Isolation of Primary Fibroblast Culture from Wildlife: the Panthera onca Case to Preserve a South American Endangered Species

Ana Cecilia Mestre‐Citrinovitz1, Adrián Jorge Sestelo2, María Belén Ceballos2, José Lino Barañao3, Patricia Saragüeta1

1 Instituto de Biología y Medicina Experimental, Buenos Aires, 2 Zoológico de Buenos Aires, Buenos Aires, 3 Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires (UBA), Buenos Aires
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 28.7
DOI:  10.1002/cpmb.25
Online Posting Date:  October, 2016
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Abstract

Cell line establishment of somatic cells is a valuable resource to preserve genetic material of rare, difficult‐to‐find, endangered and giant species like Jaguar (Panthera onca), the largest South American felid. This unit focuses on the isolation and culture of fibroblasts from Jaguar skin and muscle biopsies, and ear cartilage dissection immediately after death to preserve one of the several endangered species in this biome. These culture techniques enabled us to contribute 570 samples from 45 autochthonous and endangered species, including Jaguar. The fibroblasts obtained are a part of the Genetic Bank of Buenos Aires Zoo with the 6700 samples, including tissues such as muscle, ovarian, testicular, blood, fibroblast cultures, sperm, hair, and fluids and cells from 450 individuals of 87 different species. © 2016 by John Wiley & Sons, Inc.

Keywords: biobank; cell culture; Panthera onca

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Establishment of Primary Cell Culture from Panthera onca Skin or Muscle Biopsies by Direct Culture
  • Alternate Protocol 1: Establishment of Primary Cell Culture from Panthera onca Skin or Muscle Biopsies by Tissue Disaggregation
  • Support Protocol 1: Establishment of Primary Cell Culture Ear Cartilage from Panthera onca after Death
  • Support Protocol 2: Biopsy Samples and Cells Freezing and Defrosting
  • Support Protocol 3: Culture and Trypsinization of Passage 1 (P1 Cells)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Establishment of Primary Cell Culture from Panthera onca Skin or Muscle Biopsies by Direct Culture

  Materials
  • Tissue biopsy sampling, primary cell culture of fibroblasts from skin or muscle ( protocol 3)
  • Phosphate‐buffered saline (PBS; Irvine Scientific, cat. no. 9235), 1×
  • Dulbecco's modified Eagle medium (DMEM; with red phenol) + supplements (see recipe)
  • 0.25% Trypsin, 1× (see recipe)
  • 20‐, 200‐, and 1000‐μl pipets
  • Pliers
  • Pipet tips
  • 60‐ and 35‐mm cell culture dish
  • Scissors and scalpel
  • CO 2 incubator
  • Phase‐contrast inverted microscope
  • 50‐ml tube

Alternate Protocol 1: Establishment of Primary Cell Culture from Panthera onca Skin or Muscle Biopsies by Tissue Disaggregation

  Additional Materials
  • Collagenase (see recipe)
  • 15‐ml tubes
  • 1‐ml pipets
  • Centrifuge

Support Protocol 1: Establishment of Primary Cell Culture Ear Cartilage from Panthera onca after Death

  Materials
  • 70% ethanol
  • Saline solution
  • 40 mg/ml gentamicin (see recipe)
  • Phosphate‐buffered saline (PBS; Irvine Scientific, cat. no. 9235)
  • Dulbecco's modified Eagle medium (DMEM; with red phenol) + supplements (see recipe)
  • 200 nM L‐glutamine, 100×
  • 5 mg/ml Collagenase, 2×
  • 0.25% trypsin, 1×
  • 20‐, 200‐, 1000‐μl pipets
  • Pipet tips
  • Animal hair clippers
  • 15‐ and 50‐ml Falcon tubes
  • Self‐sealable zipper storage bags
  • Pliers
  • Scissors
  • Tweezers
  • 35‐, 60‐, and 100‐mm petri dishes
  • Scalpel
  • 37°C CO 2 incubator

Support Protocol 2: Biopsy Samples and Cells Freezing and Defrosting

  Materials
  • Biopsied skin fragments (see protocol 3)
  • Freezing solution (see recipe)
  • Liquid nitrogen
  • Cells (see protocol 5, step 4)
  • Ice
  • Phosphate‐buffered saline (PBS; Irvine Scientific, cat. no. 9235), 1×
  • Cryovials
  • Ultrafreezer (−80°C)
  • Mr. Frosty, Nalgene, freezer container (Sigma, cat. no. C1562)
  • 15‐ml tubes
  • Centrifuge
  • Pliers
  • 20‐, 200‐, 1000‐μl Pipets
  • Pipet tips
  • Scalpel
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Figures

