living body when transplant materials are autotransplanted using small animals such as mice, which are originally difficult to autotransplant. Therefore, it can be used as a substitute for autotransplant experiments that could only be performed with medium and large animals, and can follow the trend of animal welfare 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.

An outline of the test carried out in this example is shown in Figure 1. The abbreviations used in the following examples are explained below.
Terminology:
GM: Green Mouse (transgenic mouse with constitutive expression of EGFP),
ICR mice: the most prolific and popular albino strain of inbred laboratory mice.
P1, P1A: Experimental facilities that comply with specific measures (containment measures) to prevent the spread of genetically modified organisms in accordance with the international treaty "Cartagena Protocol," with P1 and P1A being the least restrictive. P1 is primarily required for handling cells, and P1A is primarily required for handling animals.
1. P1A (Animal Experiment Facility) Collection of GM-derived tissues and crude protein production The process of collecting donor-derived antigen-presenting proteins to induce immune tolerance in the recipient.
2. P1A (Animal Experiment Facility) Immune tolerance treatment for ICR mice (wild species) The process of administering antigen protein to the recipient's newborn to induce immune tolerance
3. P1A (Animal Experiment Facility)
The process of collecting transplant candidate cells or tissues from a donor.
4. P1 (Cell Experiment Facility)
A process in which collected donor-derived cells are processed as necessary or prepared as transplant materials.
5. P1A (Animal Experiment Facility)
Transplanting the material from the donor into a tolerized recipient.
6. P1A (Animal Experiment Facility)
A process of observing the condition after transplantation at the appropriate time, evaluating the performance of the transplant material, and, if necessary, inferring the design of the transplant material.

(donor)
The donor used was a genetically modified inbred mouse strain (Green Mouse: C57BL/6-(CAG-EGFP) C14-Y01-FM131Osb (RBRC00267: RIKEN Bioresearch Center)) expressing EGFP (Enhanced green fluorescence protein) throughout its body (hereafter referred to as "Green Mouse (GM)"). Note that an inbred mouse strain is a mouse strain that has been repeatedly mated with another mouse strain, and has a homology of genetic traits and their expression between generations and/or between individuals, equivalent to that of identical twins.

Figure 2 shows a photograph of the fluorescence of a donor green mouse. By irradiating the mouse with excitation light (ultraviolet light in the case of GM), green fluorescence due to EGFP within the cells can be confirmed. Fluorescence was confirmed in the whole body epidermis of the newborn mouse (left side of Figure 2), and in the excised brain (center of Figure 2) and exposed epidermis excluding body hair (right side of Figure 2) of the adult mouse.

Preparation of Crude Extracts from Donors
Tissues were collected from donor green mice and disrupted with a homogenizer (tissue disruption device), then centrifuged or filtered to recover a crude extract containing soluble proteins, and crude protein was prepared (step 1 in Figure 1). The top panel of Figure 3 shows that the crude extract in the test tube is fluorescent. In order to efficiently induce immune tolerance, it is also possible to prepare a crude extract containing the transplant candidate tissue, or to further purify the crude extract before use.

(Immune Tolerance Process)
The recipient ICR mice (wild type inbred strain) were commercially available experimental animals. The ICR mice were subjected to immune tolerance treatment (step 2 in Figure 1). The donor crude extract was initially administered subcutaneously into the abdomen of newborn ICR mice within 24 hours of birth at approximately 5% BW. Over the next 7 days after birth, the donor-derived crude extract was administered subcutaneously into the abdomen for a total of 4 times every 2 days. The subcutaneously administered crude extract contained 1/20 the protein amount of the recipient's body weight. The mice were then kept in normal conditions for more than 4 weeks.

Figure 3 shows a recipient mouse that underwent immune tolerance treatment. The donor-derived crude extract in the test tube that shows green fluorescence in the upper part of Figure 3 was administered subcutaneously to the midline of the abdomen of an ICR neonate (lower left of Figure 3). Next, when excitation light was irradiated onto the abdomen, green fluorescence could be confirmed by looking through the skin (lower right of Figure 3).

(Preparation of composite materials for transplantation)
A tubular structure with a silicon tube as a core was produced as a transplant material for transplantation. This tubular structure can be used as a pseudo-blood vessel. In addition, this tubular structure can also be used as a membrane-like structure when unfolded. In this embodiment, a tube made of biocompatible silicon (product name: Saffid silicone catheter: outer diameter 2.7 mm) (hereinafter, silicon tube) was used as a prototype. A white collagen sheet (product name: Integran: sheet type 45 x 30 mm) (hereinafter, collagen sheet), which is a self-organizing material, was wound around this silicon tube in two or more layers. Next, to prevent unwinding or unraveling, a blue-purple bioabsorbable PGA thread (PGA: polyglycolic acid) was further wound and fixed (upper left diagram in Figure 4). At this point, the wound tubular collagen sheet is not watertight, so water easily leaks.

Figure 4 is a photograph showing the state of the material being transplanted 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 the transplant material being transplanted into a green mouse. The upper right of Figure 4 shows the state one week after the transplant material was transplanted 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 the transplant material, including the transplant material that was extracted and recovered from the green mouse, under fluorescent observation.

