KR20140093811A - Method of Ovary Cryopreservation Using Antifreeze Protein - Google Patents
Method of Ovary Cryopreservation Using Antifreeze Protein Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
Description
본 발명은 동결보존액에 항동결 단백질을 첨가하여 난소를 동결보존하는 방법에 관한 것이다.The present invention relates to a method for cryopreserving ovaries by adding an anti-freeze protein to a frozen storage solution.
난소조직의 동결보존은 시험관 아기 시술 시 잉여 배아의 차후 사용을 위한 보존과 함께 암이나 기타 이유로 하여 화학치료나 방사선 치료를 받아야 하는 사람의 경우 자신의 생식 세포를 안전하게 보존하여 차후 재 사용할 수 있는 방법의 하나로 매우 유용하게 사용될 수 있다. 또한, 임신 중기 태아 난소의 경우에는 100만개 이상의 생식세포가 존재하며, 이러한 생식 세포의 존재는 현재까지 가장 적합한 난자의 저장고로 알려져 왔고, 또한 최근 줄기세포에 의한 치료법이 의학에 도입되면서 줄기세포를 만들기 위한 가장 적절한 소재의 하나로 알려지게 되어 난소조직의 냉동보존의 중요성이 새로이 부각되고 있다.Cryopreservation of ovarian tissue can be preserved for future use of surplus embryos during in vitro fertilization and can be safely stored for future use in the case of a person who needs chemotherapy or radiotherapy for cancer or other reasons Can be very useful as one of. In addition, there are more than one million germ cells in the mid-gestation fetal ovary. The existence of germ cells has been known to be the best oocyte reservoir to date, and recently, stem cell treatment has been introduced into medicine, The importance of cryopreservation of ovarian tissues is newly emerging as it is known as one of the most suitable materials to make.
동결 보존제 및 첨가물로는 프로판디올(Propanediol (PROH)), 글리콜(glycerol), 에틸렌 글리콜(ethylene glycol (EG)), dimethyl-sufoxide (DMSO) 등의 투과성 동결보존제 (Ali and Sheton, 1993; Kasai, 1997; Wright et al., 2004)와 수크로즈(sucrose), 트레할로스(threhalose), 피콜(Ficoll), 덱스트란(dextran), 폴리비닐피롤리돈(polyvinyl pyrrolidone), 폴리에틸렌 글리콜(polyethylene glyconl) (Leibo and Oda, 1992; Naitana et al., 1997; Ohboshi et al., 1997; Shaw et al., 1997; Kuleshova et al., 2001; Asada et al., 2002) 등의 비투과성 동결보존제의 혼합용액이 초급성 유리화 동결법의 동결보조제로 사용되고 있다. 그 중 EG가 낮은 분자량과 적은 독성으로 가장 많이 사용되고 있는 동결 보조제이나(Chian et al., 2004), 한편으로는 두 가지 이상의 동결 보존제를 혼합하여 사용하는 것이 더 효과적이라는 보고도 있다 (Vajta et al., 1998).Cryopreservation agents and additives include permeable cryopreservation agents such as propanediol (PROH), glycerol, ethylene glycol (EG) and dimethyl-sufoxide (Ali and Sheton, 1993; Kasai, 1997; Wright et al., 2004) and sucrose, thalhalose, Ficoll, dextran, polyvinyl pyrrolidone, polyethylene glyconl (Leibo a mixed solution of a non-permeable cryoprotectant such as Kawasaki et al., 2001; Asada et al., 2002) It is used as a freezing aid in the super-vitrification-freezing method. It has been reported that EG is the most commonly used frozen adjuvant with low molecular weight and low toxicity (Chian et al., 2004), and on the other hand, it is more effective to use two or more frozen preservatives (Vajta et al , 1998).
현재까지 기존의 동결 보존제를 사용한 난소동결의 효율은 높지 않은 상태로 임상적으로 널리 활용하기 위해서는 이의 개선이 필요한 상태이다. To date, the efficiency of ovarian freezing using conventional cryopreservation agents has not been high enough to be widely used clinically.
