Vitrification freezing method for urinary including metanephros, urinary duct, urinary bladder derived from pig for implantation, production method of vitrification frozen object, production method of urinary for implantation and vitrification frozen object

Abstract

To provide a vitrification freezing method for an organ or a tissue for implantation capable of reducing a contamination risk (for example, cell contamination) of a biological material for implantation, a production method of a vitrification frozen object, a production method of an organ or tissue for implantation, and a vitrification frozen object.SOLUTION: There is provided a vitrification freezing method for an organ or tissue for implantation comprising: a coating step for coating by a gel, at least a part of a surface of the organ or tissue for implantation; and a freezing step for bringing the organ or tissue for implantation after coated by the gel, into contact with a coolant for vitrification freezing.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for vitrifying organs or tissues for transplantation, a method for producing a vitrified frozen material, a method for producing an organ or tissue for transplantation, and a vitrified frozen material, which are suitable for vitrification and cryopreservation of regenerative medicine products, etc. In particular, the present invention relates to a method for vitrifying organs or tissues for xenotransplantation, a method for producing a vitrified frozen material, a method for producing an organ or tissue for transplantation, and a vitrified frozen material, which are suitable for vitrification and cryopreservation of regenerative medicine products, etc. [Background technology]

[0002] Rapid freezing by vitrification, which involves immersing biological materials for transplantation in a vitrification refrigerant, is essential for the long-term preservation and quality maintenance of regenerative medicine products. For example, Non-Patent Document 1 describes a technique for long-term storage of cell sheets that can be widely applied in regenerative medicine, in which the cell sheet is immersed in an equilibrium solution, then in a vitrification solution, and then exposed to liquid nitrogen vapor to vitrify and cryopreserve it. Furthermore, Non-Patent Document 2 describes xenotransplantation data in which porcine metanephroi that had undergone vitrification were transplanted into cats.

[0003] On the other hand, repeated use of vitrification-freezing refrigerants or vitrification treatment solutions or simultaneous use on multiple samples poses a risk of contamination (e.g., cell contamination). In other words, there is a risk of contamination of transplant biological materials via the vitrification-freezing refrigerants or vitrification treatment solutions, which is undesirable from the viewpoint of quality as regenerative medicine products. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] M. Maehara et al.,BMC Biotechnology 13(58),2013 [Non-patent document 2] Matsumoto K. et al.,StemCells 30(2012):1228-35.DOI:10.1002 / stem.1101. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention has been made in consideration of the problems of the conventional technology, and aims to provide a method for vitrifying organs or tissues for transplantation, a method for producing a vitrified frozen material, a method for producing an organ or tissue for transplantation, and a vitrified frozen material, which can reduce the risk of contamination (e.g., cellular contamination) of biological materials for transplantation. [Means for solving the problem]

[0006] As a result of intensive research into the above-mentioned problems, the present inventors have found that the risk of contamination (e.g., cellular contamination) of organs or tissues for transplantation can be reduced by coating with a gel at least a portion of the surface of the organ or tissue for transplantation that comes into contact with the vitrification refrigerant, etc. The present invention was completed based on the above findings. That is, the present invention is as follows.

[0007] <1> A coating step of coating at least a portion of the surface of an organ or tissue for transplantation with a gel; and A method for vitrifying an organ or tissue for transplantation, comprising a freezing step of contacting the organ or tissue for transplantation after being coated with the gel with a refrigerant for vitrification. <2> The organ or tissue for transplantation has a foreign substance injected therein and an injection mark, and the gel covers at least a part of the injection mark. <1> The method described below. <3> The method further comprises a step of contacting the transplant donor organ or tissue with a vitrification liquid after the coating step. <2> The method described below. <4> The foreign material is a cell derived from the same animal species as the recipient. <2> or <3> The method described below. <5> The organ or tissue for transplantation is a xenotransplant organ or tissue. <1> ~ <4> 10. The method according to any one of claims 1 to 9. <6> The organ or tissue for transplantation is an organ or tissue derived from a pig. <1> ~ <5> 10. The method according to any one of claims 1 to 9. <7> The gel is a hydrogel. <1> ~ <6> 10. The method according to any one of claims 1 to 9. <8> the above <1> ~ <7> A method for producing a vitrified frozen organ or tissue for transplantation, comprising the method according to any one of the above. <9> the above <8> The method for producing an organ or tissue for transplantation comprises the method described in the above item 1, further comprising, after the freezing step, the step of contacting the organ or tissue with a thawing liquid and the step of removing the gel covering the surface of the organ or tissue for transplantation. <10> A vitrified frozen organ or tissue for transplantation having a gel coating on at least a portion of its surface. <11> The organ or tissue for transplantation has a foreign substance injected therein and an injection mark, and the gel covers at least a part of the injection mark. <10> The vitrified frozen product according to claim 1. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a method for vitrifying an organ or tissue for transplantation, a method for producing a vitrified frozen material, a method for producing an organ or tissue for transplantation, and a vitrified frozen material, which can reduce the risk of contamination (e.g., cellular contamination) of biological materials for transplantation. DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments and can be implemented with appropriate modifications within the scope of the object of the present invention.

