KR102278277B1 - The development of vitrification container for cryopreservation and vitrification method using the same - Google Patents

Abstract

The present invention relates to a vitrified container used for cryopreservation, wherein when freezing and thawing cells, a handling unit and a cell loading unit are separately marked so that the possibility of cell loss when stored in a vial is significantly reduced and convenience is secured during movement, and a first absorption unit or a second absorption unit is further included to minimize ice-freezing of the cells, thereby improving the viability of cells.

Description

Preservation container for vitrification freezing and vitrification method using the same {THE DEVELOPMENT OF VITRIFICATION CONTAINER FOR CRYOPRESERVATION AND VITRIFICATION METHOD USING THE SAME}

The present invention relates to a vitrified container used for cryopreservation, and more particularly, to a preservation container and a vitrification method for vitrified freezing applied to cryopreservation of human or animal germ cells.

In general, the cryopreservation of reproductive cells such as eggs or embryos in Assisted Reproductive Technology (ART) is known as a technology that provides more chances of conception to infertile patients.

The cryopreservation technology of these germ cells can be divided into a gentle freezing method and a vitrification freezing method.

First, the gentle freezing method uses the osmotic principle to equilibrate a part of the moisture in the cytoplasm and a cryoprotectant by substituting a part of the moisture in the cytoplasm with a permeable cryoprotectant, and then slowly freezing the moisture outside the cell by a cell freezer. It is known as a technology that generates osmotic pressure by raising the concentration of the cryo inhibitor outside the cell, and uses this power to slowly remove the remaining water in the cell, leaving only the minimum amount of water and minimizing the ice crystals of the remaining water in the cell. .

However, this method requires relatively expensive equipment, a lot of time, and a large amount of liquid nitrogen. In particular, it has fatal disadvantages in the survival rate and recovery rate of germ cells after thawing due to the ice crystal phenomenon.

In contrast, the vitrification method uses a cryo inhibitor with high permeability and low toxicity or a combination of permeability and impermeable cryo inhibitor when freezing germ cells to lower the absolute concentration without changing the relative concentration, reduce toxicity to cells, and thaw It is known as a technique to enhance the efficiency of post-frozen germ cells.

In addition, since the process is simple and fast, the operation time for cryopreservation is shortened and a temperature control device is not required.

This vitrification freezing method is applied to cryopreservation of various cells or tissues, and is widely used in many fertility in vitro treatment centers and animal germ cell laboratories.

However, vitrification such as cryo-top, cryo-loop, cryo-pipet, cryo-tip, EM-grid, etc. Vitrification carriers or vitrification containers are available at a high price on the market, increasing the cost of fertility procedures and animal studies, as well as explosion of specimen tubes or straw canisters or attachment of germ cells during thawing after freezing of germ cells. It has a disadvantage in that the loss rate of germ cells is high.

Looking at the background technology to supplement this problem, in "Jig and cryopreservation method for cryopreservation of cells or tissues" of Korean Patent Publication No. 10-1962052 (hereinafter referred to as 'Document 1'), All or part of the surface layer containing the water-soluble high molecular compound on the outermost surface of the mounting part is dissolved in the lysing solution, so that cells or tissues do not adhere to the mounting part of the cryopreservation jig, and there is an advantage that it can be easily peeled off and recovered.

However, when germ cells including embryos or eggs are applied as cells of Document 1, since germ cells are larger than normal cells or cancer cells, ice crystals are formed and they can be easily damaged.

Looking at the other background art, in the "jig for cryopreservation of cells or tissues" of Korean Patent Application Laid-Open No. 10-2020-0005582 (hereinafter referred to as 'Document 2'), the mounting surface has a convex part for maintaining cells or tissues and Disclosed is a jig for cryopreservation of cells or tissues having a recess for storing a preservative solution.

When the invention of Document 2 is used, there is an advantage in that it is easy to remove the preservative solution remaining around cells or tissues by the convex portions and concave portions, but there is a disadvantage that cells may be damaged due to the concave portions and the shapes of the convex portions.

Looking at the other background art, in the "jig for cryopreservation" of Japanese Patent Application Laid-Open No. 2020-103224 (hereinafter referred to as 'Document 3'), an adhesive layer on a light-transmissive support and a storage solution absorber in which a storage solution absorbing layer is laminated on the adhesive layer having a preservative absorber Disclosed is a jig for cryopreservation.

When the invention of Document 3 is used, it has the advantage of preventing local saturation of the preservative solution and rapidly removing unnecessary preservative solution during freezing of cells or tissues, but due to the formation of ice crystals upon thawing due to the formation of an absorbent layer on the upper side of the support. There is a disadvantage that can cause cell damage.

