KR101509295B1 - Paste an embryo in the vitrification straw - Google Patents

Abstract

The present invention relates to a straw vitrification method, and more particularly, to a straw vitrification method in which frozen-thawed embryos are cultured in a paste an embryo in the vitrification straw (pV) which attaches germ cells to the inner wall of the straw, ≪ / RTI > The method of the present invention is useful not only by increasing the freezing rate by reducing the loading volume, but also by directly transferring the vitrified frozen embryo to the recipient after thawing without further experimentation.

Description

Background Art [0002] Paste an embryo in the vitrification straw, which attaches an embryo to an inner wall of a straw,

The present invention relates to a straw vitrification method, and more particularly, to a method of freezing preservation of germ cells by pasteurized embryo in vitrification straw (pV) which attaches germ cells to the inner wall of a straw .

Cryopreservation is a process in which biological materials such as cells, tissues, and individuals are kept at low temperature for a long period of time without morphological and functional changes, and are melted to restore their original function.

This cryopreservation has developed in close association with the life of mankind and has become increasingly important in recent years, coupled with the remarkable development of reproductive biology and assisted reproductive technology (ART) in humans and animals It is getting bigger. In the early days, the freezing technique, which was only available for small sperm or embryos, is expanding its indications with the storage of the largest cells, oocytes and tissues.

For the cryopreservation of embryos and oocytes, which are relatively large in size and high in water content compared to normal somatic cells, the researchers first introduced a general slow cooling-rapid thawing method. The gentle freezing method first equilibrates by replacing a part of water in the cytoplasm with a permeable cryoprotectant. Thereafter, the cells are placed in a cell freezer to slowly freeze the moisture outside the cell. The osmotic pressure is increased by increasing the concentration of the freezing inhibitor outside the cell, and the water remaining in the cells is slowly removed using this force. It is a technique to minimize the generation of crystals. Slow freezing is relatively simple and easy to use, but it has disadvantages such as relatively expensive equipment, a large amount of liquid nitrogen, a lot of time, and freezing damage caused by ice crystals.

The most common cause of cell damage during freezing is mechanical damage caused by freeze formation during freezing. Therefore, to overcome these damages, many scholars have attempted various studies on the freezing method, freezing rate, selection and melting method of freezing protection, and recently introduced vitrification freezing method in which no ice crystals are formed. Studies have shown that it is essential to reduce the exposure time or increase the cooling rate of high-concentration freeze inhibitors compared to conventional somatic cells for vitrification of relatively large, high-moisture cells such as embryos and oocytes.

Therefore, the researchers first tried to reduce the toxicity to the cells by introducing freezing inhibitors with high permeability and low toxicity, or by combining absolute permeability and nonpermeable freezing inhibitor with absolute concentration without changing the relative concentration, . Second, we tried to develop various methods to reduce the volume of freezing inhibitor and freezing inhibitor to increase the cooling rate.

1. AGCA, Y., MONSON, R.L., NORTHEY, D. L., PESCHEL, D. E., SCHAEFER, D.M. and RUTLEDGE, J. J., 1998. Normal calves from transfer of biopsied, sexed and vitrified IVP bovine embryos. Theriogenology, 50 (1), pp. 129-145. 2. AKIYAMA, K., KOBAYASHI, J., SATO, Y., SATA, R., OHASHI, M., SASAKI, E., ODA, Y., OGAWA, Y., UEDA, S., NABENISHI, H and MATOBA, S., 2010. Calf production from vitrified bovine sexed embryos following in-straw dilution. Animal science journal = Nihon chikusan Gakkaiho, 81 (4), pp. 461-466.

It is an object of the present invention to provide a paste an embryo in the vitrification straw (pV) which is easy to perform even when the survival rate of germ cells after fusion is high.

Another object of the present invention is to provide frozen frozen germ cells and their uses by the above-mentioned sticky straw vitrification freezing method.

In order to solve the above-mentioned problems, the present invention provides a method for preventing germ cells using paste-embryo in the vitrification straw (pV) method which attaches germ cells to the inner wall of a straw while using straw vitrification Thereby providing a vitrification freezing method.

As a subject, there is no limitation as long as it is a biomaterial which requires cryopreservation. Preferably, the germ cell is an oocyte, an in vitro fertilized egg or an embryo cell, more preferably a blastocyst. Seven days old blastocysts were used in one embodiment of the present invention.

