KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
권호 표지

Development of hypothermic preservation solution for the human dermal fibroblast using protein hydrolysates

단백질 가수분해 물을 이용한 인간 피부 섬유아세포의 저온 보존액 개발

  • 변순휘 (건국대학교 미생물공학과) ;
  • 최태부 (건국대학교 미생물공학과)
  • Published : 2009.06.29
Download PDF
⟨ Previous Next ⟩

Abstract

Stable cell preservation is an essential factor in the regenerative medicine for cell therapies and transplantation of biologic materials. In this study, we studied to provide more stable hypothermic preservation by protection of cell damage during the preservation at $4^{\circ}C$. The result of searching for key components that have excellent efficacy in hypothermic preservation of cells, we have identified the fact that the hypothermic preservation adding protein hydrolysates such as yeast hydrolysate is far superior to others. All protein hydrolysates that are derived from animal, plant and microbe sources have superior efficacy, especially the peptides which have molecular weights under 10 kDa have the best efficacy among the components of protein hydrolysate. The protein hydrolysates prevented the decrease of ATP level in the cells caused by hypothermic environment and they inhibited the generation of ROS. Adding antioxidants and control agents of osmotic pressure were showed to have more superior efficacy in hypothermic preservation. Finally, KUL261 solution (DMEM/F12 1 : 1 medium, yeastolate 1%, $\alpha$-tocopherol $100{\mu}M$, dextran 2.5%), the preservation solution developed in this study, showed the best efficacy in both cell viability and cell growth more than other conventional preservation solutions. In conclusion, the improved hypothermic preservation solution that contains the protein hydrolysates as a key component provide the best preservation efficacy. It provides better efficacy than other preservation solutions and will contribute to both the development of regenerative medicine and global commercialization in this therapeutic field.

치료용 단백질을 생산하는 생물의약품 산업이나 세포치료제 및 이식용 세포를 다루는 재생의학 분야 등의 세포기반 산업에서 안정적인 세포의 보존은 필수적인 요소이다. 본 연구에서는 인간 피부 섬 유아세포의 $4^{\circ}C$ 저온보존에서 우수한 성능을 나타내는 개선된 저온보존액을 개발하고, 저온에 의한 세포 손상을 보호함으로써 보다 안정적인 세포 저온보존 기술을 제공하고자 하였다. 세포의 저온보존에서 우수한 효능을 나타내는 핵심 성분을 탐색한 결과, yeast hydrolysate 등의 단백질 가수분해물을 첨가한 보존액에서 월등히 뛰어난 보존효과가 나타남을 확인하였다. 단백질 가수분해 물은 미생물, 식물, 동물유래 단백질 가수분해 물에서 모두 우수한 효과를 나타냈으며, 특히 단백질 가수분해물 성분 중 분자량 10kDa 이하의 펩타이드를 첨가한 저온보존에서 우수한 보존효과가 나타났다. 저온에 의한 세포손상에 대해 단백질 가수분해물은 세포내 ATP level의 감소를 막아주고 ROS 생성을 억제하는 것으로 나타났으며, 항산화제 및 삼투압 조절물질을 단백질 가수분해 물과 함께 첨가하였을 때 더욱 우수한 세포 보존효과를 보였다. 최종적으로 본 연구에서 개발한 KUL261 저은보존액 (DMEM/F12 1 : 1 medium, yeastolate 1%, $\alpha$-tocopherol $100{\mu}M$, dextran 2.5%)은 기존의 저온 보존액에 비해 세포 생존을 및 성장률에서 월등히 우수한 성능을 나타내었다. 결론적으로, 핵심 유효성분으로 단백질 가수분해물을 포함하는 개선된 저온보존액은 기존의 보존액보다 월등히 우수한 보존효과를 제공하며, 세포치료제 및 재생의학 분야의 발전과 글로벌 상업화에 기여할 수 있을 것이다.