이에 본 발명자들은 난소의 동결보존시 난소 조직의 생존능력을 높일 수 있는 방법을 확립하여 본 발명을 완성하게 되었다.Accordingly, the present inventors have established a method for enhancing the viability of ovarian tissues upon cryopreservation of ovaries, thereby completing the present invention.
본 발명은 생쥐 난소를 동결 보존-해동 후 이식된 난조조직의 생존능력이 높지 않아, 동결방법의 개선을 통해 난소 조직의 생존능력을 향상시키는 것을 목적으로 한다.The present invention aims to improve the viability of ovarian tissue by improving the freezing method because the viability of the hatching tissues implanted after freezing-thawing the mouse ovary is not high.
상기 목적을 달성하기 위하여 본 발명의 일 구체예에서 항동결 단백질의 농도가 3~7mg/ml가 되도록 첨가하여 동결 보존용 조성물을 제조한 후, 상기 조성물로 난소를 동결시키는 것을 특징으로 하는 난소를 동결보존하는 방법을 제공한다.In order to achieve the above object, in one embodiment of the present invention, an ovary is prepared by adding a freezing protein concentration of 3 to 7 mg / ml to prepare a composition for cryopreservation, A method for cryopreservation is provided.
본 발명에 있어서, 난소의 동결보존시 1차 난포(primary follicles) 또는 2차 난포(secondary follicles)의 보존율이 향상되는 것을 특징으로 하고, 상기 항동결 단백질은 antifreeze protein type III인 것을 특징으로 하며, 상기 조성물에 혈청, 에틸렌 글리콜, 피콜 및 수크로즈로 이루어진 군으로부터 선택되는 어느 하나 이상을 추가로 혼합하는 것을 특징으로 하며, 상기 조성물에 18~22부피%의 혈청, 38~42부피%의 에틸렌 글리콜, 15~20부피%의 피콜 및 0.2~0.4M의 수크로스로 이루어진 군으로부터 선택되는 어느 하나 이상을 추가로 혼합하는 것을 특징으로 한다.
In the present invention, the preservation ratio of primary follicles or secondary follicles is improved upon cryopreservation of ovaries. The anti-freeze protein is characterized by being an antifreeze protein type III, Wherein the composition is further mixed with at least one selected from the group consisting of serum, ethylene glycol, phycol and sucrose, wherein the composition contains 18 to 22% by volume of serum, 38 to 42% by volume of ethylene glycol , 15 to 20% by volume of phycol, and 0.2 to 0.4M of sucrose are further mixed.
일 구체예에서 3~7mg/ml의 항동결 단백질을 포함하는 난소의 동결보존용 조성물을 제공한다.In one embodiment, there is provided a composition for cryopreservation of ovaries comprising 3-7 mg / ml of an anti-freeze protein.
본 발명에 있어서, 난소의 동결보존시 1차 난포(primary follicles) 또는 2차 난포(secondary follicles)의 보존율이 향상되는 것을 특징으로 하고, 상기 항동결 단백질은 antifreeze protein type III인 것을 특징으로 하며, 상기 조성물은 혈청, 에틸렌 글리콜, 피콜 및 수크로즈로 이루어진 군으로부터 선택되는 어느 하나 이상을 추가로 포함하는 것을 특징으로 하며, 상기 조성물은 18~22부피%의 혈청, 38~42부피%의 에틸렌 글리콜, 15~20부피%의 피콜 및 0.2~0.4M의 수크로스로 이루어진 군으로부터 선택되는 어느 하나 이상을 추가로 포함하는 것을 특징으로 한다.
In the present invention, the preservation ratio of primary follicles or secondary follicles is improved upon cryopreservation of ovaries. The anti-freeze protein is characterized by being an antifreeze protein type III, Wherein the composition further comprises at least one selected from the group consisting of serum, ethylene glycol, phycol and sucrose, wherein the composition comprises 18 to 22% by volume of serum, 38 to 42% by volume of ethylene glycol , 15 to 20% by volume of phycol, and 0.2 to 0.4M of sucrose are further included.
일 구체예에서 항동결 단백질의 농도가 3~7mg/ml가 되도록 첨가하여 난소의 동결 보존용 조성물을 제조하는 방법을 제공한다.In one embodiment, a method for preparing a composition for cryopreservation of ovaries is provided by adding the anti-freezing protein at a concentration of 3 to 7 mg / ml.