Videos

Literature Cited

Literature Cited
  Beisiegel, B.M., Sana, D.A., and Moraes, E. Jr. 2012. The jaguar in the Atlantic Forest. CatNews Special Issue 7:14.
  Cullen, L. Jr. 2006. Jaguars as landscape detectives for the conservation of Atlantic Forests in Brazil. PhD Thesis, Canterbury, University of Kent, Canterbury, U.K.
  Galetti, M., Eizirik, E., Beisiegel, B., Ferraz, K., Cavalcanti, S., Srbek‐Araujo, A.C., Crawshaw, P., Paviolo, A., Galetti, P.M. Jr., Jorge, M.l., Marinho‐Filho, J., Vercillo, U., and Morato, R. 2013. Atlantic rainforest's jaguars in decline. Science 342:930. doi: 10.1126/science.342.6161.930‐a.
  Haag, T., Santos, A.S., Sana, D.A., Morato, R.G., Cullen, L. Jr., Crawshaw, P.G Jr., De Angelo, C., Di Bitetti, M.S., Salzano, F.M., and Eizirik, E. 2010. The effect of habitat fragmentation on the genetic structure of a top predator: Loss of diversity and high differentiation among remnant populations of Atlantic Forest jaguars (Panthera onca). Mol. Ecol. 22:4906‐4921. doi: 10.1111/j.1365‐294X.2010.04856.x.
  Holt, W.V., Bennett, P.M., and Volobouev, V. 1996. Genetic resource banks in wildlife conservation. J. Zool. Lond. 238:531‐544. doi: 10.1111/j.1469‐7998.1996.tb05411.x.
  Jorge, M.L.S.P., Galetti, M., Ribeiro, M.C., and Ferraz, K.M.P.M.B. 2013. Mammal defaunation as surrogate of trophic cascades in a biodiversity hotspot. Biol. Conserv. 163:49‐57 doi: 10.1016/j.biocon.2013.04.018.
  Leon‐Quinto, T., Simon, M.A., Cadenas, R., Jones, J., Martinez‐Hernandez, F.J., Moreno, J.M., Vargas, A., Martinez, F., and Soria, B. 2009. Developing biological resource banks as a supporting tool for wildlife reproduction and conservation The Iberian lynx bank as a model for other endangered species. Anim. Reprod. Sci. 112:347‐361. doi: 10.1016/j.anireprosci.2008.05.070.
  Miller, B.y. and Rabinowitz, A. 2002. ¿Porqué conservar al jaguar? pg. 210‐216 en El Jaguar en el Nuevo Milenio (R. A. Medellin et al.). Fondo de la Cultura Económica, Universidad Nacional Autónoma de México y Wildlife Conservation Society, D.F. México.
  Miller, B., Reading, R., Strittholt, J., Carroll, C., Noss, R., Soulé, M., Sanches, O., Terborgh, J., Brightsmith, D., Cheeseman, T., and Foreman, D. 1999. Using focal species in the design of nature reserve networks. Wild Earth Winter 81‐92.
  Moro, L.N., Sestelo, A.J., and Salamone, D.F. 2014. Evaluation of cheetah and leopard spermatozoa developmental capability after interespecific ICSI with domestic cat oocytes. Reprod. Domest. Anim. Vol. 49:693‐700. doi: 10.1111/rda.12355.
  Moro, L.N., Jarazo, J., Buemo, C., Hiriart, MI., Sestelo, A.J., and Salamone, D.F. 2015a. Tiger, bengal and domestic cat embryos produced by homospecific and interespecific zona‐free nuclear transfer. Reprod. Domest. Anim. Vol. 50: 849‐857. doi: 10.1111/rda.12593.
  Moro, L.N., Hiriart, MI., Buemo, C., Jarazo, J., Sestelo, AJ., Veraguas, D., Rodriguez‐Alvarez, L., and Salamone, D.F. 2015b. Cheetah interspecific SCNT followed by embryo aggregation improves in vitro development but not pluripotent gene expression. Reproduction 150:1‐10. doi: 10.1530/REP‐15‐0048.
  Noss, R.F., Quigley, H.B., Hornocker, M. G., Merrill y, T., and Paquet, P.C. 1996. Conservation biology and carnivore conservation in the Rocky Mountains. Conserv. Biol. 10:949‐963. doi: 10.1046/j.1523‐1739.1996.10040949.x.
  Ray, J. 2005. Large carnivorous animals as tools for conserving biodiversity: Assumptions and uncertainties. In Large Carnivores and the Conservation of Biodiversity (J.C. Ray et al., eds.) pp. 34‐56. Island Press, Washington, D.C.
  Ribeiro, M.C., Metzger, J.P., Martensen, A.C., Ponzoni, F.J., and Hirota, M.M. 2009. The Brazilian Atlantic Forest: How much is left, and how is the remaining forest distributed? Implications for conservation. Biol. Conserv. 142:1141‐1153. doi: 10.1016/j.biocon.2009.02.021.
  Roldán, E.R.S., and Garde, J.J. 2004. Biotecnología de la reproducción y conservación de especies en peligro de extinción. En “Los retos medioambientales del Siglo XXI. La problemática de la conservación de la biodiversidad en España” (Gomendio M ed.). Fundación BBVA.
  Sherman, J.K. 1965. Pretreatment with protective substances as a factor in freeze‐thaw survival. Cryobiology 1:249‐304. doi: 10.1016/0011‐2240(65)90039‐8.
  Watson, P.F. and Holt, W.V. 2001. Organizational issues concerning the establishment of a genetic resource bank. In Cryobanking the Genetic Resource. Wildlife Conservation the Future? (Watson, P.F., Holt, W.V., eds.), pp. 21‐46. Taylor & Francis, London.
  Wildt, D.E., Rall, W.F., Critser, J.K., Monfort, S.L., and Seal, U.S. 1997. Genome resource banks. Living collections for biodiversity conservation. Bioscience 47: 689‐698. doi: 10.2307/1313209.
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