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

The Korea Academia-Industrial cooperation Society (한국산학기술학회)
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The Effects of Polyampholyte on Vitrification Process for cryopreservation of Bovine Oviduct Epithelial Cell

Polyampholyte가 소난관상피세포의 초자화 동결방법에 미치는 영향

  • 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)
  • 김성우 (농촌진흥청 국립축산과학원 가축유전자원센터) ;
  • 이재영 (농촌진흥청 국립축산과학원 가축유전자원센터) ;
  • 김찬란 (농촌진흥청 국립축산과학원 가축유전자원센터) ;
  • 유연희 (농촌진흥청 국립축산과학원 가축유전자원센터) ;
  • 이성수 (농촌진흥청 국립축산과학원 가축유전자원센터) ;
  • 고응규 (농촌진흥청 국립축산과학원 가축유전자원센터)
  • Received : 2020.04.22
  • Accepted : 2020.06.05
  • Published : 2020.06.30
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Abstract

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.

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.

Keywords

References

  1. A. K. Belew, K. Tesfaye, G. Belay, "The state of conservation of animal genetic resources in developing countries: a review", Int. J. Pharm. Med. Biol. Sci., Vol.5, No.1, pp.58-66, Jun 2016. DOI: https://doi.org/10.18178/ijpmbs.5.1.58-66
  2. Y. Hoshino, K. Saeki, "Animal cloning by nuclear transfer using samatic cells recovered from organs frozen without cryoprotectant", J. Mamm. Ova Res., Vol.27, No.3, pp.93-100, September 2010. DOI: https://doi.org/10.1274/jmor.27.93
  3. D. N. Wells, "The integration of cloning by nuclear transfer in the conservation of animal genetic resources", F. Farm Animal. Genet. Resours., Vol.30, No.3, pp.223-241, Febuary 2018. DOI: https://doi.org/10.1017/S0263967X0004204X
  4. K. H. Campbell, "A background to nuclear transfer and its applications in agriculture and human therapeutic medicine", J. Anat., Vol.200, No.3, pp.267-275, March 2002. DOI: https://doi.org/10.1046/j.1469-7580.2002.00035.x
  5. K. Bowey-Dellinger, L. Dixon, K. Ackerman, C. Vigueira, Y. K. Suh, T. Lyda, K. Sapp, M. Grider, D. Crater, T. Russell, M. Elias, V. Mc. Coffield, V. A. Segarra, "Introducing mammalian cell culture and cell viability techniques in the undergraduate biology laboratory", J. Microbiol. Biol. Educ., Vol.18, No.2, pp.1-7, August 2017. DOI: https://doi.org/10.1128/jmbe.v18i2.1264
  6. J. G. Baust, D. Gao, J. M. Baust, "Cryopreservation An emerging paradigm change", Organogenesis, Vol.5, No.3, pp.90-96, September 2009. DOI: https://doi.org/10.4161/org.5.3.10021
  7. N. Kotobuki, M. Hirose, H. Machida, Y. Katou, K. Muraki, Y. Takakura, H. Ohgushi, "Viability and osteogenic potential of cryopreserved human bone marrow-derived mesenchymal cells", Tissue Eng., Vol.11, No.5-6, pp.663-673, May-Jun 2005. DOI: https://doi.org/10.1089/ten.2005.11.663
  8. J-E. Oh, K. Karlmark Raja, J-H. Shin, A. Pollak, M. Hengstschlager, G. Lubec, "Cytoskeleton changes following differentiation of N1E-115 neuroblastoma cell line.", Amino Acids, Vol.31, No.3, pp.289-298, October 2006. DOI: https://doi.org/10.1007/s00726-005-0256-z
  9. C. J. Hunt. "Cryopreservation: vitrification and controlled rate cooling.", Methods Mol. Biol., Vol.1590, No.3, pp.41-77, March 2017. DOI: https://doi.org/10.1007/978-1-4939-6921-0_5
  10. G. M. Fahy, D. R. MacFarlane, C. A. Angell, H. T. Meryman, "Vitrification as an approach to cryopreservation." Cryobiology, Vol.21, No.4, pp.407-426, August 1984. DOI: https://doi.org/10.1016/0011-2240(84)90079-8
  11. K. R. Fowke, J. Behnke, C. Hanson, K, Shea, LM. C. Cosentino, "Apoptosis: a method for evaluating the cryopreservation of whole blood and peripheral blood mononuclear cells." J. Immunol. Methods., Vol.244, No.1-2, pp.136-144, October 2000. DOI: https://doi.org/10.1016/s0022-1759(00)00263-5
  12. J. Yang, N. Diaz, J. Adelsberger, X, Zhou, R. Stevens, A. Rupert, J. A. Metcalf, M. Baseler, C. Barbon, T. Imamichi, R. Lempicki, L. M. Cosentino, "The effects of storage temperature on PBMC gene expression", BMC Immunol., Vol.17, No.6, March 2016. DOI: https://doi.org/10.1186/s12865-016-0144-1
  13. N. Taghizabet, M. A. Khalili, F. Anbari, A. Agha-Rahimi, S. A. Nottola, G. Macchiarelli, M. G. Palmerini, "Human cumulus cell sensitivity to vitrification, an ultrastructural study", Zygote, Vol.26, No.3, pp.224-231, Jun 2018. DOI: https://doi.org/10.1017/S0967199418000138
