JP2020096554A - Jig for cryopreservation of cells or tissues - Google Patents

Abstract

To provide a cryopreservation jig that allows easy confirmation of cells or tissues.SOLUTION: Provided is a jig for cryopreservation of cells or tissues, the jig having a substantially sheet-shaped mounting portion for mounting cells or tissues together with a preservation solution, and the contact angle of the surface of the mounting portion with respect to water being 30 degrees or less.SELECTED DRAWING: Figure 1

Description

The present invention relates to a cryopreservation jig for cells or tissues, which is used when cryopreserving cells or tissues.

Excellent preservation techniques for cells or tissues are required in various industrial fields. For example, in the embryo transfer technique for cows, the embryos are cryopreserved, the embryos are thawed according to the estrus cycle of the recipient cow, and the embryos are transferred. Further, in the treatment of human infertility, after collecting an egg or an ovary from a mother, it is cryopreserved so as to match the timing suitable for transplantation, and thawed at the time of transplantation.

In general, cells or tissues collected from the inside of a living organism gradually lose their activity even in a culture medium, and thus long-term culture of the cells or tissues in vitro is not preferable. Therefore, a technique for storing for a long time without losing bioactivity is important. Good preservation techniques allow for more accurate analysis of harvested cells or tissues. Further, it is expected that the excellent preservation technique enables cells or tissues to be used for transplantation while maintaining higher bioactivity, and that the survival rate after transplantation is improved. Furthermore, cultured skin cultured in vitro, artificial tissues for transplantation such as so-called cell sheets constructed in vitro, are sequentially produced and stored, and can be used when necessary, Not only in terms of medical treatment, but also in terms of industry, great benefits can be expected.

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 preservation solution obtained by incorporating a cryoprotectant into a physiological solution such as phosphate buffered saline. As the antifreezing agent, compounds such as glycerol and ethylene glycol are used. After immersing the cells or tissues in the preservation solution, the cells or tissues are cooled inside or outside the cells or tissues by cooling at a relatively slow cooling rate (for example, 0.3 to 0.5°C/minute) to -30 to -35°C. The internal and external solutions are sufficiently cooled and the viscosity becomes high. In such a state, when the cells or tissues in the preservation solution are further cooled to the temperature of liquid nitrogen (-196°C), the minute solution inside the cells or inside the tissues and around the outside thereof remains amorphous and solid. Vitrification occurs. When vitrification solidifies the inside or outside of cells or the inside or outside of the tissue, the movement of molecules is substantially lost. Therefore, it is considered that the vitrified cells or tissues can be semipermanently preserved in liquid nitrogen.

However, in the slow freezing method, since it is necessary to cool at a relatively slow cooling rate, it takes a long time for operation for cryopreservation. 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, since ice crystals are formed in the preservation solution outside the cells or tissues, the cells or tissues may be physically damaged by the ice crystals.

A vitrification freezing method has been proposed as a method for solving the problems in the slow freezing method. The vitrification freezing method is based on the principle that ice crystals are less likely to be formed even at a freezing point due to the freezing point depression of a storage solution containing a large amount of antifreezing agents such as glycerol, ethylene glycol, and DMSO (dimethyl sulfoxide). .. If this stock solution is rapidly cooled in liquid nitrogen, it can be solidified without forming ice crystals. This solidification is called vitrification freezing. A preservation solution containing a large amount of antifreezing agent is called a vitrification solution.

As a specific operation of the vitrification freezing method, cells or tissues are immersed in a preservation solution containing a large amount of antifreezing agent, and then cooled at the temperature of liquid nitrogen (-196°C). Since the vitrification freezing method is such a simple and quick process, it does not require a long time for operation for cryopreservation, and does not require a device or jig for temperature control. There is.

In principle, the vitrification-freezing method can avoid physical damage (freezing damage) to cells during freezing and thawing because ice crystals do not occur inside or outside the cells. In order to achieve such 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 of antifreezing agent contained in the preservation solution has high chemical toxicity for cells.

