Skip to main content

Advertisement

SpringerLink
Log in

Cold storage of biopsies from wild endangered native Chilean species in field conditions and subsequent isolation of primary culture cell lines

  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Abstract

We evaluated the possibility of deriving primary cell cultures from tissue biopsies taken in field conditions from six threaten endemic Chilean species of free-ranging mammals. Biopsies were taken either by ear punching or darts fired to animals and hold in hypothermic conditions (4° C) in defined salt solution for time periods ranging from 0 to 7 d before biopsy samples reached the cell culture laboratory. Previously, holding times were evaluated in experimental cows in controlled conditions. Two enzymatic treatments, collagenase alone or collagenase followed by tripsin, were used to disaggregate tissues for cell culture. We found that ear notches and dart-derived biopsies can be storaged at 4° C for 1 wk and still yield primary cultures. For dart-derived biopsies, there was an invert correlation between length of cold storage and cellular viability in culture. Healthy fibroblast cell lines were obtained in 92% of the biopsies taken despite the origin (punch or biopsy). We are not aware of similar study for free-ranging animals, especially for the use of darting system to biopsy wild terrestrial mammals, we believe that our results could help for a more widespread implementation of these procedures in the practice of ex situ conservation

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.

Similar content being viewed by others

References

  • Ackers J. P. Biopreservation of cells and engineered tissues. Adv. Biochem. Engin/Biotechnol 103: 157–187; 2006.

    Article  Google Scholar 

  • Adams A. M.; Pratt S.; Stice S. L. Knockdown of the Dnmt1s transcript using small interfering RNA in primary murine and bovine fibroblast cells. Mol. Reprod. Dev 723: 311–319; 2005. doi:10.1002/mrd.20357.

    Article  PubMed  CAS  Google Scholar 

  • Al-Nazhan S.; Al-Nasser A. Viability of human periodontoal ligament fibroblasts in tissue culture after exposure to different contact lens solutions. J. Contemp. Dent. Pract 74: 1–9; 2006.

    Google Scholar 

  • Barret-Lennard L. G.; Smith T. G.; Ellis G. M. A cetacean biopsy system using lightweight pneumatic darts, and its effect on the behaviour of killer whales. Mar. Mamm. Sci 12: 14–27; 1996 doi:10.1111/j.1748-7692.1996.tb00302.x.

    Article  Google Scholar 

  • Beutler E. Effect of temperature on red cell ATP. Clin. Chim. Acta 199: 117–118; 1991a doi:10.1016/0009-8981(91)90016-6.

    Article  PubMed  CAS  Google Scholar 

  • Beutler E. Principles of transfusion medicine. In: Rossi E. C.; Simon T. L.; Moss G. S. (eds) Principles of transfusion medicine. Williams & Wilkins, Baltimore, p 47; 1991b.

    Google Scholar 

  • Bowler K.; Fuller B. J. Temperature and animal cells. The Company of Biologists, Cambridge, 1987: p 460

    Google Scholar 

  • Bourne W. M. Corneal preservation: past, present, and future. Refract. Corneal 7: 60–61; 1991

    CAS  Google Scholar 

  • Bozinovic F.; Ruiz G.; Rosemann M. Energetics and torpor of a South American “living fossil” the microbiotheriid Dromiciops gliroides. J. Comp. Physiol. B 174: 293–297; 2004. doi:10.1007/s00360-004-0414-8.

    Article  PubMed  Google Scholar 

  • Bravo D.; Rigley T. H.; Gibran N.; Strong D. M.; Newman-Gage H. Effect of storage and preservation methods on viability in transplantable human skin allografts. Burns 4: 367–378; 2000 doi:10.1016/S0305-4179(99)00169-2.

    Article  Google Scholar 

  • Caudron A. K.; Negro S. S.; Muller C. G.; Boren L. J.; Gemmell N. J. Hair sampling and genotyping from hair follicles: a minimally-invasive alternative for genetics studies in small, mobile pinnipeds and other mammals. Mar. Mamm. Sci 23: 184–192; 2007 doi:10.1111/j.1748-7692.2006.00084.x.

    Article  Google Scholar 

  • Cofré H.; Marquet P. A. Conservation status, rarity, and geographic priorities for conservation of Chilean mammals: an assessment. Biol. Conserv 88: 53–68; 1999 doi:10.1016/S0006-3207(98)00090-1.

    Article  Google Scholar 

  • Doufas A. G.; Sessler D. I. Physiology and clinical relevance of induced hypothermia. Neurocrit Care 1: 489–498; 2004. doi:10.1385/NCC:1:4:489.

    Article  PubMed  Google Scholar 

  • Freshney R. I. Culture of animal cells. 3rd ed. Wiley, New York, 1994

    Google Scholar 

  • Freshney R. I. Culture of animal cells: a manual of basic technique. Wiley, Toronto, 2000.