Looking at the other background art, in the “jig for cryopreservation of cells or tissues for vitrification of cells or tissues” of Korean Patent Publication No. 10-1988140 (hereinafter referred to as 'Document 4'), a vitrification solution absorber in which an adhesive layer and a vitrification solution absorption layer are formed on a support. It discloses a jig for cryopreservation of cells or tissues characterized in that it has a portion in which an adhesive layer does not exist between a portion on which the cells or tissues of the vitrification solution absorption layer are placed and a support.

In the case of using the invention of Document 4, the vitrification absorber absorbs the excess vitrification liquid, so a separate process for removing the excess vitrification liquid is not performed, which has a simple advantage. However, the vitrification liquid absorption layer and the adhesive layer formed on the upper side of the support, etc. It is located in this narrow container and has a disadvantage in that it is structurally complicated and causes an increase in manufacturing cost.

<Background art literature>

Republic of Korea Patent Publication No. 10-1962052

Republic of Korea Patent Publication No. 10-2020-0005582

Japanese Patent Laid-Open No. 2020-103224

Republic of Korea Patent Publication No. 10-1988140

The present invention is to solve the problems of the prior art, and a cryo-top, a cryo-loop, a cryo-pipet, which are conventionally applied for cryopreservation of cells, Vitrification carriers or vitrification containers, such as cryo-tips, EM-grid, etc., are provided in the market at a high price, which increases the cost of fertility procedures and animal studies, as well as increases the cost of animal research. , An object of the present invention is to provide a storage container for vitrification freezing and a vitrification method using the same, which can compensate for the disadvantage of a high cell loss rate due to the explosion of a specimen tube or straw capsule when thawing cells after freezing.

Moreover, EM-grid is formed of metal and developed for the purpose of fixing a sample in an electron microscope to observe it under a microscope. When applied to cryopreservation, due to the affinity between the organic polymer of the cell and the metal, the cell is frozen or An object of the present invention is to provide a storage container for vitrification freezing that can minimize the disadvantage that cells are attached to metal during thawing and cell death after thawing, and a vitrification method using the same.

In addition, the possibility of death and loss of frozen cells is reduced, and a separate mark is applied to the bent handling part of the first container to minimize confusion with the location of frozen cells, and the possibility of cell loss in the process of movement or storage An object of the present invention is to provide a storage container for freezing and vitrification that can lower the temperature and a method for vitrification using the same.

Furthermore, a storage container for vitrification freezing that can minimize ice crystals and improve viability by cryopreserving cells with a smaller amount of vitrified freezing solution by optionally further including a receiving part, a first absorption part, or a second absorption part; and An object of the present invention is to provide a vitrification method using the same.

In order to achieve the above object, the present invention is characterized in that it includes a first container 100 formed in a plate shape to accommodate the cells 10 on the upper side.

In addition, the first container 100 is characterized in that it is gripped by a mechanism including tongs.

Moreover, the cell 10 is characterized in that it is a germ cell including sperm, egg or embryo applied to the in vitro fetal procedure.

In addition, the first container 100 is characterized in that it is formed of a synthetic resin,

Furthermore, the first container 100 is characterized in that it is formed of any one or more selected from polyethyleneterephthalate (PET), poly-chloro-tri-fluoro ethylene (PCTFE), polytetrafluoroethylene (PTFE), and perfluoroalkoxy (PFA).

In addition, the first container 100 is characterized in that it includes a handling unit 110 having an end bent in the upward direction.

Moreover, it is characterized in that the first display unit 111 is formed at the end of the first container 100 opposite to the part where the cells 10 are accommodated for the convenience of movement or use.

In addition, the first container 100 is formed to extend in the longitudinal direction at a position adjacent to the cell 10, it is characterized in that it includes a receiving portion 200 having a groove formed therein.

Furthermore, it is characterized in that it includes a first absorption unit 300 formed at a position adjacent to the cell 10 of the first container 100 .

In addition, it is characterized in that it includes a second absorption portion 400 formed to protrude outwardly at the lower end of the first container (100).

The present invention is a device or container for cryopreservation, such as an EM grid, which is conventionally applied for cryopreservation of cells, is formed of metal and is developed for the purpose of fixing a sample in an electron microscope to observe it under a microscope. When applied, due to the affinity between the organic polymer of the cell and the metal, the cell adheres to the metal when the cell is frozen or thawed, so the disadvantage of cell death after thawing is minimized and an instrument not suitable for the intended use is not used. There is this.

In addition, a bent handling part is formed in the first container so that the cells can be stored and moved easily in a vial when freezing or thawing, so that the handling part and the cells are attached (loading) when freezing and thawing the cells. ), the possibility of cell loss when placed in a vial and stored in a vial is markedly reduced, and has the advantage of being convenient when moving.

Furthermore, the first container optionally further includes a receiving part, a first absorption part, or a second absorption part, so that the amount of vitrified freezing liquid, which is a preservation solution, is applied to a small amount, and ice freezing of cells due to ice crystals is minimized, thereby reducing mortality or loss. By doing so, there is an advantage in that the viability of cells can be improved.

