JP2023098487A - 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 - Google Patents

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

The present invention provides a method for vitrifying and freezing an organ or tissue for transplantation, a method for producing a vitrified frozen product, a method for producing an organ or tissue for transplantation, and vitrification and freezing suitable for vitrified cryopreservation of regenerative medicine products. In particular, a method for vitrifying and freezing an organ or tissue for xenotransplantation, a method for producing a vitrified frozen product, a method for producing an organ or tissue for transplantation, and glass, which are particularly suitable for vitrified cryopreservation of regenerative medicine products Regarding frozen products.

Rapid freezing by vitrification freezing based on immersing the biological material for transplantation in a refrigerant for vitrification freezing is indispensable from the viewpoint of long-term storage stability and quality maintenance of products such as regenerative medicine.
For example, in Non-Patent Document 1, as a technique for long-term storage of cell sheets that can be widely applied to regenerative medicine, the cell sheets are immersed in an equilibrium solution, then in a vitrification solution, and then in liquid nitrogen vapor. A method of vitrification and cryopreservation by exposure is described.
In addition, Non-Patent Document 2 describes xenotransplantation data obtained by transplanting vitrified porcine metanephroi to cats.

On the other hand, vitrification-freezing refrigerants or vitrification liquids pose a risk of contamination (for example, cell contamination) when repeatedly used or simultaneously used for a plurality of samples. In other words, there is a risk of contamination of the biological material for transplantation via the vitrification freezing refrigerant or the vitrification treatment liquid, which is not preferable from the viewpoint of quality as a regenerative medicine product or the like.

M. Maehara et al. , BMC Biotechnology 13(58), 2013 Matsumoto K. et al. , Stem Cells 30 (2012): 1228-35. DOI: 10.1002/stem. 1101.

The present invention has been made in view of such problems in the prior art. An object of the present invention is to provide a method for producing a product, a method for producing an organ or tissue for transplantation, and a vitrified frozen product.

As a result of intensive studies on the above-mentioned problems, the present inventors found that by coating at least a portion of the surface of the organ or tissue for transplantation that is in contact with the vitrification freezing refrigerant or the like with a gel, the organ or tissue for transplantation is contaminated. It has been found that the risk (eg cell contamination) can be reduced. The present invention has been completed based on the above findings.
That is, the present invention is as follows.

<1> For transplantation, comprising a coating step of coating at least part of the surface of an organ or tissue for transplantation with a gel, and a freezing step of contacting the organ or tissue for transplantation coated with the gel with a refrigerant for vitrification freezing. A vitrification freezing method for an organ or tissue.
<2> The method according to <1>, wherein the organ or tissue for transplantation has a foreign substance injected therein and an injection scar, and the gel covers at least part of the injection scar.
<3> The method according to <2>, further comprising the step of contacting the transplant donor organ or tissue with a vitrification solution after the coating step.
<4> The method according to <2> or <3>, wherein the foreign substance is a cell derived from the same animal species as the recipient.
<5> The method according to any one of <1> to <4>, wherein the organ or tissue for transplantation is an organ or tissue for xenotransplantation.
<6> The method according to any one of <1> to <5>, wherein the organ or tissue for transplantation is a porcine-derived organ or tissue.
<7> The method according to any one of <1> to <6>, wherein the gel is a hydrogel.
<8> A method for producing a vitrified frozen product of an organ or tissue for transplantation, comprising the method according to any one of <1> to <7> above.
<9> For transplantation, comprising the method according to <8> above, and further comprising, after the freezing step, the step of contacting with a thawing solution, and the step of removing the gel covering the surface of the organ or tissue for transplantation. A method for producing an organ or tissue.
<10> A vitrified frozen product of an organ or tissue for transplantation having a gel coating on at least a part of the surface.
<11> The glass according to <10>, wherein the organ or tissue for transplantation has a foreign substance injected therein and an injection scar, and the gel covers at least part of the injection scar. frozen product.

According to the present invention, a method for vitrifying and freezing an organ or tissue for transplantation, a method for producing a vitrified frozen product, a method for producing an organ or tissue for transplantation, which can reduce the risk of contamination (e.g., cell contamination) of a biological material for transplantation, And, a vitrified frozen product can be provided.

Embodiments of the present invention will be described in detail below, but the present invention is by no means limited to the following embodiments, and can be carried out with appropriate modifications within the scope of the object of the present invention. .