Keywords

References

  1. Aejaz, H. M., Aleem, A. K., Parveen, N., Khaja, M.N., Narusu, M. L., and Habibullah, C. M. (2007), Stem cell therapy-present status, Transplantation Proceedings 39(3), 694-699 https://doi.org/10.1016/j.transproceed.2007.01.069
  2. Aleem Khan, A., Parveen, N., Habeeb, and M. A., Habibullah, C. M. (2006), Journey from hepatocyte transplantation to hepatic stem cells: a novel treatment strategy for liver diseases, The lndian Journal of Medical Research 123(5), 601-614
  3. Webber, D. J. and Minger, S. L. (2004), Therapeutic potential of stem cells in central nervous system regeneration, Current Opinion in lnvestigational Drugs 5(7), 714-719
  4. Yard, B., Beck, G., Schnuelle, P., Braun, C., Schaub, M., Bechtler, M., Gottmann, U., Xizo, Y., Breedijk, A., Wandschneider, S., Losel, R., Sponer, G., Wehling, M., and van der Woude F. J. (2004), Prevention of cold-preservation injury of cultured endothelial cells by catecholamines and related compounds, American Journal of Transplantation 4(1), 22-30 https://doi.org/10.1046/j.1600-6143.2003.00268.x
  5. Taylor M. J., et. al (1996), Hypothermia in relation to the acceptable limits of ischemia for bloodless surgery, In Steponkus PL, Advances in Low-temperature Biology Eds.; JAI Press, London, pp1-64
  6. Nowak, G., Ungerstedt, J., Wemerson, A., Ungerstedt, U., and Ericzon, B. G. (2003), Hepatic cell membrane damage during cold preservation sensitizes liver grafts 10 rewarmling injury, Journal of Hepato-biliary-pancreatic Surgery 10, 200-205 https://doi.org/10.1007/s00534-002-0760-4
  7. Breton, S. and Brown, D. (1998), Cold-inclduced microtubule disruption and relocalization of membrane proteins in kidney epithelial cells, Journal of the American Society of Nephrology 9(2), 155-166
  8. Mitchell, S. J., Churchill, T. A., Winslet, M. C., Fuller, B. J. (1999), Energy metabolism following prolonged hepatic cold preservation: benefits of interrupted hypoxia on the adenine nucleotide pool in rat liver, Cryobiology 39(2), 130-137 https://doi.org/10.1006/cryo.1999.2191
  9. Vajdov$\'{a}$, K , Graf, R., and Clavien, P. A. (2002), ATPsupplies in the cold-preserved liver: A long-neglected factor of organ viability, Hepatology 36(6), 1543-1552 https://doi.org/10.1002/hep.1840360633
  10. Abrahamse, S. L., van Runnard Heimel, P., Hartman, R. J., Chamuleau, R. A., and van Gulik, T. M. (2003), Induction of necrosis and DNA fragmentation during hypothermic preservation of hepatocytes in UW, HTK, and Celsior solutions, Cell Transplantation 12(1), 59-68 https://doi.org/10.3727/000000003783985160
  11. Hart, N. A., Leuvenink Henri, G. D., and Ploeg, R. J. (2002), New solutions in organ preservation, Transplantation Reviews 16(3), 131-141 https://doi.org/10.1053/trre.2002.124340
  12. Meng, Q. (2003), Hypothermic preservation of hepatocytes, Biotechnology Progress 19(4), 1118-1127 https://doi.org/10.1021/bp025628n
  13. Shirouzu, Y., Gu, Y., Koga, M., Sakurai, T., Qi, M., Hiura, A., Sumi, S., and Inoue, K. (2006), Cold preservation of islets in UW solution-with special reference to apoptosis, The Journal of Surgical Research. 133(2), 167-175 https://doi.org/10.1016/j.jss.2005.10.006
  14. Roberts, R. F ., Nishanian, G. P., Carey, J. N., Sakamaki, Y ., Starnes, V. A., and Barr, M. L. (1999), A comparison of the new preservation solution Celsior to Euro-Collins and University of Wisconsin solutions in lung reperfusion injury, Transplantation 67(1), 152-155 https://doi.org/10.1097/00007890-199901150-00025
  15. Janssen, H., Janssen, P. H., and Broelsch, C. E. (2004), UW is superior to Celsior and HTK in the protection of human liver endothelial cells against preservation injury, Liver Transplantation 10(12), 1514-1523 https://doi.org/10.1002/lt.20309