    Google Scholar 

  • Gemmell N. J.; Majluf P. Projectile biopsy sampling of fur seals. Mar. Mamm. Sci 13: 512–516; 1997 doi:10.1111/j.1748-7692.1997.tb00661.x.

    Article  Google Scholar 

  • Georgiadis N.; Bischoff L.; Templeton A.; Patton J.; Karesh W.; Western D. Structure and history of African elephant populations. I. Eastern and Southern Africa. J. Hered 85: 100–104; 1994 Medline.

    PubMed  CAS  Google Scholar 

  • Gibbons J.; Arat S.; Rzucidlo J.; Miyoshi K.; Waltenburg R.; Respess D.; Venable A.; Stice S. L. Enhanced survivability of cloned calves derived from roscovitine-treated adult somatic cells. Biol. Reprod 66: 895–900; 2002. doi:10.1095/biolreprod66.4.895.

    Article  PubMed  CAS  Google Scholar 

  • Gosden R. B.; Baird D. T.; Wade J. C.; Webb R. Restoration of fertility to oophorectomized sheep by ovarian autografts stored at -96°C. Hum. Reprod 9: 597–603; 1994Medline.

    PubMed  CAS  Google Scholar 

  • Gulliksson H. Additive solutions for the storage of platelets for transfusion. Transfus. Med 10: 257–264; 2000. doi:10.1046/j.1365-3148.2000.00262.x.

    Article  PubMed  CAS  Google Scholar 

  • Gunasena K. T.; Villines P. M.; Critser E. S.; Critser J. K. Live births after autologous transplant of cryo-preserved mouse ovaries. Hum. Reprod 12: 101–106; 1997. doi:10.1093/humrep/12.1.101.

    Article  PubMed  CAS  Google Scholar 

  • Gundersen Y.; Vaagenes P.; Pharo A.; Valo E. T.; Opstad P. K. Moderate hypothermia blunts the infl ammatory response and reduces organ injury after acute haemorrhage. Acta Anaesthesiol Scand 458: 994–1001; 2001. doi:10.1034/j.1399-6576.2001.450812.x.

    Article  PubMed  CAS  Google Scholar 

  • Hagiwara S.; Iwasaka H.; Matsumoto S.; Noguchi T. Changes in cell culture temperature alter release of inflammatory mediators in murine macrophagic RAW264.7 cells. Inflamm. Res 567: 297–303; 2007. doi:10.1007/s00011-007-6161-z.

    Article  PubMed  CAS  Google Scholar 

  • Hanks J. Conservation strategies for Africa’s large mammals. Reprod. Fertil. Dev 13: 459–468; 2001. doi:10.1071/RD01070.

    Article  PubMed  CAS  Google Scholar 

  • Harp R.; Leibach J.; Black J.; Karow A. Cryopreservation of murine ovarian tissue. Cryobiology 31: 336–343; 1994. doi:10.1006/cryo.1994.1040.

    Article  PubMed  CAS  Google Scholar 

  • Hayes O.; Ramos B.; Rodriguez L. L.; Aguilar A.; Badia T.; Castro F. O. Cell confluency is as efficient as serum starvation for inducing arrest in the G0/G1 phase of the cell cycle in granulosa and fibroblast cells of cattle. Anim. Reprod. Sci 873–4: 181–192; 2005b. doi:10.1016/j.anireprosci.2004.11.011.

    Article  PubMed  CAS  Google Scholar 

  • Hayes O.; Rodriguez L. L.; Gonzalez A.; Falcon V.; Aguilar A.; Castro F. O. Effect of cryopreservation on fusion efficiency and in vitro development into blastocysts of bovine cell lines used in somatic cell cloning. Zygote 134: 277–282; 2005a. doi:10.1017/S0967199405003278.

    Article  PubMed  CAS  Google Scholar 

  • Hogman C. F. Liquid-stored red blood cells for transfusion. A status report. Vox Sang 76: 67–77; 1999a. doi:10.1046/j.1423-0410.1999.7620067.x.

    Article  PubMed  CAS  Google Scholar 

  • Hogman C. F. Storage of blood components. Curr. Opin. Hematol 6: 427–431; 1999b doi:10.1097/00062752-199911000-00013.

    Article  PubMed  CAS  Google Scholar 

  • Holt W. V.; Pickard A. R.; Prather R. L. Wildlife conservation and reproductive cloning. Reproduction 127: 317–324; 2004. doi:10.1530/rep.1.00074.

    Article  PubMed  CAS  Google Scholar 

  • Houck M. L.; Ryder O. A.; Vahala J.; Kock R. A.; Oosterhuis J. E. Diploid chromosome number and chromosomal variation in the white rhinoceros (Ceratotherium simum). J. Hered 851: 30–34; 1994.