≪Method for Vitrification and Freezing of Organ or Tissue for Transplantation≫
A first aspect of the present invention includes a coating step of coating at least a portion of the surface of an organ or tissue for transplantation with a gel; A method for vitrifying and freezing an organ or tissue for transplantation, comprising steps.
In a first aspect, by coating at least part of the surface of the organ or tissue for transplantation with a gel, there is a risk of contamination (e.g., cell contamination) of the organ or tissue for transplantation from the refrigerant for vitrification freezing, and the above Any risk of contamination (eg, cell contamination) from the transplanted organ or tissue to the vitrification freezing refrigerant can be reduced.
Here, vitrification freezing refers to freezing in an amorphous glass state by suppressing crystallization (ice crystal formation) of water by bringing it into contact with a refrigerant for vitrification freezing (preferably freezing by so-called rapid cooling, more Preferably, it refers to freezing by so-called ultra-rapid cooling.
According to the vitrification freezing, it is possible to suppress the volume expansion due to the crystallization of water, so that the damage (freezing damage) such as breaking of the cell membrane can be suppressed.
Liquid nitrogen (boiling point −196° C.), liquid helium (boiling point −269° C.), liquid ethane (boiling point −175° C.), etc., and their vapors can be used as vitrification freezing refrigerants.
The contact with the vitrification freezing refrigerant is carried out at -273° C. to -70° C. (preferably -270° C. to -80° C., more preferably -269° C. to -150° C.), for example, from 1 second to It is preferable to carry out for 10 minutes (preferably 0.5 seconds to 1 minute, more preferably 0.1 seconds to 10 seconds).

The method of contact with the vitrification-freezing refrigerant is not particularly limited, but includes immersion or application (flash) of liquid, spraying of vapor, and the like.
Contact with the refrigerant for vitrification and freezing includes a support for vitrification and freezing, a cryotop, a straw (e.g., a straw for transplantation, a straw with a sharp tip, etc.), a capillary pipette, which can support a plurality of organs or tissues for transplantation at the same time. It may or may not be performed using a support for vitrification and freezing such as, but it is preferable to perform using the above-mentioned support.
From the standpoint that a plurality of the organs or tissues for transplantation can be frozen as one unit at a time, and the production efficiency of the transplantation material can be improved, and the risk of contamination can be suppressed by the present invention, a plurality of the organs or tissues for transplantation can be used as the support tool. More preferred is the support for vitrification and freezing, which can support organs or tissues at the same time.
As the support for vitrification and freezing, which can simultaneously support a plurality of organs or tissues for transplantation, for example, a glass plate, a metal plate, a plastic plate, or the like has a plurality of depressions on the surface thereof, and a plurality of the transplantation is performed in the depressions. Chips or plates capable of simultaneously supporting internal organs or tissues,
A mesh net, a non-woven fabric, or the like may be used as a supporting tool capable of simultaneously supporting a plurality of organs or tissues for transplantation.
For example, the present inventors have reported a method of vitrifying and freezing mouse or pig embryos in hollow fibers (H. Matsunari, et al., Journal of Reproduction and Development Vol. 58 (2012) No. 5 599-608). Such a hollow fiber containing organs or tissues for transplantation can also be used as the support for vitrification and freezing, which can support a plurality of organs or tissues for transplantation at the same time.
Moreover, CRYOTOP (registered trademark; manufactured by Kitasato Biopharma) and the like can be mentioned as a commercially available supporting tool.
After the freezing step, the organ or tissue for transplantation can be stably stored for a required period (for example, one day or more, one week or more, one month or more, one year or more). Preservation may be carried out in a vitrification cryopreservation container (for example, a Dewar bottle) while contacting with the vitrification and freezing refrigerant, or by other methods.
Although there is no particular upper limit for the vitrified cryopreservation period, examples thereof include 20 years or less, 10 years or less, and 5 years or less.
From the viewpoint of reducing the risk of contamination by the present invention while improving the production efficiency of transplant materials, the freezing step is performed by freezing a plurality of the organs for transplantation or Tissues can be contacted concurrently.
The freezing step may be performed a plurality of times or may not be performed, for example, by performing the freezing step again after the step of contacting with the melting liquid, which will be described later.

