KR100270929B1 - A freezing composite and a freezing method of immaturity yolk and thawing method - Google Patents

Abstract

PURPOSE: A freezing composition for immature follicular oocytes, and freezing and thawing method for immature follicular oocytes using the same are provided, which can freeze immature follicular oocytes effectively, and use the immature follicular oocytes effectively by thawing them if necessary. CONSTITUTION: The freezing method comprises the steps of: mixing tissue culture medium 199 with serum, adding ethylene glycol, ficoll, and trehalose, mixing, and preparing the freezing composition; putting immature follicular oocytes right after collecting that from ovary into the cryocomposition, equilibrating for 10min, putting the immature follicular oocytes into straw, and fixing; cooling down to -7deg.C, seeding, fixing, cooling down to -30deg.C, and soaking into liquid nitrogen for storage. The thawing method comprises the steps of: taking out the immature follicular oocytes from liquid nitrogen by straw, exposing to air, and shaking in 30deg.C water bath; cutting the straw, putting the immature follicular oocytes into a solution composed of tissue culture medium 199 and serum, and thawing by reabsorbing moisture for 10min.

Description

Freezing Composition of Immature Oocytes and Freezing and Thawing Methods of Immature Oocytes

The present invention relates to a freezing composition of immature follicles, and to a method of freezing and thawing immature follicles using the same, more specifically, to preserve the magnetic reproductive cells such as livestock, conventional livestock, rare livestocks having high ability genetic factors The present invention relates to a freezing composition of immature follicle eggs and the method of freezing and thawing immature follicle eggs using the same.

At present, the world needs to study the useful genetic preservation of the country, such as the Convention on Biological Diversity, and the preservation of germ cells of cancer livestock that can be born as an individual can be achieved by conventional livestock, rare animals and old animals. The need for this has been known to be of great value because the eggs of the oxen can be preserved and used when needed.

However, the preservation of immature follicles by freezing has been known to be impossible. Recently, although there have been some successful reports on the preservation of immature follicles, they have been mainly made only for experimental animals such as mice and rabbits. It is rarely done for.

The preservation by fertilization of the fertilized egg was frozen by using 1.8M Ethylene Glycol as the cryoprotectant, but after freezing it in the egg and melting it, the survival rate was less than 44%. .

This is because the structure and physiology of the fertilized egg and the unfertilized egg are different. Generally, the fertilized egg has a stable nuclear nucleus, and thus the development rate after freezing and thawing is high.

However, since the fertilized egg has an unstabilized nucleus and cumulus cells are surrounded by the egg, it should be taken into account when freezing and thawing. The method is fundamentally different.

In recent years, the success rate has increased as the maturation, in vitro fertilization, and in vitro development methods of follicular eggs collected from bovine ovaries have increased.For example, 1.8M Ethylene Glycol, 5% Polyvinylpyrrolidone An example of a calf produced by freezing-thawing and transplanting bovine eggs using "PVP" and 0.05M Trehalose has been reported to the society.

However, in the case of using the above-mentioned freezing liquid, the survival rate was 73.6%, the split rate was 28.8%, and the incidence of transplantable fertilized eggs was only 11.4%.

The present invention is to overcome the above problems to provide a composition that can effectively cryopreserved follicle eggs, to effectively cryopreserve the follicle eggs using such a composition, and to effectively fuse the cryopreserved follicles.

In the present invention, 10% (1.8Mol) Ethylene glycol, 5% Ficoll and 1.7% (5% Serum) in a mixture of 5% serum (Serum) in a known culture medium used for the culture of Tissue Culture Medium 199 or Phosphate Buffer Slaine or fertilized eggs. 0.05M) Freezing composition by further mixing of Trehalose and freezing of the immature follicles can be effectively used by fusing frozen immature follicles as needed.

In the present invention, the freezing liquid is formed as a freezing liquid is prepared as follows.

First, Tissue Culture Medium 199 (hereinafter referred to as "TCM199") or Phosphate Buffer Slaine (PBS) is used as a basic solution. Known cultures may also be used.