In a preferred embodiment, it may include forming perforations in the germ cells prior to the vitrification, and the effect of enhancing the hatchability of the embryo or the like through such perforation may be obtained.

The paste an embryo in the vitrification straw (pV) method of the present invention is characterized by using a vitrification-free composition containing the following components as main components:

Holding medium (HM: TCM199 supplemented with 25 mM Hepes and 20% FBS),

Vitrification solution 1 (VS1: HM + 10% ethylene glycol),

Vitrification solution 2 (VS2: HM + 35% ethylene glycol + 5% PVP + 0.5 M sucrose),

Dilution solution 1 (DS1: HM + 0.3 M sucrose), and

Dilution solution 2 (DS2: HM + 0.15 M sucrose).

Therefore, the above-mentioned adhesive sticking vitrification freezing composition for practicing the present invention can also be provided as the present invention.

On the other hand, the present invention provides vitrified frozen germ cells and their various uses by the above method.

The vitrified frozen germ cells exhibit excellent survival rates and pregnancy rates even after direct transplant without further processing after thawing. Accordingly, the present invention provides a germ cell transplantation method characterized in that the vitrified frozen germ cells are directly transplanted to a mammal other than a human, without further processing after thawing.

In particular, it can be used effectively in cattle management such as cattle, and can be used to increase the production of progeny with desirable characteristics.

As described above, the use of the sticky straw-freezing method of attaching germ cells to the inner wall of the straw of the present invention exerts almost the same effect as that of using a fresh embryo, so that the embryo can be efficiently used.

The paste an embryo in the vitrification straw (pV) method which attaches germ cells to the inner wall of the straw according to the present invention not only increases the freezing rate by reducing the loading volume, Thereby improving the possibility of survival after dissolution. Therefore, the vitrified frozen embryo according to the present invention is highly useful for storage and utilization of germ cells, since it is directly implanted into the recipient body (surrogate mother) after fusion without further experimental process and exhibits a high effect.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram of a paste an embryo in the vitrification straw (pV) of the present invention.
2 is a diagram of a conventional column-type vitrification straw (cV).
3 is a photomicrograph of whole cells and dead cells determined by TUNEL staining.

The term "cryopreservation" refers to cryopreservation of a biological material.

The term "freezing material" refers to any material capable of causing the biomaterials to be vitrified. In theory, any freezing medium that is sufficiently cold can be used since the sample is not in direct contact with the freezing medium. Suitable materials include, but are not limited to, liquid gases such as liquid nitrogen, liquid propane, liquid helium, ethane, and the like. In one embodiment of the present invention, liquid nitrogen was used.

The term "directly" means that the biological material is "directly" or "in contact " with the frozen material, for example when it comes into contact with the surface of the biological material, Quot; directly "means exposed.

The term "embryo" refers to embryos up to three months after the start of fertilization, when the organ and system of the organism begin to develop.

The term " blastocyst "refers to a preimplantation embryo composed of a ball or a ball of cells having an outer cell layer, a liquid filled steel and an inner cell mass.

The term "medium" means a suitable medium capable of maintaining the cells of the present invention. Refers to a composition capable of proliferating a cell, maintaining a normal karyotype, and maintaining pluripotency. A variety of media are known in the art.

"About" means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words "comprises" and "comprising ", unless the context requires otherwise, include the steps or components, or groups of steps or elements, Steps, or groups of elements are not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method of straw vitrification. More particularly, the present invention relates to a method for freezing germ cell vitrification using paste an embryo in the vitrification straw (pV) method, which is characterized by attaching germ cells to the inner wall of the straw.

In the vitrification method, many factors such as the type of frozen solution, the exposure temperature of the frozen solution, the thermal conductivity of the storage container, the amount of frozen solution, the timing and quality of embryo development, etc., .

The inventors of the present invention have found that, instead of the conventional column-forming vitrification straw (cV) method for forming a column, a paste an embryo in the vitrification straw which is characterized by attaching a reproductive cell to the inner wall of the straw , pV) method, an excellent effect of improving the cooling rate and improving the survival rate after freezing-thawing of the embryo was confirmed.

object

There is no limitation to the biological material which requires cryopreservation as the object of the present invention, but the biomaterial is a living cell or a cell, particularly a cell derived from a germ cell or a germ cell. The cell may be in any developmental stage. Preferably the cell is a mammalian source such as, but not limited to, a human, non-human primate such as a monkey, a laboratory animal such as a rat, a mouse and a hamster, an agricultural livestock such as a pig, , Cattle, goats, and horses.

