JP7292637B1 - Cryopreservation device for oocytes or fertilized eggs - Google Patents

Abstract

A cryopreservation tool for oocytes or fertilized eggs according to the present invention comprises a main body (10 ), and a tubular cover portion (20) in which the main body portion is accommodated, and the opening size of the mesh of the ovum or the like holding portion is 170 μm or more. As a result, the amount of cryoprotectant around the ovum can be adjusted to an appropriate amount without performing complicated work, and the ovum can be easily removed during thawing.

Description

The present invention relates to a cryopreservation tool for cryopreserving ova or fertilized eggs.

Vitrification is known as a technique for cryopreserving eggs or fertilized eggs (embryos) in the field of human infertility treatment and the like. In the vitrification freezing method, eggs or fertilized eggs (hereinafter collectively referred to as "eggs, etc.") are immersed in liquid nitrogen together with a small amount of cryoprotectant (vitrification liquid) to rapidly cool them. This is a method of freezing in an amorphous glass state without crystallizing the water inside and outside the cells. According to this method, it is possible to suppress the damage to the eggs and the like due to ice crystal formation, and to increase the survival rate of the eggs and the like during subsequent melting.

In such a vitrification freezing method, cryopreservation tools are widely used that include a rod-shaped main body provided with a thin plate-shaped mounting portion for placing eggs and the like on the tip, and a cylindrical cover that accommodates the main body. used (see, for example, Patent Document 1). When freezing ova or the like using such a cryopreservation tool, first, the ova that have been immersed in a cryoprotectant in advance are collected with a pipette or the like, and placed on the mounting section together with a small amount of the cryoprotectant. place. Then, after the tip of the main body including the mounting portion is immersed in liquid nitrogen for rapid cooling, the main body is fitted with the cover and stored in a liquid nitrogen tank.

However, in the vitrification freezing method, if there is a large amount of cryoprotectant around the ovum, the rate of cooling by liquid nitrogen may decrease, and the viability of the ovum may decrease. Therefore, in the cryopreservation tool as described above, when the amount of cryoprotectant placed on the mounting part together with the ovum or the like is large, it is necessary to remove the excess cryoprotectant using a pipette or the like to adjust the amount appropriately. However, skill was required to adjust the appropriate amount of cryoprotectant without damaging or drying the oocytes.

Therefore, Patent Literature 2 proposes a cryopreservation tool from which excess cryoprotectant can be removed with a simple operation. In this cryopreservation tool, the placement section for placing the ova and the like is formed in a mesh shape. Excess cryoprotectant can be removed by manipulation.

JP-A-2002-315573 WO2011/070973

However, in a cryopreservation tool having a net-like placement part as described above, when the cryopreservation tool is removed from the liquid nitrogen tank after cryopreservation and the placement part is immersed in the melting liquid to thaw the ova and the like, In addition, there are cases where it is difficult for the ovum or the like to come off from the placing portion. In such a case, it is necessary for the operator to aspirate the ovum with a pipette or the like and remove it from the placement unit, but there is a risk of damaging the ovum at this time, and the burden on the operator increases. I had a problem.

The present invention has been made in view of the above points, and its object is to make the amount of cryoprotective solution around eggs, etc. appropriate without performing complicated work, and to thaw them. To provide a cryopreservation tool from which an ovum or the like can be easily removed from a placing part.

The oocyte or fertilized egg cryopreservation tool according to the present invention, which was made to solve the above problems,
a main body having a bar-shaped handle, and a mesh-shaped ovum or the like holding section provided at the tip of the handle;
a tubular cover portion in which the main body portion is accommodated;
, and the opening size of the mesh in the ovum or the like holding portion is 170 μm or more.

According to the cryopreservation tool for oocytes or fertilized eggs according to the present invention having the above configuration, it is possible to make the amount of cryoprotectant around the oocytes appropriate without performing complicated work, and furthermore, during thawing, Eggs and the like can be easily removed from the cryopreservation device.

1 is a schematic diagram showing the overall configuration of a cryopreservation tool according to one embodiment of the present invention. FIG. The figure which expands and shows the front-end|tip part of the main-body part in the said embodiment. FIG. 2 is a schematic diagram showing a procedure for freezing ova or fertilized eggs using the cryopreservation tool. FIG. 3 is a schematic diagram showing a procedure for thawing oocytes or fertilized eggs cryopreserved using the cryopreservation tool. Photographs taken from directly above showing the state in which blastocysts are held in various holding parts for ova and the like having different mesh hole sizes (opening dimensions). A photograph taken from the side showing a state in which a blastocyst is held in an ovum holding part having a mesh opening size of 200 μm. A photograph taken from directly above of various fertilized eggs held in an ovum holding unit having a mesh opening size of 200 μm.

