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

  • 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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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.
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