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생쥐배포의 유리화 동결보존에 관한 연구

Vitrification of Mouse Blastocyst

김미영|이석원|이여일|이유진
대한산부인과학회 (Korean Soceity of Obstetrics and Gynecology)|2004.07|Obstetrics & Gynecology Science|제47권 제7호|pp. 1348-1354

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Vitrification생쥐배포EFS40용액VS용액유리화동결Mouse blastocystEFS40 solutionVS solution
주제분류산부인과학

초록

목적 : 본 연구는 생쥐 배포기 배아인 배포를 이용하여 두 가지 동결보호제로 유리화 동결 보존 및 해동 후 배아의 생존율과 발달률을 비교하고자 시행하였다. 연구 방법 : 배포를 획득하기 위하여 4-6주된 ICR계 계통의 암컷과 8-12주령의 수컷 생쥐를 이용하여 PMSG와 hCG로 과배란 유도한 후 수정 48시간 째에 난관을 통해 2세포기의 수정란을 회수하여 3일 동안 10% Serum Substitute Supplement (SSS)가 첨가된 Human Tubal Fluid (HTF) 배양액으로 체외배양 시켰으며, 배포기로 성장한 배아를 선별하여 동결하였다. 동결시 사용되는 동결보호제로는 40% ethylene glycol, 18% ficoll, 0.5 M sucrose가 함유된 EFS40용액과 20% ethylene glycol, 20% DMSO, 10% 1,3-butanediol이 함유된 VS용액을 사용하였다. 각각의 용액에 노출시켜 배포에 대한 독성을 점검한 후에, EFS40용액은 3단계의 동결법으로 유리화동결 후 0.5 M sucrose의 농도를 5단계로 낮춰주면서 해동을 실시하였고, VS용액은 2단계의 동결법으로 유리화동결 후 1.0 M의 trehalose를 이용하여 해동을 실시하고, 회수된 배포의 회수율 및 생존율과 부화 배포로의 배아 발달률인 부화율을 비교하였다. 결과 : 각각의 용액에 동결없이 노출 후 발달을 유도한 결과 EFS40군에서는 95%의 생존율과 100%의 부화율을 보인 반면, VS군에서는 100%의 생존율과 87.5%의 부화율을 보였다. 각 동결보호제를 이용하여 동결보존 및 해동시킨 후 그 발달률을 관찰해 본 결과, 먼저 회수율에서 EFS40군은 73.7%, VS군은 66.5%를 보였고, 생존율은 24시간 배양 후 EFS40군은 80.5%, VS군은 66%, 48시간 배양 후 EFS40군은 69%, VS군은 58.3%의 결과를 보였다. 부화율은 24시간 배양 후 EFS40군에서는 20.7%, VS군에서는 0%, 48시간 배양 후 EFS40군은 33.3%, VS군은 1.9%를 보여 EFS40군이 VS군에 비해 생존율과 부화율에서 각각 유의하게 높았다. 결론 : 이러한 결과로 미루어 보아 생쥐 배포의 유리화동결을 위한 동결보호제로는 VS용액보다 EFS40용액이 더 적합할 것으로 사료된다

Abstract

Objective : The aim of this study was to compare the survival and developmental rate of two vitrification solutions for the vitrification of mouse expanded blastocysts. Methods : Mouse embryos were obtained at 2 cell stage and cultured to expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% serum substitute supplement (SSS). The vitrification solutions used were EFS40 and VS. EFS40 consisted of 40% ethylene glycol, 18% ficoll, and 0.5 M sucrose while VS consisted of 20% ethylene glycol, 20% DMSO, and 10% 1,3-butanediol diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% calf serum (CS). Toxicity was tested by exposing expanded blastocysts to vitrification solution. The vitrification procedure used for EFS40 was performed in three steps, after which they were warmed in 5 steps with 0.5 M sucrose. VS was performed in two steps, after which they were warmed with 1.0 M trehalose. Recovery, survival and hatching rate per expanded blastocysts recovered were compared between two groups. Results : In toxicity test, survival and hatching rate of EFS40 group were 95% and 100%, respectively. In contrast, survival and hatching rate of VS group were 100% and 87.5%, respectively. After vitrification and warming in solution, recovery rate for EFS40 group was 73.7% whereas recovery rate for VS group was 66.5%. After 24 h culture, survival and hatching rate were 80.5% and 20.7% for EFS40 and 66% and 0% for VS group, respectively. After 48 h culture, survival and hatching rate were 69% and 33.3% for EFS40 and 58.3% and 1.9% for VS group, respectively. Survival and hatching rate in EFS40 group were significantly higher than those found in VS group. Conclusion : The EFS40 solution was better than VS solution for vitrification of mouse expanded blastocysts.

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