JP2020039261A - Cryopreservation liquid - Google Patents

Abstract

To provide a cell or tissue cryopreservation liquid that can easily and reliably perform cell or tissue cryopreservation work.SOLUTION: A cryopreservation liquid contains a modified polyvinyl alcohol having an ethylene oxide group.SELECTED DRAWING: None

Description

  The present invention relates to a cryopreservation solution used for cryopreservation of cells or tissues.

  Excellent techniques for preserving cells or tissues of organisms are required in various industrial fields. In general, cells or tissues collected from a living body gradually lose their activity even in a culture solution, so that long-term culture of cells or tissues in vitro is not preferable. Therefore, a technique for preserving the bioactivity for a long time without losing its biological activity is important. By transplanting cells or tissues while maintaining higher biological activity by using excellent preservation techniques, it is expected that the survival rate after transplantation will be improved. Furthermore, an excellent preservation technique is to sequentially produce and store artificial tissues for transplantation, such as cultured skin cultured in vitro and so-called cell sheets constructed in vitro, and use them when necessary. It is possible to achieve great benefits not only in research and medicine but also in industry. In addition, an excellent preservation technique enables more accurate analysis of the collected cells or tissues.

  As a method for preserving cells or tissues, for example, a slow freezing method is known. In this method, first, cells or tissues are immersed in a cryopreservation solution obtained by including a freeze-resistant agent in a physiological solution such as a phosphate buffered saline. Compounds such as glycerol and ethylene glycol are used as the antifreezing agent. After immersing the cells or tissues in the preservative solution, the cells or tissues are cooled to −30 to −35 ° C. at a relatively slow cooling rate (for example, a rate of 0.3 to 0.5 ° C./min), so that the inside or outside of cells or The solution inside and outside is sufficiently cooled, and the viscosity of the storage solution increases. In such a state, when the cells or tissues in the storage solution are further cooled to the temperature of liquid nitrogen (−196 ° C.), a glass in which the micro solution in the cells or in the tissues and the surroundings are solidified in an amorphous state. Transformation occurs. When the inside and outside of the cell or the inside of the tissue is solidified by vitrification, the movement of the molecule is substantially stopped. Therefore, it is considered that the vitrified cell or the tissue can be stored semipermanently by storing it in liquid nitrogen.

  However, the slow freezing method has a problem that the operation for cryopreservation requires a long time because it is necessary to cool at a relatively low cooling rate. Further, there is a problem that a device or a jig for controlling the cooling rate is required. In addition, in the slow freezing method, ice crystals are formed in the extracellular or extracellular preservation solution, and cells or tissues may be physically damaged by the ice crystals.

  As a method for solving the problems of the slow freezing method described above, a vitrification cryopreservation method has been proposed. The vitrification cryopreservation method is based on the principle that freezing points of a cryopreservation solution containing a large amount of a freezing agent such as glycerol, ethylene glycol, and DMSO (dimethyl sulfoxide) are reduced so that ice crystals are hardly formed even below the freezing point. When this cryopreserved solution is rapidly cooled in liquid nitrogen, it can be solidified without forming ice crystals. This solidification is called vitrification freezing.

  As a specific operation of the vitrification cryopreservation method, cells are immersed in a cryopreservation solution, and then cooled at the temperature of liquid nitrogen (−196 ° C.). The vitrification cryopreservation method does not require a long time for the operation for cryopreservation because it is such a simple and quick process, and does not require a device or a jig for temperature control. There are advantages.

  When the vitrification freezing method is used, in principle, ice crystals are not generated inside or outside the cells, so that physical damage (freezing damage) to the cells at the time of freezing and thawing can be avoided. In order to achieve effective vitrification and freezing, the concentration of the antifreezing agent contained in the preservation solution used for vitrification must be high. On the other hand, the high-concentration cryoprotectant contained in the cryopreservation solution has high chemical toxicity to the cells. Therefore, from the viewpoint of reducing cytotoxicity, it is preferable to reduce the concentration as much as possible. It is known that in order to perform vitrification freezing with a cryopreservation solution containing a low-concentration antifreeze, it is necessary to increase the freezing speed.