(Transplantation process)
The process of transplanting the above-mentioned tubular structure (transplant material) into a donor green mouse is shown in Figure 4 (lower left diagram in Figure 4). A skin incision of about 1 cm was made on the back of an adult green mouse that was to serve as a donor, and then a tunnel-like subcutaneous layer was peeled off about 3 cm long 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.

One week after subcutaneous implantation, the skin of the transplanted green mouse was incised and turned over enough to see the entire tubular structure, and the entire tubular structure was confirmed (Figure 4, top right). The wrapped collagen sheet was moistened with interstitial fluid and covered with cells derived from the green mouse, making it semi-transparent. It was connected to the surrounding bed tissue by membranous connective tissue. Using this connective tissue as a base, abundant bridging of new blood vessels was observed in the collagen tube, and it was confirmed that fine blood vessels had developed all around it.

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). An incision was made at the part of the connective tissue connected to the bed tissue, and the collagen tube was extracted without damaging it.

When excitation light was irradiated onto the excised tubular structure, the transplant material emitted fluorescence, revealing that the collagen tubes were evenly seeded with green mouse-derived cells. It was also separately confirmed that at this stage, the collagen sheet had already achieved watertightness with no water leakage. Furthermore, the cells seeded on the collagen sheet by culturing from the excised collagen tubes were live cells that could be cultured.

(Transplantation process)
The transplant material containing the tubular structure recovered from the green mouse was subcutaneously transplanted in a wild-type ICR mouse that had no marker and had immune tolerance induced in the green mouse, by carrying out the same procedure as described above (preparation of composite material for transplantation). Specifically, a skin incision of about 1 cm was made on the back of the ICR mouse, and the subcutaneous tissue was peeled off in a tunnel-like manner 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 inserted subcutaneously through this (Figure 5). When the entire inserted tubular structure could be comfortably embedded subcutaneously, the mantle tube was pulled out, and the insertion hole was sutured closed.

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). The wrapped collagen sheet was moistened with interstitial fluid and was covered with tissue cells derived from ICR mice from above the cell layer derived from Green Mouse, reducing the transparency of visible light. It was firmly connected to the surrounding bed tissue by connective tissue. Using this connective tissue as a base, abundant bridging of new blood vessels was observed in the tubular structure, and it was confirmed that new blood vessels derived from Green Mouse had further developed, providing blood vessels around the entire circumference.

Furthermore, because the fluorescence characteristic of green mice was confirmed by irradiating them with excitation light (right side of Figure 6), it was confirmed that the cells derived from green mice were able to engraft without immune rejection even in the subcutaneous tissue, which is particularly prone to eliciting a strong immune response, inside the bodies of immune-tolerized wild-type ICR mice. Furthermore, live cells that could be cultured were confirmed in the tissue taken from the tubular structures transplanted into ICR mice, and the coexistence of green fluorescent cells and non-fluorescent cells was confirmed.

Figure 5 shows the state of transplantation of a transplant material containing a tubular structure into a recipient mouse.

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) observed with fluorescence one month after transplantation. One month after transplantation, the distribution of donor-derived cells and tissues attached to the tubular structure, as well as new cells and tissues that developed from them, was observed by fluorescence observation.

The present invention can be used for a highly reliable and reproducible transplantation experimental system. In addition, compared to conventional animal experiments (such as experimental systems using immunodeficient animals) with similar purposes, the present invention allows experiments to be performed under conditions closer to actual clinical medical care. Based on this, it is most important in regenerative medicine to ensure better performance and quality as transplant medical materials, including regenerative medicine products. Furthermore, since widely available and inexpensive experimental materials (experimental animals such as mice and their breeding materials) and research facilities (clean rooms and facilities to prevent the spread of genetically modified animals) can be selected, it is possible to significantly reduce the necessary expenses for conducting experiments. In addition, since there is no need to use medium-sized and large animals that are essential for conventional autotransplantation experimental systems, it does not conflict with social trends such as animal welfare, and it is easy to obtain a number of experimental cases in terms of cost and labor, and since the life span is short, it can be adapted to different biological conditions at each age of life, making it possible to design and evaluate the performance of various transplant medical materials under different conditions.

Claims (5)

subcutaneously administering a crude protein solution extracted from cells or tissues of a donor expressing a labeled marker to a newborn of a recipient to obtain a recipient that is immunotolerant to the donor;
and a step of transplanting a transplant material comprising an artificial transplant material coated, adhered, infiltrated or contained with tissue or cells of the labeled marker-expressing donor into the immune-tolerant recipient,
The method of production, wherein the donor and the recipient are inbred animals (excluding humans) . The method for producing an immune tolerant recipient for evaluating a transplant material according to claim 1, wherein the transplant material is a tubular structure, a membrane structure, or an artificial organ. The method for producing an immune tolerance recipient for evaluating a transplant material according to claim 1 or 2, wherein the transplant material is transplanted into the body of the donor, left in place for 2 to 30 days, and then recovered. The method for producing an immune tolerant recipient for evaluating a transplant material according to claim 4, wherein the labeling marker is a fluorescent protein.
The method for producing an immune tolerant recipient for evaluating a transplant material according to claim 1, wherein the transplant material is a collagen sheet seeded with donor-derived cells.

Images