Furthermore, the portion that can accommodate the cells in the first container is formed to be larger than the conventionally applied EM-grid, so there is an advantage that the loss of cells due to the user or external stimuli can be significantly reduced.

1 is a perspective view of a vitrification container used for cryopreservation according to the present invention.
2 is a perspective view according to an embodiment of the present invention.
3 is a cross-sectional view according to an embodiment of the present invention.
4 is a perspective view according to an embodiment of the present invention.
5 is a cross-sectional view according to an embodiment of the present invention.
6 is a cross-sectional view according to an embodiment of the present invention.
7 is a perspective view according to an embodiment of the present invention.
8 is a perspective view according to an embodiment of the present invention.
9 is a perspective view according to an embodiment of the present invention.
10 is a perspective view according to an embodiment of the present invention.
11 is a result value of Experimental Example 1.
12 is a result value of Experimental Example 1.
13 is a result value of Experimental Example 1.
14 is a result value of Experimental Example 1.
15 is a result value of Experimental Example 2.
16 is a cross-sectional view according to an embodiment of the present invention.
17 is a cross-sectional view according to an embodiment of the present invention.
<Explanation of code>
10: cell, 20: bottom surface
100: first container, 110: handling unit, 111: first display unit
200: receiving unit, 210: receiving path
300: first absorption unit
400: second absorption unit

Hereinafter, the present invention will be described in detail through examples.

Objects, features and advantages of the present invention will be easily understood through the following examples.

The present invention is not limited to the embodiments disclosed herein, and may be embodied in other forms. The embodiments disclosed herein are provided so that the spirit of the present invention can be sufficiently conveyed to those of ordinary skill in the technical field to which the present invention belongs, and all transformations included in the technical spirit and scope of the present invention , should be understood to include equivalents or substitutes.

Therefore, the present invention should not be limited by the following embodiments, and it should be understood that all transformations included in the technical spirit and scope of the present invention are included. That is, a person of ordinary skill in the art to which the present invention pertains can variously modify or modify the present invention by adding, changing, deleting or adding components within the scope that does not depart from the spirit of the present invention described in the claims. It will be possible to change, it will also be said to be included within the scope of the present invention.

Since the present invention is capable of various modifications and various embodiments, specific embodiments are illustrated in the drawings and described in detail. In the drawings, the size of the elements or the relative sizes between the elements may be exaggerated for a clear understanding of the present invention. In addition, the shape of the element shown in the drawings may be slightly changed due to variations in the manufacturing process or the like.

Therefore, the embodiments disclosed in the present specification should not be limited to the shapes shown in the drawings unless otherwise noted, and should be understood to include some degree of deformation.

On the other hand, various embodiments of the present invention may be combined with any other embodiments unless clearly indicated to the contrary. Any feature indicated as particularly preferred or advantageous may be combined with any other feature and features indicated as preferred or advantageous. That is, the various aspects, features, embodiments or implementations of the present invention may be used alone or in various combinations.

It should be understood that the terminology used in this specification is for the purpose of describing specific embodiments and is not intended to be limited by the claims, and all technical and scientific terms used in this specification are those of ordinary skill unless otherwise noted. has the same meaning as is commonly understood by a person with The singular expression includes the plural expression unless the context clearly dictates otherwise.

In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

<Example 1>

The vitrification container (hereinafter, referred to as 'container' for convenience of explanation) used for cryopreservation according to the present invention is to be formed including the cells 10 and the first container 100, as shown in FIG. can

More specifically, the container according to the present invention may be formed including the first container 100 formed in a plate shape to accommodate the cells 10 on the upper side.

In this case, the cell 10 is a cell 10 preserved in the preservative solution, and germ cells such as sperm, egg, embryo, or blastocyst, which are embryos in a pre-implantation state, applied to in vitro fetal procedures may be applied.

Preferably, the cell 10 may be a germ cell such as a sperm, an egg, or an embryo, but is not limited thereto.

In this way, the cells 10 are preserved in the preservative solution so that the cells are not damaged or lost when moving to the first container 100 .

Next, the first container 100 may be formed in a plate shape to accommodate the cells 10 on the upper side.

At this time, as shown in FIG. 1 , by stably loading the cell 10 on one end of the first container 100 , the cell 10 is not only stably positioned, but also the cell 10 . There is an advantage that does not stick to the first container (100).

To this end, the first container 100 may be formed of a synthetic resin to stably support the cells 10, preferably PET (polyethyleneterephthalate), PCTFE (Poly-Chloro-Tri-Fluoro Ethylene), PTFE (Polytetrafluoroethylene). ) or PFA (perfluoroalkoxy) may be formed of any one or more selected from.

Most preferably, it may be formed of any one or more materials selected from PET or PCTFE, which are known to have excellent rigidity, electrical properties, oil resistance, chemical resistance, tensile strength, heat resistance, and transparency, but is not limited thereto.