From the above background, in the cryopreservation of cells or tissues, from the viewpoint of avoiding the toxicity derived from the high concentration of antifreeze contained in the preservation solution, the time during which the cells or tissues are exposed to the preservation solution (that is, frozen) It is preferable that the time until the operation) is short, and the operator is required to perform a quick operation.

Further, it is known that the faster the freezing rate is, the more preferable it is because vitrification and freezing can be appropriately performed. Further, it is known that the faster the thawing rate is, the more preferable from the viewpoint of suppressing the formation of re-ice crystals in cells or tissues even at the time of thawing after freezing.

Regarding cryopreservation of cells or tissues using the vitrification freezing method, examples using various types of cells or tissues by various methods have been shown. For example, Patent Document 1 shows that application of the vitrification freezing method to animal or human germ cells or somatic cells is extremely useful in terms of survival rate after cryopreservation and thawing.

The vitrification-freezing method is a technology that has been developed mainly using human germ cells, but recently, its application to iPS cells and ES cells has been widely studied. In addition, Non-Patent Document 1 shows that the vitrification-freezing method was effective for the preservation of Drosophila embryos. Further, Patent Document 2 shows that the vitrification-freezing method is effective in preserving plant cultured cells and tissues. Thus, the vitrification-freezing method is known to be useful for the preservation of cells and tissues of a wide variety of species.

In Patent Document 3 and Patent Document 4, a cryopreservation method used in the field of human infertility treatment is used to freeze-preserve eggs using a strip-shaped flexible, colorless and transparent film as a strip for retaining eggs. A method has been proposed in which an egg or an embryo is placed on the film together with an extremely small amount of a preservation solution under microscope observation using a tool and then frozen and preserved.

Patent Document 5 proposes a biological sample storage device having a structure including a biological sample mounting portion and a cryopreservation container capable of housing the sample mounting portion. It is described that the material of the sample mounting part in this document preferably has a pure water contact angle of about 60 to 90 degrees from the viewpoint of preventing the biological sample from falling off.

Japanese Patent No. 3044323 JP, 2008-5846, A JP-A-2002-315573 JP 2006-271395A International Publication 2006/059626 Pamphlet

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

Patent Documents 3 and 4 propose a method of cryopreserving an egg or an embryo together with a small amount of a preservative solution by limiting the width of a film on which the egg or the embryo is placed to obtain an excellent survival rate. There is. This method has a problem in that it is necessary to put an egg or an embryo on a film together with a small amount of a preservation solution by an operator's operation, which makes the operation difficult. Moreover, in order to confirm under the microscope that the ovum or embryo was placed on the film, once the egg or embryo was placed on the film together with the preservation solution, the excess preservation solution was aspirated from the film. Although a complicated operation such as removal may be performed, there is a problem that the operation is still difficult.

Patent Document 5 proposes a biological sample storage device having a structure including a biological sample mounting portion and a cryopreservation container capable of housing the sample mounting portion. Similarly, in order to reliably confirm that the sample has been placed on the sample placing section, it may be necessary to suck and remove the excess storage solution.

It is a main object of the present invention to provide a jig for cryopreservation of cells or tissues, which enables easy and reliable cryopreservation of cells or tissues. More specifically, after immersing the cell or tissue in a preservation solution, when the cell or tissue is dropped and adhered to the mounting portion under the microscope observation, the mounted cell or tissue can be confirmed with good visibility. It is to provide a cryopreservation jig that can be used.

The present inventor, as a result of extensive studies to solve the above problems, a jig for cryopreservation of cells or tissues having the following constitution (in the present specification, "jigs for cryopreservation of cells or tissues", It was found that the above-mentioned problems can be solved simply by “a jig for cryopreservation”.

A treatment for cryopreservation of cells or tissues, comprising a substantially sheet-shaped mounting part for mounting cells or tissues together with a preservation solution, and a contact angle of water on the mounting part surface is 30 degrees or less. Ingredient

According to the above invention, a cryopreservation jig capable of confirming a mounted cell or tissue with good visibility when the cell or tissue is dropped and adhered onto the mounting portion under microscope observation. Can be provided.