The present invention can be used for cryopreserving spermatozoa, oocytes, embryos and other reproductive cells, more preferably eggs, embryos and embryos.

Although the method of the present invention can be used regardless of what stage they are in, the "blastocyst" of the embryo has a large number of cells and a small size. Therefore, the embryo of the entire nucleus, The survival rate after cryopreservation is higher and the development potential is higher, it is most preferable to be a cryopreservation target. The use of blastocysts also has the advantage of maintaining adequate pregnancy rates and reducing multiple pregnancies when a small number of embryos are transplanted after thawing. Seven days old blastocyst cells were used in one embodiment of the present invention.

Cultivation for the development of an embryo can be carried out by a person skilled in the art with reference to known methods.

The possible period may be from 10 seconds to 20 minutes, since at this stage the point of view is to promote equilibration with the solution and allow the freeze protection to be applied sufficiently, but in the presence of DMSO the cells may be in a somewhat toxic DMSO It should not be exposed too long.

Typically from about 10 seconds to about 15 minutes, from about 15 seconds to about 10 minutes, from about 20 seconds to about 7.5 minutes, from about 30 seconds to about 5 minutes, from about 40 seconds to about 10 minutes, From about 50 seconds to about 3 minutes, from about 30 seconds to about 2 minutes, from about 45 seconds to about 1.5 minutes, or from about 1 minute to about 37 < 0 > C.

The number of oocytes and embryos in mammals, including humans, is very low and because of their embryological importance, the need for cryopreservation is higher than any other cell. In other words, successful cryopreservation of human embryos and embryos can be used not only as an important means of preserving fertility, but also in controlling the number of embryos produced and transplanted, thereby contributing to the resolution of ethical or legal problems.

In the present invention, it is preferable that punctures are formed on the germ cells before vitrification and freezing.

Preferably, the germ cell can be an embryo, more preferably a blastocyst, and the perforation can be appropriately used in the art. In one embodiment of the present invention, perforations were formed by collapsing the bag with a fine pasteur pipette. By forming perforations before vitrification, hatching of the germ cells and developmental competence of the embryos can be increased.

Vitrification  Freezing

Eggs, and embryos are bulkier than ordinary cells and have a very high moisture content. Therefore, in order to effectively cryopreserve them, a cryopreservation technique that minimizes ice crystals is required. In this respect, a vitrification method is introduced.

Freezing of vitrification induces cryopreservation by directly exposing cells to liquid nitrogen (LN ₂ ) at -196 ° C. However, in reality, ice crystals are not formed in the cell during freezing, and moisture is amorphous As shown in FIG. This state is referred to as " vitrification ", and it is necessary for the intracellular water to be replaced with a high concentration of freezing inhibitor, and a very high cooling rate (more than 3,000 DEG C per minute) is required. The advantage of vitrification is that it is simple to operate and does not require expensive equipments, and most of all it is typical that ice crystals are not originally formed and injuries can be minimized

The method of the present invention basically relates to such a vitrification freezing method, among which the Straw method is used, so that the usual processes necessary for the freezing process are naturally included. For example, the method includes sealing the straw and supporting it on liquid nitrogen (LN ₂ ). Hereinafter, the features of the present invention, which are distinguished from the conventional methods, will be mainly described.

(i) a composition for vitrification and freezing

The vitrified composition of the present invention may contain one or more cryoprotectants or a mixture of cryoprotectants.

Of course, non-toxic cryoprotectants are preferred. Cryoprotectants help to minimize shrinkage by reducing the mole fraction of other solutes remaining in the unfrozen water. This suppresses the formation of crystalline ice, thus lowering the freezing point of water. The above can also prevent protein denaturation by hydrogen bonding with the binding water. As the cell cools, the solvent water turns into extracellular ice, and the increasing extracellular concentration of the impermeable electrolyte or non-electrolyte damages the cell.

When cells are treated with a cryoprotectant, the salt concentration of untreated cells is not reached until a much lower temperature is reached. The chemical reaction proceeds very slowly at such a low temperature, and consequently, cell damage is minimized.