Optionally, in order to increase the visibility of the cells 10, the first container 100 may be formed of a transparent material, but is not limited thereto.

Moreover, the first container 100 may be formed in a plate shape to stably support the cells 10 positioned on the upper side.

To this end, the length of the first container 100 may be formed to be less than 50 mm, but is preferably formed to be 20 to 30 mm so that the operator can easily move the first container 100 as well as the cells 10 . It has the advantage of being able to provide a length sufficient to accommodate the

More preferably, it may be formed of 25 mm, but is not limited thereto.

Furthermore, the width of the first container 100 may be formed in a width of 0.5 to 5 mm, preferably in a width of 1 to 3 mm, and most preferably in a width of 2 mm.

If the width of the first container 100 is formed to be less than 0.5 mm, not only the cells 10 by the width of the first container 100, but also the preservative solution is separated to the outside of the first container 100 and the cells ( 10) has a high possibility of damage, and when it is formed to be more than 5 mm, there is a disadvantage in that the manufacturing cost increases due to the wide width.

In addition, the first container 100 may be formed to be gripped by a mechanism including tongs.

In more detail, the end of the side opposite to the position of the cell 10 in the first container 100 is gripped by the mechanism for gripping the first container 100 so that it can be moved to a location desired by the user. .

To this end, as the mechanism for holding the first container 100, forceps may be applied, preferably tweezers may be applied, and most preferably, medical forceps may be applied, but the present invention is not limited thereto. to be.

<Experimental Example 1>

1) Mouse embryo culture

1-1) 7.5 IU of PMSG (pregnant mare serum gonadotropin) was injected into 6-week-old B6D2 F1 mice, and after 48 hours, 7.5 IU of hCG was injected to induce superovulation.

1-2) In order to induce in vivo fertilization, male mice of the same strain were mated.

1-3) After 20 hours of hCG injection, female mice's plugs were inspected to determine whether mating occurred, and the mice were slaughtered by dislocating the cervical spine.

1-4) Only the ovaries were removed from the slaughtered mice, and then transferred to a dish containing MTF (mouse tubal fluid) culture solution.

1-5) Under a dissecting microscope, a syringe containing the MTF culture medium maintained at 37° C. was put at one end of the fallopian tube, and the embryo was induced to come out to the other end while carefully inserting the culture solution into the fallopian tube.

1-6) The cumulus cell mass was placed in a culture medium containing hyaluronidase and the cumulus cell was removed.

1-7) The mouse embryos were recovered, washed twice with fresh culture medium, and then cultured in an incubator.

1-8) About 100 embryos each were vitrified and frozen by dividing 6-8 cell stage embryos obtained by culturing in vitro for 48 hours into 3 types of cryopreservation containers (EM grid, cryotop, TPS).

At this time, the storage container for vitrification freezing according to the present invention was applied to the TPS.

1-9) The blastocyst embryos obtained by culturing in vitro for 96 hours were divided into three types of cryopreservation containers (EM grid, Cryotop, TPS), and about 100 embryos each were vitrified and frozen.

1-10) The shelled or shelled blastocyst embryos cultured for more than 120 hours were divided into two types of cryopreservation containers (EM grid, TPS), and about 80 each were vitrified and frozen.

2) Freezing mouse embryos

2-1) Vitrification solution 1 formed of 7.5% ethylene glycol, 7.5% DMSO (dimethyl sulfoxide) and 20% SSS (serum substitute supplement) in D-PBS was prepared.

2-2) Vitrification solution 2 formed with 15% ethylene glycol, 15% DMSO (Dimethyl sulfoxide), 10μg/ml Ficoll, 0.65M sucrose, 20% SSS (serum substitute supplement) in D-PBS was prepared.

2-3) After allowing the embryo to stand in vitrification solution 1 for 2 minutes and then in vitrification solution 2 for 45 seconds, the embryos exposed to vitrification solution 2 were quickly transferred to an EM grid, cryotop, or TPS.

3) Thaw mouse embryos

3-1) A thawing solution 1 (warming solution 1) formed of 0.25M sucrose and 20% SSS in D-PBS was prepared.

3-2) A thawing solution 2 (warming solution 2) formed of 0.125M sucrose and 20% SSS in D-PBS was prepared.

3-3) Place the EM grid, Cryotop, and TPS in thawing solution 1 for 2 minutes.

3-4) It was allowed to stand in thawing solution 2 for 3 minutes.

3-5) After washing 3 times, cultured.

4) Cell death test after freezing and thawing of mouse embryos

4-1) Mouse embryos were placed in 4% paraformaldehyde at room temperature for 30 minutes.

4-2) Washed 3 times with 0.3% polyvinylalcohol.

4-3) After permeating 0.1% Triton X-100 at 4°C for 10 minutes, it was washed three times with 0.3% polyvinylalcohol.

