Clinical and Experimental Reproductive Medicine

The Korean Society for Reproductive Medicine (대한생식의학회)
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Effect on Survival and Developmental Competence of Vitrified Mouse Embryos Using Various Cryoprotectants and Cooling Speeds

생쥐 배아의 유리화 동결에 동결액의 조성과 냉각속도의 영향

  • Park, Jae-Kyun (Fertility Center of CHA Gangnam Medical Center, CHA University College of Medicine) ;
  • Go, Young-Eun (Department of Biomedical Science, College of Life Science, CHA University) ;
  • Eum, Jin-Hee (Fertility Center of CHA Gangnam Medical Center, CHA University College of Medicine) ;
  • Won, Hyung-Jae (Fertility Center of CHA Gangnam Medical Center, CHA University College of Medicine) ;
  • Lee, Woo-Sik (Fertility Center of CHA Gangnam Medical Center, CHA University College of Medicine) ;
  • Yoon, Tae-Ki (Fertility Center of CHA Gangnam Medical Center, CHA University College of Medicine) ;
  • Lee, Dong-Ryul (Fertility Center of CHA Gangnam Medical Center, CHA University College of Medicine)
  • 박재균 (차의과학대학교 강남차병원 여성의학연구소) ;
  • 고영은 (차의과학대학교 의생명과학과) ;
  • 엄진희 (차의과학대학교 강남차병원 여성의학연구소) ;
  • 원형재 (차의과학대학교 강남차병원 여성의학연구소) ;
  • 이우식 (차의과학대학교 강남차병원 여성의학연구소) ;
  • 윤태기 (차의과학대학교 강남차병원 여성의학연구소) ;
  • 이동률 (차의과학대학교 강남차병원 여성의학연구소)
  • Received : 2010.11.16
  • Accepted : 2010.11.29
  • Published : 2010.12.31
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Abstract

Objective: Vitrification requires a high concentration of cyroprotectant (CPA) and an elevated cooling speed to avoid ice crystal formation. We have evaluated the effect of different combinations of cooling rate and CPA on embryonic integrity (developmental competence) in order to increase the efficiency of vitrification without impairing embryo viabilit. We hypothesized that the combination of CPA or the increase of cooling rates can reduce the concentration of toxic CPA for vitrification. As consequently, we performed experiments to evaluate the effect of various composition of CPA or slush nitrogen ($SN_2$) on the mouse embryonic development following vitrification using low CPA concentration. Methods: Vitrification of mouse embryos was performed with EM grid using liquid nitrogen ($LN_2$) or $SN_2$ and different composition of CPAs, ethylene glycol (EG) and dimethylsulfoxide (DMSO). After vitrification-warming process, their survival and blastocyst formation rates were examined. For analyzing long-term effect, these blastocysts were transferred into the uterus of foster mothers. Results: Survival and blastocyst formation rates of vitrified embryos were higher in EG+DMSO group than those in EG only. Furthermor, the group using $SN_2$ with a lower CPA concentration showed a higher survival of embryos and developmental rates than group using $LN_2$. Conclusion: The combination of EG and DMSO as CPAs may enhance the survival of mouse embryos and further embryonic development after vitrification. $SN_2$ can generate high survival and developmental rate of vitrified/warmed mouse embryos when a lower concentration of CPA was applied. Therefore, these systems may contribute in the improvement of cryopreservation for fertility preservation.

목적: 유리화 동결액의 조성조절과 냉각속도 증진을 통한 동결보호제의 농도를 낮추는 전략을 통해 세포에 미치는 독성을 감소시켜 유리화 동결 및 융해 후 생쥐 배아의 생존율 및 발생률을 증진시키고, 궁극적으로 배아의 유리화 동결법을 개선하고자 하였다. 연구방법: 생쥐 배아와 포배기를 그리드를 이용한 유리화 동결법을 이용하여 동결/융해하였다. 동결액 내 ethylene glycol와 dimethylsulphoxide (DMSO)의 농도와 당의 농도를 조절하여 생쥐 배아의 융해 후 생존율과 발생률을 관찰하였고, 냉각속도의 증가와 동결억제제의 농도와의 상관관계를 포배기의 융해 후 생존율과 발생률에 따라 비교하였다. 또한 융해 후 배아를 대리모에 이식하여 산자를 생산함으로 냉각속도의 효율성을 알아보았다. 결과: EG를 단독으로 사용한 동결보존액 보다는 DMSO와 혼합된 동결보존액의 사용이 보다 유리하다는 결과를 얻을 수 있었다. 슬러시 질소에 의한 냉각속도의 증가가 동결보존의 대상의 상해를 줄여 유리화 동결의 효율성을 증진시키는 것으로 생각된다. 결론: 혼합된 동결보호제를 사용하였을 때 생쥐 배아의 유리화 동결 후 생존과 발생률이 증진되었다. 슬러시 질소를 이용한 유리화 동결의 도입은 냉각속도의 증가를 통해 기존 유리화 동결방법의 효율을 증진시켜 생존율과 융해후 발생률, 임신율 증진에 기여하였다. 또한 냉각속도의 증진은 유리화 동결의 필수요건인 고농도의 동결억제제에 대한 노출을 감소시킬 수 있었다. 이러한 노력은 생식력의 보전을 위한 유리화 동결법의 효율 향상에 기여할 것이다.

Keywords

References

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