  From the viewpoint of increasing the freezing rate of the cells or tissues, it is preferable that the amount of the cryopreservation solution existing around the cells or tissues during cryopreservation is small. The smaller the amount of cryopreservation solution present around the cells or tissues, the smaller the heat capacity of the object to be frozen, the faster the freezing rate of the cells or tissues, which is preferable for vitrification freezing. Furthermore, the fact that the amount of cryopreservation solution present around cells or tissues is small means that even during thawing after freezing, the cryopreservation solution is promptly diluted in the thawing solution, and re-ice crystals form in cells or tissues. It is preferable from the viewpoint of suppression. Furthermore, the concentration of the antifreezing agent mixed into the melt at the time of thawing can be reduced, which is preferable from the viewpoint of reducing the chemical toxicity derived from the antifreezing agent.

  Regarding the cryopreservation of cells or tissues using the vitrification cryopreservation method, examples using various types of cells or tissues by various methods are shown. For example, Patent Document 1 shows that application of the vitrification cryopreservation method to germ cells or somatic cells of animals or humans is extremely useful in terms of the survival rate after cryopreservation and thawing, A cryopreservation solution containing 5.5M ethylene glycol and 1M sucrose, a cryopreservation solution containing 40% by mass ethylene glycol and 0.3M trehalose, and the like are described.

  The vitrification cryopreservation method is a technique that has been developed mainly using human germ cells. Recently, application to iPS cells and ES cells has been widely studied. Non-patent Document 1 shows that the vitrification cryopreservation method was effective for preserving Drosophila embryos. Further, Patent Document 2 discloses that the vitrification cryopreservation method is effective in preserving cultured plant cells and tissues. In the former, a cryopreservation solution containing ethylene glycol, glycol, propylene glycol, glycerol, and DMSO as a cryoprotectant is described, and in the latter, DMSO, as a cryoprotectant (cryoprotectant) contained in the cryopreservation solution, Propylene glycol, glycerol, polyethylene glycol, butanediol, formamide, propanediol, sorbitol, mannitol and the like are exemplified. As described above, the vitrification cryopreservation method is known to be useful for preserving a wide variety of cells and tissues.

  In Patent Document 3, the eggs or embryos are placed on a preservative remover together with a cryopreservation solution and aspirated from below to remove excess cryopreservation solution attached to the periphery of the eggs or embryos, resulting in excellent survival. A cryopreservation method has been proposed, wherein the cryopreservation solution contains ethylene glycol, glycerol and sucrose.

  Further, Patent Document 4 discloses a cryopreservation solution containing 1.5% of polyvinyl alcohol as a cryopreservation solution for cells or tissues in a slow freezing method, mainly for protection of cells, that is, for achieving excellent viability. A preservation solution has been proposed, and Patent Literatures 5 and 6 each disclose polyvinyl alcohol as a cell protection substance or a cryoprotection substance contained in the cryopreservation liquid in the vitrification cryopreservation method. On the other hand, Patent Literature 7 describes a cryopreservation jig having a outermost layer which is a dry film containing a modified PVA having an ethylene oxide group on a mounting portion on which cells or tissues are mounted. By dissolving in the melt, the recoverability of cells or tissues (the detachability of cells or tissues) is enhanced.

Japanese Patent No. 3044432 JP 2008-5846 A International Publication No. 2011/070973 pamphlet JP 2005-261413 A JP 2010-213692 A JP 2013-1111017 A JP 2017-60457 A

Steponkus et al. , Nature 345: 170-172 (1990).

  As described above, in vitrification cryopreservation, by placing cells or tissues together with a small amount of cryopreservation solution, excellent viability of the cells or tissues is achieved. When the tissue is placed and frozen, when thawing after freezing, the ovum or embryo on the sheet often adheres to the surface of the placement part, depending on the type or state of the cell or tissue, and is high when these are collected. There was a problem that skill was required. Furthermore, as the worker freezes the cells or tissues with a smaller amount of cryopreservation solution, which is a preferable state for freezing, the problem that the cells or tissues adhere to the surface of the mounting portion when the cells or tissues are thawed after freezing is more remarkable. Met.