4-4) Incubated with TUNEL reaction solution at 37°C for 1 hour in a dark place.

4-5) The slide was fixed with 4,6-diamidino-2-phenyldole.

4-6) A slide cover was put on and observed with a fluorescence microscope, and the total number of cells was observed in blue and dead cells were observed in green.

As shown in FIG. 11, when metaphase embryos were frozen and then thawed using EM grid, cryotop, and TPS, survival rates of embryos (Survived embryos), blastocysts (produced blastocysts) and apoptotic cells No significant difference was observed between the three groups.

At this time, the values shown in FIG. 11 were expressed as numbers (%) or mean±SD, and it was determined that there was no significant difference between values with other superscripts in the column at ρ>0.05.

Moreover, Survived embryos, produced blastocysts and hatching-stage blastocysts are values by chi-square test, respectively, and No. of total cells, No. of apoptotic cells is a value by analysis of variance (Tukey's test).

In addition, the produced blastocysts were cultured in vitro for 48 hours after warming, and the rate at which they reached the blastocyst stage was evaluated.

Moreover, hatching-stage blastocysts were cultured in vitro for 72 hours after warming and the hatchability of embryos was evaluated.

In addition, as shown in FIG. 12, when thawed after freezing using hatching mouse blastocysts, there was no significant difference in re-expanded blastocysts and hatched blastocysts between TPS and EM grids.

However, the survival rate of embryos was significantly higher than that of the EM grid when TPS was used (ρ<0.05).

At this time, the value shown in FIG. 12 is expressed as a number (%), the re-expanded blastocysts are the evaluation of the re-expanding rate of embryos after culturing the embryos in vitro for 3 hours after warming, and the Hatched blastocysts are 24 embryos after warming. The rate of incubation in vitro over time and reaching the hatching stage was evaluated.

Moreover, as shown in FIG. 13, when fully hatched blastocysts were frozen and then thawed, no difference in survival rates between TPS and EM grids was observed, but the re-expanded blastocysts were the same when using TPS. It was observed to be significantly higher than when the EM grid was used (ρ<0.05).

At this time, the value shown in FIG. 13 is expressed as a number (%), and the re-expanded blastocysts evaluate the re-expanding rate of the embryo by culturing the embryo in vitro for 3 hours after warming.

Next, as shown in FIG. 14 , when thawing mouse blastocyst embryos after freezing, the survival rates of cryotop and TPS were significantly higher than when EM grid was used when surviving embryos between EM grid, Cryotop, and TPS were examined. appeared high.

At this time, the difference in the re-expanded blastocysts of the surviving mouse blastocysts was not significantly different between the three groups.

However, the difference between the number of hatching-stage blastocysts, Total No. of cells, and No. of apoptotic cells that reached the hatching stage after re-expansion was observed using the EM grid. A significantly lower one could be observed (ρ<0.05).

Therefore, it was found that TPS showed a higher survival rate of embryos during freezing and thawing than the conventional EM grid, and the tendency for embryos to continue to develop after thawing was high, and the number of cells killed after thawing was also low.

In other words, TPS shows much better results than the EM grid that is used as a refrigeration carrier, and it has enough value as a new refrigeration carrier in that it shows the same results as Cryotop imported and used overseas. could infer.

At this time, the values shown in FIG. 14 are expressed as numbers (%) or mean±SD, and Survived embryos, produced blastocysts and hatching-stage blastocysts are values by chi-square test, respectively, and No. of total cells, No. of apoptotic cells is a value by analysis of variance (Tukey's test).

Moreover, No. (%) of re-expanded blastocysts was evaluated by evaluating the rate of re-expanded embryos after culturing the embryos in vitro for 3 hours after warming. (%) of hatching-stage blastocysts is an evaluation of the hatching rate of embryos after warming and culturing them in vitro for 24 hours.

<Experimental Example 2>

1) Freezing and thawing human metaphase or blastocyst embryos

1-1) vitrification basic media is formed of PBS, 20% SSS, vitrification solution 1 is formed of 20% ethylene glycol, vitrification basic media, vitrification solution 2 is formed of 40% ethylene glycol, 18% Ficoll, 0.3M sucrose, A culture medium for freezing formed of vitrification basic media was prepared.

1-2) Thawing solution 1 was prepared with vitrification basic media, and thawing solution 2 was prepared with 0.5M sucrose and vitrification basic media for thawing.

2) Human metaphase or blastocyst embryo vitrification freezing process

2-1) The embryos to be frozen were taken out of the incubator and observed under a microscope to determine and record the number of frozen embryos, the quality of the embryos, and the number of times of freezing.

2-2) The blastocysts to be frozen were removed from the incubator and shrunk using a 29-G insulin syringe, and this process was omitted for metaphase embryos.

2-3) After checking whether the shrink is complete, the culture dish containing the embryo to be frozen was moved to the embryo freezer.

2-4) The embryos were transferred to vitrification basic media and left at room temperature for 30 seconds.