본 발명에 있어서, 난소의 동결보존시 1차 난포(primary follicles) 또는 2차 난포(secondary follicles)의 보존율이 향상되는 것을 특징으로 하고, 상기 항동결 단백질은 antifreeze protein type III인 것을 특징으로 하며, 상기 조성물에 혈청, 에틸렌 글리콜, 피콜 및 수크로즈로 이루어진 군으로부터 선택되는 어느 하나 이상을 추가로 혼합하는 것을 특징으로 하며, 상기 조성물에 18~22부피%의 혈청, 38~42부피%의 에틸렌 글리콜, 15~20부피%의 피콜 및 0.2~0.4M의 수크로스로 이루어진 군으로부터 선택되는 어느 하나 이상을 추가로 혼합하는 것을 특징으로 한다.
In the present invention, the preservation ratio of primary follicles or secondary follicles is improved upon cryopreservation of ovaries. The anti-freeze protein is characterized by being an antifreeze protein type III, Wherein the composition is further mixed with at least one selected from the group consisting of serum, ethylene glycol, phycol and sucrose, wherein the composition contains 18 to 22% by volume of serum, 38 to 42% by volume of ethylene glycol , 15 to 20% by volume of phycol, and 0.2 to 0.4M of sucrose are further mixed.
본 발명에서 난소의 보존 정도 평가는 형태학적 평가 및 세포 자연사 여부 평가의 두 가지 방법으로 진행되었다.
In the present invention, evaluation of the degree of preservation of ovaries was performed by two methods, morphological evaluation and evaluation of cell death.
형태학적 평가는 난소를 포르말린 용액에 고정시킨 후 파라핀 블록을 만들어 단면을 얻은 후 헤마톡실린-에오진(H&E) 염색을 시행하여 형태학적 이상 여부를 광학 현미경으로 관찰한 것으로, 난소조직 중 난자의 생성에 가장 중요하며 직접적인 관련이 있는 난포의 형태를 Primordial, Primary, Secondary 및 Antral로 분류하고(Lundy et al., 1999 참고) grade를 평가하였다. Grade 1에 해당하는 난포를 손상되지 않은 난포로 정의하여 이의 비율을 평가지표로 사용하였다(Gandolfi et al., 2006 참고).
The morphological evaluation was made by fixing the ovary in a formalin solution and then making a paraffin block to obtain a cross section. The hematoxylin-eosin (H & E) staining was performed and the morphological abnormality was observed with an optical microscope. The most important and directly related follicular forms of development were classified as Primordial, Primary, Secondary, and Antral (see Lundy et al., 1999) and graded. Grade 1 follicles were defined as uninjured follicles and their ratio was used as an indicator (see Gandolfi et al., 2006).
세포 자연사(apoptosis) 여부 평가는 세포의 자연사 여부를 평가할 수 있는 방법인 TUNEL assay를 이용하여 세포 자연사가 발생한 난포의 비율을 평가하여 이를 난소 보존상태의 평가지표로 사용하였다. TUNEL assay의 방법은 기존에 상업화되어 판매되고 있는 “In Situ Cell Death Detection kit”를 사용하였다. 결과적으로 세포 자연사가 발생한 세포는 녹색의 형광을 발현하게 됨. 이를 바탕으로 30% 이상의 난포세포에서 녹색 형광을 발현하는 난포를 세포자연사가 발생한 난포로 평가하였다.
The rate of apoptosis was assessed by using the TUNEL assay, a method of assessing the natural history of the cells, and the rate of ovarian follicle development was evaluated as an indicator of ovarian reserve status. For the TUNEL assay, a commercially available "In Situ Cell Death Detection kit" was used. As a result, cells that develop cell death cause green fluorescence. Based on this, follicles expressing green fluorescence in more than 30% of the follicular cells were evaluated as follicles that developed cell death.