It is an overall view showing an example of a jig for cryopreservation of the present invention.

The cryopreservation jig of the present invention is used for cryopreserving cells or tissues. In the present invention, a cell includes not only a single cell but also a cell population of an organism composed of a plurality of cells. The cell population composed of a plurality of cells may be a cell population composed of a single type of cell or a cell population composed of a plurality of types of cells. In addition, the tissue may be a tissue composed of a single type of cell or a tissue composed of a plurality of types of cells, and contains a non-cellular substance such as extracellular matrix in addition to the cells. Anything is fine.

The cryopreservation work according to the present invention includes freezing work for freezing cells or tissues using a cryogenic refrigerant, storage work for storing cells or tissues in a cryogenic refrigerant, and thawing cells or tissues in a melt. It should include a series of melting operations.

The cryopreservation jig of the present invention is used for cryopreservation work of cells or tissues, and preferably for vitrification cryopreservation work of cells or tissues. Specifically, the cryopreservation jig of the present invention is for immersing cells or tissues immersed in a preservation solution in a cooling solvent such as liquid nitrogen for freezing. When the cells or tissues placed on the placing part are thawed, the cells or tissues are taken out from the cooling medium together with the cryopreservation jig and immersed in the thaw solution to thaw. When the cryopreservation jig of the present invention is used, it is possible to confirm the placed cells or tissues with good visibility when the cells or tissues are dropped and attached to the placing portion under microscopic observation. That is, at the time of freezing work, the work of placing cells or tissues under observation using a microscope becomes easy.

The cryopreservation jig of the present invention has a substantially sheet-shaped mounting portion on which cells or tissues are mounted together with a storage solution. The substantially sheet shape in the present invention means a shape having a flat surface capable of holding at least cells or tissues, such as a flat sheet shape, a curved sheet shape, a corrugated sheet shape, and a V-shaped sheet shape. The shape is illustrated.

The contact angle of water on the surface of the mounting portion of the cryopreservation jig of the present invention is 30 degrees or less. When the contact angle is 30 degrees or less, when the cells or tissues are dropped and adhered to the mounting portion, the preservation solution dropped and adhered together with the cells or tissues is uniformly spread on the surface of the mounting portion, and a thin film is formed. Form. Therefore, the cells or tissues in the preservative solution that have been dropped and attached can be confirmed with good visibility under a microscope. More preferably, the contact angle of the surface of the mounting portion with water is 15 degrees or less. On the other hand, when the contact angle of the surface of the mounting portion with respect to water is more than 30 degrees, the preservative solution dropped and attached together with the cells or tissues does not spread to the surface of the mounting portion and remains relatively large around the cells or tissues. The visual refraction or the like on the surface of the preservation solution inhibits the visual recognition of cells or tissues in the preservation solution. In such a case, change the setting of the microscope (contrast or how to apply illumination) or remove the preservative solution that was once dropped to remove the dropped cells or tissues under the microscope. It will be necessary to confirm with.

The substantially sheet-shaped mounting portion of the cryopreservation jig of the present invention may have a contact angle of water on the surface of the mounting portion of 30 degrees or less, and forms the mounting portion surface having the contact angle. As a method of doing so, in addition to forming the placing portion using a highly hydrophilic material having the above-mentioned contact angle, a placing portion in which the contact angle of the substrate surface is modified by a general coating method or the like is used. It can be illustrated.

Glass can be exemplified as the above-mentioned material having high hydrophilicity.

When the contact angle to water is adjusted to 30 degrees or less by the above coating method, various resin films, rubbers, metals, etc. are exemplified as the substrate to be surface-modified. Further, two or more kinds of substrates may be used in combination as long as the effects of the present invention are exhibited. Among them, the resin film is preferably used because it is easy to handle. Specific examples of the resin film include polyester resins such as polyethylene terephthalate (PET) and polyethylene naphthalate (PEN), acrylic resins, epoxy resins, silicone resins, polycarbonate resins, diacetate resins, triacetate resins, polyarylate resins, polychlorinated resins. Examples of the resin film include vinyl resin, polysulfone resin, polyether sulfone resin, polyimide resin, polyamide resin, polyolefin resin, cyclic polyolefin resin and the like.