[0010] <Method for vitrification of organs or tissues for transplantation> The first aspect of the present invention includes a coating step of coating at least a part of the surface of an organ or tissue for transplantation with a gel; The method for vitrifying an organ or tissue for transplantation comprises a freezing step of contacting the organ or tissue for transplantation after being coated with the gel with a refrigerant for vitrification. In the first aspect, by coating at least a portion of the surface of an organ or tissue for transplantation with a gel, it is possible to reduce both the risk of contamination (e.g., cellular contamination) from the vitrification freezing refrigerant to the organ or tissue for transplantation, and the risk of contamination (e.g., cellular contamination) from the organ or tissue for transplantation to the vitrification freezing refrigerant. Here, vitrification freezing refers to contacting a material with a refrigerant for vitrification freezing to suppress water crystallization (ice crystal formation) and freezing it in an amorphous glassy state (preferably, freezing by so-called rapid cooling, more preferably, freezing by so-called ultra-rapid cooling). Vitrification freezing can suppress volume expansion that accompanies water crystallization, and therefore can suppress damage (freezing damage) such as cell membrane rupture. Examples of refrigerants for vitrification freezing include liquid nitrogen (boiling point -196°C), liquid helium (boiling point -269°C), liquid ethane (boiling point -175°C), and the like, as well as vapors of these. The contact with the vitrification freezing refrigerant is preferably carried out at a temperature of -273°C to -70°C (preferably -270°C to -80°C, more preferably -269°C to -150°C), for example, for 1 second to 10 minutes (preferably 0.5 seconds to 1 minute, more preferably 0.1 seconds to 10 seconds).

[0011] The method of contacting the vitrification-freezing refrigerant is not particularly limited, but examples include immersion or application (flushing) of a liquid, spraying of steam, and the like. The contact with the vitrification / freezing refrigerant may or may not be performed using a vitrification / freezing support device capable of simultaneously supporting multiple organs or tissues for transplantation, such as a cryotop, a straw (e.g., a transplant straw, a straw with a sharp tip, etc.), or a capillary pipette, but is preferably performed using the support device. As the support device, the vitrification / freezing support device that can simultaneously support multiple organs or tissues for transplantation is more preferable, from the viewpoint that multiple organs or tissues for transplantation can be frozen as a single unit at once, thereby improving the production efficiency of transplant materials and also reducing the risk of contamination using the present invention. Examples of the vitrification / freezing support device capable of simultaneously supporting a plurality of the above-mentioned organs or tissues for transplantation include a chip or plate having a plurality of recesses on a glass plate, metal plate, plastic plate, etc., in which a plurality of the above-mentioned organs or tissues for transplantation can be simultaneously supported; Examples of such supports include mesh nets, nonwoven fabrics, etc. that can simultaneously support a plurality of the above-mentioned organs or tissues for transplantation. For example, the present inventors have reported a method for vitrifying and freezing mouse or pig embryos in hollow fibers (H. Matsunari, et al., Journal of Reproduction and Development Vol. 58 (2012) No. 5 pp. 599-608). Such hollow fibers containing the organ or tissue for transplantation can also be used as the vitrification support device capable of simultaneously supporting multiple organs or tissues for transplantation. In addition, examples of commercially available supports include CRYOTOP (registered trademark; manufactured by Kitasato Biopharma). After the freezing step, the organ or tissue for transplantation can be stably stored for a required period (e.g., one day or more, one week or more, one month or more, one year or more). Storage can be performed in a vitrification cryopreservation container (e.g., a Dewar vessel) while in contact with the vitrification freezing refrigerant, or by other methods. There is no particular upper limit to the vitrification cryopreservation period, but examples include 20 years or less, 10 years or less, and 5 years or less. From the viewpoint of improving the production efficiency of transplant materials while also reducing the risk of contamination by the present invention, the freezing process allows multiple transplant organs or tissues to be brought into contact with the vitrification freezing refrigerant present in the same space simultaneously and in parallel. The freezing step may be carried out multiple times, for example, by carrying out the freezing step again after the step of contacting the sample with a melting liquid described below, or may not be carried out at all.