The above organ or tissue for transplantation may be derived from a fetus, a juvenile, or an adult, but from the viewpoint of low immunogenicity, an organ or tissue derived from a fetus is preferable. In particular, fetal-derived organs or tissues are preferred for xenotransplantation.
Organs or tissues for transplantation include internal organs (e.g., pancreas, kidney, ureter, bladder, liver, heart, stomach, intestine, etc.), reproductive organs (e.g., ovary, testis, etc.), fertilized eggs, embryos, fetuses, bone marrow. (e.g., hematopoietic organs), brain, eyes, nose, mouth, skin, nerves, or tissues derived from them, or artificial tissues (cell sheets such as chondrocyte sheets, organoids, etc.), internal organs, reproductive organs, Fertilized eggs, embryos and fetuses are preferred, viscera and reproductive organs are more preferred, pancreas (eg pancreatic islets) and kidneys (eg metanephros, especially urinary organs including metanephros, ureters and bladders) are even more preferred.
Examples of animals that provide organs or tissues for transplantation include pigs, cows, horses, sheep, goats, primates (e.g., humans, apes (chimpanzees, monkeys, etc.)), rodents (e.g., mice, rats), and the like. and preferably mammals that are closer to humans in characteristics such as physique than rodents, more preferably pigs, sheep, goats, primates (e.g., humans, apes), and pigs (i.e., the transplantation Pig-derived organs or tissues are more preferred.
The organ or tissue for transplantation is preferably a xenotransplantation organ or tissue.

The method of coating at least part of the surface of the organ or tissue for transplantation with a gel includes a method of embedding or embedding the organ or tissue for transplantation in the gel, and a sol (sol before gelation of the gel). Examples include a method in which the organ or tissue for transplantation is immersed in a liquid containing the sol (preferably a hydrosol) and then gelled, and a method in which the organ or tissue for transplantation is coated with a liquid containing the sol and then gelled. The liquid containing the sol and the gel may or may not contain a basal medium or a basal medium, which will be described later.
Gelation of the sol can be performed by any method such as addition of polyvalent metal ions such as calcium ions and barium ions, cooling, and the like.
From the viewpoint of achieving the object of the present invention more reliably, it is preferable to coat the entire surface of the organ or tissue for transplantation with the gel.
The gel is not particularly limited as long as the object of the present invention can be achieved, but there is no actual harm even if the gel cannot be completely removed and a small amount remains, and the gel remains coated without being removed. A pharmacologically acceptable gel (a pharmaceutically acceptable non-toxic gel) is preferable, and a hydrogel is more preferable, from the viewpoint of portability.
In addition, the above gel is a gel in which a sol-gel phase transition occurs due to ionic cross-linking, or a temperature , 20-25° C., 25°-30° C., 30°-35° C. or 35°-40° C.) undergoing a sol-gel phase transition are preferred.
A gel that undergoes a sol-gel phase transition due to the above ionic cross-linking is, for example, ethylenediaminetetraacetic acid (EDTA), ethylenediaminetetraacetic acid disodium ethylenediaminetetraacetic acid (EDTA.2Na). can become

Examples of the hydrogel include alginic acid or salts thereof (e.g., 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. , calcium alginate, and polyvalent metal salts of barium alginate are preferred.), gelatin, carrageenan, agar (agarose gel), pectin, chitosan, silicone hydrogel, konjac, and other polysaccharides. A hydrogel in which a sol-gel phase transition occurs due to temperature or a temperature-sensitive hydrogel in which a sol-gel phase transition occurs due to temperature is preferred. Alginic acid or its salts (preferably polyvalent metal salts) are examples of hydrogels in which sol-gel phase transition occurs due to ionic cross-linking, and temperature-sensitive hydrogels in which sol-gel phase transition occurs due to temperature include gelatin. , carrageenan, agar (agarose gel), pectin and the like. Gelatin is preferred because it can be solified at a relatively low temperature (for example, 35° C. or lower) and causes less damage to the above organs.
For example, 5-25% (w/v) gelatin solutions can be used, 7-20% (w/v) gelatin solutions are preferred, and 10-15% (w/v) gelatin solutions are more preferred.
The coating step may or may not be performed multiple times using one or more gels.

The covering gel may be one layer or two or more layers.
When the gel to be coated has a plurality of layers, a gel with a relatively low sol temperature (eg, gelatin) is used as the outer layer (eg, the outermost layer), and a gel with a higher sol temperature and a sol is formed by treatment with the chelating agent. A soluble gel (eg, alginic acid) or a pharmacologically acceptable gel is preferably used as the inner layer (eg, the innermost layer).
When gel-coating a plurality of organs or tissues for transplantation in parallel, the outer layer (for example, the outermost layer) may be common among the plurality of organs or tissues for transplantation. A plurality of units of organ or tissue and an inner layer (eg, innermost layer) may be brought together by an outer layer (eg, outermost layer).
For example, the organ or tissue for transplantation and a plurality of units of the inner layer (e.g., the innermost layer) are dispersed in a bag filled with the sol that forms the outer layer (e.g., the outermost layer). A common outer layer (for example, the outermost layer) can be formed among a plurality of transplant organs or tissues.
Each thickness of the coating formed of the gel is not particularly limited as long as the above problems can be solved, but examples thereof include 0.1 mm to 5 mm, preferably 0.5 mm to 2 mm.