    PubMed  CAS  Google Scholar 

  • Jamieson N. V.; Sundberg R.; Lindell S.; Claesson K.; Moen J.; Vreugdenhil P. K.; Wight D. G.; Southard J. H.; Belzer F. O. Preservation of the canine liver for 24–48 h using simple cold storage with UW solution. Transplantation 464: 517–522; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Karesh W. B.; Smith F.; Frazier-Taylor H. A remote method for obtaining skin biopsy samples. Conserv. Biol 1: 261–262; 1987 doi:10.1111/j.1523-1739.1987.tb00041.x.

    Article  Google Scholar 

  • Kreeger T. Handbook of wildlife chemical inmobilization. Wildlife Pharmaceuticals, Colorado, 1999: p 342.

    Google Scholar 

  • Lakey J. R.; Tsujimura T.; Shapiro A. M.; Kuroda Y. Preservation of the human pancreas before islet isolation using a two-layer (UW solution-perfluorochemical) cold storage method. Transplantation 74: 1809–1811; 2002. doi:10.1097/00007890-200212270-00031.

    Article  PubMed  CAS  Google Scholar 

  • Lambertson R. A biopsy system for large whales and its use for cytogenetics. J. Mammal 68: 443–445; 1987 doi:10.2307/1381495.

    Article  Google Scholar 

  • Lincoln C. K.; Gabridge M. G. Cell culture contamination: sources, consequences, prevention, and elimination. Methods Cell Biol 57: 49–65; 1998. doi:10.1016/S0091-679X(08)61571-X.

    Article  PubMed  CAS  Google Scholar 

  • Loskutoff N. M. Role of embryo techniques in genetic management and conservation of wildlife. In: Holt W. V.; Pickard A. R.; Roger J. C.; Wildt D. E. (eds) Reproductive Science and Integrated Conservation. Cambridge University, Cambridge, pp 183–194; 2003.

    Google Scholar 

  • Loskutoff N. M.; Bartels P.; Meintjes M.; Godke R. A.; Schiewe M. C. Assisted reproductive technology in nondomestic ungulates: a model approach to preserving and managing genetic diversity. Theriogenology 43: 3–12; 1995 doi:10.1016/0093-691X(94)00005-F.

    Article  Google Scholar 

  • Margules C. R.; Pressey R. L. Systematic conservation planning. Nature 6783: 243–253; 2000 doi:10.1038/35012251.

    Article  Google Scholar 

  • Marsili L.; Fossi M. C.; Neri G.; Casini S.; Gardi C.; Palmeri S.; Tarquini E.; Panigada S. Skin biopsies for cell cultures from Mediterranean free-ranging cetaceans. Mar. Environ. Res 501–5: 523–536; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Mathew A. J.; Baust J. M.; Van Buskir R. G.; Baust J. G. Preservation in reparative and regenerative medicine: evolution of individualized solution composition. Tissue Eng 1: 1662–1671; 2004 doi:10.1089/ten.2004.10.1662.

    Article  Google Scholar 

  • Mathews E. A.; Keller S.; Weiner D. B. A method to collect and process skin biopsies for cell culture from free-ranging gray whales (eschrichtius robustus). Mar. Mamm. Sci 41: 1–12; 1988 doi:10.1111/j.1748-7692.1988.tb00178.x.

    Article  Google Scholar 

  • Prithiviraj F.; Pfrender M. E.; Encalada S. E.; Lande R. Mitochondrial DNA variation, phylogeography and population structure of the Asian elephant. Heredity 84: 362–372; 2000 Medline.

    Article  Google Scholar 

  • Pukazhenthi B.; Comizzoli P.; Travis A. J.; Wildt D. E. Applications of emerging technologies to the study and conservation of threatened and endangered species. Reprod. Fertil. Dev 18: 77–90; 2006. doi:10.1071/RD05117.

    Article  PubMed  Google Scholar 

  • Rauen U.; de Groot H. Mammalian cell injury induced by hypothermia—the emerging role for reactive oxygen species. Biol. Chem 3833–4: 477–488; 2002. doi:10.1515/BC.2002.050.

    Article  PubMed  CAS  Google Scholar 

  • Ryder O. A.; Benirschke K. The potential use of ‘cloning’ in the conservation effort. Zoo. Biol 16: 295–300; 1997 doi:10.1002/(SICI)1098-2361(1997)16:4<295::AID-ZOO1>3.0.CO;2-5.

    Article  Google Scholar 

  • Ryder O. A.; McLaren A.; Brenner S.; Zhang Y.-P.; Benirschke K. DNA banks for endangered animal species. Science 2888: 275–277; 2000 doi:10.1126/science.288.5464.275.