  14. T. H. Jang, S. C. Park, J. H. Yang, J. Y. Kim, J. H. Seok, U. S. Park, C. W. Choi, S. R. Lee, J. Han, "Cryopreservation and its clinical applications", Integr. Med. Res., Vol.6, No.1, pp.12-18, March 2017. DOI: https://doi.org/10.1016/j.imr.2016.12.001
  15. P. Mazur, "Cryobiology: The freezing of biological systems", Science, Vol.168, No.3934, pp.939-949, May 1970. DOI: https://www.jstor.org/stable/1729310 https://doi.org/10.1126/science.168.3934.939
  16. D. Gao, J. K. Critser, "Mechanisms of cryoinjury in living cells", ILAR J., Vol.41, No.4, pp.187-196, October 2000. DOI: https://doi.org/10.1093/ilar.41.4.187
  17. H. J. Kim, J. H. Lee, Y. B. Hur, C. W. Lee, S-H. Park, B-W. Koo. "Marine antifreeze proteins: structure, function, and application to cryopreservation as a potential Cryoprotectant", Mar. Drugs, Vol.15, No.2, Article 27, Febuary 2017. DOI: https://doi.org/10.3390/md15020027
  18. R. Gupta, R. Deswal, "Antifreeze proteins enable plants to survive in freezing conditions", J. Boisci., Vol.39, No.5, pp.931-944, December 2014. DOI: https://doi.org/10.1007/s12038-014-9468-2
  19. R. Surís-Valls, I. K. Voets, "Peptidic antifreeze materials: prospects and challenges", Int. J. Mol. Sci., Vol.20, No.20, Article 5149, October 2019. DOI: https://doi.org/10.3390/ijms20205149
  20. K. Matsumura, J. Y. Bae, S. H. Hyon, "Polyampholytes as cryoprotective agents for mammalian cell cryopreservation", Cell Transplantation, Vol.19, No.6, pp.691-699, Jun 2010. DOI: https://doi.org/10.3727/096368910X508780
  21. K. Matsumura, K. Kawamoto, M. Takeuchi, S. Yoshimura, D. Tanaka, S. H. Hyon, "Cryopreservation of a two-dimensional monolayer using a slow vitrification method with polyampholyte to inhibit ice crystal formation", ACS Biomater. Sci. Eng., Vol.2, No.6, pp.1023-1029, April 2016. DOI: https://doi.org/10.1021/acsbiomaterials.6b00150
  22. R. C. Deller, M. Vatish, D. A. Mitchell, M. I. Gibson, "Glycerol-free cryopreservation of red blood cells enabled by ice-recrystallization-inhibiting polymers", ACS Biomater. Sci. Eng., Vol.1, No.9, pp.789-794, April 2015. DOI: https://doi.org/10.1021/acsbiomaterials.5b00162
  23. B. D. Bavister, "Role of oviductal secretions in embryonic growth in vivo and in vitro", Theriogenology, Vol.29, No.1, pp.143-154, January 1988. DOI: https://doi.org/10.1016/0093-691X(88)90037-4
  24. C. Polge, A. U. Smith, A. S. Parkes, "Revival of spermatozoa after vitrification and dehydration at low temperatures." Nature, Vol.164, pp.666, October 1949. DOI: https://doi.org/10.1038/164666a0
  25. J. E. Lovelock, M. W. H. Bishop, "Prevention of freezing effect of antifreeze glycopeptides on membrane potential damage to living cells by dimethyl sulphoxide." Nature, Vol.183, pp.1394-1395, May 1959. DOI: https://doi.org/10.1038/1831394a0
  26. G. S. Jiang, K. H. Bi, T. H. Tang, J. W. Wang, Y. K. Zhang, W. Zhang, H. Q. Ren, H. Q. Bai, Y. S. Wang, "Down-regulation of TRRAP-dependent hTERT and TRRAP-induced CAD activation by Myc/Max contributes to the differentiation of HL60 cells after exposure to DMSO." Int. Immunopharmacol., Vol.6, No.7, pp.1204-1213, August 2006. DOI: https://doi.org/10.1016/j.intimp.2006.02.014
  27. B. R. Chakravarthy, R. Tremblay, P. Macdonald, V. Krsmanovic, J. F. Whitfield, J. P. Durkin, "The activation of inactive membrane-associated protein kinase C is associated with DMSO-induced erythroleukemia cell differentiation." Biochm. Biophys Act ., Vol.1136, No.1, pp.83-90, October 1991. DOI: https://doi.org/10.1016/0167-4889(92)90088-S
  28. M. Ankarcrona, J. M. Dypbukt, E. Bonfoco, B. Zhivotovsky, S. Orrenius, S. A. Lipton, P. Nicotera, "Glutamate-induced neuronal death: A Succession of necrosis or apoptosis depending on mitochondrial function." Neuron, Vol.15, No.4, pp.961-973, October 1995. DOI: https://doi.org/10.1016/0896-6273(95)90186-8
  29. S. W. Kim, N. Sharma, I-S. Hwang, C. Choe, D. Kim, H-H Seong, D. K. Jeong, "Application of saponin on differential staining examination in animal blastocysts", Ind. J. Anim. Sci., Vol.87, No.9, pp.1081-1086, September 2017. http://epubs.icar.org.in/ejournal/index.php/IJAnS/article/view/74292