Mouse Embryo Fibroblast (MEF) Feeder Cell Preparation

David A. Conner1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 23.2
DOI:  10.1002/0471142727.mb2302s51
Online Posting Date:  May, 2001
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Abstract

Mitotically inactive mouse embryo fibroblasts (MEFs) are commonly used as feeder layers to prevent the differentiation of mouse embryonic stem (ES) cells. This unit describes the isolation of MEFs and the use of g‐irradiation or mitomycin C to produce inactivated feeders suitable for the culture of ES cells.

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of Primary Mouse Embryo Fibroblasts
  • Basic Protocol 2: Mitotic Inactivation of MEFs with γ‐Irradiation
  • Alternate Protocol 1: Mitotic Inactivation of MEFs with Mitomycin C
  • Support Protocol 1: Freezing and Thawing MEFs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Isolation of Primary Mouse Embryo Fibroblasts

  Materials
  • Mouse embryos, 12.5 to 13.5 days postcoitum (Hogan et al., )
  • DPBS (see reciperecipe), sterile
  • Trypsin/EDTA solution (see reciperecipe)
  • MEF medium (see reciperecipe) with penicillin/streptomycin
  • Laminar flow hood
  • Inverted microscope
  • 100‐mm tissue culture dish
  • Dissecting forceps and fine scissors, sterilized by autoclaving or ethanol flaming
  • 10‐ml syringe and 16‐G needle
  • 100‐mm tissue culture plates or 75‐cm2 flasks
  • Additional reagents and equipment for passaging and freezing MEFs (see protocol 4)

Basic Protocol 2: Mitotic Inactivation of MEFs with γ‐Irradiation

  Materials
  • Frozen MEF culture (see protocol 1)
  • MEF medium (see reciperecipe) without penicillin/streptomycin
  • Ca2+‐ and Mg2+‐free HBSS (see reciperecipe)
  • 100‐mm tissue culture plates or 75‐cm2 flasks
  • 150‐cm2 tissue culture flasks
  • 100‐mm Petri dishes
  • γ‐Radiation source
  • Additional reagents and equipment for passaging, freezing, and thawing MEFs (see protocol 4)

Alternate Protocol 1: Mitotic Inactivation of MEFs with Mitomycin C

  •  1 mg/ml mitomycin C (Sigma) stock solution, filter sterilized (store at 4°C protected from light)

Support Protocol 1: Freezing and Thawing MEFs

  Materials
  • Plates containing MEFs (see Basic Protocols protocol 11 and protocol 22; see protocol 3)
  • Ca2+‐ and Mg2+‐free HBSS (see reciperecipe)
  • Trypsin/EDTA solution (see reciperecipe)
  • Freezing medium (see reciperecipe)
  • MEF medium (see reciperecipe) with or without penicillin/streptomycin
  • Cryovials
  • Additional reagents and equipment for counting cells with a hemacytometer ( appendix 3F)
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Figures

Videos

Literature Cited

   Coté, R. 2000. Assessing and controlling microbial contamination in cell cultures. In Current Protocols in Cell Biology (J.S. Bonifacino, M. Dasso, J.B. Harford, J. Lippincott‐Schwartz, and K.M. Yamada, eds.) pp. 1.5.1‐1.5.18. John Wiley & Sons, New York.
   Hogan, B., Beddington, R., Constantini, F., and Lacy, E. 1994. Manipulating the Mouse Embryo: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
   Martin, G.R. and Evans, M.J. 1975. Differentiation of clonal lines of teratocarcinoma cells: Formation of embryoid bodies in vitro. Proc. Natl. Acad. Sci. U.S.A. 72:1441‐1445.
   Robertson, E.J. 1987. Embryo‐derived stem cell lines. In Teratocarcinomas and Embryonic Stem Cells: A Practical Approach (E.J. Robertson, ed.) pp. 71‐112. IRL Press, Oxford.
   Wurst, W. and Joyner, A.L. 1993. Production of targeted embryonic stem cell clones. In Gene Targeting: A Practical Approach (A.L. Joyner, ed.) pp. 33‐61. IRL Press, Oxford.
Key References
   Hoganet al., 1994. See above.
  Provides additional or alternative protocols and defines the context for use of MEFs during gene targeting in ES cells.
Internet Resources
   www.biosupplynet.com
  Search this Web site for “embryonic stem cell reagents” to obtain a current list of suppliers that provide medium and MEFs suitable for ES cell culture.
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