A mode for carrying out the present invention will be described with reference to the drawings.

As shown in FIG. 1, the oocyte or fertilized egg cryopreservation tool (hereinafter simply referred to as "cryopreservation tool") according to the present embodiment includes a main body 10 and a cylindrical cover part that accommodates the main body 10. 20 and.

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

As shown in FIG. 2, the ovum holding part 12 is composed of a mesh made of fibers 13 and has a flat plate shape with a width of 3 mm to 10 mm and a length of 5 mm to 30 mm as a whole. As the fiber 13, synthetic resin such as polyester can be suitably used, but the fiber is not limited to this, and for example, metal such as stainless steel may be used. If the mesh holes of the ovum holding part 12 are too small, it becomes difficult for the ova to come out of the mesh holes during melting. Furthermore, if the mesh holes are too small, when the cryoprotective liquid, which is a relatively highly viscous liquid, is placed on the ovum holding unit 12 together with the ova, etc., the cryoprotective liquid (and the ova, etc.) will be absorbed through the mesh holes (mesh). There is also a risk that it will not be able to freeze properly because it will be in a state of being on a mesh without getting inside. Therefore, the size of the mesh holes (opening size) of the ovum holding portion 12 in the present embodiment, that is, the interval between adjacent fibers 13 constituting the mesh (dimension A in FIG. 2) is 170 μm. 190 μm or more (preferably 190 μm or more). The upper limit of the opening size is not particularly limited as long as the cryoprotectant can be retained by surface tension. It is desirable that the thickness is 250 μm or less (more preferably 250 μm or less). The wire diameter (dimension B in FIG. 2) of the fibers 13 forming the mesh 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, a synthetic resin such as polypropylene, or a metal such as stainless steel). It's becoming In addition, the cover portion 20 has a length that can accommodate the entire ovum holding portion 12 of the main body portion 10 and at least a portion of the handle portion 11 .

A procedure for freezing ova or fertilized eggs using the cryopreservation tool according to the present embodiment will be described with reference to FIG. The cryopreservation tool according to the present embodiment can be suitably used particularly for cryopreservation of human ova or fertilized eggs, but is not limited to this, and is not limited to this, and can be used for ova or fertilized eggs of animals other than humans. It can also be used for cryopreservation. In addition, the "egg" to be frozen using the cryopreservation tool according to the present embodiment can include mature eggs, and the "fertilized egg" includes pronuclear stage embryos, division stage embryos (early embryos ), morula, or blastocyst, but are not limited thereto.

First, an egg or a fertilized egg (hereinafter referred to as "egg 30") to be cryopreserved is immersed in the cryoprotectant 41 so that the cryoprotectant 41 permeates the ovum 30 in advance. Then, the operator collects the ovum or the like 30 together with a small amount of the cryoprotectant 41 using the pipette 42, and discharges them onto the ovum or the like holding portion 12 of the cryopreservation tool according to the present embodiment under a microscope. do. As a result, the ova and the like 30 and a small amount of the cryoprotectant 41 are retained in the meshes (mesh holes) of the ovum and the like holding section 12 due to the surface tension of the cryoprotectant 41 . At this time, the surplus cryoprotectant 41 that cannot be retained by the surface tension falls through the mesh of the mesh and falls below the ovum holding section 12. 41 need not be removed. In addition, in the cryopreservation tool according to the present embodiment, since the mesh size of the mesh forming the ovum holding unit 12 is relatively large, the operator can visually recognize the ova 30 held in the ovum holding unit 12. It also has the advantage of being easy. Subsequently, the operator grasps the handle 11 and immerses the tip of the main body 10 in the liquid nitrogen 43, thereby rapidly cooling the ova 30 and the cryoprotectant 41 held in the ovum holding unit 12. . In the cryopreservation device according to the present embodiment, the ovum or the like 30 is held in the mesh of the mesh by the surface tension of the cryoprotectant 41, and is cooled from all directions. It is possible to improve the freezing speed of the ovum or the like 30. After that, the body portion 10 is housed in the cover portion 20 and the body portion 10 and the cover portion 20 are stored in the liquid nitrogen tank 44 .

Next, the procedure for thawing the frozen-preserved ova 30 will be described with reference to FIG. First, an operator takes out the cryopreservation tool according to the present embodiment from the liquid nitrogen tank 44 , removes the cover part 20 from the body part 10 , and then immerses the tip of the body part 10 in the melting liquid 45 . In the cryopreservation tool according to the present embodiment, the mesh size of the mesh forming the ovum holding section 12 is relatively large, and the ova 30 can be retained by simply immersing the ovum holding section 12 in the lysing liquid 45. Since it is separated from the part 12, it is not necessary for the operator to manually remove the ovum or the like 30 from the ovum or the like holding part 12 using a pipette or the like.

