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대한생식의학회지 제28권 제3호 2010년
생쥐 1-세포기 수정란의 동결방법에 있어서 초자화동결과 완만동결의 비교
상지대학교 생명과학과1, 연세대학교 원주기독병원 산부인과2
이지향, 이지향1,한혁동2,구혜영1
Comparison of Vitrification and Slow Freezing-thawing Method on 1-cell Zygotes
Ji-Hyang Lee1, Hyuck-Dong Han2, Hye-Young Koo1
Yonsei University, Wonju College of Medicine, Wonju, Korea
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Objective: This study was conducted to examine the effect of vitrification on the survival and in vitro development of mice 1-cell zygotes. Method: Effects of exposure to vitrification solution and vitrification, with different concentrations of the cryoprotectant solution, were examined. The 1-cell zygotes were also subjected to a slow freezing- thawing method to compare with vitrification method. Solution composed of ethylene glycol (6.0 M, 5.0 M, 4.0 M) and sucrose (1.0 M) were used as cryopropectant. The experiments employed the method loading the embryos on electron microscope grids. Results: I. The effects of exposure in vitrification solution 1-cell zygotes were non-toxic at all concentrations of the vitrification solution showing the survival rate between 88.1% and 97.5%. Development into 2-cell was more successful in the higher concentrations of the vitrification solution. Therefore, higher concentrations of the vitirification solution do not seem to cause any problems in vitrification procedure. II. The effects of vitrification method 1-cell zygotes showed the survival rate between 78.8% and 92.4%. The lowest and the highest survival rate was observed in the 6.0 M and 4.0 M vitrification solution, respectively. 2-cell development rates varied from 77.6% to 91.3%. Blastocyst development rate was shown highest in 5.0 M and the lowest in 4.0 M solution. Therefore, the highest 2-cell and blastocyst development rate was observed in 5.0 M solution. III. Comparison of vitrification and slow freezing-thawing method on 1-cell zygotes This experiment showed that 1-cell zygotes had the highest survival and development rates in 5.0 M vitrification solution. Vitrified group of 1-cell zygotes, in the 5.0 M vitrification solution, were compared with the group processed in slow freezing-thawing method. The development rate into 2-cell and blastocyst as well as the survival rate were higher in the vitrified group than in the slowly freezed group. Conclusion: 1. The results demonstrate that the best cryoprotectant is a 5.0 M vitrification solution for 1-cell zygotes. 2. Vitrification method significantly increases the survival rate of the 1-cell zygote and its development into 2-cell and blastocyst. Equilibration and exposure time during the vitrification was remarkerbly short in this experiment. Total time, from the exposure to vitirification solution to storage in the liquid nitrogen, was taken only 90 seconds. In contrast, the slow freezing-thawing method have taken more than four hours. Taken together, we presume that the overall time used for the procedure contributes to the results as an important parameter. 3. The loading of 1-cell zygotes on the EM grid is technically more simple and takes less time than the straw or cryo vial method.
키워드 : Cryoprotectant, Vitrification, Ethylene glycol, Electron microscope grid
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