본 발명에서, “항동결 단백질”은 영하의 온도에서 식물 또는 동물이 동결되는 것을 방지하는 기능을 가지는 단백질을 의미하며, 통상 극지방 해양어류의 체내 또는 추운 지방의 식물에서 발견된다. 또한, 이에 한정하지 않지만, 예를 들어, 항동결 단백질은 AFGP, Type 1,2,3,4를 포함한다.In the present invention, the term " anti-freezing protein " means a protein having a function of preventing a plant or animal from freezing at a subzero temperature, and is usually found in plants of the body or cold fat of polar marine fish. For example, the freeze-frozen protein includes AFGP, Type 1, 2, 3, 4.
본 발명에 의하면 난소 조직의 동결 보존 후 해동 및 이식 시에, 난포 보존율이 향상되어 난소의 생존율을 높일 수 있다.According to the present invention, at the time of frozen storage of ovarian tissue and thawing and transplantation, the follicle preservation rate can be improved and the survival rate of ovary can be increased.
이하, 본 발명을 하기의 실시예에 의해 상세히 설명한다. 다만, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.
Below, The present invention is described in detail by the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.
실시예Example
실시예Example
1. 기존 동결보존제의 효용성 비교 1. Comparison of efficacy of existing cryopreservatives
20% 소태아혈청(fetal bovine serum, FBS)을 포함하는 Dulbecco? phosphate buffered saline (DPBS) 용액에 20% 에틸렌 글리콜(ethylene glycol, EG)을 첨가하여 평형용액을 제조한 후, 4주령의 생쥐 (ICR mouse)로부터 난소를 적출하여 10분간 평형용액에 침전 시켰다.Dulbecco? Containing 20% fetal bovine serum (FBS)? To the equilibrium solution, 20% ethylene glycol (EG) was added to phosphate buffered saline (DPBS) solution. Ovaries were removed from ICR mice at 4 weeks of age and precipitated in equilibrium solution for 10 minutes.
그 후 난자를 분리하고, 이를 20% 소태아혈청(fetal bovine serum, FBS)을 포함하는 Dulbecco? phosphate buffered saline (DPBS) 용액에 40% 에틸렌 글리콜, 18% 피콜(Ficoll, Pharmacia) 및 0.3M 수크로즈를 첨가하여 제조한 동결 보존액에 5분간 침전 시켰다. 상기 동결용액상의 난자를 1.2-mL cryotube에 담아 액체질소에 침전시켜 보관하여 대조군 1(상기 실시예 2의 동결보존제에서 항동결 단백질 제외)을 제조하였다.
The oocytes were then separated and plated on a Dulbecco ® (Invitrogen) containing 20% fetal bovine serum (FBS). phosphate buffered saline (DPBS) solution for 5 minutes in a frozen storage solution prepared by adding 40% ethylene glycol, 18% Ficoll, Pharmacia and 0.3M sucrose. The oocytes in the above-mentioned frozen solution were placed in a 1.2-mL cryotube and precipitated in liquid nitrogen and stored to prepare Control 1 (except freezing protein in the cryopreservative of Example 2).
20% 소태아혈청(fetal bovine serum, FBS)을 포함하는 Dulbecco? phosphate buffered saline (DPBS) 용액에 7.5% 에틸렌 글리콜(ethylene glycol, EG)을 첨가하고, 7.5% DMSO용액을 첨가하여 평형용액을 제조한 후, 4주령의 생쥐 (ICR mouse)로부터 난소를 적출하여 10분간 평형용액에 침전시켰다.Dulbecco? Containing 20% fetal bovine serum (FBS)? (EG) was added to phosphate buffered saline (DPBS) solution, and 7.5% DMSO solution was added to prepare an equilibrium solution. Ovaries were extracted from ICR mice at 4 weeks of age to obtain 10 Lt; RTI ID = 0.0 > equilibrium < / RTI >
그 후 난자를 분리하고, 이를 20% 소태아혈청(fetal bovine serum, FBS)을 포함하는 Dulbecco's phosphate buffered saline (DPBS) 용액에 20% 에틸렌 글리콜, 20% DMSO 및 0.5M 수크로즈를 첨가하여 제조한 동결 보존액에 5분간 침전시켰다. 상기 동결용액상의 난자를 1.2-mL cryotube에 담아 액체질소에 침전시켜 2주 동안 보관하여 대조군 2를 제조하였다.
