CN117980460A

CN117980460A - Cryopreservation tool for ovum or fertilized ovum

Info

Publication number: CN117980460A
Application number: CN202280057097.2A
Authority: CN
Inventors: 谷口义隆; 盐谷雅英
Original assignee: Cstec Co ltd
Current assignee: Cstec Co ltd
Priority date: 2021-08-23
Filing date: 2022-07-26
Publication date: 2024-05-03
Also published as: JPWO2023026747A1; JP7292637B1; WO2023026747A1

Abstract

The cryopreservation tool for ovum or fertilized ovum of the present invention is characterized by comprising: a main body (10) comprising a rod-shaped shank (11) and a net-shaped egg or other holding part (12) provided at the tip of the shank; and a tubular cover part (20) which accommodates the main body part and the net of the retaining part such as ovum, and the opening size of the net is more than 170 μm. Thus, the amount of the cryoprotectant around the ovum and the like can be made appropriate without performing a complicated operation, and the ovum and the like can be easily detached at the time of thawing.

Description

Cryopreservation tool for ovum or fertilized ovum

Technical Field

The present invention relates to a cryopreservation tool for cryopreserving an ovum or fertilized ovum.

Background

In the field of infertility treatment of humans, a vitrification freezing method is known as a technique for cryopreserving an ovum or fertilized ovum (embryo). The vitrification freezing method is such that: an ovum or fertilized ovum (hereinafter, may be referred to as "ovum or the like" in a general term) is immersed in a small amount of a cryoprotectant (vitrification liquid) together with liquid nitrogen and rapidly cooled, whereby water inside and outside the cell is frozen in an amorphous glass state without being crystallized. According to this method, damage to the ovum or the like caused by ice crystal formation can be suppressed, and the survival rate of the ovum or the like at the time of subsequent thawing can be improved.

In such a vitrification freezing method, a freezing preservation tool is widely used, which comprises: a rod-shaped main body portion having a thin plate-shaped placement portion at a front end thereof for placing an ovum or the like; and a tubular cover that houses the main body (see patent document 1, for example). When freezing an ovum or the like using such a cryopreservation tool, an ovum immersed in a cryoprotectant solution in advance is first extracted by a pipette or the like and placed on the placement unit together with a small amount of cryoprotectant solution. Then, after the tip of the body portion including the placement portion is immersed in liquid nitrogen and rapidly cooled, the cover is attached to the body portion and stored in a liquid nitrogen tank.

However, in the vitrification freezing method, if the amount of the cryoprotectant present around the ovum or the like is large, the cooling rate of liquid nitrogen may be decreased, and the survival rate of the ovum or the like may be decreased. Therefore, in the above-described cryopreservation tool, when the amount of the cryoprotectant liquid to be placed on the placement unit together with the ovum or the like is large, it is necessary to remove the surplus cryoprotectant liquid by using a pipette or the like and adjust it to an appropriate amount.

Then, patent document 2 proposes a cryopreservation tool capable of removing an excessive cryoprotectant by a simple operation. In this cryopreservation tool, a placement portion for placing an ovum or the like is formed in a net shape, and after placing the ovum or the like and the cryoprotectant liquid on the net-shaped placement portion, the cryoprotectant liquid can be removed by performing a suction operation from below the placement portion.

Prior art literature

Patent literature

Patent document 1: japanese patent laid-open No. 2002-315573

Patent document 2: international publication No. 2011/070973

Disclosure of Invention

Problems to be solved by the invention

However, in the cryopreservation tool including the above-described mesh-shaped placement portion, when the cryopreservation tool is taken out from the liquid nitrogen tank after cryopreservation and the placement portion is immersed in the thawing liquid to thaw the ovum or the like, the ovum or the like may not be easily detached from the placement portion. In such a case, the worker needs to suck the ovum with a pipette or the like and remove the ovum from the placement portion, but in this case, there is a problem that the ovum or the like may be damaged, and the load of the worker increases.

The present invention has been made in view of the above-described points, and an object thereof is to provide a cryopreservation tool as described above: the cryoprotectant around the ovum and the like can be properly used without complex operation, and the ovum and the like can be easily separated from the loading part during thawing.