[0012] The organ or tissue for transplantation may be derived from a fetus, a young animal, or an adult, but from the viewpoint of low immunogenicity, an organ or tissue derived from a fetus is preferred, particularly in the case of xenotransplantation. Examples of the organ or tissue for transplantation include internal organs (e.g., pancreas, kidney, ureter, bladder, liver, heart, stomach, intestine, etc.), reproductive organs (e.g., ovaries, testes, etc.), fertilized eggs, embryos, fetuses, bone marrow (e.g., hematopoietic organs), brain, eyes, nose, mouth, skin, nerves, or tissues derived therefrom, or artificial tissues (cell sheets such as chondrocyte sheets, organoids, etc.), with internal organs, reproductive organs, fertilized eggs, embryos, and fetuses being preferred, internal organs and reproductive organs being more preferred, and pancreas (e.g., pancreatic islets), kidneys (e.g., metanephros, particularly the urinary tract including the metanephros, ureters, and bladder) being even more preferred. Animals that provide the organ or tissue for transplantation include any mammal, such as pigs, cows, horses, sheep, goats, primates (e.g., humans, apes (chimpanzees, monkeys, etc.)), and rodents (e.g., mice and rats). Mammals with physical characteristics such as those closer to humans than rodents are preferred, with pigs, sheep, goats, and primates (e.g., humans and apes) being more preferred, and pigs (i.e., the organ or tissue for transplantation is derived from a pig) being even more preferred. The organ or tissue for transplantation is preferably an organ or tissue for xenotransplantation.

[0013] Methods for coating at least a portion of the surface of the organ or tissue for transplantation with a gel include burying or embedding the organ or tissue for transplantation in the gel, immersing the organ or tissue for transplantation in a liquid (preferably a hydrosol) containing a sol (the sol before gelation) and then gelling it, applying a liquid containing the sol to the organ or tissue for transplantation and then gelling it, etc. The liquid containing the sol or the gel may or may not contain a basal medium or basal liquid medium described below. The gelation of the sol can be carried out by any method, such as adding polyvalent metal ions such as calcium ions or barium ions, or by cooling. In order to more reliably achieve the object of the present invention, it is preferable to cover the entire surface of the organ or tissue to be transplanted with the gel. There are no particular restrictions on the gel as long as it can achieve the object of the present invention, but a pharmacologically acceptable gel (a pharmaceutically acceptable, non-toxic gel) is preferred, and a hydrogel is more preferred, from the viewpoint that there is no actual harm if a small amount of the gel remains if it cannot be completely removed, and that the gel can be used for transplantation in its coated state without being removed. Furthermore, the above gel is preferably a gel in which a sol-gel phase transition occurs due to ionic crosslinking, or a temperature-sensitive gel in which a sol-gel phase transition occurs depending on temperature (for example, a phase transition temperature of 5 to 40°C, preferably 5 to 10°C, 10 to 15°C, 15 to 20°C, 20 to 25°C, 25°C to 30°C, 30°C to 35°C, or 35°C to 40°C). The gel undergoing the sol-gel phase transition due to the ionic crosslinking can be converted into a sol by chelating with a chelating agent such as ethylenediaminetetraacetic acid (EDTA) or disodium ethylenediaminetetraacetic acid (EDTA·2Na), which breaks down the ionic crosslinking.

[0014] Examples of the hydrogel include alginic acid or its salts (for example, polyvalent metal salts such as calcium alginate and barium alginate, and monovalent metal salts such as sodium alginate and potassium alginate; from the viewpoint of achieving gelation more reliably, polyvalent metal salts of calcium alginate and barium alginate are preferred), gelatin, carrageenan, agar (agarose gel), pectin, chitosan, silicone hydrogel, konjac, and other polysaccharides. Among these, hydrogels in which a sol-gel phase transition occurs due to the above-mentioned ionic crosslinking, or thermosensitive hydrogels in which a sol-gel phase transition occurs due to temperature, are preferred. Examples of hydrogels that undergo a sol-gel phase transition due to the above-mentioned ionic crosslinking include alginic acid or its salts (preferably polyvalent metal salts), and examples of temperature-sensitive hydrogels that undergo a sol-gel phase transition due to temperature include gelatin, carrageenan, agar (agarose gel), pectin, etc., with gelatin being preferred because it can be converted into a sol at a relatively low temperature (e.g., 35°C or below) and causes less damage to the above-mentioned organs, etc. For example, a 5 to 25% (w / v) gelatin solution can be used, a 7 to 20% (w / v) gelatin solution is preferred, and a 10 to 15% (w / v) gelatin solution is more preferred. The coating step may or may not be carried out multiple times using one or more types of gel.

[0015] The coated gel may be one layer or two or more layers. When the coated gel has multiple layers, it is preferable to use a gel with a relatively low solation temperature (e.g., gelatin) as the outer layer (e.g., the outermost layer), and a gel with a higher solation temperature that can be solated by treatment with the above-mentioned chelating agent (e.g., alginic acid) or a pharmacologically acceptable gel as the inner layer (e.g., the innermost layer). When multiple organs or tissues for transplantation are gel-coated simultaneously, the outer layer (e.g., the outermost layer) may be common to the multiple organs or tissues for transplantation, or multiple units of the organs or tissues for transplantation and the inner layer (e.g., the innermost layer) may be combined into one by the outer layer (e.g., the outermost layer). For example, by dispersing multiple units of the organ or tissue for transplantation and the inner layer (e.g., the innermost layer) in a bag filled with a sol that forms the outer layer (e.g., the outermost layer), a common outer layer (e.g., the outermost layer) can be formed among multiple organs or tissues for transplantation. There are no particular limitations on the thickness of each coating formed from the gel as long as it can solve the above problems, but it is, for example, 0.1 mm to 5 mm, and preferably 0.5 mm to 2 mm.

