Culture of Clonal Lines in Porcine Fetal Fibroblast Cells

돼지 태아섬유아세포 Clonal Lines의 배양

  • Kwon, D. J. (College of Animal Resource Science, Kangwon National University) ;
  • Park, C. K. (College of Animal Resource Science, Kangwon National University) ;
  • B. K. Yang (College of Animal Resource Science, Kangwon National University) ;
  • Kim, C. I. (College of Animal Resource Science, Kangwon National University) ;
  • H. T. Cheong (College of Animal Resource Science, Kangwon National University)
  • Published : 2004.03.01

Abstract

This study was performed to establish the effective culture condition for the establishment of clonal lines from porcine fetal fibroblast cells. Fibroblasts derived from a pig fetus (Day 50) were cultured and passaged two times before use. A single cell was seeded in 96-well plates, cultured in medium supplemented with different concentrations of FBS, catalase or $\beta$-mercaptoethanol ($\beta$ME), and classified by cell size and morphology. Cells were passaged two times into 4-well dish before freezing. The establishment efficiencies were not different among different concentrations of FBS (0.3 to 5.1%). However, population doubling time (PDT) was significantly decreased by increasing the FBS concentration (P<0.05). The establishment efficiency of $\beta$ME-added group (10.4%) was significantly higher than those of catalase-added and control groups (3.5%, and 3.5%, respectively, p<0.05), and PDT was significantly decreased (23.6 vs 28.1, and 25.5 h, respectively, p<0.05). However, catalase did not show a positive effect on the establishment efficiency. Cell size and morphology did not affect the establishment efficiency and PDT of clonal lines. The result of present study shows that the establishment efficiency of clonal cell lines can be enhanced by the culture in media supplemented with 30% FBS and $\beta$ME.