Usually, a combination of cryoprotectants is used, because there may be differences between the different types. The cryoprotectants may also act as an osmotic agent.

Suitable cryoprotectants may be selected from the group consisting of ethylene glycol, propylene glycol, dimethylsulfoxide, glycerol, propanediol, sugars, such as sucrose, trehalose, maltose, lactose and methylpentanediol.

In particular, the composition used in the vitrification freeze used in the present invention preferably comprises the following components:

Holding medium (HM: TCM199 supplemented with 25 mM Hepes and 20% FBS),

Vitrification solution 1 (VS1: HM + 10% ethylene glycol),

Vitrification solution 2 (VS2: HM + 35% ethylene glycol + 5% PVP + 0.5 M sucrose),

Dilution solution 1 (DS1: HM + 0.3 M sucrose), and

Dilution solution 2 (DS2: HM + 0.15 M sucrose).

The vitrification-free composition of the present invention comprises two types of vitrification freezing solutions (VS), wherein VS1 and VS2 are such that the concentration of the individual agonists contained in the VS2 solution is typically between 5 and 50% v / v, For example, from about 5% to about 40% v / v, such as from about 5% to about 25% v / v (VSl solution) and from about 5% to about 30% v / v (VS2 solution). Typically, the total concentration of the cryoprotectant in the VS2 solution (calculated as v / v, w / v or M) is greater than the concentration in the VS 1 solution. The VS1 and VS2 solutions may contain one or more cryoprotectants that are the same or different. The concentration of one or more cryoprotectants in the VS1 and VS2 solutions may be the same or different and typically the total concentration of the cryoprotectant in the VS2 solution is greater than the concentration in the VS1 solution.

In one embodiment of the invention, sucrose may be used as a diluent. The concentration of sucrose contained in the vitrified frozen composition is typically from about 0.02 M to about 1 M, such as from about 0.05 M to about 0.9 M, from about 0.1 M to about 0.8 M, from about 0.2 M to about 0.7 M, From about 0.3 M to about 0.65 M, from about 0.4 M to about 0.6 M, from about 0.45 M to about 0.55 M.

Sucrose is a unique natural disaccharide, found in hundreds of plants and animals. Sucrose is an important energy source and a key factor in the stabilization of organisms during the freezing season. Sucrose has been shown to lower the phase transition temperature of the cell membrane so that the cell membrane can remain in liquid state even during drying. This is presumed to prevent cell membrane leakage during rehydration and preserve cell growth potential. Regarding proteins, sucrose inhibits protein denaturation by water exclusion from the protein surface when the cells are hydrated.

The composition for vitrification of the present invention is most preferably composed of the composition described above, but the concentration of the composition can be suitably adjusted by those skilled in the art as needed.

In addition, optionally, the vitrified composition for freezing may comprise one or more viscosity modifiers.

As viscosity adjusting agents, there may be selected from the group consisting of Ficoll, Percoll, Hyaluronic Acid, Albumin, Polyvinylpyrrolidone, Alginic acid, Gelatin and Glycerol and the like. The concentration of the one or more viscosity modifiers may be the same or different

(ii) freezing process

The germ cells to be frozen and the mixture of the vitrified composition for freezing are loaded into a straw for freezing. At this time, using a glass pipette (Pasteur, VWR International) or the like, the mixture is pasted on the surface of the inner wall of the straw without forming a column as conventionally. These differences can be seen also in Figs. 1 and 2.

Because of this characteristic of attaching reproductive cells to the inner wall of the straw, the present invention was named "paste an embryo in the vitrification straw (pV)" method. Then, the straw is sealed and carried on liquid nitrogen (LN ₂ ).

One of the factors affecting the viability of frozen embryos is the cooling rate. The method of the present invention can increase the freezing rate by reducing the loading volume of the vitrification solution on the straw. It also improves post-thaw survivability by reducing the recrystallization of the vitrification solution containing the embryo.

(iii) Removal of fusing and cryoprotectant

In order to utilize the vitrification-preserved embryo in the above method, a thawing procedure is required.

The thawing method consists of rapid thawing in which the embryo is taken out from LN ₂ and thawed at room temperature or 37 ° C. and embryos taken from LN ₂ are placed in a freezer adjusted to -80 ° C. to -100 ° C. and slowly melted slow thawing. In one embodiment of the present invention, a rapid melting method is used. During embryo melting process, the most damage occurs. Especially, it is easy to cause re-icing phenomenon when melting.