The thickness of these substrates is preferably 10 to 1000 μm.

When the surface of the substrate is modified by the coating method, the surface modification can be performed by using a coating liquid that exhibits high hydrophilicity on the coated surface. Examples of the coating liquid exhibiting high hydrophilicity on the surface include, for example, a coating liquid mainly composed of silicon oxide fine particles and a binder component of 5% by mass or less based on the solid content of the silicon oxide fine particles, a layered silicate. And a coating liquid containing a hydrophilic polymer compound composed of an extract derived from fish, a coating liquid containing finely dispersed cellulose, and the like. You may use these together. From the viewpoint of exhibiting particularly high hydrophilicity, it is preferable to use a coating liquid containing silicon oxide fine particles as a main component and a binder component of 5% by mass or less based on the solid content of the silicon oxide fine particles. The term “mainly” means that 50% by mass or more of the total solid content of the coating liquid is silicon oxide fine particles.

You may add the auxiliary agent which improves a coating property to the above-mentioned coating liquid in the range which does not affect surface characteristics. In addition, an undercoat layer may be provided on the surface of the substrate for the purpose of controlling the coatability. After coating, various processes such as light irradiation and heat treatment may be performed to further surface-modify the surface of the mounting portion.

The placing portion included in the jig for cryopreservation of the present invention preferably has a total light transmittance of 80% or more. It is more preferably 85% or more. The haze value of the light-transmitting substrate is preferably 10% or less. This facilitates the confirmation work of the cells or tissues placed on the placing part (the work of observing the cells or tissues through the placing part and observing the cells or tissues with a transmission microscope). be able to.

In the present invention, the area of the placing portion may be appropriately set according to the amount of the preservative solution dropped together with the cells or tissues, and is not particularly limited. For example, 100 μm ² or more per 1 μL of preservative solution to be dropped. It is preferable. Further, from the viewpoint of workability, it is more preferable that the area of the placing portion included in the jig for cryopreservation of the present invention is 5 to 400 mm ² .

The cryopreservation jig of the present invention may have a configuration in which one end of the placing part is grasped and used. However, when the one end of the placing part is connected to the grasping part, workability at the time of freezing/thawing Is preferable, which is preferable.

FIG. 1 is an overall view showing an example of a cryopreservation jig of the present invention. In FIG. 1, the cryopreservation jig 3 has a grip portion 1 and a mounting portion 2. The grip 1 is preferably made of a liquid nitrogen resistant material. As such a material, for example, various metals such as aluminum, iron, copper, and stainless alloy, ABS resin, polypropylene resin, polyethylene resin, fluorine resin, various engineered plastics, and glass can be preferably used. The mounting portion 2 has a substantially sheet shape.

A method of joining the grip portion 1 and the placing portion 2 in FIG. 1 will be described. When the grip portion 1 is made of resin, for example, the mounting portion 2 can be joined to the grip portion 1 by insert molding during molding. It is also possible to make an insertion part (not shown) in the grip part 1 and join the mounting part 2 with an adhesive. Although various adhesives can be used, a silicon-based or fluorine-based adhesive resistant to low temperature can be preferably used.

When cells or tissues are cryopreserved for a long period of time using the cell or tissue cryopreservation jig of the present invention, a cap is provided on the mounting portion of the cryopreservation jig shown in FIG. It is also possible to cover. Further, normally, liquid nitrogen is not sterilized, and when it is brought into direct contact with liquid nitrogen to be frozen, the sterilized state may not be guaranteed even if the cryopreservation jig is sterilized. Therefore, before the freezing, the placing part to which the cells or tissues are attached may be capped and frozen without directly contacting with the liquid nitrogen. It is also useful from the viewpoint of physically protecting the mounting part to which cells or tissues are attached. The cap is preferably made of various metals, which are liquid nitrogen resistant materials, various resins, glass, ceramics and the like. The shape may be any shape, such as a pencil cap or a cylindrical straw cap, as long as it does not come into contact with the mounting portion and can be shielded from the outside world.

