Preparation of Rodent Testis Co‐Cultures

Susanna Wegner1, Sungwoo Hong1, Xiaozhong Yu2, Elaine M. Faustman1

1 Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, 2 Department of Environmental Health Science, College of Public Health, University of Georgia, Athens, Georgia
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 16.10
DOI:  10.1002/0471140856.tx1610s55
Online Posting Date:  February, 2013
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Abstract

Male reproductive development is a complex process that is sensitive to disruption by a range of toxicants. There is a great need for in vitro models that can evaluate potential male reproductive toxicants. The current unit presents a protocol for preparation of a three‐dimensional in vitro model of male reproductive development that reduces the number of animals required for evaluation of toxicants. A Matrigel overlay provides a three‐dimensional extracellular matrix that improves cell attachment, viability, and communication, and makes the model more reflective of in vivo environments. Curr. Protoc. Toxicol. 55:16.10.1‐16.10.7. © 2013 by John Wiley & Sons, Inc.

Keywords: male reproductive development; three‐dimensional in vitro models; primary cell culture

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Three‐Dimensional Testicular Cell Co‐Culture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Three‐Dimensional Testicular Cell Co‐Culture

  Materials
  • Enzyme digestion cocktail A (5 ml) and cocktail B (10 ml) (see reciperecipes)
  • Complete cell culture medium (see recipe)
  • Matrigel (BD Biosciences; Matrigel should have protein concentration ranging from 9.2 to 10 µg/ml and endotoxin <1.5 EU/ml)
  • Ice and dry ice
  • Minimum essential medium (MEM), phenol‐free
  • Male rat pups
  • Trypsin inhibitor solution (see recipe)
  • 0.05% Trypsin and EDTA
  • 37°C water bath
  • Mid‐sized (e.g., 60 mm × 15–mm) cell culture dishes for testis dissection
  • Dissecting tools (two pairs of fine forceps per person dissecting)
  • Dissecting microscope and light
  • Waste beaker
  • CO 2 chamber
  • Surgical instruments including:
    • Large scissors
    • Small scissors
    • Fine forceps
  • 15‐ml plastic conical tubes
  • 3‐ml plastic transfer pipets
  • Pulled glass pipets
  • Nylon mesh cell strainer (100‐µm)
  • 50‐ml conical tubes
  • Hemacytometer
  • Primeria‐coated 35 mm ×10–mm dishes or 96‐well plates for plating cells (Falcon)
  • Pipet and pipet tips for plating cell suspension
  • Repeat pipets and pipet tips (any size appropriate for 30‐µl aliquots)
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Figures

Videos

Literature Cited

Literature Cited
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   Bilinska, B. 1989. Interaction between Leydig and Sertoli cells in vitro. Cytobios 60:115‐126.
   Bilinska, B., Viklicky, V., Draber, P., and Wojtusiak, A. 1989. Luteinizing hormone‐induced modifications of the cytoskeleton in cultured mouse Leydig cells. Cytobios 58:25‐34.
   Bonde, J.P. 2010. Male reproductive organs are at risk from environmental hazards. Asian J. Androl. 12:152‐156.
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   Gray, T.J. 1986. Testicular toxicity in vitro: Sertoli‐germ cell co‐cultures as a model system. Food Chem. Toxicol. 24:601‐605.
   Gregotti, C., Di Nucci, A., Costa, L.G., Manzo, L., Scelsi, R., Berte, F., and Faustman, E.M. 1992. Effects of thallium on primary cultures of testicular cells. J. Toxicol. Environ. Health 36:59‐69.
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   Lejeune, H., Sanchez, P., and Saez, J.M. 1998. Enhancement of long‐term testosterone secretion and steroidogenic enzyme expression in human Leydig cells by co‐culture with human Sertoli cell‐enriched preparations. Int. J. Androl. 21:129‐140.
   Li, L.H., Jester, W.F. Jr., and Orth, J.M. 1998. Expression of 140‐kDa neural cell adhesion molecule in developing testes in vivo and in long‐term Sertoli cell‐gonocyte cocultures. J. Androl. 19:365‐373.
   Orth, J.M., Jester, W.F., Li, L.H., and Laslett, A.L. 2000. Gonocyte‐Sertoli cell interactions during development of the neonatal rodent testis. Curr. Top. Dev. Biol. 50:103‐124.
   Rider, C.V., Wilson, V.S., Howdeshell, K.L., Hotchkiss, A.K., Furr, J.R., Lambright, C.R., and Gray, L.E. Jr. 2009. Cumulative effects of in utero administration of mixtures of “antiandrogens” on male rat reproductive development. Toxicol. Pathol. 37:100‐113.
   Saldutti, L.P., Beyer, B., Breslin, W., Brown, T., Chapin, R.E., Campion, S., Enright, B., Faustman, E.M., Foster, P., Kelce, W., Kim, J.H., Loboa, E.G., Piersma, A., Sasaki, J.C., Seyler, D., Turner, K., Yu, H., and Yu, X. 2013. Proceedings Paper from Testicular Toxicity In Vitro Models Workshop: Opportunities for Advancement via Biomedical Engineering Techniques.
   Sidhu, J.S., Farin, F.M., and Omiecinski, C.J. 1993. Influence of extracellular matrix overlay on phenobarbital‐mediated induction of CYP2B1, 2B2, and 3A1 genes in primary adult rat hepatocyte culture. Arch. Biochem. Biophys. 301:103‐113.
   Yu, X., Sidhu, J.S., Hong, S., and Faustman, E.M. 2005. Essential role of extracellular matrix (ECM) overlay in establishing the functional integrity of primary neonatal rat Sertoli cell/gonocyte co‐cultures: An improved in vitro model for assessment of male reproductive toxicity. Toxicol. Sci. 84:378‐393.
   Yu, X., Hong, S., Moreira, E.G., and Faustman, E.M. 2009. Improving in vitro Sertoli cell/gonocyte co‐culture model for assessing male reproductive toxicity: Lessons learned from comparisons of cytotoxicity versus genomic responses to phthalates. Toxicol. Appl. Pharmacol. 239:325‐336.
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