After preparation of the organ or tissue for transplantation (e.g., after extraction), before or after the coating step (preferably after the coating step), and before the step of contacting with a vitrification solution described later, An organ or tissue for transplantation is placed in an equilibrium solution at −5° C. to 37° C. (preferably 0° C. to 35° C.), for example, for 1 minute to 3 hours (preferably 5 minutes to 1 hour, more preferably 2 minutes). minutes to 30 minutes) contact (immersion, coating, etc.) step (so-called pretreatment step) may or may not be included. The pretreatment step can alleviate osmotic shock, rapid concentration change, etc., and improve the survival rate.
Examples of the equilibrium solution include a solution (preferably an aqueous solution) containing at least one agent selected from the group consisting of a cell-permeant cryoprotectant and a cell-impermeant cryoprotectant. and a cell-impermeant cryoprotective agent at a concentration lower than that of the vitrification solution described below, preferably a cell-permeable cryoprotective agent and a cell-impermeable cryoprotective agent. More preferably, the solution contains at least one agent selected from the group consisting of agents (in the case where multiple agents are included, each agent) in an amount of 10% by volume or less.
The equilibrium solution is preferably a solution containing a cell-permeable cryoprotectant.
The equilibrium solution may contain a single cell-permeable cryoprotectant or may contain two or more cell-permeable cryoprotectants.
The equilibrium solution may contain a single cell-impermeant cryoprotectant or may contain two or more cell-impermeant cryoprotectants.
Specific examples of the cell-permeable cryoprotectant and the cell-impermeable cryoprotectant are described below.
The equilibrium solution may or may not further contain a basal medium or basal medium.
The basal medium or basal medium is not particularly limited, but includes any synthetic medium such as TCM199 (preferably 10 to 35 mM HEPES-buffered TCM199), PBS (phosphate buffer), and MEM. It may or may not contain bovine serum (CS), fetal calf serum (FCS), serum replacement, etc. (for example, 10 to 20% by mass).
The step of contacting with the equilibrium solution may or may not be performed multiple times (for example, stepwise) using one or more equilibrium solutions.

Before or after the coating step, the organ or tissue for transplantation is placed in a 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) preferably includes a step of contact (immersion, coating, etc.).
The step of contacting with the vitrification solution may be before or after the coating step, but from the viewpoint of promoting penetration of the vitrification solution, the organ for transplantation or the It preferably includes the step of contacting the tissue with the vitrification fluid.
On the other hand, from the viewpoint of reducing the risk of contamination, it is preferable to include a step of contacting with the vitrification liquid after the coating step.
Examples of the vitrification solution include a solution (preferably an aqueous solution) containing a cell-permeable cryoprotectant and a cell-impermeable cryoprotectant.
The content of the above-mentioned cell-permeable cryoprotectant (total amount when multiple types are contained) is 25% by volume or more and 60% by volume or less, 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) is 0.2 to 2M, preferably 0.4 to 1M.
Examples of the cell-permeable cryoprotectant include dimethylsulfoxide (DMSO), ethylene glycol (EG), propanediol, glycerin and the like.
Examples of the cell-impermeable cryoprotectant include saccharides such as sucrose, trehalose, sorbitol and dextran, carboxylated polylysine, polyvinyl alcohol, polyvinylpyrrolidone, antifreeze proteins and the like.
The vitrification solution may or may not further contain the basal medium or basal medium, and from the viewpoint of improving survival rate, may further contain any serum, polyvinylpyrrolidone, or any thickening agent. may or may not be present.
The step of contacting with the vitrification liquid may or may not be performed multiple times (for example, stepwise) using one or more vitrification liquids.

From the viewpoint of more reliably avoiding the above-described risks, the above-described contact with the vitrification-freezing refrigerant is performed after the above-described coating and the above-described After housing the organ or tissue after contact with the vitrification liquid, the contact may be made via the container, or after the organ or tissue after the coating and after contact with the vitrification liquid is surrounded by an arbitrary film. (After wrapping), contact may be made via the film.
After the freezing step, the organ or tissue after vitrification freezing (preferably after vitrification cryopreservation) is placed in a thawing solution for, for example, 30 seconds to 30 minutes (preferably 40 seconds to 10 minutes, more preferably 50 seconds to 5 minutes) contact (immersion, application, etc.) may or may not be included, but it is preferable to include the frozen organ or tissue in contact with the thawing solution or diluted. However, it is more preferable to warm to room temperature (eg, 25°C) to 38°C.
The melting liquid is not particularly limited and may be any melting liquid. liquid).
The melt may or may not contain the basal medium or basal medium.
The step of contacting with the melt may or may not be performed multiple times (for example, stepwise) using one or more melts.