The method of cryopreserving cells or tissues using the jig for cryopreservation of the present invention is not particularly limited, and for example, cells or tissues immersed in a preservation solution are first dropped onto a mounting portion together with the preservation solution with a micropipette. .. When the preservation solution is dropped, the cells or tissues on the mounting portion are observed under a microscope with good visibility to confirm the presence or absence of cells or tissues. Next, the cells or tissues are frozen by immersing the cryopreservation jig in liquid nitrogen or the like while holding the cells or tissues on the mounting portion. At this time, a cap capable of blocking the cells or tissues on the mounting portion from the outside can be attached to the mounting portion and immersed in liquid nitrogen or the like. As the preservation solution, one that is usually used for freezing cells such as eggs and embryos can be used. For example, a physiological solution such as phosphate buffered saline contains a cryoprotectant (glycerol, ethylene glycol, etc.). A preservative solution or a preservative solution containing a large amount of various antifreezing agents such as glycerol, ethylene glycol, and DMSO (dimethyl sulfoxide) (at least 10% by mass or more, and more preferably 20% by mass or more based on the total mass of the preservative solution). Can be used. At the time of thawing work, the cryopreservation jig is taken out from a cooling solvent such as liquid nitrogen, and the placing part on which the frozen cells or tissues are placed is immersed in the thawing solution, and then the cells or tissues are removed. to recover.

Cells that can be cryopreserved using the jig for cryopreservation of the present invention include, for example, mammalian (eg, human (human), bovine, porcine, equine, rabbit, rat, mouse, etc.) eggs, embryos, sperms. And the like; artificial pluripotent stem cells (iPS cells), embryonic stem cells (ES cells) and other pluripotent stem cells. In addition, cultured cells such as primary cultured cells, subcultured cells, and cell line cells are also included. Further, the cell, in one or more embodiments, fibroblasts, cancer-derived cells such as pancreatic cancer and liver cancer cells, epithelial cells, vascular endothelial cells, lymphatic endothelial cells, nerve cells, chondrocytes, tissue stem cells, And adhesive cells such as immune cells. Furthermore, the tissues that can be cryopreserved include tissues composed of cells of the same or different types, for example, tissues such as ovary, skin, corneal epithelium, periodontal ligament, myocardium. The present invention is particularly suitable for cryopreservation of a tissue having a sheet-like structure (eg, cell sheet, skin tissue, etc.). The jig for cryopreservation of the present invention is not limited to a tissue directly collected from a living body, but is, for example, a cultured skin that is cultured and proliferated in vitro, a so-called cell sheet constructed in vitro, JP 2012-205516 A. It can also be preferably used for cryopreservation of artificial tissues such as the proposed tissue model having a three-dimensional structure. The cryopreservation jig of the present invention is suitably used as a cryopreservation jig for cells or tissues as described above.

Hereinafter, the present invention will be described in more detail with reference to Examples and the present invention will be specifically described, but the present invention is not limited to the following Examples. The contact angles described in the examples are dynamic contact angles measured using a fully automatic contact angle meter CA-W (Kyowa Interface Science Co., Ltd.) and multifunction integrated analysis software FAMAS (Kyowa Interface Science Co., Ltd.). Is.

(Example 1)
A transparent PET film (total light transmittance 89%, haze value 2.3%) is used as a substrate, and silicon oxide fine particles are mainly contained on the substrate, and the binder component is 1 mass with respect to the solid content of the silicon oxide fine particles. % Or less Cell Face Coat (manufactured by Marusho Sangyo Co., Ltd.) is applied so that the solid content when dried is 10 mg/m ² , dried at room temperature, and further dried at 60° C. for 1 hour to prepare a mounting portion. did. The contact angle of the mounting portion in Example 1 was 10 degrees. This mounting portion was cut into a size of 2 mm×20 mm (40 mm ² ) and joined to an ABS resin gripping portion to manufacture a cryopreservation jig of Example 1 in the form shown in FIG.

