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In vitro culture of fibroblast-like cells from postmortem skin of Katahdin sheep stored at 4°C for different time intervals

  • Mahipal Singh
  • Xiaoling Ma
  • Eugene Amoah
  • Govind Kannan
Report

Abstract

Live animals have been produced recently from animal tissues preserved for decades at frozen temperatures with or without cryoprotectants. However, the tissues in these studies were cryopreserved within few hours of animal death to obtain culturable live cells as nuclear donors. How long the tissues can be left unfrozen after animal death, without losing the viability and potential to in vitro culture with comparable morphology and proliferative rate as the fresh tissues, is not completely understood. To understand this phenomenon, ear skin samples from individual sheep (n = 3) were procured from slaughter plant and stored at 4°C. After various intervals (2, 8, 24, 32, 48, and 56 h after slaughter), 2–3 mm2 pieces (n = 10) of skin samples were cultured for 12 d on two dishes (60 mm) for each sheep. Outgrowth of fibroblast-like cells was observed as early as day 4 of culture and was visible on dishes of all time points including 56 h by day 10. The number of outgrowing cells decreased with increasing time interval between animal slaughter and culture initiation. Secondary cultures were successfully established for all the time points. All cultures proliferated well and were apparently normal. Passage 2 cultures of 2 h and 56 h interval for one of the three sheep were compared for their growth pattern and proliferation rates. The population doubling time of 2 h and 56 h intervals was 33.12 and 34.8 h, respectively, and both the lines exhibited similar cell morphology and an “S”-shaped growth curve. These results suggest that skin tissues of sheep and perhaps other animal species with superior traits are effectively preserved at cellular level at least for 56 h at normal refrigerating conditions, without need of complicated cryopreservatives/cryotanks that are usually not available at small farms.

Keywords

Sheep Fibroblast-like cells Postmortem Skin Small ruminants Cell culture 

Notes

Acknowledgments

We would like to thank the Georgia Small Ruminant Research and Extension Center staff for help in procuring skin samples. This work was partly supported by Evans-Allen funds.

References

  1. Carter D. A.; Mayer E. J.; Dick A. D. The effect of postmortem time, donor age and sex on the generation of neurospheres from adult human retina. Br J Ophthalmol 91: 1216–1218; 2007.PubMedCrossRefGoogle Scholar
  2. Erker L.; Azuma H.; Lee A. Y.; Guo C.; Orloff S.; Eaton L.; Benedetti E.; Jensen B.; Finegold M.; Willenbring H.; Grompe M. Therapeutic liver reconstitution with murine cells isolated long after death. Gastroenterology 139: 1019–1029; 2010.PubMedCrossRefGoogle Scholar
  3. Freshney R. I. Culture of Animal Cells: a manual of basic techniques. Wiley-Liss, INC, New York; 2000.Google Scholar
  4. Hoshino Y.; Hayashi N.; Taniguchi S.; Kobayashi N.; Sakai K.; Otani T.; Iritani A.; Saeki K. Resurrection of a bull by cloning from organs frozen without cryoprotectant in a −80 degrees c freezer for a decade. PLoS ONE 4: e4142; 2009.PubMedCrossRefGoogle Scholar
  5. Mastromonaco G. F.; Perrault S. D.; Betts D. H.; King W. A. Role of chromosome stability and telomere length in the production of viable cell lines for somatic cell nuclear transfer. BMC Dev Biol 6: 41; 2006.PubMedCrossRefGoogle Scholar
  6. Prows J.; Nathan P. Evaluation of storage conditions for refrigerated rabbit skin. Cryobiology 17: 125–129; 1980.PubMedCrossRefGoogle Scholar
  7. Roth V (2006) calculating doubling time. In: . http://www.doubling-time.com/compute.php.
  8. Silvestre M. A.; Saeed A. M.; Cervera R. P.; Escriba M. J.; Garcia-Ximenez F. Rabbit and pig ear skin sample cryobanking: effects of storage time and temperature of the whole ear extirpated immediately after death. Theriogenology 59: 1469–1477; 2003.PubMedCrossRefGoogle Scholar
  9. Silvestre M. A.; Sanchez J. P.; Gomez E. A. Vitrification of goat, sheep, and cattle skin samples from whole ear extirpated after death and maintained at different storage times and temperatures. Cryobiology 49: 221–229; 2004.PubMedCrossRefGoogle Scholar
  10. Strober W (2001) Trypan blue exclusion test of cell viability. Curr. Protoc. Immunol Appendix 3:Appendix 3BGoogle Scholar
  11. Wakayama S.; Ohta H.; Hikichi T.; Mizutani E.; Iwaki T.; Kanagawa O.; Wakayama T. Production of healthy cloned mice from bodies frozen at −20 degrees C for 16 yr. Proc Natl Acad Sci USA 105: 17318–17322; 2008.PubMedCrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2011

Authors and Affiliations

  • Mahipal Singh
    • 1
  • Xiaoling Ma
    • 1
  • Eugene Amoah
    • 1
  • Govind Kannan
    • 1
  1. 1.Animal Science Division, Agricultural Research StationFort Valley State UniversityFort ValleyUSA

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