Solution for solving the problem

The cryopreservation tool for ovum or fertilized ovum of the present invention, which has been completed to solve the above problems, is characterized in that,

The cryopreservation tool comprises:

A main body portion including a rod-shaped shank portion and a net-shaped ovum holding portion provided at a tip end of the shank portion; and

A tubular cover portion for accommodating the main body portion,

The mesh of the egg holding portion has an opening size of 170 μm or more.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the cryopreservation tool for ovum or fertilized ovum of the present invention having such a structure as described above, the appropriate amount of the cryoprotectant around the ovum or the like can be obtained without performing complicated work, and the ovum or the like can be easily detached from the cryopreservation tool at the time of thawing.

Drawings

Fig. 1 is a schematic view showing the overall structure of a cryopreservation tool according to an embodiment of the present invention.

Fig. 2 is an enlarged view of the front end portion of the main body portion according to the embodiment.

FIG. 3 is a schematic view showing a freezing step of an ovum or fertilized ovum by using the cryopreservation tool.

FIG. 4 is a schematic view showing a thawing step of an ovum or fertilized ovum frozen and preserved by the freezing preservation apparatus.

Fig. 5 is a photograph of a state in which a blastocyst is held by holding portions such as various eggs having different sizes (opening sizes) of mesh holes from directly above.

FIG. 6 is a photograph of a state in which a blastocyst is held by a holding portion such as an ovum having an opening size of 200 μm from the front side.

FIG. 7 is a photograph of a state in which various fertilized eggs are held by a holding portion such as an ovum having an opening size of 200 μm from directly above.

Detailed Description

The mode for carrying out the invention will be described with reference to the accompanying drawings.

As shown in fig. 1, the cryopreservation tool for ovum or fertilized ovum (hereinafter simply referred to as "cryopreservation tool") of the present embodiment includes a main body 10 and a tubular cover 20 that houses the main body 10.

The main body 10 includes a shank 11 and a holding portion 12 such as an ovum provided at the tip of the shank 11. The shank 11 is a rod-shaped member made of a low-temperature-resistant material. The length of the shank 11 is not particularly limited, but is desirably 9cm to 11cm. As a material constituting the handle 11, for example, a metal such as stainless steel, a synthetic resin, or the like can be used.

As shown in fig. 2, the retaining portion 12 such as an ovum is formed of a net made of fibers 13, and has a flat plate shape having a width of 3mm to 10mm and a length of 5mm to 30mm as a whole. As the fibers 13, fibers made of synthetic resin such as polyester can be preferably used, but the present invention is not limited thereto, and fibers made of metal such as stainless steel can be used. If the mesh size of the holding portion 12 such as an ovum is too small, the ovum is less likely to separate from the mesh during thawing. If the mesh size is too small, when the cryoprotectant liquid, which is a relatively high viscosity liquid, is placed on the ovum or other holding portion 12 together with the ovum or other material, the cryoprotectant liquid (and the ovum or other material) may not enter the inside of the mesh (or mesh) but be placed on the mesh, and may not be properly frozen. Therefore, the size (opening size) of the mesh hole of the retaining portion 12 such as an ovum of the present embodiment, that is, the interval between adjacent fibers (dimension a in fig. 2) among the fibers 13 constituting the mesh is 170 μm or more (preferably 190 μm or more). The upper limit of the opening size is not particularly limited as long as it is within a range in which the cryoprotectant can be held by the surface tension, but is desirably 300 μm or less (more desirably 250 μm or less) from the viewpoint of suppressing the amount of the cryoprotectant held in the holding portion 12 such as an ovum. The diameter of the fibers 13 constituting the web (dimension B in fig. 2) is not particularly limited, but is, for example, 40 μm to 70 μm (more preferably 50 μm to 60 μm).

The cover portion 20 is a thin tubular member made of a low-temperature resistant material (for example, synthetic resin such as polypropylene or metal such as stainless steel), and has an inner diameter slightly larger than the largest dimension of the main body portion 10 in the width direction. The cover 20 has a length capable of accommodating at least a part of the whole of the holder 12 such as an ovum of the body 10 and the handle 11.