2-5) Transfer the embryo to Vitrification solution 1 and let it stand at room temperature for 50 seconds.

2-6) Prepare EM-grid or TPS and immerse cane and cryovial in liquid nitrogen.

2-7) After transferring the embryo to vitrification solution 2, it was allowed to stand at room temperature for 20 seconds.

2-8) After the treatment with Vitrification solution 2 was completed, the embryos were placed on a TPS or EM grid under a microscope.

2-9) The TPS or EM grid was placed in a cryovial pre-cooled in liquid nitrogen and stored in a cryopreservation tank (LN2 tank).

3) Thaw human metaphase or blastocyst embryos

3-1) Each 0.5ml of the thawing culture solution was put into a thawing dish.

3-2) The cane and cryovial were taken out from the liquid nitrogen container in which the embryos to be thawed were stored and placed in a nitrogen tank for transport.

3-3) Take out the TPS or EM grid in the cryovial, immerse it in thawing solution 2, and let it stand at room temperature for 5 minutes.

3-4) The embryos were transferred to thawing solution 1 and left at room temperature for 5 minutes.

3-5) After thawing, the embryos were transferred to a culture dish, washed several times, and then transferred to a culture dish for incubation.

3-6) Viability of thawed embryos and embryo quality were observed.

In this case, the culturing of the embryos, the culturing of the embryos, CO ₂ :O ₂ :N ₂ A ratio of 6:5:89 may be performed.

In addition, the metaphase embryos were frozen on the 3rd day after collection, and the quality of the embryos was evaluated according to the degree of fast or slow blastocyst division and the uniformity of the blastomeres based on the 8-cell stage.

Furthermore, blastocysts were graded into A, B, and C grades according to the degree of expansion, the quality of the inner cell mass, and the quality of the vegetative epithelial cell appearance.

Based on normal ovulation patients, 10 mg of estrogen was administered 2 weeks after menstruation, and progesterone was administered when the endometrium was 8 mm or more thick, and then embryo transplantation was performed.

Pregnancy was confirmed by measuring serum hCG concentration 14 days after embryo transfer.

As shown in FIG. 15 , when freezing and thawing of metaphase embryos and blastocysts using TPS and EM grids, respectively, the survival rate of embryos and the average number of transplanted embryos did not differ significantly.

However, when freezing and thawing were performed using TPS and EM grid, the clinical pregnancy rate was higher when using TPS in metaphase embryos and blastocysts than when using EM grid.

As such, in the present invention, devices or containers for cryopreservation, such as an EM grid, which are conventionally applied for cryopreservation of cells, are formed of metal, so that the cells 10 are attached to the metal when the cells 10 are frozen or thawed. Accordingly, there is an advantage of minimizing the disadvantage that the cells 10 die after thawing and not using an instrument that is not suitable for the intended use.

Furthermore, since the portion capable of accommodating the cells 10 in the first container 100 is formed larger than the conventionally applied EM-grid, there is an advantage that the loss of cells due to the user or external stimuli can be significantly reduced.

<Example 2>

2 and 3 , the first container 100 may further include a handling unit 110 having an end bent in an upward direction.

Preferably, the handling unit 110 may be bent upwardly at the end of the side opposite to the position where the cell 10 is accommodated.

Since the handling unit 110 is formed, the operator can easily determine a position in which the cells 10 can be accommodated.

Furthermore, since the handling part 110 is bent upward with respect to the bottom surface 20, the first container 100 is not contaminated by the tongs for holding the first container 100 and the cleanliness is maintained. At the same time, there is an advantage of easy gripping.

To this end, the handling unit 110 may be bent by θ with respect to the bottom surface 20 .

At this time, θ may be formed at an angle of 1 to 90° with respect to the bottom surface 20 , preferably at an angle of 10 to 45°.

If θ is formed to be 1° or less with respect to the bottom surface 20, it is not possible to easily grip the handling unit 110 with forceps due to a low angle, and it is confused with the position where the cells 10 are accommodated, so that the operator There is a disadvantage that can make an error of positioning the cells in the handling unit 110, and when it is formed at 90° or more, the operator places the cells 10 on the upper side of the first container 100, and then, by using forceps There is a disadvantage in that the cell 10 may be damaged when the handling unit 110 is gripped.

Optionally, a first display unit 111 may be formed at the end of the first container 100 opposite to the portion in which the cells 10 are accommodated for convenience of movement or use.

In more detail, the first display unit 111 is formed at the opposite end of the part where the cell 10 is accommodated, so that a part that the user can hold with forceps, etc. and a part where the cell 10 can be positioned are separated. It has the advantage of being able to identify it at a glance.

To this end, the first display unit 111 may be formed in a color contrasting from the first container 100 , but the present invention is not limited thereto.

For example, when the first container 100 is formed to be transparent, the first display unit 111 may be formed to be colored.