  In particular, in Patent Document 3 described above, by using a vitrified preservative removal material in a portion where an embryo or an egg is placed, it is not necessary for an operator to remove an excess cryopreservation liquid attached around cells or tissues. Although good workability can be obtained in that freezing preservation solution is removed and freezing is performed with a small amount of cryopreservation solution, when the preservation solution absorber absorbs the preservation solution, the cells or tissues are absorbed by the preservation solution absorber. In particular, and may require particularly high skill in recovering cells or tissues. In the freezing operation, cells or tissues are frozen after being pipetted several times in a cryopreservation solution, and this operability is also important.

  The main object of the present invention is to provide a cryopreservation solution for cells or tissues, which can perform the cryopreservation work of cells or tissues easily and reliably. More specifically, it shows good workability in the pipetting operation during the freezing operation, and allows the cells or the tissue to be easily collected during the thawing operation without the cells or the tissue sticking to the surface of the mounting portion. It is an object to provide a cryopreservation solution that can be used.

  As a result of intensive studies to solve the above problems, the present inventors have found that the above problems can be solved by the following means.

  (1) A cryopreservation solution containing a modified polyvinyl alcohol having an ethylene oxide group.

  According to the invention of the above (1), when the cells or tissues are vitrified and cryopreserved, good workability is exhibited in the pipetting operation at the time of freezing, and the cells or tissues are placed on the surface of the mounting portion at the time of thawing. It is possible to provide a cryopreservation solution from which cells or tissues can be easily collected without sticking to the cells.

  The cryopreservation solution of the present invention is used when cryopreserving cells or tissues. In the present invention, a cell includes a single cell or a cell population of an organism composed of a plurality of cells. The cell population composed of a plurality of cells may be a colony-shaped or cluster-shaped cell population composed of a single type of cells, or may be a colony-shaped or cluster-shaped cell population composed of a plurality of types of cells. . In addition, the tissue may be a tissue composed of a single type of cells or a tissue composed of a plurality of types of cells, and includes a non-cellular substance such as an extracellular matrix in addition to the cells. It may be something.

  The cryopreservation solution of the present invention is used for cryopreservation work, preferably for vitrification cryopreservation work. More specifically, the cryopreservation solution of the present invention can be preferably used for cryopreservation of embryos or eggs, including the Cryotop (registered trademark) method.

  The freezing operation in the cryotop method is generally performed according to the following procedure. After immersing cells or tissues such as embryos and ova in the equilibration solution, the cells or tissues are taken out together with a small amount of the equilibration solution using a tubular jig such as a pipette and moved into a cryopreservation solution. After a cell or tissue is immersed in a cryopreservation solution for a predetermined time, the cell or tissue is taken out together with a small amount of cryopreservation solution, and the cell or tissue is stored in a cryopreservation jig sheet (for example, JP-A-2002-315573 and On a colorless and transparent egg attachment holding strip described in JP-A-2006-271395 or a vitrification stock removal material described in Patent Document 3, a small amount of a cryopreservation solution is dropped and adhered. At this time, when a large amount of the cryopreservation solution is dropped, an operation of removing an excess cryopreservation solution is performed using a thin tubular jig such as a pipette. Thereafter, the sheet on which the cells or tissues are placed is immersed in a cooling medium such as liquid nitrogen and frozen. In the freezing operation, for the purpose of dehydrating the water in the cells or tissues and replacing the cells or tissues with a solution containing a cryoprotectant, the cells or tissues in the equilibration solution or the cryopreservation solution are formed into a tubular jig. In some cases, the pipetting operation is performed by using the pipette. According to the present invention, the pipetting operation can be performed with good workability. Next, the melting operation will be described. When thawing cells or tissues, the cells or tissues placed on the sheet are taken out of the cooling medium and immersed in a melt to melt. When the cryopreservation solution of the present invention is used, the cells or tissues can be easily collected from the mounting portion at the time of thawing, so that the operation related to the cryopreservation of the cells or tissues can be performed easily and reliably.