[0016] After preparation of the organ or tissue for transplantation (e.g., after extraction), before or after the coating step (preferably after the coating step), but before the step of contacting with a vitrification solution described below, a step of contacting (immersing, applying, etc.) the organ or tissue for transplantation with an equilibrium solution at -5°C to 37°C (preferably 0°C to 35°C) for, for example, 1 minute to 3 hours (preferably 5 minutes to 1 hour, more preferably 2 minutes to 30 minutes) (so-called pretreatment step) may or may not be included. The pretreatment step can alleviate osmotic shock, sudden changes in concentration, etc., and improve survival rates. The equilibration solution may be a solution (preferably an aqueous solution) containing at least one agent selected from the group consisting of cell-permeable cryoprotectants and cell-impermeable cryoprotectants, preferably a solution containing at least one agent selected from the group consisting of cell-permeable cryoprotectants and cell-impermeable cryoprotectants at a lower concentration than the vitrification solution described below, and more preferably a solution containing at least one agent selected from the group consisting of cell-permeable cryoprotectants and cell-impermeable cryoprotectants at 10% by volume or less (each of which is present when multiple types are contained). The equilibration solution is preferably a solution containing a cell-permeable cryoprotectant. The equilibration solution may contain a single cell-permeable cryoprotectant or may contain two or more cell-permeable cryoprotectants. The equilibration solution may contain a single cell-impermeant cryoprotectant or may contain two or more cell-impermeant cryoprotectants. Specific examples of the cell-permeable cryoprotectants and the cell-non-permeable cryoprotectants are as described below. The balance solution may or may not further contain a basal medium or a basal medium solution. The basal medium or basal liquid medium is not particularly limited, and examples thereof include any synthetic medium such as TCM199 (preferably 10 to 35 mM HEPES-buffered TCM199), PBS (phosphate buffered saline), and MEM, which may or may not contain calf serum (CS), fetal calf serum (FCS), serum substitutes, etc. (for example, 10 to 20% by mass). The step of contacting with the equilibrium solution may or may not be carried out multiple times (for example, stepwise) using one or more types of equilibrium solution.

[0017] It is preferable to include a step before or after the coating step in which the organ or tissue for transplantation is brought into contact (immersed, coated, etc.) with the vitrification solution under temperature conditions such as -5°C to 37°C (preferably, 0°C to 30°C) for, for example, 1 minute to 90 minutes (preferably, 1 minute to 60 minutes, more preferably, 1 minute to 30 minutes). The step of contacting the organ or tissue for transplantation with the vitrification liquid may be carried out either before or after the coating step. However, from the viewpoint of promoting the penetration of the vitrification liquid, it is preferable to include a step of contacting the organ or tissue for transplantation with the vitrification liquid before the coating step. On the other hand, from the viewpoint of reducing the risk of contamination, it is preferable to include a step of contacting the substrate with the vitrification liquid after the coating step. The vitrification solution includes a solution (preferably an aqueous solution) containing a cell-permeable cryoprotectant and a cell-impermeable cryoprotectant. The content of the cell-permeable cryoprotectants (the total amount when multiple types are contained) is 25% by volume or more and 60% by volume or less, and preferably 30% by volume or more and 50% by volume or less. The content of the cell-impermeable cryoprotectant (total amount when multiple types are contained) may be 0.2 to 2M, and preferably 0.4 to 1M. Examples of the cell-permeable cryoprotectants include dimethyl sulfoxide (DMSO), ethylene glycol (EG), propanediol, and glycerin. Examples of the cell-impermeable cryoprotectants include sugars such as sucrose, trehalose, sorbitol, and dextran, carboxylated polylysine, polyvinyl alcohol, polyvinylpyrrolidone, and antifreeze proteins. The vitrification solution may or may not further contain the basal culture medium or basal medium solution, and from the viewpoint of improving the survival rate, may or may not further contain any serum, polyvinylpyrrolidone, or any thickener. The step of contacting with the vitrification liquid may or may not be carried out multiple times (for example, stepwise) using one or more types of vitrification liquid.

[0018] In order to more reliably avoid the above-mentioned risks, the above-mentioned contact with the vitrification freezing refrigerant may be carried out by placing the coated organ or tissue in a container (e.g., a bag) for containing the organ or tissue, and then contacting the coated organ or tissue after contacting the vitrification liquid through the container, or by surrounding (wrapping) the coated organ or tissue after contacting the vitrification liquid with any film, and then contacting the organ or tissue through the film. After the freezing step, the process may or may not include a step of contacting (immersing, applying, etc.) the organ or tissue after vitrification freezing (preferably after vitrification cryopreservation) with a thawing solution for, for example, 30 seconds to 30 minutes (preferably, 40 seconds to 10 minutes, more preferably, 50 seconds to 5 minutes), but it is preferable to include such a step.It is more preferable to warm the frozen organ or tissue to room temperature (e.g., 25°C) to 38°C while contacting or diluting it with the thawing solution. The melting liquid is not particularly limited and may be any melting liquid, for example, a solution (preferably an aqueous solution or a phosphate buffer) containing 0.5 to 3 M (preferably 0.7 to 2 M) of sucrose. The thawing solution may or may not contain the basal medium or basal liquid medium. The step of contacting with the melt may or may not be carried out multiple times (for example, stepwise) using one or more melts.