The melted embryo should remove the cryoprotectant introduced into the cell. Various methods are used to remove the intracellular cryoprotectant. Typically, a solution containing sugar such as sucrose is used to remove the cryoprotectant without cytotoxic damage of the melted embryo. That is, a method of removing the cryoprotectant in the embryonic cytoplasm while minimizing the damage of the cell membrane due to the rapid osmotic pressure change during the fusion process by preparing a sucrose solution containing various concentrations at the time of melting and treating the embryo at a high concentration to a low concentration Be used

In the present invention, the contents of the straw are merely melted, and the temperature may be room temperature to 40 ° C. Higher temperatures may cause thermal shock upon fusion of the cells if they are not rapidly removed from the water bath after thawing.

Use

In addition, the present invention includes germ cells frozen and vitrified by an adherent straw-freezing (pV) method in which germ cells are attached to the inner wall of the straw and the use thereof.

The method of the present invention not only increases the freezing rate by reducing the loading volume for the frozen straw to be vitrified comprising the embryo but also the vitrified frozen embryo which has undergone such a method can be used directly It also improves survival rate, post-thaw incidence, and pregnancy rate after transplantation. Therefore, it can be useful for processes such as artificial insemination ("AI") and in vitro fertilization ("IVF").

In one embodiment, the present invention includes a germ cell transplantation method, wherein the vitrified frozen germ cell is directly transplanted to a mammal other than a human, without further processing after thawing.

Mammals other than humans include, but are not limited to, non-human primates such as monkeys, laboratory animals such as rats, mice and hamsters, agricultural livestock such as pigs, sheep, .

That is, the vitrification freezing method of the present invention is an effective assisted reproductive technique that provides an opportunity for pregnancy to an infertile patient by efficiently using surplus embryos, and can be effectively used, for example, in animal management, Can be used to increase offspring production. In particular, this would allow producers of animals such as cows or edible wines to increase livestock production efficiency and quality.

<Examples>

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

The experimental process was approved by the Accreditation Committee of the Division of Animal Laboratory (ACDAL) of Gyeongsang National University and all compounds and media were purchased from Sigma Chemical Co. (St. Louis, Mo.).

The results are expressed as mean ± SD, and P <0.05 is considered statistically significant unless otherwise noted. Data were analyzed using one-way ANOVA (SPSS Inc., Chicago, IL). Significant differences between groups were detected using Duncan's multiple range tests.

< Example  1>

Animal preparation and embryo manufacturing

1-1. Animal preparation

Recipient cows were purchased from private farms. (Korean traditional endemic species) were synchronized using the GnRH-PGF2a-GnRH protocol (Momcilovic D, et al., 1998; Thatcher WW, Santos JE.

1-2. Embryo manufacturing

To produce bovine IVP blastocyst cells, ovaries were collected from local slaughterhouses and transported for 4 hours. In vitro embryo generation protocols are described in Jeong et al. 2009 document.

Briefly, cumulus-oocyte complexes (COC) were recovered from 2 to 8 mm diameter hair follicles using an 18 gauge needle attached to a vacuum pump. The eggs with 3-5 layers in dense cumulus cells and cytoplasm were suspended in TL-HEPES (114 mM sodium chloride, 3.2 mM potassium chloride, 2 mM sodium bicarbonate, 0.34 mM sodium phosphate, 10 mM sodium lactate, 0.5 mM magnesium chloride, , 2.0 mM CaCl 2, 10 mM HEPES, 1 μL / mL phenol red, 100 IU / mL penicillin, and 0.1 mg / mL of streptomycin) and finally in IVM (in vitro maturation) Containing TCM 199 supplemented with 700 μL of IVM medium [10% (vol / vol) FBS, 1 μg / mL estradiol-17β, 10 μg / mL FSH, 0.6 mM cysteine, and 0.2 mM sodium pyruvate] Transferred to a well of a 4-well dish.