A freeze/thaw experiment on fertilized eggs performed to confirm the influence of the size of the mesh holes will be described below. In this experiment, five types of cryopreservation tools having different opening sizes of the mesh constituting the ovum holding part were used. The structure of each cryopreservation device was almost the same as that shown in FIG. 1, and the meshes constituting the ovum holding part were all made of polyester fibers with a wire diameter of 55 μm. The opening size of the mesh was 100 μm (Comparative Example 1), 150 μm (Comparative Example 2), 200 μm (Example 1), 250 μm (Example 2), and 300 μm (Example 3). Human blastocysts were used as fertilized eggs to be frozen, and the blastocysts were frozen and thawed according to the procedure shown in FIGS. n=8 for the opening dimensions of 100 μm, 150 μm, 250 μm, and 300 μm, and n=10 for the opening size of 200 μm). At that time, "Vit Kit-Freeze NX" manufactured by FUJIFILM Wako Pure Chemical Co., Ltd. was used as the cryoprotectant, and "Vit Kit-Warm NX" manufactured by FUJIFILM Wako Pure Chemical Co., Ltd. was used as the thawing liquid. "It was used.

The photograph of FIG. 5 is taken from directly above the state in which the blastocyst is held in the ovum and the like holding section made of mesh with each opening size. In addition, the photograph of FIG. 6 is a side view of the blastocyst and the cryoprotectant held in the ovum holding part with an opening size of 200 μm. As shown in these photographs, the blastocyst and the cryoprotectant could be retained in the meshes of the ovum, etc., regardless of the opening size of the ovum holder. However, when the frozen blastocyst and cryoprotectant were thawed, the embryos with the opening size of 200 μm, 250 μm, and 300 μm (that is, Examples 1 to 3) were immediately immersed in the thawing solution. Although the cysts detached from the ovum-holding part, the blastocysts of 100 μm and 150 μm (ie, Comparative Examples 1 and 2) sometimes did not detach even when immersed in the lysate.

Furthermore, using the cryopreservation device of Example 1 (that is, one with an opening size of 200 μm), freezing/thawing experiments were conducted on various human fertilized eggs. Freezing and thawing of the fertilized eggs were performed in the same manner as described above. The photographs of FIG. 7 are taken from directly above the cryopreservation tool of Example 1, in which various fertilized eggs are held together with a cryoprotectant. FIG. 7(a) shows the retention of early embryos 2 days after fertilization (D2), and FIG. 7(b) shows the retention of early embryos 3 days after fertilization (D3). In addition, FIG. 7(c) shows grade 3 (G3) blastocysts (BL embryos) retained, and FIG. 7(d) shows grade 4 (G4) blastocysts. ) shows grade 5 (G5) blastocysts, and FIG. 7(f) shows grade 6 (G6) blastocysts. Also, the size of each fertilized egg is indicated at the top of each figure. As is clear from these photographs, when using a cryopreservation tool equipped with an ovum holding part with an opening size of 200 μm, fertilized eggs of various types and sizes can be stored in the mesh of the mesh. , was confirmed to be retained by the surface tension of the cryoprotectant. Also, in any case, the fertilized egg could be removed from the holding part for ova and the like simply by immersing the holding part for ova and the like after freezing in the thawing solution.

A cryopreservation tool having a mesh opening size of 167 μm and a cryopreservation tool having a mesh opening size of 183 μm are prepared, and human fertilized eggs are frozen using these cryopreservation tools. / Melting experiments were performed. The structure of each cryopreservation device is almost the same as that shown in Fig. 1, but for the cryopreservation device with an opening size of 167 µm, the mesh that constitutes the ovum holding part is made of polyester fiber with a wire diameter of 45 µm. A cryopreservation tool with an opening size of 183 μm was made of polyester fiber with a wire diameter of 71 μm. As fertilized eggs to be frozen, use undivided fertilized eggs (with a diameter of about 150 μm to 160 μm) or dividing fertilized eggs (with a diameter of about 150 μm to 175 μm), and freeze and thaw them as described above. It was carried out in the same way (the number of samples was set to n=8 when using either cryopreservation device). As a result, the fertilized eggs and the cryoprotectant could be held and frozen in the mesh of the mesh using either of the cryopreservation devices. However, since the mesh opening size is smaller than that of the cryopreservation tools of Examples 1 to 3 (that is, the mesh opening size is 200 μm, 250 μm, or 300 μm), it is difficult to hold the fertilized egg. There was an impression that it was difficult to visually check the mesh holes when confirming whether or not the was held. In addition, when the opening size is 167 μm, it may not be possible to hold the fertilized egg and the cryoprotectant in the mesh holes at the same time. A two-stage operation was required, first holding the fertilized egg and then placing the fertilized egg in the mesh hole. On the other hand, when thawing fertilized eggs after freezing, it was possible to instantly remove the fertilized eggs from the ovum holder simply by immersing them in the thawing solution with an eye opening size of 183 μm, but the eye opening size was 167 μm. In some cases, the fertilized egg cannot be removed from the mesh just by immersing it in the thawing solution. became necessary.