  The vitrification freezing in the present specification is different from the so-called slow freezing method in which the freezing is performed at a relatively low cooling rate (for example, a rate of 0.3 to 0.5 ° C./min), and a cryogenic cooling medium (for example, liquid nitrogen) is used. ) Is a method of freezing cells or tissues at a high cooling rate while suppressing ice crystal formation. The cooling rate in the vitrification freezing is, for example, a cooling rate from 0 ° C. to −150 ° C. measured using a sheath type thermocouple manufactured by Chino Corporation (tip outer diameter 0.3 mm) is 200 ° C./min or more. is there.

  The cryopreservation solution of the present invention contains a modified polyvinyl alcohol having an ethylene oxide group.

  In a cryopreservation solution, polyvinyl alcohol is known to have a role as a cell protective substance or a cryoprotective substance, and is known to be contained mainly for the purpose of improving the viability after freeze-thaw. According to the above-mentioned Patent Document 4, it is described that there is an action of maintaining a skeleton of an embryo cell as a substitute for a protein similarly added as a cryoprotectant.

  The polyvinyl alcohol contained in the cryopreservation solution of the present invention is a modified polyvinyl alcohol having an ethylene oxide group. The modified polyvinyl alcohol having an ethylene oxide group has excellent hydrophilicity and moderate solubility, and the cryopreservation solution containing the polyvinyl alcohol is placed when dropped onto the above-mentioned sheet together with cells or tissues. The solution remains and adheres to the surface of the mounting portion in a solution state, and covers the surface of the mounting portion. The periphery of the cell or the tissue placed on the surface of the placement section is covered with the polyvinyl alcohol. The coating formed by these processes effectively acts to prevent the cell or tissue from sticking to the mounting portion, and as a result, the cell or tissue can be easily collected. As the modified polyvinyl alcohol having an ethylene oxide group, for example, Gosenex (registered trademark) WO-320N and Gosenex WO-320R manufactured by Nippon Synthetic Chemical Industry Co., Ltd. can be used.

  The content of the modified polyvinyl alcohol having an ethylene oxide group contained in the cryopreservation solution of the present invention is preferably 0.1 to 3% by mass. If the content is less than 0.1% by mass, good releasability during melting may not be obtained. On the other hand, if the content is more than 3% by mass, the viscosity of the cryopreservation solution is too high, which may hinder handling such as operation of a tubular jig such as a pipette. In some cases, the workability of pipetting work or removing cells or tissue from a cryopreservation solution may be reduced. A more preferred content of the modified polyvinyl alcohol having an ethylene oxide group is 0.7 to 2.2% by mass.

  The cryopreservation solution of the present invention preferably contains an organic solvent-based antifreeze. As the organic solvent-based antifreeze, an organic solvent-based antifreeze such as ethylene glycol, DMSO (dimethyl sulfoxide), glycerol, propylene glycol, or propanediol can be suitably used. The organic solvent-based antifreeze may be used alone or in combination of two or more. The concentration of the organic solvent-based antifreeze in the cryopreservation solution of the present invention is preferably 20 to 50% by volume. More preferably, the content is 25 to 45% by volume.

  The cryopreservation solution of the present invention can contain a saccharide from the viewpoint of adjusting osmotic pressure and imparting vitrification performance (freezing resistance). As the saccharide, sucrose, trehalose, glucose, raffinose, lactose, maltose, mannose, galactose, and fructose can be suitably used. The saccharides may be used alone or in combination of two or more. The concentration of the saccharide in the vitrified cryopreservation solution of the present invention is preferably from 0.2 to 1M, more preferably from 0.3 to 0.7M.

  The cryopreservation solution of the present invention can contain a polymer compound other than polyvinyl alcohol having an ethylene oxide group from the viewpoint of viscosity adjustment and cell protection effect. As the high molecular compound, for example, a high molecular compound such as polyethylene glycol, ficoll (copolymer of sucrose and epichlorohydrin), dextran, polyvinylpyrrolidone, albumin, or hyaluronic acid, gellan gum, xanthan gum, carrageenan, sodium alginate Alginic acid derivatives and salts thereof, and thickening polysaccharides such as cellulose derivatives such as methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose and carboxymethylcellulose and salts thereof. The content of these polymer compounds is preferably 100% by mass or less based on polyvinyl alcohol having an ethylene oxide group.

  The cryopreservation solution of the present invention may further contain a biological material such as serum. Serum has been widely used for its cytoprotective effect during freezing, although its components have not yet been completely elucidated.