    Article  Google Scholar 

  • Simione F. P. Jr. Key issues relating to the genetic stability and preservation of cells and cell banks. J. Parenter. Sci. Technol 46: 226–232; 1992 Medline.

    PubMed  Google Scholar 

  • Snyder K. K.; Baust J. M.; Mathew A. J. Biological packaging for the global cell and tissue therapy markets. Bioprocessing J May/June: 1–7; 2004.

    Google Scholar 

  • Southard J. H.; Belzer F. O. Organ preservation. Annu. Rev. Med 46: 235–247; 1995. doi:10.1146/annurev.med.46.1.235.

    Article  PubMed  CAS  Google Scholar 

  • Stefanovich P.; Ezzell R. M.; Sheehan S. J.; Tompkins R. G.; Yarmush M. L.; Toner M. Effects of hypothermia on the function, membrane integrity, and cytoskeletal structure of hepatocytes. Cryobiology 32: 389–403; 1995. doi:10.1006/cryo.1995.1039.

    Article  PubMed  CAS  Google Scholar 

  • St Peter S. D.; Imber C. J.; Friend P. J. Liver and kidney preservation by perfusion. Lancet 9306: 604–613; 2002 doi:10.1016/S0140-6736(02)07749-8.

    Article  Google Scholar 

  • Taylor M. J. Physico chemical principles in low temperature. In: Grout B. W. W.; Morris G. J. (eds) The effects of low temperatures on biological systems. Arnold, London, p 3; 1987.

    Google Scholar 

  • Trent J. F.; Kirsner R. S. Tissue engineered skin: Apligraf, a bi-layered living skin equivalent. Int. J. Clin. Pract 6: 408–413; 1998.

    Google Scholar 

  • Wigg A. J.; Phillips J. W.; Berry M. N. Maintenance of integrity and function of isolated hepatocytes during extended suspension culture at 25 degrees C. Liver Int 23: 201–211; 2003. doi:10.1034/j.1600-0676.2003.00817.x.

    Article  PubMed  Google Scholar 

  • Winn H. E.; Bishoff W. L.; Taruski A. G. Cytological sexing of Cetacea. Marine Biology 23: 343–346; 1973 doi:10.1007/BF00389342.

    Article  Google Scholar 

  • Wiig O.; Berg V.; Gjertz I.; Seagars J.; Skaare J. U. Use of skin biopsies for assessing levels of organochlorines in walruses (Odobenus rosmarus). Polar Biol 23: 272–278; 2000 doi:10.1007/s003000050444.

    Article  Google Scholar 

  • Worthington-Wilmer J.; Allen P. J.; Pomeroy P. P.; Twiss S. D.; Amos W. Where have all the fathers gone? An extensive microsatellite analysis of paternity in the grey seal (Halichoerus grypus). Mol. Ecol 8: 1417–1429; 1999. doi:10.1046/j.1365-294x.1999.00705.x.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

Authors wish to acknowledge M.L. Houck from CRES of the San Diego Zoo and L Nel-Thermat from Audubon Center for Research of Endangered Species in Louisina for help with detailed protocols and encouraging support. We also wish to thank Mr. Oscar Guineo for his help in huemul’s field work. This research was partially funded by FIA from Ministry of Agriculture of Chile, under grant number: PIC-2005-1-P-097. Appropriate permits for capturing wild species were obtained from Servicio Agrícola y Ganadero (SAG) of Chile (# 1804 from 04.19.2006 for kod kod; # 1337 from 03.20.2006 for Chilean Shrew opposum; # 1851 from 04.21.2006 for huemul; # 1963 from 05.02.2006 for chinchilla) and from CONAF (Corporación Nacional Forestal de Chile) under permit numbers: (335 from 04.25.2006 for Chilean Shrew opposum; 010 from 05.30.2006 for chinchilla; and 1182 from 04.02.2007 for huemul).

Author information

Authors and Affiliations

  1. University of Concepcion, Avenida Vicente Méndez 595, Chillan, Chile

    Heribelt Tovar, Felipe Navarrete, Lleretny Rodríguez, Oscar Skewes & Fidel Ovidio Castro

Authors
  1. Heribelt Tovar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Felipe Navarrete
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lleretny Rodríguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Oscar Skewes
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Fidel Ovidio Castro
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fidel Ovidio Castro.

Additional information

Editor: J. Denry Sato

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tovar, H., Navarrete, F., Rodríguez, L. et al. Cold storage of biopsies from wild endangered native Chilean species in field conditions and subsequent isolation of primary culture cell lines. In Vitro Cell.Dev.Biol.-Animal 44, 309–320 (2008). https://doi.org/10.1007/s11626-008-9124-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11626-008-9124-y

Keywords

Search

Navigation