Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
권호 표지

Modification of Efficient Vitrification Method by Using Open Pulled Straw (OPS) and EM Grid as Vehicles in Human Embryonic Stem Cell

인간 배아 줄기세포의 OPS와 Grid를 이용한 유리화 동결법의 효율성 비교

  • 박규형 (포천중문의과대학교 생명과학대학원 분자발생과) ;
  • 최성준 (포천중문의과대학교 생명과학대학원 분자발생과) ;
  • 김희선 (서울대학교 의학연구원) ;
  • 오선경 (서울대학교 의학연구원) ;
  • 문신용 (서울대학교 의학연구원) ;
  • 차광렬 (포천중문의과대학교 세포 및 유전자치료연구소) ;
  • 정형민 (포천중문의과대학교 세포 및 유전자치료연구소)
  • Published : 2003.12.01
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Abstract

Human embryonic stem (hES) cell lines have been derived from human blastocysts and are expected to have far-reaching applications in regenerative medicine. The objective of this study is to improve freezing method with less cryo-injuries and best survival rates in hES cells by comparing various vitrification conditions. For the vitrifications, ES cells are exposed to the 4 different cryoprotectants, ethylene glycol (EG), 1,2-propanediol (PROH), EG with dime-thylsulfoxide (DMSO) and EG with PROH. We compared to types of vehicles, such as open pulled straw (OPS) or electron microscopic cooper grids (EM grids). Thawed hES cells were dipped into sequentially holding media with 0.2 M sucrose for 1 min, 0.1 M sucrose for 5 min and holding media for 5 min twice and plated onto a fresh feeder layer. Survival rates of vitrified hES cells were assessed by counting of undifferentiated colonies. It shows high survival rates of hES cells frozen with EG and DMSO (60.8％), or EG and PROH(65.8％) on EM grids better than those of OPS, compared to those frozen with EG alone (2.4％) or PROH alone (0％) alone. The hES cells vitrified with EM grid showed relatively constant colony forming efficiency and survival rates, compared to those of unverified hES cells. The vitrified hES cells retained the normal morphology, alkaline phosphates activity, and the expression of SSEA-3 and 4. Through RT-PCR analysis showed Oct-4 gene expression was down-regulated and embryonic germ layer markers were up-regulated in the vitrified hES cells during spontaneous differentiation. These results show that vitrification method by using EM grid supplemented with EG and PROH in hES cells may be most efficient at present to minimize cyto-toxicity and cellular damage derived by ice crystal formation and furthermore may be employed for clinical application.

Keywords

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