  In the freezing operation using the cryopreservation solution of the present invention, the time during which cells or tissues are immersed in the cryopreservation solution (the time from contacting the cells with the cryopreservation solution to starting cooling with the cooling solvent) is used. Although it depends on the composition of the cryopreservation solution, in most cases, it is preferably within 3 minutes from the viewpoint of avoiding the chemical toxicity of the cryopreservation solution. More preferably, it is within 1 minute and 30 seconds.

  The cryopreservation operation using the cryopreservation solution of the present invention involves, after the cells or tissues are equilibrated, immersing the cells or tissues in the cryopreservation solution, and then vitrifying the cells using a cryogenic cooling medium. Preferably. By freezing at a rapid cooling rate, it is expected that the cells will be cryopreserved in a state where the formation of ice crystals is small. It is preferable to use liquid nitrogen as the cooling medium.

  In order to freeze at a rapid cooling rate, it is common to freeze cells or tissues with a small amount of cryopreservation solution, thereby achieving excellent cell or tissue viability. On the other hand, when cells or tissues are placed and frozen with a small amount of preservation solution, when thawing after freezing, cells or tissues on the sheet often adhere to the surface of the placement part depending on the type or state of the cells or tissues. Therefore, there is a problem that a high skill is required when collecting these. In particular, in the method proposed in Patent Document 3 for removing excess vitrification liquid using a vitrification storage liquid removing material (for example, an absorbent such as a membrane filter, a wire mesh, or filter paper), an excellent cooling rate is obtained. On the other hand, there is a problem that the fixation of cells or tissues becomes more remarkable, but the use of the cryopreservation solution of the present invention allows the cells or tissues to be easily peeled off at the time of thawing, and thus can be suitably used.

  The cryopreserved cells can be stored semipermanently by storing them in an extremely low temperature environment in which the vitrified state of the cells is maintained. The temperature at which the vitrification state is maintained varies depending on the composition of the cryopreservation solution, but in most cases, it is preferably -150 ° C or lower. The environment in which such a temperature is maintained is preferably in a liquid nitrogen storage container or a gas phase nitrogen storage container.

  The freezing operation in the cryopreservation method of the present invention has been described above. Next, the melting operation will be described.

  Thawing of cells or tissues using the cryopreservation solution of the present invention can be performed by a generally known method such as a cryotop method. The cells or tissues placed on the sheet are taken out of the cooling solvent and are brought into direct contact with the melt at 37 ° C. to melt the cryopreservation solution and the cells or tissues and dilute the cryopreservation solution. I do. At this time, cells or tissues are peeled from the sheet in the melt. It is preferable to gently peel cells or tissues from the sheet from the viewpoint of viability after freezing and thawing. After a predetermined time, the detached cells or tissues are transferred from the melt to a diluent, soaked for a predetermined time, and further transferred to a washing solution. By such an operation, an operation of gradually changing the osmotic pressure, diluting / removing the antifreeze in the cells or tissues, and returning to a mild culture condition is performed.

  Cells that can be cryopreserved using the cryopreservation solution of the present invention include, for example, mammalian (eg, human (human), cow, pig, horse, rabbit, rat, mouse, etc.) eggs, embryos, sperm, etc. Germ cells; pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells). In addition, cultured cells such as primary cultured cells, subcultured cells, and cell lines can be used. In one or more embodiments, the cells are fibroblasts, cancer-derived cells such as pancreatic cancer / hepatic cancer cells, epithelial cells, vascular endothelial cells, lymphatic endothelial cells, nerve cells, chondrocytes, tissue stem cells, And adherent cells such as immune cells. Furthermore, tissues that can be cryopreserved include tissues composed of cells of the same or different kinds, for example, tissues such as ovaries, skin, corneal epithelium, periodontal ligament, and cardiac muscle. The present invention is particularly suitable for cryopreservation of a tissue having a sheet-like structure (eg, a cell sheet, skin tissue, etc.). The jig for cryopreservation of the present invention is not only a tissue directly collected from a living body, but also, for example, a cultured skin grown and cultured in vitro, a so-called cell sheet constructed in vitro, JP-A-2012-205516. Cryopreservation of an artificial tissue such as a proposed tissue model having a three-dimensional structure can also be suitably used. The cryopreservation solution of the present invention can be suitably used as a cryopreservation solution for cells or tissues as described above.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention will be described in detail. However, the present invention is not limited to the following examples.