The procedure for freezing an ovum or fertilized ovum by using the cryopreservation tool according to the present embodiment will be described with reference to fig. 3. The cryopreservation tool according to the present embodiment can be used particularly preferably for cryopreservation of human ova or fertilized ova, but is not limited to this, and can be used for cryopreservation of ova or fertilized ova of animals other than humans. The "ovum" to be subjected to the freezing treatment using the freezing preservation tool of the present embodiment may be a mature ovum, and the "fertilized ovum" may be a prokaryotic embryo, a mitotic embryo (early embryo), a morula, a blastula, or the like, but is not limited thereto.

First, an ovum or fertilized ovum (hereinafter referred to as "ovum or the like 30") to be frozen is immersed in a cryoprotectant 41, and the cryoprotectant 41 is infiltrated into the ovum or the like 30 in advance. Then, the worker extracts the ovum or the like 30 together with a small amount of the cryoprotectant 41 by using the pipette 42, and discharges the same under a microscope onto the ovum or the like holding portion 12 of the cryopreservation tool according to the present embodiment. Thus, the ovum or the like 30 and a small amount of the cryoprotectant 41 are held in the mesh (mesh opening) of the net constituting the ovum or the like holding portion 12 by the surface tension of the cryoprotectant 41. At this time, the surplus cryoprotectant liquid 41 which is not held by the surface tension falls down below the holding portion 12 such as an ovum through the mesh of the net, and therefore, the worker does not need to remove the surplus cryoprotectant liquid 41 by using a pipette or the like. In the cryopreservation tool according to the present embodiment, since the mesh size of the net constituting the egg or the like holding portion 12 is relatively large, there is also an advantage that the operator can easily visually check the egg or the like 30 held in the egg or the like holding portion 12. Next, the operator holds the handle 11 and dips the tip of the body 10 into liquid nitrogen 43, thereby rapidly cooling the ovum and the cryoprotectant 41 held in the ovum and other holding portion 12. In the cryopreservation tool of the present embodiment, the ovum or the like 30 is held in the mesh of the net by the surface tension of the cryoprotectant 41 and is cooled from the entire circumferential direction, and therefore, the freezing speed of the ovum or the like 30 can be increased as compared with the conventional cryopreservation tool. After that, the main body 10 is housed in the cover 20, and the main body 10 and the cover 20 are stored in the liquid nitrogen tank 44.

Next, a procedure for thawing the ovum or the like 30 frozen by the above procedure will be described with reference to fig. 4. First, the worker removes the cryopreservation tool according to the present embodiment from the liquid nitrogen tank 44, removes the cover portion 20 from the body portion 10, and then dips the tip of the body portion 10 into the melting liquid 45. In the cryopreservation tool of the present embodiment, the mesh size of the net constituting the egg and other holding portion 12 is relatively large, and the egg and other 30 can be separated from the egg and other holding portion 12 by immersing the egg and other holding portion 12 in the thawing liquid 45, so that the worker does not need to manually remove the egg and other 30 from the egg and other holding portion 12 using a pipette or the like.

Examples

Hereinafter, a freezing/thawing test of fertilized eggs performed to confirm the effect of the size of the mesh hole will be described. In this experiment, 5 kinds of cryopreservation tools having different opening sizes of the net constituting the holding portion such as an ovum were used. The structure of each cryopreservation tool was substantially the same as that shown in fig. 1, and a net made of polyester fiber having a wire diameter of 55 μm was used as the net constituting the holding portion for eggs and the like. The mesh opening sizes were set to 100 μm (comparative example 1), 150 μm (comparative example 2), 200 μm (example 1), 250 μm (example 2), 300 μm (example 3). As fertilized eggs to be frozen, human blasts were used, and freezing and thawing of the blasts were performed in accordance with the procedure shown in fig. 3 and 4 described above (n=8 in the case where the mesh opening sizes were 100 μm, 150 μm, 250 μm, and 300 μm, and n=10 in the case of 200 μm, respectively, for the number of samples). In this case, as the cryoprotectant, a "Vit Kit-Freeze NX" manufactured by Fuji film and Wako pure chemical industries, ltd was used, and as the thawing solution, a "Vit Kit-Warm NX" manufactured by Fuji film and Wako pure chemical industries, ltd was used.