[0019] After the step of contacting with the thawing solution, the method may or may not include a step of contacting (immersing, applying, etc.) the organ or tissue with the dilution solution under temperature conditions such as 0°C to 37°C (preferably 10°C to 35°C) for, for example, 30 seconds to 30 minutes (preferably 40 seconds to 10 minutes, more preferably 50 seconds to 5 minutes), but it is preferable to include such a step. The diluent is not particularly limited and may be any diluent, for example, a solution (preferably an aqueous solution or a phosphate buffer) containing 0.1 to 2 M (preferably 0.3 to 1 M) sucrose. The diluent may or may not further contain a penetrating cryoprotectant such as ethylene glycol. The dilution liquid may or may not further contain the basal medium or basal liquid medium. The step of contacting with the diluent may or may not be carried out multiple times (for example, stepwise) using one or more diluents. Each of the above steps may or may not be performed in an atmospheric (air) atmosphere, or may or may not be performed in a low-oxygen atmosphere (for example, an atmosphere of 5% oxygen, 5% carbon dioxide, and 90% nitrogen, or an atmosphere of 5% carbon dioxide and 95% air, etc.).

[0020] The above series of steps may or may not be carried out consecutively in a container (for example, a bag) that contains the organ or tissue. The container for containing the organ or tissue is preferably a perfusion container comprising a container body for containing the organ or tissue, an injection member for injecting liquid from outside the container into the container, and a discharge member for discharging liquid from inside the container to outside the container, and it is more preferable that the injection member and the discharge member are positioned so that a liquid flow occurs between them and that this liquid flow comes into contact with the organ or tissue contained in the container body. The container body may be of any shape as long as it can accommodate a biological sample, but a bag-shaped container is preferred. The bag-shaped container preferably has an opening for inserting and removing the organ or tissue, and this opening is preferably openable and closable with a zipper or the like. A commercially available zippered storage bag can be used as the bag-shaped container. The container body is not limited to a bag-shaped container and may be, for example, a container with a picture frame-like frame having films adhered to the upper and lower surfaces. The container body may have any shape, but a rectangular shape is preferred for ease of installation of the inlet and outlet members. The container body may also be made of any material, but a transparent material with good thermal conductivity is preferred. When the container body is a bag-shaped container, preferred materials include low-density polyethylene, medium-density polyethylene, ultra-high molecular weight polyethylene, polyethylene-polytetrafluoroethylene copolymer, and polyethylene-1,2-dichloroethane copolymer. The size of the container body can be determined depending on the organ or tissue to be accommodated. For example, when the container body is a rectangular bag-shaped container, the rectangle can have a long side of 30 to 350 mm and a short side of 10 to 100 mm.

[0021] After the above treatment, the organ or tissue for transplantation may or may not be cultured in the above-mentioned basal medium or basal liquid medium before transplantation (preferably xenotransplantation).

[0022] For example, when the transplant organ or tissue is used as a regenerative medicine product, it may be necessary to inject the foreign substance (e.g., cells derived from the same animal species as the recipient) into the transplant organ or tissue, and then vitrify and store the organ or tissue. On the other hand, if there are injection marks as described below, there is a problem that the foreign substance leaks out of the organ or tissue for transplantation during the freezing process (and the process of contacting with the vitrification liquid) due to the osmotic pressure gradient between the inside of the organ or tissue for transplantation and the external solution.

[0023] In view of the above problems, one preferred embodiment is one in which the organ or tissue for transplantation has a foreign substance injected therein and an injection mark, and the gel covers at least a portion of the injection mark. Here, "injection marks" refers to holes formed to artificially inject the foreign substance into the organ or tissue for transplantation, and refers not only to holes on the surface of the organ or tissue for transplantation, but also to communicating holes that reach the injection target region inside the organ or tissue for transplantation. The "injection marks" can be formed, for example, by a needle-shaped member. The diameter (e.g., average diameter) of the holes is not particularly limited, and may be, for example, 0.05 mm to 3 mm, preferably 0.1 mm to 2 mm, and more preferably 0.5 mm to 1.5 mm. According to the preferred embodiment, at least a portion of the injection mark formed by the hole in the surface and the communication hole, and the hole in the surface can be covered with the gel. This makes it possible to prevent leakage of the foreign substance due to the osmotic pressure gradient between the inside of the organ or tissue for transplantation and the external solution (for example, the vitrification solution, the equilibration solution, etc.).