(Example 1)
A commercially available Medium 199 medium (manufactured by Life Technologies) containing L-glutamine, phenol red, and 25 mM HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) is used as a base solution, and ethylene glycol is used as an organic solvent-based antifreeze agent. % By volume, 15% by volume of DMSO (dimethyl sulfoxide), 0.5 M of sucrose as a saccharide, 50 mg / L of gentamicin as an antibiotic, and Gosenex WO of Nippon Synthetic Chemical Industry Co., Ltd., a modified polyvinyl alcohol having an ethylene oxide group. The cryopreservation solution of Example 1 containing 0.5% by mass of -320R (degree of saponification: 88 mol%) was prepared.

(Example 2)
A cryopreservation solution of Example 2 was prepared in the same manner as in Example 1, except that the content of GOHSENX WO-320R was changed to 0.5% by mass and 0.9% by mass.

(Example 3)
A cryopreservation solution of Example 3 was prepared in the same manner as in Example 1 except that the content of GOHSENX WO-320R was changed to 1.8% by mass instead of 0.5% by mass.

(Example 4)
A cryopreservation solution of Example 4 was prepared in the same manner as in Example 1, except that the content of GOHSENX WO-320R was changed to 0.5% by mass and 2.5% by mass.

(Example 5)
The procedure was carried out with the exception of adding 0.5% by mass of Gosenex WO-320N (a saponification degree of 99 mol%), a modified polyvinyl alcohol having an ethylene oxide group, without adding Gosenex WO-320R. In the same manner as in Example 1, the cryopreservation solution of Example 5 was prepared.

(Example 6)
A cryopreservation solution of Example 6 was prepared in the same manner as in Example 5, except that the content of GOHSENX WO-320N was changed to 0.5% by mass and 0.9% by mass.

(Example 7)
A cryopreservation solution of Example 7 was prepared in the same manner as in Example 5 except that the content of GOHSENX WO-320N was changed to 1.8% by mass instead of 0.5% by mass.

(Example 8)
A cryopreservation solution of Example 8 was prepared in the same manner as in Example 5, except that the content of GOHSENX WO-320N was changed to 0.5% by mass and 2.5% by mass.

(Comparative Example 1)
A cryopreservation solution of Comparative Example 1 was prepared in the same manner as in Example 1, except that GOHSENX WO-320R was not added.

(Comparative Example 2)
Gosenol (registered trademark) EG05P (a saponification degree: 88 mol%), which is a polyvinyl alcohol having no ethylene oxide group and made by Nippon Synthetic Chemical Industry Co., Ltd., containing 0.9% by mass without adding Gosenex WO-320R. A cryopreservation solution of Comparative Example 2 was prepared in the same manner as in Example 1 except for the above.

<Adjustment of sphere>
Spheres used for evaluation of cell or tissue detachability were adjusted as follows. After culturing MEF cells, which are mouse embryonic fibroblasts, on a culture dish, the cells were detached and collected by trypsin treatment. Thereafter, the cells were seeded at a cell number of 50 cells / well on PrimeSurface (registered trademark) 96U plate manufactured by Sumitomo Bakelite Co., Ltd., and sphere formation was induced by suspension culture. After 3 days of culture, spheres having a diameter of about 100 μm were obtained.

<Preparation of jig for cryopreservation>
A cryopreservation jig used for cryopreservation of cells or tissues was prepared as follows. A hot melt urethane resin Purmelt (registered trademark) QR 170-7141P manufactured by Henkel Japan Co., Ltd. was applied as an adhesive layer on the transparent PET film so as to have a solid content of 30 g / m ² when dried. The application of the adhesive layer was not performed on the mounting area, but was performed only on the periphery. Before the adhesive layer is completely cured, a hydrophilic polytetrafluoroethylene porous material (pore diameter 0.2 μm, porosity 71%, thickness 35 μm) manufactured by Advantech Toyo Co., Ltd. is applied as a storage liquid absorber. This was used as a mounting portion for mounting cells or tissues. This mounting portion was cut into a size of 1.5 mm × 20 mm, and the short side of the mounting portion was joined to a stick-shaped ABS resin holding portion to prepare a jig for cryopreservation.