5% serum, 10% (1.8Mol) Ethylene glycol, 5% Ficoll and 1.7% (0.05M) Trehalose are mixed in the base solution to form a freeze composition for freezing of follicular eggs.

The serum used in the cryogenic composition of the present invention has been used in the culture of fertilized eggs, which is used for buffering and preventing the effect of the egg on the straw during freezing thawing of the egg.

The above 10% (1.8Mol) Ethylene glycol, 5% Ficoll and 1.7% (0.05M) Trehalose is mixed. When the concentration is higher than this, the osmotic pressure and the temperature of the ice crystal are changed to affect the survival of the egg. There is a concern.

After forming a freeze composition in the present invention will be described in detail for the method of freezing the immature follicle using it.

First, immature follicles immediately after collection from the ovary are put into the above-mentioned freeze composition and allowed to equilibrate for 10 minutes.

At this time, moisture is dehydrated.

In this state, the immature oocytes are put into a 0.25 ml straw, which is commonly used, and left to stand at 0 ° C. for 2 minutes in a liquid nitrogen freezer. After 2 minutes, take Seeding.

Thereafter, the mixture is cooled down to -30 ° C at a rate of 0.3 to 0.5 ° C / min, immersed in liquid nitrogen, and stored.

In the freezing method of the present invention,

The immature follicles are placed in a 0.25 ml straw, which is normally used, for 2 minutes at 0 ° C. to increase the survival rate of the eggs by protecting the impact of the eggs due to the rapid cooling of the temperature.

In addition, after standing as described above, the temperature is lowered to -7 ° C at a rate of 1 ° C / min. Since this is a temperature close to the freezing point, when the temperature is lowered more rapidly, sudden ice crystals are formed, which causes internal and external immature follicles. Ice crystals are formed, resulting in destruction of cell membranes.

In addition, if the temperature is lowered to less than 1 ° C./min to -7 ° C., the immature follicle may survive due to the impact of the osmotic pressure from the freezing composition on the cell membrane and the internal microstructure of the immature follicle. There is a problem that there is no.

The temperature is lowered to -7 ℃ at the rate of 1 ℃ / min, and then cooled at -7 ℃ for 2 minutes. This is because the concentration of the frozen composition is relatively high. To give.

This is to forcibly form the ice in advance to avoid the synergistic effect of the temperature in the process of changing the liquid into solid ice at the temperature at which the ice is formed (about -10 ° C) when the temperature falls below -7 ° C.

After standing still at -7 ° C for 8 minutes after cooling, cooling down to -30 ° C at a rate of 0.3 ° C / min and immersing in liquid nitrogen is below a rate of 0.3 to 0.5 ° C / min up to -30 ° C or When cooled down to a higher speed and immersed in liquid nitrogen, the survival of immature follicles is greatly affected, and survival after fusion becomes opaque, which affects survival by prolonged exposure time to the impact of osmotic pressure.

After cryopreservation of immature follicles by such a method, the thaw should be thawed when it is used, and the thawing method according to the present invention will be described in detail.

As described above, the immature follicles in the cryopreservation state are taken out of the liquid nitrogen with a straw and exposed to air for 5 to 10 seconds by the freezing method of the present invention.

After such exposure, shake for 30 seconds in hot water at 30 ° C, melt, and then cut the straw, and put immature oocytes into the composition solution composed of TCM 199 and 5% serum to induce moisture resorption for 10 minutes. .

After this melting process, the cultures are taken and used for their intended purpose.

In the present invention, the immature follicles are removed from the liquid nitrogen and exposed to air for 5 seconds to blow off the liquid order attached to the outside of the straw, and the straw is broken or broken when the straw is immediately put in hot water at 30 ° C. This is to prevent this.

Shake for 10 seconds in hot water at 30 ℃ to minimize the exposure of eggs to high concentrations of freeze composition. If melted for more than 10 seconds, 10 seconds is most suitable because of detrimental effects on egg survival. .