[0024] Here, the foreign substance refers to a substance derived from a species other than the organ or tissue for transplantation, and examples of the foreign substance include substances derived from the same animal species as the recipient (transplant recipient individual), substances derived from the same animal species as the donor (transplant source individual) but a different individual from the donor, substances derived from an animal species different from both the recipient and the donor, and drugs (e.g., pharmaceuticals). As substances derived from animal species, substances derived from the same animal species as the recipient are preferred from the viewpoint of low immunogenicity. Examples of substances include cells, growth factors, hormones, cytokines, etc., with cells being preferred among them. The cells may or may not be artificial cells (e.g., genetically modified cells, ES cells, iPS cells). For example, in the case of xenotransplantation in which a pig organ or tissue is transplanted into a human, the above-mentioned "cells derived from the same animal species as the recipient" include "cells derived from a human." In one preferred embodiment, from the viewpoint of suppressing leakage of the foreign substance due to the osmotic pressure gradient, it is preferable to further include a step of contacting the organ or tissue for transplantation with a vitrification liquid after the coating step.

[0025] When injecting the foreign substance, the organ or tissue for transplantation is positioned by contacting it with a gel similar to the gel described above (i.e., using the gel as a positioning jig), and the foreign substance may or may not be injected into the organ or tissue for transplantation after positioning. If a gel similar to the gel described above is used as a positioning jig, it is preferable to position the organ or tissue to be transplanted by contacting it with a gel similar to the gel described above, from the viewpoint that after the injection of the foreign substance, the organ or tissue to be transplanted, which remains in contact with the gel before the gel used as the positioning jig is removed, can be subjected to the above-mentioned coating process with the same gel. Furthermore, from the viewpoint that both the gel used as the positioning jig and the gel used in the coating step can be removed together in the removal step described below, which removes the coating gel, it is preferable to position the organ or tissue for transplantation by bringing it into contact with a gel similar to the gel described above. Here, "positioning" refers to grasping (holding firmly) and positioning the organ or tissue to be transplanted so that it does not move during the process of injecting the foreign substance, thereby enabling the positional relationship of the organ or tissue to be transplanted relative to the gel to be maintained with high precision.

[0026] <Method for producing vitrified frozen organs or tissues for transplantation, and method for producing organs or tissues for transplantation> A second aspect of the present invention is a method for producing a vitrified frozen organ or tissue for transplantation, which comprises the vitrification method according to the first aspect. The method for producing a vitrified frozen product according to the second aspect may or may not include a removal step of removing the gel covering the surface of the organ or tissue for transplantation after the freezing step, but it is preferable that the removal step be included from the viewpoint that the organ or tissue for transplantation after the removal can be used for transplantation. A third aspect of the present invention is a method for producing an organ or tissue for transplantation, which includes the method for producing a vitrified frozen material according to the second aspect, and further includes a step of contacting the organ or tissue with a melting liquid and a step of removing the gel that has coated the surface of the organ or tissue for transplantation. The coated gel can be removed by heating to a sol-forming temperature, by any sol-forming process such as the chelate treatment, or by applying physical pressure. Furthermore, washing with any aqueous solution or any washing liquid such as phosphate buffer, (running water) or the like may or may not be performed. The removal step may or may not be carried out multiple times (for example, stepwise).

[0027] When a multi-layer gel is coated with two or more layers, with an outer layer (e.g., outermost layer) being a gel with a relatively low solation temperature (e.g., gelatin) and an inner layer (e.g., innermost layer) being a gel with a higher solation temperature that can be solated by treatment with the above-mentioned chelating agent (e.g., alginic acid) or a pharmacologically acceptable gel, It is preferable to remove the gel in two steps: first, removing the outer layer (e.g., the outermost layer) by thermal solation and physical pressure, which pose a high risk of damage, and then soling the inner layer (e.g., the innermost layer) by the above-mentioned chelation treatment. In this way, the gel in the inner layer (for example, the innermost layer) acts as a barrier, making it difficult for damage caused when the outer layer (for example, the outermost layer) is removed to be transmitted to the organ or tissue for transplantation. Furthermore, since the chelate treatment is mild, damage to the organ or tissue for transplantation during the solation process can be minimized. Furthermore, the pharmacologically acceptable gel does not necessarily have to be removed at the time of implantation. In this case, damage caused by gel removal can be more reliably avoided. Furthermore, the outer layer gel acts as a barrier to the source of contamination, preventing it from reaching the inner layer (e.g., the innermost layer), so there is no problem even if the gel in the inner layer (e.g., the innermost layer) is left in place during transplantation.