  16. McAnulty, J. F. and Huang, X. Q. (1997), The efficacy of antioxidants administered during low temperature storage of warm ischemic kidney tissue slices, Cryobiology 34(4), 406-415 https://doi.org/10.1006/cryo.1997.2011
  17. Chiang, C. H., Wu, K., Yu, C. P., Pemg, W. C., Yan, H. C., Wu, C. P., Chang, D. M., and Hsu, K. (1998), Protective agents used as additives in University of Wisconsin solution to promote protection against ischaemia-reperfusion injury in rat lung, Clinical Science 95(3), 369-376 https://doi.org/10.1042/CS19980032
  18. Mosbah, I. B., Saidane, D., Peralta, C., Rosell$\'{o}$-Catafau J., and Abdennebi, H. B. (2005), Effiicacy of polyethyleneglycols in University of Wisconsin preservation solutions: a study of isolated perfused rat liver, Transplantation Proceedings 37(9), 3948-3950 https://doi.org/10.1016/j.transproceed.2005.10.038
  19. Surowka, K. and Zmudzinski, D. (2004), Functional properties modification of extruded soy protein concentrate using Neutrase, Czech Journal of Food Sciences 22(5), 163-174
  20. Crouch, S. P., Kozlowski, R , Slater, K. J., and Fletcher, J. (1993), The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity, Journal of lmmunological Methods 160(1), 81-88 https://doi.org/10.1016/0022-1759(93)90011-U
  21. Carter, W. O., Narayanan, P. K., and Robinson, J. P. (1994), Intracellular hydrogen peroxide and superoxide hydroon detection in endothelial cells, Journal of Leukocyte Biology 55(2), 253-258 https://doi.org/10.1002/jlb.55.2.253
  22. Poullain, M. G., Fautrel, A., Guyomard, C., Chesne, C., Grislain, L., and Guillouzo, A. (1992), Viability and primary culture of rat hepatocytes aftter hypothermic preservation: the superiority of the Leibovitz medium over the University of Wisconsin solution for cold storage, Hepatology 15, 97-106 https://doi.org/10.1002/hep.1840150118
  23. Mamprin, M. E., Vega, F., and Rodriguez, J. V. (2005), Adenosine 5'triphosphate transport and accumulation during the cold preservation of rat hepatocytes in University of Wisconsin solution, World Journal of Gastroenterology 11(13), 1957-1964 https://doi.org/10.3748/wjg.v11.i13.1957
  24. Richter, C., Schweizer, M., Cossarizza, A., and Franceschi, C. (1996), Control of apoptosis by the cellular ATP level, FEBS Letters 378(2), 107-110 https://doi.org/10.1016/0014-5793(95)01431-4
  25. Leist, M., Single, B., Castoldi, A. F., K$\"{u}$hnle, and S., Nicotera, P. (1997), Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis, The Journal of Experimental Medicine 185(8), 1481-1486 https://doi.org/10.1084/jem.185.8.1481
  26. Byoun, S. H., Park, H. W., and Choe, T. B. (2006), Short-term hypothermic preservation of CHO Cells Using Sεrum-Free Media, Korean J. Biotechnol. Bioeng. 21(4), 306-311
  27. Giampiero Girolomoni, Maria Lucia Santantonio, Saveria Pastore, Paul R. Bergstresser, Alberto Giannetti, and Ponciano D. Cruz Jr (1993), Epidermal Langerhans Cells Are Resistant to the Permeabilizing Effects of Extracellular ATP: In Vitro Evidence Supporting a Protective Role of Membrane ATPase, Journal of lnvestigative Dermatology 100, 282-287 https://doi.org/10.1111/1523-1747.ep12469769
  28. Kl$\"{o}$ppel, K., Gerlach, J., and Neuhaus, P. (1994), Addition of an osmotic agent to liver preservative solutions in a model of in vitro preservation of hepatocytes, Langenbecks Archiv fiir Chirurgie 379(6), 329-334
  29. Bessems, M., Doorschodt, B. M., van Vliet, A. K., and van Gulik T. M. (2004), Preservation of rat livers by cold storage: a comparison between the University of Wisconsin solution and Hypothermosol, Annals of Transplantation 9(2), 35-37