For this reason, when the first container 100 is lost due to the carelessness of the operator, there is an advantage that can be easily found by the first display unit 111 .

In addition, a bent handling part 110 is formed in the first container 100 so that the cells 10 can be easily stored and moved in a vial when freezing or thawing, so that the cells 10 are frozen. During the thawing operation, the handling unit 110 and the cell loading site are separated and marked, so that the possibility of cell 10 loss when stored in a vial is significantly reduced, and there is a convenient advantage when moving. .

<Example 3>

4, 5 and 16, the first container 100 is formed to extend in the longitudinal direction at a position adjacent to the cell 10, and includes a receiving part 200 having a groove formed therein. can be

More specifically, the accommodating part 200 is dug at a position adjacent to the cell 10 , or is recessed or penetrated to accommodate the preservation solution accommodated together with the cell 10 on the upper side of the first container 100 . can be

Preferably, as shown in FIG. 16 , the receiving part 200 is penetratingly formed in a position adjacent to the cell 10 so that the aqueous solution can be easily discharged or received to the outside, but is not limited thereto. Of course.

At this time, the preservative solution may be accommodated in the accommodating part 200 by capillary action.

For this reason, since the cells 10 and an appropriate amount of the preserving solution are present on the upper side of the first container 100 , there is an advantage in that it is possible to minimize the formation of ice crystals by the preserving solution during cryopreservation of the cells 10 .

That is, the surplus preservative solution removed by being accommodated by the cells 10 and the receiving unit 200 or discharged to the outside may be present on the upper side of the first container 100 .

To this end, the receiving unit 200 is formed to be spaced apart from the position where the cells 10 are accommodated, so that when the first vessel 100 and the cells 10 are frozen together, the preservation solution introduced into the receiving unit 200 is formed. It may be formed so as to prevent damage to the cells 10 by freezing.

Optionally, as shown in FIGS. 6 and 17 , an accommodating passage 210 extending in the longitudinal direction may be further included at the end of the accommodating part 200 .

In detail, the receiving passage 210 may be formed to extend from the end of the receiving unit 200 in a direction in which the cells 10 are located.

At this time, the receiving passage 210 is formed to be inclined upward from the end of the receiving part 200 , so that the cells 10 are positioned in the first container 100 and the preservation solution is accommodated by the inclination of the receiving passage 210 at the same time. It may be formed so as to stably flow into the part 200 .

To this end, the diameter of the end of the receiving path 210 adjacent to the cell 10 is 5 mm or less, and the end of the cell 10 facing the side may be the same as or smaller than the receiving unit 200 .

For this reason, the cell 10 is not accommodated in the receiving unit 200 due to the diameter of the receiving passage 210 and may be formed to selectively introduce the preservation solution into the receiving unit 200 .

At this time, the length of the receiving passage 210 is sufficiently extended in the longitudinal direction to selectively and sufficiently accommodate the preservative solution in the receiving portion 200, and when the first container 100 and the cells 10 are frozen together, the reception It may be formed to prevent damage to the cells 10 by freezing of the preservative solution introduced into the unit 200 .

<Example 4>

As shown in FIG. 7 , it may be formed to include the first absorption unit 300 formed at a position adjacent to the cell 10 of the first container 100 .

In more detail, as shown in FIG. 7 , the first absorption unit 300 is formed on the upper side of the first vessel 100 by being spaced apart from the cells 10 located on the upper side of the first vessel 100 by a predetermined distance. can

As the first absorption unit 300 is formed, the preservative solution located on the upper side of the first container 100 together with the cells 10 is absorbed into the first absorption unit 300, and the cells 10 and an appropriate amount of the preservative solution are prepared. 1 The container 100 is located on the upper side and the cryopreservation can be performed at the same time.

To this end, the first absorption part 300 may be formed to be detachable from the first container 100 .

In detail, the lower surface of the first absorbent part 300 is formed of an adhesive layer, so that after absorbing a certain amount of the preservative, the operator can simply peel and remove the first absorbent part 300 with tongs. can be formed.

Preferably, the first absorption part 300 may be formed to be smaller or larger than the width of the first container 100 so that it can be easily peeled off from the first container 100 by tongs, but is not limited thereto. Of course not.

For this reason, the cells 10 and an appropriate amount of the preservative solution are present on the upper side of the first container 100 , and at the same time, there is an advantage in that damage to the cells 10 can be minimized due to the formation of ice crystals in the preservative solution during cryopreservation.

The first absorbent unit 300 thus formed may be formed of fibers capable of absorbing a preservative solution including Whatman paper or Korean paper in order to easily absorb the preservative solution, but is not limited thereto.

Optionally, as shown in FIGS. 8 to 10 , a second absorption part 400 protruding outwardly below the end of the first container 100 may be included.