[0028] <Vitrified frozen organs or tissues for transplantation> A fourth aspect of the present invention is a vitrified frozen organ or tissue for transplantation having a gel coating on at least a portion of its surface. The vitrified frozen product according to the fourth embodiment has at least a portion of its surface coated with gel, and therefore can be cryopreserved in a state where the risk of contamination (for example, cell contamination) is reduced. Furthermore, by carrying out the above-mentioned removal step, the tissue can be used as a transplant donor. Specific and preferred examples of the organs or tissues for transplantation are as described above. It is preferable that the organ or tissue for transplantation has a foreign substance injected inside and an injection mark, and that the gel covers at least a part of the injection mark, it is more preferable that the gel covers the entire injection mark, and it is even more preferable that the gel covers the entire surface of the organ or tissue for transplantation. Specific and preferred examples of the foreign substance are as described above. The injection marks are as described above. [Example]

[0029] The present invention will be explained in more detail below by showing examples of the present invention, but the present invention is not limited to these examples and various applications are possible within the scope of the technical idea of ​​the present invention.

[0030] <Examples 1 to 4 and Comparative Example 1> (material) We prepared a capillary tube with a sharply polished tip into which GFP-expressing human mesenchymal stem cells that express green fluorescent protein (GFP) and emit green fluorescence were aspirated. Of the 40 metanephroi extracted from 20 pig fetuses obtained by Caesarean section from five wild-type sows (identical clones) under DPF management, 24 were placed on a 35 mm dish (manufactured by Iwaki Co., Ltd.) and placed under the capsule of each metanephroi with the capillaries as foreign bodies. The human mesenchymal stem cells were injected. In addition, 16 of the 40 metanephros that were removed were semi-embedded in 10% (w / v) gelatin gel (i.e., there was an exposed surface of the metanephros that was not in contact with the gel, while the part of the metanephros that was in contact with the gel was embedded), and each metanephros semi-embedded in the gel was placed and positioned on a 35 mm dish (manufactured by Iwaki Co., Ltd.), and the human mesenchymal stem cells were injected as a foreign substance into the exposed surface of the nephrogenic zone under the capsule of each semi-embedded metanephroi using the capillary.

[0031] Of the 24 metanephros that had injection marks after injection and were not semi-embedded, eight were pretreated by immersion in 4.5 ml of an equilibration solution containing 7.5% EG and 7.5% DMSO as cryoprotectants at room temperature (25-27°C) for 25 minutes prior to the coating process described below. They were then immersed in a vitrification solution containing twice the volume of the equilibration solution (15% EG and 15% DMSO by mass, plus 0.5 M sucrose) for 30 minutes at room temperature. The eight metanephros that had injection marks and were pretreated and immersed in the vitrification solution prior to the coating process described below were designated the metanephros group of Example 1.

[0032] (Coating process) Gelatin powder (Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in HEPES-buffered TCM199 medium at pH 7.2 to prepare a gelatin solution with a concentration of 10 to 15% (w / v). For the eight metanephros of Example 1 that had the injection mark, were not semi-embedded, and had been subjected to the pretreatment, the 10-15% (w / v) gelatin solution was dripped onto them, or the metanephros was placed in a drop of gelatin solution, and the metanephros was coated with gelatin gel so that only the injection mark area of ​​the metanephros and its surrounding area (a part of the surface of the metanephros that includes the injection mark area) were covered. Furthermore, for eight of the 16 metanephros that had the injection scar after injection, were not semi-embedded, and had not been subjected to the pretreatment step, the gelatin solution was dropped onto them or the metanephros was placed in a drop of gelatin solution, and the metanephros was coated with gelatin gel so that only the injection scar and its vicinity were covered. The eight metanephros that had not been subjected to the pretreatment step before the coating step and that only the injection scar and its vicinity were covered were used as the metanephros group for Example 2. On the other hand, the remaining 8 of the 16 metanephros that had injection marks after injection and were not semi-embedded were not subjected to the gelatin gel coating process. The 8 metanephros that had injection marks after injection and were not subjected to the coating process were used as the metanephros group for Comparative Example 1. Furthermore, for eight of the 16 metanephros that had the injection scar after injection, were semi-embedded, and had not been subjected to the pretreatment process, the gelatin solution was dropped onto them or the metanephros was placed in a drop of gelatin solution, and the gelatin gel was applied to cover only the injection scar and its vicinity. The eight metanephros that had not been subjected to the pretreatment process before the coating process, were semi-embedded, and only the injection scar and its vicinity were covered, and were designated the metanephros group of Example 3. Furthermore, for the remaining 8 of the 16 metanephros that had injection marks after injection, were semi-embedded, and had not been subjected to the pretreatment process, the gelatin solution was dropped onto them or the metanephros was placed in a drop of gelatin solution, and the entire metanephros, including the injection marks, was covered with gelatin gel. The group of 8 metanephros that were semi-embedded and entirely covered, including the injection marks, was designated the metanephros group of Example 4. (Pretreatment process) Twenty-four metanephros from Examples 2 to 4 after being coated with the gelatin gel and eight metanephros from Comparative Example 1 that were not coated with the gelatin gel were pretreated by immersing them in 4.5 ml of equilibration solution containing 7.5% by mass EG and 7.5% by mass DMSO as cryoprotectants at room temperature (25 to 27°C) for 25 minutes. (vitrification freezing process, thawing process and dilution process) Next, after pretreatment, 24 metanephroi from Examples 2 to 4 and 8 metanephroi from Comparative Example 1, which were not coated with the gelatin gel, were immersed in a vitrification solution containing twice the amount of EG, 15% by mass, DMSO, and 0.5M sucrose at room temperature for 30 minutes. After immersion in the vitrification solution, the 32 metanephros of Examples 1 to 4 and the 8 metanephros of Comparative Example 1 that were not coated with the alginate gel were placed on a support device "Cryotop" (Kitasato Biopharmaceuticals) for the contamination risk test evaluation described below. The tissue was then placed in liquid nitrogen intentionally contaminated with E. coli (a pathogen) and cooled extremely rapidly, resulting in vitrification. The organs were transferred to a vitrification cryopreservation container and stored in liquid nitrogen for 10 days. After that, they were thawed by immersing them in a thawing solution containing 1 M sucrose at 37°C for 1 minute. Then, they were washed twice, one time for 3 minutes in a dilution solution containing 0.5 M sucrose at room temperature and the other for 5 minutes in a washing solution, to dilute and remove the cryoprotectant.