After IVM, the male and female spermatozoa and COCs were thawed in a hot water bath at 36 ° C for 1 minute, and washed and then pelleted by 750 × g centrifugation in Dulbecco's PBS (D-PBS) at room temperature for 5 minutes. The pellet was carefully diluted in IVF (in vitro fertilization) medium (Tyrode's lactate solution supplemented with 6 mg / mL BSA, 22 μg / mL sodium pyruvate, 100 IU / mL penicillin, and 0.1 mg / mL streptomycin) They were incubated in a humidified atmosphere of 5% CO ₂ for 15 min to allow sperm to have fertility.

Sperm supposed to have fertility were diluted in IVF medium at a density of 1 × 10 ⁶ spermatozoa / mL and matured oocytes were transferred to IVF medium (700 μL) containing sperm for 18-20 hours.

The cumulus cells were removed by pipetting from a TL-HEPES in a 35-mm dish (SPL Life Sciences Co. Ltd., Gyeonggi-do, Republic of Korea) which was conjugated with TL-HEPES.

Denuded conjugates were incubated in 700 μL CR1-aa medium supplemented with 44 μg / mL sodium pyruvate, 14.6 μg / mL glutamine, 10 μL / mL penicillin / streptomycin, 3 mg / mL BSA and 310 μg / mL glutathione al., 1993) (IVC-I).

They were cultured until day 7 of embryonic development (d 0; day of IVF) except that BSA was replaced with 10% (vol / vol) FBS in the same composition medium (IVC II). During IVM, IVF, and IVC, the culture conditions were atmospheric 5% CO ₂ at 38.5 ° C under maximum moisture. The development rate of blastocysts was assessed on day 7 as the proportion of conjugates transferred into IVC-I medium. Expanded and / or intermediate expanded blastocysts were selected for good and / or good case and vitrified for 7 days

1-3. Blatant  boring( Puncture )

Choi YH. et al. (2010), a 7-day-old embryo bag was pipetted 2-3 times with a fine pasteur pipette to disrupt the embryo bag. Adjust the internal stiffness of the pasture pipette to about 120 μm and trim the end of the pipette to fire

< Example  2>

Vitrification freezing  And melting process

The vitrification solution was added to a holding medium (HM: TCM199 with 25 mM Hepes plus 20% FBS), vitrification freeze solution 1 (VS1: HM plus 10% ethylene glycol), vitrification freeze solution 2 (VS2: HM plus 35% 5% PVP + 0.5 M sucrose), diluted solution 1 (DS1: HM plus 0.3 M sucrose) and diluted solution 2 (DS2: HM plus 0.15 M sucrose).

Good morphologically superior IVP blastocysts were selected and distributed randomly to each treatment group. The blots were exposed to HM and transferred to VS1 with a fine glass pasteur pipette.

After 5 minutes at VS1, the blastocysts were transferred to VS2 and loaded onto a 0.25 mL straw with a microglass passive pipette to attach on the surface of the inner wall of the straw without making a column (psV, FIG. 1); Or 0.25 mL straw as a conventional straw loading method to freeze the column straw vitrification.

The loaded straw was sealed with a heated forceps for about 10 seconds. The sealed straw was temporarily carried on liquid nitrogen LN2, and the side of the embryo-containing straw was first carried thereon for one minute, and then the whole straw was slowly carried on liquid nitrogen LN2. This prevents the frozen straw from breaking down. For ddV, a blastocyst was loaded into a fine-paste pipette and dropped directly onto LN2.

The vitrified frozen straw was taken out of the LN2 tank and exposed to room temperature for 10 seconds and then immersed in water at 37 DEG C for 30 seconds. Clean the molten straw with a paper towel, shake the straw up to collect the entire medium at the top, and then shake the straw down to gather the entire medium at the bottom. After dilution, the sealed top of the straw was cut and poured into a 30 mL Petri dish and washed with HM

pV and cV were diluted inside the straw and dV droplets were sequentially diluted in 0.3 M sucrose for 5 minutes and 0.15 M sucrose for 3 minutes in a 30-mL culture dish.

Recovery rates of blastocysts after thawing were counted and cultured in CR1aa supplemented with 10% (v / v) FBS for analysis of viability after thawing (24 h re-expansion and hatching and / or hatching development at 36 h) At this time, live embryos of arbitrarily selected groups were subjected to TUNEL staining according to Deb GK et al., (2011) to evaluate apoptotic cells and overall evaluation of the blastocysts.