After the step of contacting with the lysing solution, the organ or tissue is placed in a diluent under a temperature condition of 0° C. to 37° C. (preferably 10° C. to 35° C.), for example, for 30 seconds to 30 minutes (preferably may or may not include a step of contact (immersion, coating, etc.) for 40 seconds to 10 minutes, more preferably 50 seconds to 5 minutes, but preferably includes.
The diluent is not particularly limited, and may be any diluent. liquid). The diluent may or may not additionally contain a penetrating cryoprotectant such as ethylene glycol.
The diluent may or may not further contain the basal medium or basal medium.
The step of contacting with the diluent may or may not be performed 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 in a low oxygen atmosphere (for example, an atmosphere of 5% oxygen, 5% carbon dioxide and 90% nitrogen, 5% carbon dioxide gas It may or may not be carried out under a carbon dioxide gas atmosphere of 95% air).

Each of the above series of steps may or may not be performed continuously in a container (for example, bag) containing the organ or tissue.
The container for containing the organ or tissue includes a container body for containing the organ or tissue, an injection member for injecting liquid from the outside of the container into the inside of the container, and a discharge member for discharging the liquid from the inside of the container to the outside of the container. wherein the injection member and the discharge member are positioned such that a liquid flow occurs between the injection member and the discharge member, and the liquid flow is contained in the container body More preferably, it is installed at a position that contacts the organ or tissue.
The container body may be of any type as long as it can contain the biological sample, but a bag-like container is preferred. The bag-like container is preferably provided with an opening through which the organ or tissue is taken in and out, and the opening is preferably openable and closable with a zipper or the like. As this bag-like container, a commercially available storage bag with a zipper can be used. The container body is not limited to a bag-like container, and may be, for example, a container in which films are adhered to the upper and lower surfaces of a picture-frame-like frame. The shape of the container body may be any shape, but a rectangular shape is preferable in terms of ease of installation of the injection member and the discharge member. Any material may be used for the container body, but a material that is transparent and has good thermal conductivity is preferable. When the container body is a bag-like container, the material of the container body is, for example, low-density polyethylene, medium-density polyethylene, ultra-high-molecular-weight polyethylene, polyethylene-polytetrafluoroethylene copolymer, polyethylene-1,2- A dichloroethane copolymer and the like are preferred. The size of the container body can be determined according to the organ or tissue to be accommodated. For example, when the container body is a rectangular bag-like container, the rectangle can have a long side of 30 to 350 mm and a short side of 10 to 100 mm.

The above-treated organ or tissue for transplantation may or may not be cultured in the above basal medium or basal medium before transplantation (preferably xenotransplantation).

For example, when the organ or tissue for transplantation is used as a regenerative medicine product, the foreign substance (for example, cells derived from the same animal species as the recipient) is injected into the organ or tissue for transplantation, and then vitrified and frozen. may be required to be stored as
On the other hand, if there is an injection scar, which will be described later, in the freezing step (and the step of contacting with the vitrification solution), the osmotic pressure gradient between the inside of the organ or tissue for transplantation and the external solution causes the foreign matter to be transferred to the transplanted organ or tissue. There is a problem of leakage from the organ or tissue used.

In view of the above problems, it is also preferable that the organ or tissue for transplantation has a foreign substance injected inside and an injection scar, and the gel covers at least a part of the injection scar. morphology.
Here, the term "injection scar" means a hole formed for artificially injecting the foreign substance into the organ or tissue for transplantation, and not only the hole on the surface of the organ or tissue for transplantation. , means a communicating hole leading to the injection target area inside the organ or tissue for transplantation. The "injection scar" may be formed by, for example, a needle-like member or the like. The pore diameter (eg, average diameter) of the pores is not particularly limited, and is, 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 part of the injection marks made up of the holes and the communication holes on the surface and the holes on the surface may be covered with the gel.
This makes it possible to suppress leakage of the foreign substance due to an osmotic pressure gradient between the inside of the organ or tissue for transplantation and an external solution (for example, the vitrification solution, the equilibrium solution, etc.).