Keywords

References

  1. Allen, R. G. 1998. Oxidative stress and superoxide dismutase in development, aging and gene regulation. Age 21:47-76 https://doi.org/10.1007/s11357-998-0007-7
  2. Ames, B. N. and Gold, L. S. 1991. Endogenous mutagens and the causes of aging and cancer. Mutat. Res. 251:3-16
  3. Baguisi, A., Behboodi, E., Melican, D. T., Pollock, J. S., Destrempes, M. M., Cammuso, C., Williams, J. L., Nims, S. D., Porter, C. A., Midura, P., Palacios, M. J., Ayres, S. L., Denniston, R. S., Hayes, M. L., Ziomek, C. A., Meade, H. M., Godke, R. A., Gavin, W. G., Overstrom, E. W. and Echelard, Y. 1999. Production of goats by somatic cell nuclear transfer. Nat. Biotechnol. 17:456-461 https://doi.org/10.1038/8632
  4. Balin, A. K., Goodman, D. B. P., Rasmussen, H. and Cristofalo, V. J. 1976. The effect of oxygen tension of the growth and metabolism of WI-38 cells. J. Cell. Physiol. 89:235-250 https://doi.org/10.1002/jcp.1040890207
  5. Balin, A. K., Prett, L. and Allen, R. G. 2002. Effects of ambient oxygen concentration on the growth and antioxidant defences of human cell cultures established from fetal and postnatal skin. Free Radic. BioI. Med. 22(3):257-267
  6. Bradley, T. R., Hodgson, G. S. and Rosendaal, M. 1978. The effect of oxygen tension on haemopoietic and fibroblast cell proliferation in vitro. J. Cell. Physiol. 97:517-522 https://doi.org/10.1002/jcp.1040970327
  7. Chance, B. 1947. An intermediate compound in the catalase-hydrogen peroxide reaction. Acta Chem. Scand. 1:236-267 https://doi.org/10.3891/acta.chem.scand.01-0236
  8. Chance, B., Sies, H. and Boveris, A. 1979. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59:527-905
  9. Dai, Y., Vaught, T. D., Boone, J., Chen, S. H., Phelps, C. J., Ball, S., Monahan, J. A, Jobst, P. M., McCreath, K. J., Lamborn, A. E., Cowell-Lucero, J. L., Wells, K. D., Colman, A., Polejaeva, I. A. and Ayares, D. L. 2002. Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs. Nat. Biotechnol. 20:251-255 https://doi.org/10.1038/nbt0302-251
  10. Gardner, C. S. and Reed, D. J. 1994. Status of glutathione during oxidant-induced oxidative stress in the preimplantation mouse embryo. BioI. Reprod. 51:1307-1314 https://doi.org/10.1095/biolreprod51.6.1307
  11. Gille, J. J. P., van Berkel, C. G. M. and Joenje, H. 1992. Effect of iron chelators on the cytotoxic and genotoxic action of hyperoxia in Chinese hamster ovary cells. Mutat. Res. 275:31-39
  12. Honda, H. and Matsuo, M. 1980. The sensitivity to hyperbaric oxygen of human diploid fibroblasts during ageing in vitro. Mech. Ageing Dev. 12:31-37
  13. Honda, S. and Matsuo, M. 1988. Relationships between the cellular glutathione level and in vitro life span of human diploid fibroblasts. Exp. Gerontol. 23:81-86
  14. Horikoshi, T., Balin, A. K. and Carter, D. M. 1986. Effect of oxygen on the growth of human epidermal keratinocytes. J. Invest. Dermatol. 86:424-427 https://doi.org/10.1111/1523-1747.ep12285695
  15. Ishii, T., Bannai, S. and Sugita, Y. 1981. Mechanism of growth stimulation of L1210 cells by 2-mercaptoethanol in vitro. J. BioI. Chem. 256:12387-12392
  16. Junod, A. F., Petersen, H. and Jornot, L. 1987. Thymidine kinase, thymidylate synthase, and endothelial cell growth under hyperoxia J. Appl. Physiol. 62:10-14
  17. Kato, Y., Tani, T., Sotomaru, Y., Kurokawa, K., Kato, J., Doguchi, H., Yasue, H. and Tsunoda, Y. 1998. Eight calves cloned from somatic cells of a single adult. Science 282:2095-2098
  18. Kim, P. M., DeBoni, U. and Wells, P. G. 1997. Peroxidase-dependent bioactivation and oxidation of DNA and protein in benzo[$\alpha$]pyrene-initiated micronucleus formation. Free Rad. Biol. Med. 23:579-596. https://doi.org/10.1016/S0891-5849(97)00012-9
  19. Lai, L., Kolber-Simonds, D., Park, K. W. Cheong, H. T., Greenstein, J. L., Im, G. S., Samuel, M., Bonk, A., Rieke, A., Day, B. N., Murphy, C. N., Carter, D. B., Hawley, R. J. and Prather, R. S. 2002. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295: 1089-1092 https://doi.org/10.1126/science.1068228
  20. Larson, R. A., Lloyd, R. A., Marley, K. A. and Tuveson, R. W. 1992. Ferric-ion-photosensitized damage to DNA by hydroxyl and non-hydroxyl radical mechanism, J. Photo-chem. Photobiol. B. BioI. 14:245-247