< Example  3>

Embryo implantation Embryo transfer ) And pregnancy diagnosis

The fresh and post-thaw blast of the psV group was directly implanted into the synchronized uterine ovary of recipients with functional good progenitor (CL). One blast (fresh or vitrified) was loaded per straw, and one embryo was transplanted each recipient. All recipients except for those who returned to the estrus period were diagnosed with pregnancy by promoting the 60th day of transplantation.

< Experimental Example  1>

small Blatant After melting  About the viability Straw Loading method  effect

Seven days old blastocysts were incubated in the same manner as described in Example 1 except that the cells were incubated in an incubation temperature of 30 ° C, It was vitrified and frozen in three different ways.

Individual shoots were vitrified at each time with all three vitrification methods. The vitrified frozen blastocysts were stored in a liquid nitrogen (LN ₂ ) container for at least one week and were used for the recovery after fusion, for re-expansion at 24 hours or for evaluation of hatching and / or hatching at 36 hours, CR1aa (IVC2 medium). All data were compared to blastocysts (control) of non-vitrified frozen control. The experiment was repeated.

As a result, the recovery rates after vitrification of frozen blastocysts were higher in psV, csV and ddV (Table 1).

Loading type Number of vitrified frozen embryos Restored embryo (%) Developed embryos (%) Re-expansion rate (24 h) denaturalization pV 37 37 (100) 37 (100.0%) ^{a, b} - cV 25 21 (84.0) 12 (57.14%) ^c 9 (42.9) dV 26 16 (61.5) 15 (93.75%) ^b 1 (6.3) Control 20 - 20 (100.0%) ^{a, b} -

The psV and ddV methods showed similar re-dilatation rates (P> 0.05) as the control, non-vitrified frozen blastocysts. However, the cV method showed a low reexpansion rate when compared with pV, dV and control (P <0.05)

The post-thawed vitrified frozen blots and controls according to pV were not degenerated until 24 hours in culture, whereas approximately 42.9% cV and 6.3% dV groups were denatured. The post-thawed vitrified frozen tobacco according to pV showed a lower total cell count per bladder (114 ± 48.1 in psV vs. 102 ± 35.1 in cV vs. 105.6 ± 33.9 in dV) (P <0.05) compared with control blast (134.4 ± 38.9) were not related to the vitrification method.

Among the three vitrification methods, the embryos of the psV group showed higher total cell numbers than the other two groups (cV and dV) (P <0.05). The number of apoptotic cells per bladder was higher (P <0.05) in post-thawed vitrified frozen blastocysts than in control blastocysts (0.4 ± 1.4). The number of apoptotic cells was 6.6 ± 9.5 (pV) vs. 14.5 ± 9.5 (sV) vs. 10.9 ± 9.6 (dV).

Loading type Number of embryos counted The number of counted nuclei (mean ± SE) Whole cell Dead cell pV 13 114.0 ± 48.1 ^b 6.6 ± 9.5 ^{a, b} cV 10 102.0 + - 35.1 ^{b, c} 14.5 ± 9.5 ^b dV 10 105.6 ± 33.9 ^{b, c} 10.9 ± 9.6 ^b Control 13 134.4 ± 38.9 ^a 0.4 ± 1.4 ^a

< Experimental Example  2>

small Blatant  The possibility of survival after thawing Foamed steel  Impact of Perforation

Seven-day old blastocysts were divided into shredding groups and non-shredding groups to assess whether blastocyst fractures mediated the beneficial effects of psV by perforating them with a micropascopic pipette prior to vitrification. Both of these groups were vitrified using the same protocol of the psV method. All vitrified frozen bracts were melted and cultured to count recovery rates, 24 hour re-expansion or hatching and / or 36 hour hatching of blastocysts, and total number of dead cells. All data were compared to blastocysts (control) of non-vitrified frozen control. The experiment was repeated.

As a result, post-thaw re-expanded pulp development at 24 hrs after culture was not affected by shattering of pre-vitrification freezing (Table 3).

