한국산학기술학회논문지 (Journal of the Korea Academia-Industrial cooperation Society)

  • 제21권6호
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  • Pages.527-535
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  • 2020
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  • 1975-4701(pISSN)
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  • 2288-4688(eISSN)
한국산학기술학회 (The Korea Academia-Industrial cooperation Society)
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Polyampholyte가 소난관상피세포의 초자화 동결방법에 미치는 영향

The Effects of Polyampholyte on Vitrification Process for cryopreservation of Bovine Oviduct Epithelial Cell

  • 김성우 (농촌진흥청 국립축산과학원 가축유전자원센터) ;
  • 이재영 (농촌진흥청 국립축산과학원 가축유전자원센터) ;
  • 김찬란 (농촌진흥청 국립축산과학원 가축유전자원센터) ;
  • 유연희 (농촌진흥청 국립축산과학원 가축유전자원센터) ;
  • 이성수 (농촌진흥청 국립축산과학원 가축유전자원센터) ;
  • 고응규 (농촌진흥청 국립축산과학원 가축유전자원센터)
  • Kim, Sung Woo (Animal Genetic Resources Research Center, National Institute of Animal Science, RDA) ;
  • Lee, Jae-Yeong (Animal Genetic Resources Research Center, National Institute of Animal Science, RDA) ;
  • Kim, Chan-Lan (Animal Genetic Resources Research Center, National Institute of Animal Science, RDA) ;
  • Yu, Yeonhee (Animal Genetic Resources Research Center, National Institute of Animal Science, RDA) ;
  • Lee, Sung Soo (Animal Genetic Resources Research Center, National Institute of Animal Science, RDA) ;
  • Ko, Yeoung-Gyu (Animal Genetic Resources Research Center, National Institute of Animal Science, RDA)
  • 투고 : 2020.04.22
  • 심사 : 2020.06.05
  • 발행 : 2020.06.30
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초록

본 연구의 목적은 가축의 조직에서 유래한 동물세포를 재생산하기 위하여 세포를 동결하는 방법으로 초자화 동결법을 간단하게 이용하는 조건을 확립하고자 하였다. 소난관상피세포를 초자화 동결법에 적용하기 위하여 소 난관상피세포를 0.25ml 스트로에 밀봉하여 액체질소에 직접 노출하였다. 발정 3.5일자의 난관에서 추출된 소 난관상피세포는 polyampholyte가 주성분인 StemCell KeepTM을 구매하여 초자화 동결을 유도하였고 5, 10, 25, 50, 75 및 100% 농도에서 생존율을 조사하였다. 세포의 생존성은 트립판 블루염색기법과 SYTO-13/PI 핵 염색시약을 이용하여 차별적 생사염색기법을 이용하여 분석하였다. Trypan blue 염색법에서는 각각 5.6±11.8, 12.5±7.2, 53.0±2.7, 85.1±6.9, 79.8±0.6 및 60.7±6.7%의 생존율이 관찰되었고, SYTO-13/PI 염색시약에서는 각각 4.6±2.5, 30.8±12.1, 58.4±2.5, 85.5±1.2, 79.8±0.6 및 71.2±1.2%의 생존율이 관찰되었다. 이러한 결과는 소 난관상피세포는 50% StemCell KeepTM 농도의 동결배양액을 이용하여 동결 보존하는 것이 가장 우수한 생존율을 얻을 수 있었고 세포 유전자원 은행을 위한 영구보존에 적절할 것으로 판단된다.

The purpose of this study was to establish a simple vitrification protocols to preserve animal cell lines derived from tissues of livestock that could be recultured. Bovine oviduct epithelial cells (BOEC) were used for the vitrification process using a 0.25 ml straw to increase cryopreservation efficiency. BOEC was cultured from the oviduct of 3.5-day estrus state, and the commercially available polyampholyte StemCell KeepTM was used as a cryoprotective agent. Using different concentrations, the viability rates of BOEC in 5, 10, 25, 50, 75, and 100% in freezing media were investigated. Survivability was determined using a differential staining technique using a trypan blue test and a CYTO-13/PI staining protocol. The viability rates of BOEC in the trypan blue test were 5.6±11.8, 12.5±7.2, 53.0±2.7, 85.1±6.9, 79.8±0.6, and 60.7±6.7% with a respective concentration of StemCell KeepTM. The viability rates in CYTO-13/PI staining were 4.6±2.5, 30.8±12.1, 58.4±2.5, 85.5±1.2, 79.8±0.6, and 71.2±1.2%, respectively. These results indicate that BOEC could be preserved with StemCell KeepTM without toxicity in a 0.25-ml straw. The optimal concentration of vitrification solution with StemCell KeepTM was determined to be 50% and can be considered as a proper preservation method for cryobanking.

키워드

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