The oocytes were then separated and prepared by adding 20% ethylene glycol, 20% DMSO and 0.5 M sucrose to a Dulbecco's phosphate buffered saline (DPBS) solution containing 20% fetal bovine serum (FBS) It was settled in the frozen storage solution for 5 minutes. The oocyte in the above-mentioned frozen solution was placed in a 1.2-mL cryotube and precipitated in liquid nitrogen and stored for 2 weeks to prepare Control 2.
상기 제조한 대조군 1과 대조군 2를 2주 동안 동결보존한 후 상기 실시예 2의 방법으로 해동하며 그 결과를 관찰하였다.
The control group 1 and the control group 2 prepared above were cryopreserved for 2 weeks and thawed by the method of Example 2, and the results were observed.
항동결 단백질이 투여되지 않은 대조군 1 및 2의 동결보존제의 조성에 따른 미손상 난포(grade 1)의 비율과 세포자연사 미발생 난포(non-apoptotic follicle)의 비율을 비교한 결과, 미손상 난포의 비율이 차이가 없었다. 즉, 상기 실시예 2의 결과는 항동결 단백질을 제외한 동결보존제의 다른 조성물의 영향을 받지 않았음을 알 수 있었다(표 1 참조).The proportion of uninfected follicles (grade 1) and the proportion of non-apoptotic follicles according to the composition of the cryoprotectants of the control groups 1 and 2 without the anti-freezing protein was compared with that of uninfected follicles There was no difference in the ratio. That is, the results of Example 2 above were not influenced by other compositions of the cryoprotectant except the anti-freeze protein (see Table 1).
실시예Example
2. 2.
항동결Freeze
단백질 첨가에 의한 난소의 동결보존 Cryopreservation of ovary by addition of protein
20% 소태아혈청(fetal bovine serum, FBS)을 포함하는 Dulbecco's phosphate buffered saline (DPBS) 용액에 20% 에틸렌 글리콜(ethylene glycol, EG)을 첨가하여 평형용액을 제조한 후, 4주령의 생쥐(ICR mouse)로부터 난소를 적출하여 10분간 평형용액에 침전시켰다.After equilibrium solution was prepared by adding 20% ethylene glycol (EG) to Dulbecco's phosphate buffered saline (DPBS) solution containing 20% fetal bovine serum (FBS), 4-week old mice mouse), and the ovaries were precipitated in an equilibrium solution for 10 minutes.
그 후 난자를 분리하고, 이를 20% 소태아혈청(fetal bovine serum, FBS)을 포함하는 Dulbecco's phosphate buffered saline (DPBS) 용액에 40% 에틸렌 글리콜, 18% 피콜(Ficoll, Pharmacia) 및 0.3M 수크로즈를 첨가하여 제조한 동결 보존액에 5분간 침전시켰다. 상기 동결용액상의 난자를 1.2-mL cryotube에 담아 액체 질소에 침전시켜 보관하였다.The oocytes were then separated and seeded in Dulbecco's phosphate buffered saline (DPBS) solution containing 20% fetal bovine serum (FBS), supplemented with 40% ethylene glycol, 18% Ficoll, Pharmacia, Was added to the frozen storage liquid for 5 minutes. The oocytes in the frozen solution were placed in a 1.2-mL cryotube and stored in liquid nitrogen.
이 때, 항동결 단백질(AntiFreeze Protein Type III)을 동결보존액에 각각 0(대조군), 5mg/mL, 20mg/mL를 첨가하여 2주 동안 동결 보존하며, 이에 따른 결과를 비교하였다.
At this time, 0 (control), 5 mg / mL, and 20 mg / mL of anti-freeze protein (Type III) were stored in the frozen storage solution for 2 weeks.
실시예Example 3. 3. 항동결Freeze 단백질의 중량비에 따른 난소의 동결보존 Cryopreservation of ovary by weight of protein 결과result
상기 실시예 1에서 2주 동안 동결 보존한 난소를 해동하여 그 결과를 관찰하였다. The ovaries stored frozen for two weeks in Example 1 were thawed and the results were observed.