<Freezing work>
The spheres obtained as described above were recovered from the culture solution, and moved into the equilibration solution together with a small amount of the culture solution using a stripper pipettor (hereinafter, pipette) which is a thin tubular jig. The composition of the equilibrium solution was such that the above-mentioned Medium 199 medium was used as a base solution and contained 7.5% by volume of ethylene glycol, 7.5% by volume of DMSO (dimethyl sulfoxide), and 50 mg / L gentamicin. After the spheres were immersed in the equilibration solution for 3 minutes, they were transferred to the cryopreservation solutions of Examples 1 to 8 or Comparative Examples 1 and 2 using a pipette together with a small amount of the equilibration solution. After pipetting the spheres several times in the cryopreservation solution, at a timing 40 seconds after immersion in the cryopreservation solution, using a pipette on the mounting portion of the jig for cryopreservation, the spheres were removed in a very small amount. Attached dropwise together with the cryopreservation solution. After adhering the spheres, confirming that the cryopreservation solution is automatically absorbed by the preservation solution absorber under a microscope. The sphere on the mounting part of the tool was immersed in liquid nitrogen and frozen. After pipetting the sphere several times in the cryopreservation solution, the time required for immersion in liquid nitrogen was about 1 minute. The frozen cryopreservation jig was stored in liquid nitrogen until the thawing operation was performed.

<Evaluation of workability in freezing operation>
The workability at the time of pipetting in a cryopreservation solution in the freezing operation was evaluated according to the following criteria. These results are shown in Table 1 under the item "Evaluation of workability in freezing operation".

The workability in the freezing operation was evaluated according to the following criteria.
：: Pipetting was easy, and workability such as pipetting in a cryopreservation solution was good.
Δ: The pipetting operation was slightly affected, but a series of operations such as pipetting in a cryopreservation solution were possible.
X: The pipetting operation was hindered, and the pipetting operation was difficult.

<Melting work>
The spheres that had been frozen with the cryopreserved solutions of Examples 1 to 8 or Comparative Examples 1 and 2 were each thawed by the following operation. The cryopreservation jig containing the frozen sphere was taken out of the liquid nitrogen, and the mounting portion of the cryopreservation jig on which the sphere was mounted was immersed in the melt at 37 ° C. The composition of the melt was 1 M sucrose and 50 mg / L gentamicin, based on the Medium 199 medium. While observing the mounting part immersed in the melt under a microscope, an attempt was made to peel the sphere from above the mounting part. The sphere peeling operation was performed as follows. After immersing the mounting portion in the melt, the sphere was observed to peel off from the mounting portion while standing still for 60 seconds. When the sphere did not peel after 60 seconds from the immersion in the melt, the mounting portion was shaken to attempt to peel the sphere.

<Evaluation of peelability in melting operation>
The peelability of the sphere from the mounting part in the melting operation was evaluated according to the following criteria. These results are shown in Table 1 under the item "Evaluation of peelability in melting operation".

The peelability in the melting operation was evaluated according to the following criteria.
A: The sphere peeled off from the mounting portion within 60 seconds after immersion in the melt.
：: 60 seconds after the immersion in the melt, the sphere could be peeled off from the mounting part by shaking the mounting part.
×: The spheres were adhered to such an extent that the spheres could not be easily collected, and could not be peeled off, or the cells constituting the spheres were separated at the time of collection.

  From the results in Table 1, it can be seen that the cryopreservation solution of the present invention is excellent in workability during the freezing operation and excellent peelability during the thawing operation.

  The present invention is not limited to embryo transplantation and artificial insemination of livestock and animals such as cattle and animals, artificial insemination to humans, iPS cells, ES cells, generally used cultured cells, and inspection or transplantation collected from a living body. It can be used for cryopreservation of cells or tissues, cells or tissues cultured in vitro, and the like.

Claims (1)

  A cryopreservation solution containing a modified polyvinyl alcohol having an ethylene oxide group.