The second absorption unit 400 is formed to protrude outward from the lower end of the first container 100 , to extend the cells 10 to remove the surplus preservative solution formed after receiving the cells 10 , and to extend the second absorption unit 400 . ) has the advantage that the surplus preservative can be easily absorbed and removed by making it in contact with the end.

To this end, the second absorption unit 400 may be formed to extend from the lower side of the end of the first container 100 on the side in which the cells 10 are accommodated, in any one or more selected from the length or width direction.

In this case, an adhesive layer may be formed on at least one selected from the upper side of one or more ends of the second absorption unit 400 selected from one side or the other side or one or more selected from the lower side of the end of the first container 100 .

Accordingly, there is an advantage in that the second absorption unit 400 can be removed by a simple operation by simply peeling the second absorption unit 400 with tongs or the like.

Moreover, the second absorption part 400 may be formed to be equal to or larger than the width of the first container 100 so that it can be easily removed while being firmly attached to the lower side of the first container 100 .

If, as shown in FIG. 9 , when the second absorption unit 400 extends from the end of the first vessel 100 in the left or right direction, the first vessel 100 of the second absorption unit 400 is formed. ) and the end of the side to be adhered may be formed to be positioned on a straight line with the end of the first container 100, but is not limited thereto.

Moreover, as shown in FIG. 10 , the second absorption unit 400 may be formed to extend in length, left and right directions around the end of the first container 100 , but is not limited thereto.

In this case, the second absorbing part 400 may be formed of the same material as the first absorbing part 300 , but the present invention is not limited thereto.

As described above, since the second absorption unit 400 is formed, it is not only easy to quickly process by the second absorption unit 400 when the preservative solution escapes, which may be generated during the movement of the cells 10 , but also contains an appropriate amount of the preservative solution. The cells 10 are located on the upper side of the first container 100 to minimize the formation of ice crystals by the preservative, and there is an advantage in that the second absorption unit 400 can be peeled off and removed by a simple operation.

Furthermore, as described above, the first container 100 optionally further includes the accommodating part 200 , the first absorbing part 300 or the second absorbing part 400 to reduce the amount of vitrified freezing liquid as a preservative solution. There is an advantage in that the survival rate of the cells 10 can be improved by minimizing the ice-freezing of the cells 10 due to ice crystals while reducing the death or loss.

The present invention is an invention that can be used in industries such as cryopreservation of cells because the possibility of cell loss in the course of movement or storage of cells is significantly reduced by storing them in a vial together with cells.

Claims (10)

It includes a first container 100 formed in a plate shape to accommodate the cells 10 on the upper side,
The first container 100 includes a handling part 110 bent in an upward direction including a first display part 111 at an end opposite to the cell 10 so that it can be gripped by an instrument including forceps, ,
The first container 100 is formed to extend in the longitudinal direction at a position adjacent to the cell 10, and includes a receiving part 200 having a groove formed therein,
The accommodating part 200 is a vitrified container used for cryopreservation, characterized in that it is formed through the longitudinal direction from the end of the first container 100 so that the preservation solution can be discharged.
The method according to claim 1,
A vitrified container used for cryopreservation, characterized in that the length of the first container 100 is 50 mm or less, and the width is 0.5-5 mm.
The method according to claim 1,
The cell 10 is a vitrification container used for cryopreservation, characterized in that the germ cells including sperm, egg or embryo applied to the in vitro fertilization procedure.
The method according to claim 1,
The first container 100 is a vitrified container used for cryopreservation, characterized in that formed of a synthetic resin.
The method according to claim 1,
The first container 100 is used for cryopreservation, characterized in that it is formed of any one or more selected from PET (polyethyleneterephthalate), PCTFE (Poly-Chloro-Tri-Fluoro Ethylene), PTFE (Polytetrafluoroethylene), and PFA (perfluoroalkoxy). vitrified container.
The method according to claim 1,
A vitrified container used for cryopreservation, characterized in that it comprises a first absorption part (300) formed at a position adjacent to the cells (10) of the first container (100).
The method according to claim 1,
A vitrified container used for cryopreservation, characterized in that it comprises a second absorption part (400) formed to protrude outwardly at the lower end of the first container (100).
It includes a first container 100 formed in a plate shape to accommodate the cells 10 on the upper side,
The first container 100 includes a first absorption part 300 formed at a position adjacent to the cell 10 or a second absorption part formed to protrude outwardly below the end of the first container 100 so that the preservative solution can be absorbed. 400),
The first absorbent part 300 or the second absorbent part 400 is adhered from the first container 100 so that it is detachably formed of fibers including Whatman paper or Korean paper including an adhesive layer on the lower side. A vitrified container used for cryopreservation.
9. The method of claim 8,
The cell 10 is a vitrification container used for cryopreservation, characterized in that the germ cells including sperm, egg or embryo applied to the in vitro fertilization procedure.
9. The method of claim 8,
The first container 100 is a vitrified container used for cryopreservation, characterized in that formed of a synthetic resin.

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