액체 질소에서 꺼낸 난소 조직을 37.0℃ 의 항온조에 담근 후 하기 표 2의 a, b 및 c 의 순서대로 침전시켜 해동하였다.The ovary tissues taken out from the liquid nitrogen were immersed in a thermostatic bath at 37.0 ° C and thawed in the order of a, b, and c in Table 2 below.
또한, 상기와 같은 방법으로 동결된 난소를 2주간 보존한 후 해동하여 난소를 적출한 생쥐의 등쪽 피하조직에 재이식하고, 2주 후에 난소를 적출하여 결과를 비교하였다.
In addition, the frozen ovaries were preserved for 2 weeks and then thawed. The ovaries were re-transplanted into the dorsal subcutaneous tissues of the ovariectomized mice and the ovaries were harvested two weeks later to compare the results.
그 결과, 동결 후 해동시킨 난소의 경우 하기 표 3에 나타난 바와 같이, 항동결 단백질의 투여 시 난소기능의 보존에 가장 중요한 난포인 원시 난포(primordial follicle)의 형태학적인 보존이 유의하게 향상되는 것을 알 수 있었다. 이러한 결과는 항동결 단백질의 농도를 높임에 따라 그 효과가 유의하게 향상됨을 알 수 있었다.
As a result, it was found that morphological preservation of primordial follicle, which is the most important follicle for preserving ovarian function, was significantly improved in the case of ovaries thawed after freezing, as shown in Table 3 below I could. These results indicate that the effect of freezing protein is significantly improved with increasing concentration of freezing protein.
또한, 난포의 발달 단계가 Primary 및 Secondary일 때는 항동결 단백질을 5mg/ml를 투여했을 때 미손상 난포의 비율의 임계적 의의를 갖음을 알 수 있었다.
In addition, when the developmental stage of the follicles was Primary and Secondary, it was found that the ratio of uninfected follicles was critical when 5 mg / ml of anti-freeze protein was administered.
또한, 전체 난포의 형태학적 보존율에서도 항동결 단백질을 20mg/mL투여한 군에서 대조군에 비해 유의하게 난포 보존율이 향상되었고, 세포자연사 미발생 난포의 비율 역시 항동결 단백질을 투여함에 따라 유의하게 향상되고 있으며, 그 용량을 높임에 따라 이러한 효과가 더 커짐을 알 수 있었다.
In addition, the morphological retention rate of total follicles was significantly improved in the group treated with 20 mg / mL of anti - freeze protein compared to the control group, and the proportion of follicle - stimulating follicles was also significantly increased , And it was found that the effect becomes larger as the capacity is increased.
(Grade I follicles)Intact follicle
(Grade I follicles)
(Primordial)Primordial follicles
(Primordial)
(Primary)Primary follicle
(Primary)
(Secondary)Secondary follicle
(Secondary)
(Antral)Tertiary follicle
(Antral)
(Total)Sum
(Total)
결론적으로, 난소조직의 동결 보존 시 동결 보존액에 각각 항동결 단백질 (AFP type III)을 첨가하여 동결보존시, 해동 후 및 이식 후 난포의 보존율이 향상됨을 알 수 있었다.In conclusion, the addition of anti-freeze protein (AFP type III) to frozen storage of ovarian tissue improves preservation of follicle after thawing and transplantation.
Claims (15)
Freezing protein to a concentration of 3 to 7 mg / ml to prepare a composition for cryopreservation, and then freezing the ovary with the composition.
난소의 동결보존시 1차 난포(primary follicles) 또는 2차 난포(secondary follicles)의 보존율이 향상되는 것을 특징으로 하는 난소를 동결보존하는 방법.
The method according to claim 1,
Wherein the preservation rate of primary follicles or secondary follicles is improved upon cryopreservation of the ovary.
상기 항동결 단백질은 antifreeze protein type III인 것을 특징으로 하는 난소를 동결보존하는 방법.
The method according to claim 1,
Wherein the anti-freeze protein is an antifreeze protein type III.
상기 조성물에 혈청, 에틸렌 글리콜, 피콜 및 수크로즈로 이루어진 군으로부터 선택되는 어느 하나 이상을 추가로 혼합하는 것을 특징으로 하는 난소를 동결보존하는 방법.