Here, the foreign substance means a substance derived from other than the above-mentioned organ or tissue for transplantation, and the foreign substance includes a substance derived from the same animal species as the recipient (transplantation recipient individual), a donor (transplantation source individual), ), but derived from an individual different from the donor, substances derived from an animal species different from both the recipient and the donor, drugs (eg, pharmaceuticals), and the like. As a substance derived from an animal species, a substance derived from the same animal species as the recipient is preferable from the viewpoint of low immunogenicity. Substances include, for example, cells, growth factors, hormones, cytokines, etc. Among them, cells are preferred. The cells may or may not be artificial cells (eg, genetically modified cells, ES cells, iPS cells).
For example, in the case of xenotransplantation in which porcine organs or tissues are transplanted into humans, the above-mentioned "cells derived from the same animal species as the recipient" include "human-derived cells".
In the above preferred embodiment, from the viewpoint of suppressing leakage of the foreign matter 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 solution after the coating step.

When injecting the foreign material, the organ or tissue for transplantation is positioned by contacting a gel similar to the gel described above (that is, the gel is used as a positioning jig), and the organ or tissue for transplantation after positioning is positioned. A foreign substance may or may not be injected into the tissue.
If a gel similar to the gel described above is used as a positioning jig, the organ or tissue for transplantation remains in contact with the gel before removing the gel used as the positioning jig after the injection of the foreign substance. From the viewpoint that the organ or tissue for transplantation can be subjected to the above-described coating step with the same gel, it is preferable to position the organ or tissue for transplantation in contact with the same gel as the gel described above.
In addition, in the following removal step of removing the coated gel, both the gel used as the positioning jig and the gel used in the coating step can be removed together in the following removal step. Preferably, the tissue is positioned in contact with a gel similar to that described above.
Here, the term "positioning" refers to grasping (firmly holding) and positioning the organ or tissue for transplantation so that it does not move during the process of injecting the foreign substance, and the transplantation of the tissue to the gel. The positional relationship of the internal organs or tissues can be maintained with high accuracy.

<<Method for Producing Vitrified Frozen Organ or Tissue for Transplantation, and Method for Producing Organ or Tissue for Transplantation>>
A second aspect of the present invention is a method for producing a vitrified frozen product of an organ or tissue for transplantation, including the vitrification freezing method according to the first aspect.
The method for producing a vitrified frozen material 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 the removal From the viewpoint that the organ or tissue for later transplantation can be used for transplantation, it is preferable that the removal step is included.
A third aspect of the present invention includes a method for producing a vitrified frozen material according to the second aspect, comprising a step of contacting with a thawing solution, and a step of removing the gel covering the surface of the organ or tissue for transplantation. A method for producing an organ or tissue for transplantation, further comprising
The coated gel can be removed by heating to a sol-forming temperature, arbitrary sol-forming such as the chelate treatment, or physical pressure.
Furthermore, it may or may not be washed with any washing liquid such as any aqueous solution or phosphate buffer (running water).
The removal step may or may not be performed multiple times (eg, stepwise).

A gel with a relatively low solation temperature (e.g., gelatin) is used as the outer layer (e.g., the outermost layer), and a gel (e.g., alginic acid) or a drug with a higher solation temperature that can be solated by treatment with the chelating agent. When two or more layers of gel with a physically acceptable gel as the inner layer (for example, the innermost layer) are coated,
After removing the outer layer (e.g., the outermost layer) by thermal solation and physical pressure, which have a high risk of damage, the inner layer (e.g., the innermost layer) is solated by the above chelate treatment. The gel is removed in two steps. is preferred.
According to this, the inner layer (for example, the innermost layer) gel acts as a barrier, and the damage during removal of the outer layer (for example, the outermost layer) is less likely to be transmitted to the transplant organ or tissue.
In addition, since the chelation treatment is mild, damage to the organ or tissue for transplantation during the solation process can be minimized.
Also, the pharmacologically acceptable gel does not necessarily have to be removed at the time of implantation. In this case, damage due to gel removal can be more reliably avoided.
In addition, the contamination source is the outer layer gel that acts as a barrier and does not reach the inner layer (e.g., innermost layer). Problems may occur even if the inner layer (e.g., innermost layer) gel is left in place when transplanted. do not have.

≪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 part of the surface.
Since at least part of the surface of the vitrified frozen material according to the fourth aspect is gel-coated, it can be cryopreserved with reduced risk of contamination (eg, cell contamination).
Furthermore, by performing the removal step, it can be used as a transplant donor.
Specific examples and preferred examples of the organ or tissue for transplantation are as described above.
Preferably, the organ or tissue for transplantation has a foreign substance injected therein and an injection scar, and the gel covers at least a part of the injection scar, and the gel covers the injection scar. More preferably, the gel covers the entire surface of the organ or tissue for transplantation.
Specific examples and preferred examples of the foreign matter are as described above.
The injection marks are as described above.