Next, a cryopreservation tool with a mesh opening size of 200 μm (ie, the cryopreservation tool of Example 1) and a cryopreservation tool with a mesh opening size of 250 μm (ie, the cryopreservation tool of Example 2). was used to evaluate the culture performance of early stage embryos after freezing and thawing. Specifically, first, a frozen mouse early embryo (Day 3 embryo, 4 to 8 cells) is thawed, left to stand for about 10 minutes, and then placed in the cryopreservation tool of Example 1 or the cryopreservation of Example 2. The embryos were frozen using a tool and then thawed immediately. Freezing and thawing were performed in the same manner as described above. After that, the thawed embryos were cultured for 3 days, and the embryos on the 2nd and 3rd days of culture (that is, Day 5 embryos and Day 6 embryos) were observed. Embryos were cultured at 37.0° C. using SAGE 1 step medium (manufactured by Origio). Table 1 shows the culture results of embryos after freezing and thawing using the cryopreservation tool of Example 1, and Table 2 shows the culture results of embryos after freezing and thawing using the cryopreservation tool of Example 2. . Note that the number of samples is n=20.

In the above table, "BL incidence" indicates the ratio of embryos that have grown to blastocyst, and "good BL incidence" means the ratio of embryos with a grade of 3AA or higher. there is As shown in Tables 1 and 2, even when using any of the cryopreservation tools of Examples 1 and 2, 90% of the embryos grew to good grade blastocysts on Day 5, and cracked zona pellucida. , cell leakage, and fracture failure did not occur.

Furthermore, when the mouse embryo grown to the blastocyst was frozen and thawed using the cryopreservation device of Example 1 or the cryopreservation device of Example 2, the culture performance of the embryo after thawing was evaluated. Specifically, first, a frozen mouse early embryo (same as above) was thawed and cultured for 1 or 2 days until the early embryo grew into a blastocyst. The culture was performed under the same conditions as above. The blastocysts were then frozen and thawed using the cryopreservation device of Example 1 or the cryopreservation device of Example 2. Freezing and thawing were performed in the same manner as described above. After that, the thawed embryos were cultured for 1 day under the same conditions as above, and the cultured embryos (that is, Day 5 embryos and Day 6 embryos) were observed. Table 3 shows the culture results of embryos frozen and thawed using the cryopreservation tool of Example 1, and Table 4 shows the culture results of embryos frozen and thawed using the cryopreservation tool of Example 2. The number of samples is n=51 for the former and n=55 for the latter.

In the table above, "hatching" means that the blastocyst reaches stage G5, and "complete escape" means that the blastocyst reaches stage G6. As is clear from Tables 3 and 4, even when using any of the cryopreservation tools of Examples 1 and 2, 90% of the embryos on Day 5 grew into good-grade blastocysts. , zona pellucida fissures, cell leakage, and fracture failure did not occur.

DESCRIPTION OF SYMBOLS 10... Main body part 11... Handle part 12... Egg holding part 13... Fibers 20... Cover part 30... Eggs, etc. 41... Cryoprotectant liquid 42... Pipette 43... Liquid nitrogen 44... Liquid nitrogen tank 45... Melting liquid

Claims (4)

a main body having a bar-shaped handle, and a mesh-shaped ovum or the like holding section provided at the tip of the handle;
a tubular cover portion in which the main body portion is accommodated;
with
A cryopreservation tool for ova or fertilized ova , wherein the mesh of the ovum or the like holding part is formed by arranging fibers having a circular cross section in a grid pattern, and the opening size of the mesh is 170 μm or more and 300 μm or less . 2. The cryopreservation tool for oocytes or fertilized eggs according to claim 1, wherein the opening size of the mesh of said oocyte holding part is 190 μm or more. 2. The cryopreservation tool for ova or fertilized ova according to claim 1 , wherein the opening size of the mesh of said ovum holding part is 250 μm or less. The cryopreservation tool for ova or fertilized ova according to claim 1, wherein the fiber has a diameter of 40 µm to 70 µm.

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