The method according to claim 1,
Wherein the composition is further mixed with at least one selected from the group consisting of serum, ethylene glycol, phycol and sucrose.
상기 조성물에 18~22부피%의 혈청, 38~42부피%의 에틸렌 글리콜, 15~20부피%의 피콜 및 0.2~0.4M의 수크로스로 이루어진 군으로부터 선택되는 어느 하나 이상을 추가로 혼합하는 것을 특징으로 하는 난소를 동결보존하는 방법.
The method according to claim 1,
The composition may further contain at least one selected from the group consisting of 18 to 22% by volume of serum, 38 to 42% by volume of ethylene glycol, 15 to 20% by volume of phycol and 0.2 to 0.4 M of sucrose A method of cryopreserving a characteristic ovary.
A composition for cryopreservation of ovaries comprising 3-7 mg / ml of an anti-freeze protein.
난소의 동결보존시 1차 난포(primary follicles) 또는 2차 난포(secondary follicles)의 보존율이 향상되는 것을 특징으로 하는 난소의 동결보존용 조성물.
The method according to claim 6,
Wherein the preservation ratio of primary follicles or secondary follicles is improved upon cryopreservation of the ovary.
상기 항동결 단백질은 antifreeze protein type III인 것을 특징으로 하는 난소의 동결보존용 조성물.
The method according to claim 6,
Wherein the anti-freeze protein is an antifreeze protein type III.
상기 조성물은 혈청, 에틸렌 글리콜, 피콜 및 수크로즈로 이루어진 군으로부터 선택되는 어느 하나 이상을 추가로 포함하는 것을 특징으로 하는 난소의 동결보존용 조성물.
The method according to claim 6,
Wherein the composition further comprises at least one selected from the group consisting of serum, ethylene glycol, phycol and sucrose.
상기 조성물은 18~22부피%의 혈청, 38~42부피%의 에틸렌 글리콜, 15~20부피%의 피콜 및 0.2~0.4M의 수크로스로 이루어진 군으로부터 선택되는 어느 하나 이상을 추가로 포함하는 것을 특징으로 하는 난소의 동결보존용 조성물.
10. The method of claim 9,
The composition may further comprise at least one selected from the group consisting of 18 to 22% by volume of serum, 38 to 42% by volume of ethylene glycol, 15 to 20% by volume of phycol and 0.2 to 0.4 M of sucrose Wherein the ovary is freeze-dried.
Freezing protein to a concentration of 3 to 7 mg / ml to prepare a composition for cryopreservation of ovaries.
난소의 동결보존시 1차 난포(primary follicles) 또는 2차 난포(secondary follicles)의 보존율이 향상되는 것을 특징으로 하는 난소의 동결 보존용 조성물을 제조하는 방법.
12. The method of claim 11,
Wherein the preservation ratio of primary follicles or secondary follicles is improved upon cryopreservation of the ovary.
상기 항동결 단백질은 antifreeze protein type III인 것을 특징으로 하는 난소의 동결 보존용 조성물을 제조하는 방법.
12. The method of claim 11,
Wherein the anti-freeze protein is an antifreeze protein type III.
상기 조성물에 혈청, 에틸렌 글리콜, 피콜 및 수크로즈로 이루어진 군으로부터 선택되는 어느 하나 이상을 추가로 혼합하는 것을 특징으로 하는 난소의 동결 보존용 조성물을 제조하는 방법.
12. The method of claim 11,
Wherein the composition is further mixed with at least one member selected from the group consisting of serum, ethylene glycol, phycol and sucrose.
상기 조성물에 18~22부피%의 혈청, 38~42부피%의 에틸렌 글리콜, 15~20부피%의 피콜 및 0.2~0.4M의 수크로스로 이루어진 군으로부터 선택되는 어느 하나 이상을 추가로 혼합하는 것을 특징으로 하는 난소의 동결 보존용 조성물을 제조하는 방법.15. The method of claim 14,
The composition may further contain at least one selected from the group consisting of 18 to 22% by volume of serum, 38 to 42% by volume of ethylene glycol, 15 to 20% by volume of phycol and 0.2 to 0.4 M of sucrose ≪ / RTI > wherein the ovary is freeze-dried.
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