The photograph in fig. 5 is a photograph of a state in which a blastocyst is held by a holding portion such as an ovum made of a net having the above-mentioned opening dimensions, taken from immediately above. The photograph of FIG. 6 is a photograph of a state in which blasts and cryoprotectant liquid are held by a holding portion such as an ovum having an opening size of 200 μm from the front side. As shown in these photographs, when using any holding portion such as an ovum having an opening size, the blastula and the cryoprotectant liquid can be held in the mesh of the net. However, in the case of thawing the frozen blastula and the cryoprotectant, the blastula was separated from the holding portion such as the ovum at the moment of immersion in the thawing solution in the cases where the opening sizes were 200 μm, 250 μm and 300 μm (i.e., examples 1 to 3), but the blastula was not separated even when immersed in the thawing solution in the cases where the opening sizes were 100 μm and 150 μm (i.e., comparative examples 1 and 2).

Then, using the cryopreservation tool of example 1 (that is, the cryopreservation tool having an opening size of 200 μm), a freezing/thawing test was performed for various fertilized eggs of human. The fertilized egg is frozen and thawed in the same manner as described above. Fig. 7 is a photograph of a state in which various fertilized eggs and a cryoprotectant liquid are held in the cryopreservation tool of example 1 from immediately above. Fig. 7 (a) is a photograph of an early embryo held 2 days after fertilization (D2), and fig. 7 (b) is a photograph of an early embryo held 3 days after fertilization (D3). Fig. 7 (c) is a photograph of a blastocyst (BL embryo) holding level 3 (G3), fig. 7 (d) is a photograph of a blastocyst holding level 4 (G4), fig. 7 (e) is a photograph of a blastocyst holding level 5 (G5), and fig. 7 (f) is a photograph of a blastocyst holding level 6 (G6). The size of each fertilized egg is shown in the upper part of each figure. From these photographs, it was clearly confirmed that fertilized eggs of various types and various sizes can be held in the meshes of the net by the surface tension of the cryoprotectant when using a cryopreservation tool having a holding portion such as an ovum with an opening size of 200 μm. In either case, the fertilized egg can be separated from the retaining portion such as an ovum by immersing the retaining portion such as a frozen ovum in the thawing liquid.

A cryopreservation tool having an opening size of 167 μm and a cryopreservation tool having an opening size of 183 μm were prepared, and human fertilized eggs were subjected to freezing/thawing experiments using these cryopreservation tools. The structure of each cryopreservation tool was substantially the same as that shown in fig. 1, but in the cryopreservation tool having an opening size of 167 μm, a net made of polyester fiber having a wire diameter of 45 μm was used as a net constituting a holding portion for ova or the like, and in the cryopreservation tool having an opening size of 183 μm, a net made of polyester fiber having a wire diameter of 71 μm was used. As fertilized eggs to be frozen, fertilized eggs before division (diameter about 150 μm to 160 μm) or fertilized eggs during division (diameter about 150 μm to 175 μm) were used, and freezing and thawing were performed in the same manner as described above (in addition, the number of samples was n=8 in any of the cases where a cryopreservation tool was used). As a result, even when any cryopreservation tool is used, fertilized eggs and the cryoprotectant can be held and frozen in the mesh of the net. However, since the mesh has smaller opening sizes than the cryopreservation tool of examples 1 to 3 (i.e., 200 μm, 250 μm, or 300 μm), there is an impression that it is difficult to visually confirm the mesh openings when the fertilized eggs are held or not when confirming whether the fertilized eggs are held. In addition, when the opening size is 167 μm, there is a case where fertilized eggs and a cryoprotectant solution cannot be held in the mesh at one time, and in such a case, it is necessary to perform a two-stage operation of holding only the cryoprotectant solution in the mesh first and then storing the fertilized eggs in the mesh. On the other hand, when thawing frozen fertilized eggs, the fertilized eggs can be instantaneously separated from the egg holding portion only by immersing in the thawing liquid when the opening size is 183 μm, but when the opening size is 167 μm, the fertilized eggs cannot be separated from the net only by immersing in the thawing liquid, and in such a case, it is necessary to shake the tip of the cryopreservation tool in the thawing liquid to separate the fertilized eggs from the net.