(Example 2)
Smecton (registered trademark) (manufactured by Kunimine Industries Co., Ltd.) containing a layered silicate was applied as a coating liquid so that the solid content at the time of drying was 200 g/m ^2, and dried at 60° C. for 24 hours. A cryopreservation jig of Example 2 was produced in the same manner as in Example 1 except for the above. The contact angle of the mounting portion in Example 2 was 20 degrees.

(Example 3)
SAM-M02 (manufactured by Taki Chemical Co., Ltd.), which is a gelatin derived from fish-derived extract, is dissolved in water to give a coating solution, which is applied so that the dry solid content is 1 g/m ² and dried at room temperature. After that, a jig for cryopreservation of Example 3 was produced in the same manner as in Example 1 except that it was further dried at 30° C. for 12 hours. The contact angle of the mounting portion in Example 3 was 18 degrees.

(Example 4)
BiNFi-s (registered trademark) (manufactured by Sugino Machine Co., Ltd.), which is a finely dispersed cellulose dispersion, is used as a coating liquid and coated so that the solid content at the time of drying becomes 100 g/m ^2, and it is sufficiently applied at room temperature A cryopreservation jig of Example 4 was produced in the same manner as in Example 1 except that it was dried. The contact angle of the mounting portion in Example 4 was 20 degrees.

(Comparative Example 1)
Freezing of Comparative Example 1 was carried out in the same manner as in Example 1 except that the transparent PET film (total light transmittance 89%, haze value 2.3%) which was the substrate was used as it was as the mounting portion of Comparative Example 1. A storage jig was produced. The contact angle of the mounting portion of Comparative Example 1 was 60 degrees.

(Comparative example 2)
Same as Example 1 except that polyvinyl alcohol PVA103 (manufactured by Kuraray Co., Ltd.) was dissolved in water to obtain a coating solution, which was applied so as to have a solid content of 100 g/m ² when dried and dried at room temperature. Then, the frozen preservation jig of Comparative Example 2 was produced. The contact angle of the mounting portion of Comparative Example 2 was 40 degrees.

<Evaluation of visibility>
While observing with an epi-illumination type upright optical microscope VC4500-S1 (OMRON Corporation) on the mounting portion of each cryopreservation jig of Examples 1 to 4 and Comparative Examples 1 and 2, mouse 8-cell stage embryos Was added drop-wise with 0.1 μL of the preservative solution, and the operation of removing the preservative solution after the drop-adhering was not performed. The following criteria were evaluated. These results are shown in the item "Evaluation of visibility" in Table 1. In addition, the preservation solution was a commercially available Medium 199 medium (Life Technologies, Inc.) containing L-glutamine, phenol red, and 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazinecinethenesulfonic acid) as a basic solution, and ethylene glycol was 15 times. A solution containing 15% by volume of DMSO (dimethyl sulfoxide), 0.5 M of sucrose and 50 mg/L of gentamicin was used.

◯: Presence of embryos on the placing part is clearly visible.
Δ: Presence of embryos on the placing part can be visually confirmed.
X: Presence of the embryo on the placing part cannot be visually recognized or is extremely difficult to visually recognize.

From the above results, the cryopreservation jig of the present invention is favorable without performing an operation of removing an excess preservation solution after the cells or tissues are dropped and adhered onto the placing portion under the microscope observation during the freezing operation. It can be seen that it is possible to perform confirmation work with good visibility.

INDUSTRIAL APPLICABILITY The present invention is applied to livestock such as cattle and embryo transfer and artificial insemination of animals, artificial insemination to humans, iPS cells, ES cells, commonly used cultured cells, tests collected from living bodies including embryos or ova It can be used for cryopreservation of cells or tissues for transplantation or transplantation, cells or tissues cultured in vitro.

1 gripping part 2 mounting part 3 jig for cryopreservation

Claims (1)

A treatment for cryopreservation of cells or tissues, comprising a substantially sheet-shaped mounting part for mounting cells or tissues together with a preservation solution, and a contact angle of water on the mounting part surface is 30 degrees or less. Ingredient