[0033] (Gel removal process) After the thawing and dilution of the cryoprotectant, 32 metanephros from Examples 1 to 4 and 8 metanephros from Comparative Example 1 were immersed in HEPES-buffered TCM199 medium at 37°C for 30 minutes to convert the gelatin gel into a sole, and then the gelatin was removed by washing twice with washing solution for 5 minutes each time. For the 16 metanephroi groups in Examples 3 and 4, which were semi-embedded in gelatin gel for positioning purposes during the injection of foreign substances, it was confirmed that the gelatin gel used for semi-embedding for positioning purposes could also be removed in the gel removal process along with the gel covering the injection marks. The metanephroi groups obtained above in Examples 1 to 4 and Comparative Example 1 were subjected to the following contamination risk test and foreign substance leakage test for evaluation.

[0034] <Contamination risk test> The metanephroi of Examples 1 to 4 and Comparative Example 1 were tested for the presence or absence of E. coli using a primer pair (forward and reverse) for the verotoxin gene of E. coli (pathogen).

[0035] <Foreign substance leakage test> The metanephros of Examples 1 to 4 and Comparative Example 1 were subjected to a test to detect the green fluorescence derived from the human mesenchymal stem cells. Examples 1 to 4 and Comparative Example 1, as well as the test results, are summarized in Table 1 below.

[0036] [Table 1]

[0037] As is clear from the results shown in Table 1, in the metanephros groups of Examples 1 to 3, where only the injection site and its vicinity were coated with gel, almost no E. coli was detected, indicating that the risk of contamination was avoided by the gel coating. In particular, in the metanephros group of Example 4, where the entire organ was coated with gel, no E. coli was detected at all, indicating that the risk of contamination was completely avoided. On the other hand, in the metanephros group of Comparative Example 1, E. coli was detected in all four metanephros, which means that they were contaminated with E. coli.

[0038] In each of the metanephros in the metanephros groups of Examples 2 to 4, green fluorescence derived from the human mesenchymal stem cells was detected. On the other hand, in the metanephros group of Example 1 and the metanephros group of Comparative Example 1, almost no green fluorescence derived from the human mesenchymal stem cells was detected, which suggests that most of the human mesenchymal stem cells leaked out from the injection site.

Claims

1. A coating step of coating at least a portion of the surface of an organ or tissue for transplantation with a gel; and A method for vitrifying and freezing an organ or tissue for transplantation, comprising a freezing step of contacting the organ or tissue for transplantation after being coated with the gel with a refrigerant for vitrification and freezing.

2. 2. The method of claim 1, wherein the transplant organ or tissue has a foreign substance injected therein and an injection mark, and the gel covers at least a portion of the injection mark.

3. The method of claim 2, further comprising the step of contacting the organ or tissue for transplantation with a vitrification liquid after the coating step.

4. The method of claim 2, wherein the foreign material is a cell derived from the same animal species as the recipient.

5. The method of claim 1 , wherein the organ or tissue for transplantation is a xenotransplant organ or tissue.

6. The method according to claim 1, wherein the organ or tissue for transplantation is an organ or tissue of pig origin.

7. The method of claim 1 , wherein the gel is a hydrogel.

8. A method for producing a vitrified frozen organ or tissue for transplantation, comprising the method according to any one of claims 1 to 7.

9. The method according to claim 8, further comprising the step of removing the gel coating the surface of the organ or tissue for transplantation after the freezing step.

10. A vitrified frozen organ or tissue for transplantation having a gel coating on at least a portion of its surface.

11. The vitrified frozen material according to claim 10, wherein the organ or tissue for transplant has a foreign substance injected therein and an injection mark, and the gel covers at least a portion of the injection mark.