Next, the results of culturing early embryo after freeze thawing were evaluated using a cryopreservation tool having a mesh opening size of 200. Mu.m (i.e., the cryopreservation tool of example 1) and a cryopreservation tool having a mesh opening size of 250. Mu.m (i.e., the cryopreservation tool of example 2). Specifically, first, after melting and leaving frozen early mouse embryos (Day 3 embryos, 4 cells to 8 cells) for about 10 minutes, the embryos were frozen using the cryopreservation tool of example 1 or the cryopreservation tool of example 2, and then immediately melted. The freezing and thawing were performed in the same manner as described above. Thereafter, the thawed embryos were cultured for 3 days, and the embryos at Day 2 and Day3 of culture (i.e., day5 embryos and Day6 embryos) were observed. The embryo was cultured at 37.0℃using SAGE 1step medium (Origio Co.). The results of culturing the embryos after freezing and thawing using the cryopreservation tool of example 1 are shown in table 1, and the results of culturing the embryos after freezing and thawing using the cryopreservation tool of example 2 are shown in table 2. The number of samples was n=20.

TABLE 1

TABLE 2

In the above table, "BL formation rate" means the rate of embryos that develop into blasts (blastocyst), and "good BL formation rate" means the rate of embryos that are rated 3AA or more. As shown in tables 1 and 2, in both cases of using the cryopreservation tools of examples 1 and 2, 90% of embryos developed to good grade blasts at Day5, and no zona pellucida cracks, cell leakage and disruption disorder occurred.

The culture results of the thawed embryos were evaluated when the mouse embryos developed to blasts were frozen and thawed using the cryopreservation tool of example 1 or the cryopreservation tool of example 2. Specifically, first, frozen mouse early embryos (similar to the above) were thawed and cultured for 1 or 2 days until the early embryos developed into blasts. The culture was performed under the same conditions as described above. Thereafter, the blastula was frozen and thawed using the cryopreservation tool of example 1 or the cryopreservation tool of example 2. The freezing and thawing were performed in the same manner as described above. Thereafter, the thawed embryos were cultured under the same conditions as described above for 1 Day, and the cultured embryos (i.e., day5 embryos and Day6 embryos) were observed. The results of culturing the embryos frozen and thawed using the cryopreservation apparatus of example 1 are shown in Table 3, and those of culturing the embryos frozen and thawed using the cryopreservation apparatus of example 2 are shown in Table 4. In addition, the former is set to n=51 and the latter is set to n=55 for the number of samples.

TABLE 3

TABLE 4

In the above table, "hatch" refers to the blastula reaching stage G5, and "complete escape" refers to the blastula reaching stage G6. As apparent from tables 3 and 4, in both cases of using the cryopreservation tools of examples 1 and 2, 90% of embryos developed to good grade blasts at Day5, and no zona pellucida cracks, cell leakage and disruption disorder occurred.

Description of the reference numerals

10. A main body portion; 11. a handle; 12. an egg holding part; 13. a fiber; 20. a cover section; 30. ovum, etc.; 41. freezing the protective liquid; 42. a pipette; 43. liquid nitrogen; 44. a liquid nitrogen tank; 45. and (5) melting the solution.

Claims

1. A cryopreservation tool for ovum or fertilized ovum, wherein,

The cryopreservation tool comprises:

A tubular cover portion for accommodating the main body portion,

The mesh of the egg holding portion has an opening size of 170 μm or more.

2. The cryopreservation tool for an ovum or fertilized ovum according to claim 1, wherein,

The mesh of the egg holding portion has an opening size of 190 μm or more.

3. The cryopreservation tool for ovum or fertilized ovum according to claim 1 or 2, wherein,

The mesh of the egg holding portion has an opening size of 300 μm or less.

4. The cryopreservation tool for an ovum or fertilized ovum according to claim 3, wherein,

The mesh of the egg holding portion has an opening size of 250 μm or less.