  21. Liu, L. and Wells, P. G. 1995. DNA oxidation as a potential molecular mechanism mediating drug-induced birth defects: Phenytoin and structurally related teratogens initiate the formation of 8-hydroxy-2'-deoxyguanosine in vitro and in vivo in murine maternal hepatic and embryonic tissues. Free Rad. BioI. Med. 19:639-648
  22. McBride, T. J., Preston, B. D. and Loeb, L. A. 1991. Mutagenic spectrum resulting from DNA damage by oxygen radicals. Biochemistry 30:207-213 https://doi.org/10.1021/bi00215a030
  23. Meister, A. 1983. Selective modification of glutathione metabolism. Science 220:472-477
  24. Phillips, B. J., James, T. E. B. and Anderson, D. 1984. Genetic damage in CHO cells exposed to enzymetically generated active oxygen species. Mutat. Res. 126:265-271 https://doi.org/10.1016/0027-5107(84)90006-X
  25. Polejaeva, I. A., Chen, S. H., Vaught, T. D., Page, R. L., Mullins, J., Ball, S., Dai, Y, Boone, J., Walker, S., Ayares, D. L., Colman, A. and Campbell, K. H. S. 2000. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407:86-90
  26. Rathbun, W. B. and Murray, D. L. 1991. Age-related cysteine uptake as rate limiting in glutathione synthesis and glutathione half-life in the cultured human lens. Exp. Eye Res. 53:205-212 https://doi.org/10.1016/0014-4835(91)90075-P
  27. Rhaese, H. and Freese, E. 1968. Chemical analysis of DNA alteration, base liberation and backbone breakage of DNA and oligodeoxyadelylic acid induced by $H_2O_2$ and hydroxyl-amine. Biochem. Biophys. Acta 155:476-490
  28. Rossman, T. G. and Goncharova, E. I. 1998. Spontaneous mutagenesis in mammalian cells is caused mainly by oxidative events and can be blocked by antioxidants and metallothioein. Mutat. Res. 402:103-110 https://doi.org/10.1016/S0027-5107(97)00287-X
  29. Shin, T., Kraemer, D., Pryor, J., Liu, L., Rugila, J., Howe, L., Buck, S., Murphy, K., Lyons, L., and Westhusin, M. 2002. A cat cloned by nuclear transplantation. Nature 415:859
  30. Suzuki, K. and Hei, T. K. 1996 Induction of heme oxygenase in mammalian cells by mineral fibers: Distinctive effect of reactive oxygen species. Carcinogenesis 17:661-667
  31. Takahashi, M., Nagai, T., Hamano, S., Kuwayama, M., Okamura, N. and Okano, A. 1993. Effect of thiol compounds on in vitro development and intracellular glutathione content of bovine embryos. BioI. Reprod. 49: 228-232
  32. Takahashi, H., Kuwayarna, M., Hamano, S., Takahashi, M., Okano, A., Kadokawa, H., Kariya, T. and Nagai, T. 1996. Effect of $\alpha$-mercaptoethanol on the viability of IVM/IVF/IVC bovine embryos during long-distance transportation in plastic straws. Theriogenology 46:1009-1015
  33. Tzaki, M. G., Byrne, P. J. and Tanswell, A. K. 1988. Cellular interactions in pulmonary oxygen toxicity in vitro: II. Hyperoxia causes adult rat lung fibroblast cultures to produce apparently autocrine growth factors. Exp. Lung Res. 14:403-419 https://doi.org/10.3109/01902148809087817
  34. Wakayama, T., Perry, A. C. F., Zuccotti, M., Johnson, K. R. and Yanagimachi, R. 1998 Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394:369-374
  35. Wells, D. N., Misica, P. M. and Tervit, H. R. 1999. Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells. BioI. Reprod. 60:996-1005 https://doi.org/10.1095/biolreprod60.4.996
  36. Wilmut, I., Schnieke, A. E., MeWhir, J., Kind, A. J. and Campbell, K. H. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature 385:810-813 https://doi.org/10.1038/385810a0
  37. Winn, L. M. and Wells, P. G. 1995. Phenytoin-initiated DNA oxidation in murine embryo culture, and embryoprotection by the antioxidative enzymes superoxide dismutase and catalase: Evidence for reactive oxygen species mediated DNA oxidation in the molecular mechanism of phenytoin teratogenicity. Mol. Pharmacol. 48:112-120
  38. Yuan, H., Kaneko, T., Kaji, K., Kondo, H. and Matsuo, M. 1995. Species difference in the resistibility of embryonic fibroblasts against oxygen-induced growth inhibition. Comp. Biochem. Physiol. B: Biochem. Mol. BioI. 110:145-154 https://doi.org/10.1016/0305-0491(94)00137-J