According to the present invention, the immature follicle was frozen and frozen after freezing based on the freeze composition according to the present invention to compare the survival rate, egg split rate and development rate of the implantable embryo.

Comparative Example 1

Comparison of survival rate of eggs

comparison target Egg survival rate (%) The present invention 78.1 EFS 60.3 EPT 73.6 EPS 74.7

* EFS: Freeze composition of 1.8M Ethylene glycol, 5% Ficoll and 0.05M Sucrose.

* EPT: A freeze composition of 1.8M Ethylene glycol, 5% PVP and 0.05M Trehalose.

* EPS: Frozen composition of 1.8M Ethylene glycol, 5% PVP and 0.05M Sucrose.

Comparative Example 2

Dividing rate

comparison target Dividing rate (%) The present invention 72.6 EFS 21.13 EPT 28.8 EPS 17.1

* EFS: Freeze composition of 1.8M Ethylene glycol, 5% Ficoll and 0.05M Sucrose.

* EPT: A freeze composition of 1.8M Ethylene glycol, 5% PVP and 0.05M Trehalose.

* EPS: Frozen composition of 1.8M Ethylene glycol, 5% PVP and 0.05M Sucrose.

Comparative Example 3

Development Rate of Implantable Embryos

comparison target Development rate of implantable fertilized eggs (%) The present invention 8.8 EFS 13.3 EPT 11.4 EPS 5.3

* EFS: Freeze composition of 1.8M Ethylene glycol, 5% Ficoll and 0.05M Sucrose.

* EPT: A freeze composition of 1.8M Ethylene glycol, 5% PVP and 0.05M Trehalose.

* EPS: Frozen composition of 1.8M Ethylene glycol, 5% PVP and 0.05M Sucrose.

As described above, it can be seen that the freezing and thawing method of the follicular egg according to the present invention has a remarkable effect on the survival rate, egg split rate and development rate of the implantable fertilized egg.

As described in detail above, the present invention can effectively cryopremature immature follicles, thereby effectively preserving conventional livestock, rare animals and high-potency eggs to create very useful effects that can be used when needed. do.

Claims (3)

In preparing a freeze composition by mixing 5% serum in Tissue Culture Medium 199 or Phosphate Buffer Slaine or a known culture medium used for the culture of fertilized eggs as a basic solution, 10% (1.8Mol) Ethylene glycol, 5% Ficoll and 1.7% (0.05M) Trehalose in a mixture of 5% Serum with Tissue Culture Medium 199 or Phosphate Buffer Slaine or a known culture medium used for culturing fertilized eggs. Cryocomposition of immature follicles characterized in that the addition is made by mixing. 10% (1.8Mol) Ethylene glycol, 5% Ficoll and 1.7% (0.05M) Trehalose were mixed with 5% serum in Tissue Culture Medium 199 or Phosphate Buffer Slaine or a known culture medium used for cultivating fertilized eggs. After adding the immature follicles immediately after collection from the ovary to equilibrate for 10 minutes in the frozen composition obtained by further mixing, put the immature follicles in a 0.25ml straw, which is commonly used, and leave them at 0 ° C for 2 minutes in a liquid nitrogen freezer. After descending at a rate of 1 ° C / min to -7 ° C, and then take ice after 2 minutes at -7 ° C. A further method of freezing immature follicular eggs, which is further cooled for 8 minutes and then cooled down to −30 ° C. at a rate of 0.3˜0.5 ° C./min to be immersed in liquid nitrogen for storage. The immature follicle in the cryopreservation state is taken out of the liquid nitrogen with a straw by the freezing method of claim 2 and exposed in the air for 5 to 10 seconds. After exposure, it is melted by shaking for 30 seconds in warm water at 30 ° C. A method of fusion of immature follicles characterized in that the immature follicles are melted in a composition solution composed of Tissue Culture Medium 199 and 5% serum to induce water resorption for 10 minutes.