Examples of the present invention are shown below to describe the present invention more specifically, but the present invention is not limited to these, and various applications are possible without departing from the technical idea of the present invention. is.

<Examples 1 to 4 and Comparative Example 1>
(material)
A capillary tube with a sharply polished tip that expresses green fluorescent protein (GFP) and into which GFP-expressing human mesenchymal stem cells that emit green fluorescence were aspirated was prepared.
24 out of 40 metanephros extracted from a total of 20 pig fetuses obtained by cesarean section from 5 DPF-controlled wild-type sows (same clone) were each placed on a 35 mm dish (manufactured by Iwaki). The above-mentioned human mesenchymal stem cells were injected as a foreign object with the above-mentioned capillaries under the capsule of each of the above-mentioned metanephros.
In addition, 16 of the 40 metanephros removed were each semi-embedded in 10% (w/v) gelatin gel (i.e., while there was an exposed surface of the metanephroi not in contact with the gel). , the portion of the metanephro in contact with the gel is embedded), each semi-embedded metanephro in the gelatin gel is placed on a 35 mm dish (manufactured by Iwaki) and positioned, and the semi-embedded The human mesenchymal stem cells were injected as an exogenous substance into the exposed surface with the capillaries in the nephrogenic zone under the capsule of each metanephroi.

8 of the 24 metanephros that had injection scars after the injection and were not semi-embedded were treated with 7.5% by weight EG and 7.5% by weight DMSO prior to the coating step described below. A pretreatment step was carried out by immersing the sample in 4.5 ml of the equilibration solution containing the freeze protection agent at room temperature (25 to 27°C) for 25 minutes, followed by double the amount of 15 mass% EG and 15 mass% DMSO and 0.5M. It was immersed in a vitrification solution containing sucrose for 30 minutes at room temperature. A group of 8 metanephros having the above-described injection scars, undergoing the above-described pretreatment process prior to the coating process described below, and immersed in the vitrification solution was used as the metanephro group of Example 1.

(Coating process)
Gelatin powder (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in HEPES-buffered TCM199 medium of pH 7.2 to prepare a gelatin solution with a concentration of 10-15% (w/v).
The above 10-15% (w/v) gelatin solution is added dropwise to the 8 metanephros of Example 1 that have the injection scar, are not semi-embedded, and are subjected to the pretreatment, or The metanephros are placed in a drop of gelatin solution, and the gelatin gel is applied so that only the injection scar of the metanephros and the vicinity thereof (part of the surface of the metanephros and including the injection scar) are coated. coated with
In addition, for 8 of the 16 metanephros that had injection scars after the injection, were not semi-embedded, and were not subjected to the pretreatment step, the gelatin solution was dripped or the gelatin solution was added. Metanephros were put into the drops and covered with gelatin gel so that only the injection scars of the metanephros and the vicinity thereof were covered. The metanephro group of Example 2 was defined as the metanephro group of 8 mice which were not subjected to the pretreatment process before the coating process and were coated only at the injection site and its vicinity.
On the other hand, the remaining 8 out of the 16 metanephros that had injection scars after the injection and were not semi-embedded were not subjected to the coating step with the gelatin gel. A group of eight metanephros that had injection scars after the above injection and were not subjected to the above-described coating step were used as the metanephro group of Comparative Example 1.
In addition, for 8 of the 16 metanephros that have injection scars after the injection, are semi-embedded, and have not been subjected to the pretreatment step, the gelatin solution is dropped or added to the gelatin solution drop. The metanephros were put in and covered with gelatin gel so that only the injection scars of the metanephros and the vicinity thereof were covered. The 8 metanephros group, which was not subjected to the pretreatment process before the coating process, was semi-embedded, and was coated only at the injection site and the vicinity thereof, was used as the metanephro group of Example 3.
In addition, for the remaining 8 of the 16 metanephros that have injection scars after the injection, are semi-embedded, and have not been subjected to the pretreatment step, the gelatin solution is dripped or the gelatin solution is added. The metanephros were put into the drops, and the entire metanephros including the injection scars were covered with gelatin gel. A group of 8 semi-embedded metanephros in which the entire metanephroi was covered including the injection scar was used as the metanephro group of Example 4.
(Pretreatment step)
For the 24 metanephros groups of Examples 2 to 4 after being coated with the gelatin gel and the 8 metanephros groups of Comparative Example 1 not coated with the gelatin gel, 7.5 mass % EG and 7.5 wt. Pretreatment was carried out by immersing in 4.5 ml of an equilibrium solution containing 5 mass % DMSO as a freeze protection agent at room temperature (25 to 27° C.) for 25 minutes.
(Vitrification freezing step, melting step and dilution step)
Then, for the 24 metanephros of the metanephros group of Examples 2 to 4 after pretreatment and the 8 metanephros of the metanephros group of Comparative Example 1 not coated with the gelatin gel, 15 masses, which is twice the above amount, % EG and 15 mass % DMSO and 0.5 M sucrose in a vitrification solution for 30 minutes at room temperature.
Then, after immersion in the vitrification solution, the 32 metanephrine groups of Examples 1 to 4 and the 8 metanephrogroups of Comparative Example 1 that were not coated with the alginate gel were attached to the support "cryotop (manufactured by Kitasato BioPharma) and placed in liquid nitrogen that was intentionally contaminated with E. coli (pathogen) for the contamination risk test evaluation described later.
The vitrified and frozen organs were transferred to a vitrified cryopreservation container, frozen in liquid nitrogen for 10 days, and thawed by immersion in a thawing solution containing 1M sucrose at 37°C for 1 minute. Then, the plate was washed twice with a diluent containing 0.5 M sucrose for 3 minutes at room temperature and twice with a washing solution for 5 minutes to dilute and remove the cryoprotectant.