Perforation Vitrification freezing
Number of embryos Re-Expanded BL Development (%) 24 h Both in hatching or in hatching Puncture pV 32 32 (100) ^a 20 (62.5) ^a Non-puncture pV 32 32 (100) ^a 12 (37.5) ^b Control 17 17 (100) ^a 14 (82.3) ^a

However, crushing of the blastocyst by perforation increased the developmental competence to total hatching and incubated blastocyst at 36 hours (P <0.05). This was similar to the control (62.5% in pV vs. 37.5% in cV vs. 82.3% in control). When pored prior to freezing by pV, the total number of cells per bladder increased (152.4 ± 39.6 punched group vs. 117.5 ± 14.4 non-punched group) (P <0.05) (152.4 ± 39.6 vs. 160 ± 48.3, respectively)

The number of apoptotic cells was not significantly different between perforated pV, non-perforated pV and control group (34.5 ± 20.1 vs. 34.6 ± 40.4 vs. 21.3 ± 12.0).

Perforation A counted embryo The number of counted nuclei (mean ± SE) Whole cell Dead cell Puncture pV 11 152.4 ± 39.6 ^{a, b} 34.5 ± 20.1 Non-puncture pV 11 117.5 ± 14.4 ^b 34.6 ± 40.4 Control 12 160.4 ± 48.3 ^a 21.3 ± 12.0

< Experimental Example  3>

Fresh embryo implantation pV Vitrified  Comparison of embryo pregnancy rate

In this experiment, the pV method was evaluated to see if the vitrified frozen embryos recovered after pregnancy in the synchronized recipient after being melted into the uterus.

A single blastocyst (fresh or frozen) was implanted into the upper part of the uterus. Fresh or frozen blastocysts were randomly selected, frozen in vitrification and fresh blastocysts were transplanted, and the pregnancy rate in the blastocysts was compared.

That is, the vitrified and fresh embryos were transplanted into the synchronized pool on day 7 and each recipient was given one fresh blast (7 days) or perforated pV vitrified frozen blast. pV blastocysts were performed in liquid nitrogen (LN ₂ ) and directly transferred to the synchronized recipient uterus after straw fusion. On the other hand, a single fresh blastula was loaded into the straw in a flask at 37 &lt; 0 &gt; C. Fresh blastocysts were transplanted into 50 recipients and pV blastocysts were transplanted into 45 surrogates.

Sixty days after embryo transfer, pregnancy was diagnosed by vaginal palpation method.

As a result, the pregnancy rate was not significantly different between fresh and pV groups (54.4% vs. 53.3%) (P> 0.05)

Embryo type Number of implanted embryos 60 days of pregnancy Fresh 50 27 (54.0) pV 45 24 (53.3)

In other words, it was confirmed that the use of the sticky straw-freezing method for attaching germ cells to the inner wall of the straw of the present invention exhibits almost the same effect as the case of using a fresh embryo.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. It will be clearly understood by those skilled in the art that such an excellent effect of the present invention is readily applicable to other biological materials such as horses, chlorine, pigs, etc., including humans.

Claims (11)

A method for freezing a germ cell vitrification using an adhesive paste embryo in the vitrification straw (pV) for attaching germ cells to the inner wall of a straw, comprising the step of forming a perforation in the germ cell prior to vitrification Characterized by a germ freezing vitrification method. The method according to claim 1,
Wherein the germ cell is an oocyte, an in vitro fertilized egg or an embryonic cell. 3. The method of claim 2,
Wherein the embryonic cell is a blastocyst. delete The method according to claim 1,
A holding medium consisting of TCM199 supplemented with 25 mM Hepes and 20% FBS,
The vitrification solution 1 consisting of the above holding medium and 10% ethylene glycol,
The above-mentioned holding medium, vitrification solution 2 consisting of 35% ethylene glycol, 5% PVP and 0.5 M sucrose,
A dilution solution 1 consisting of the above holding medium and 0.3 M sucrose, and
And a dilution solution 2 consisting of the above holding medium and 0.15 M sucrose is used for freezing and vitrification. The method according to claim 1,
The gonad cell freezing method
A method of freezing a germ-line vitrification characterized by comprising sealing a straw with germ cells attached to its inner wall and carrying it on liquid nitrogen (LN ₂ ). The method of germ cell transplantation according to claim 1, characterized in that the frozen frozen germ cells are directly transplanted to a mammal other than a human, without further processing after thawing. 8. The method of claim 7,
Wherein the germ cell is an oocyte, an in vitro fertilized egg or an embryo cell. 9. The method of claim 8,
Wherein the embryonic cell is a blastocyst. 8. The method of claim 7,
Wherein the mammal other than human is a bovine. delete

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