(Gel removal step)
After the thawing and dilution of the cryoprotectant, the 32 metanephros groups of Examples 1 to 4 and the 8 metanephros groups of Comparative Example 1 were immersed in HEPES-buffered TCM199 medium at 37°C for 30 minutes. After sol-forming the gelatin gel, the gelatin was removed by washing twice with a washing solution for 5 minutes each.
For the 16 metanephros of the above metanephros group of Examples 3 and 4, which were semi-embedded in gelatin gel for positioning at the time of injection of the foreign substance, the gelatin gel that was semi-embedded for positioning was It was confirmed that the injection trace portion can be removed together with the coated gel in the gel removing step.
The metanephros groups of Examples 1 to 4 and Comparative Example 1 obtained above were subjected to the following contamination risk test and foreign matter leakage test and evaluated.

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

<Foreign matter leakage test>
The metanephros of Examples 1 to 4 and Comparative Example 1 were tested for detection of green fluorescence derived from the human mesenchymal stem cells.
Examples 1 to 4 and Comparative Example 1, and the test results are summarized in Table 1 below.

As is clear from the results shown in Table 1 above, almost no E. coli was detected in the metanephros group of Examples 1 to 3, in which only the injection scar and its vicinity were gel-coated. It can be said that the risk of contamination was avoided. In particular, in the metanephros group of Example 4 in which the entire organ was gel-coated, no E. coli was detected, and the risk of contamination was completely avoided.
On the other hand, in the metanephros group of Comparative Example 1, the above E. coli was detected in all four metanephros, and it can be said that the E. coli was contaminated.

Green fluorescence derived from the human mesenchymal stem cells was detected in each metanephro in the metanephros of Examples 2-4.
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, and the human mesenchymal stem cells were detected from the injection scar. Most of it is said to have leaked.

Claims (10)

A coating step of coating at least a portion of the surface of a urinary tract for transplantation including metanephros, ureters and bladders from a porcine with a gel, and contacting the urinary tract for transplantation after coating with the gel with a vitrification freezing refrigerant. The method for vitrifying and freezing the urinary organ for transplantation, comprising a freezing step. 2. The method of claim 1, wherein the urinary implant has a foreign object injected therein and an injection scar, the gel covering at least a portion of the injection scar. 3. The method of claim 2, further comprising contacting the urinary implant with vitrifying fluid after the coating step. 4. The method according to claim 2 or 3, wherein the foreign substance is a cell derived from the same animal species as the recipient. The method of any one of claims 1-4, wherein the urinary transplant is a xenotransplant urinary. The method of any one of claims 1-5, wherein the gel is a hydrogel. A method for producing a vitrified frozen urinary organ for transplantation, comprising the method according to any one of claims 1 to 6. A method for producing a urinary organ for implantation, comprising the method according to claim 7, further comprising the steps of contacting the urinary organ for implantation with a thawing solution after the freezing step, and removing the gel covering the surface of the urinary organ for implantation. A vitrified frozen urinary tract, including metanephros, ureters and bladder, from porcine for transplantation, having a gel coating on at least a portion of the surface. 10. The vitrification cryopreservate according to claim 9, wherein the urinary organ for transplantation has a foreign object injected therein and an injection scar, and the gel covers at least a part of the injection scar.