Isolation and Propagation of Mouse Embryonic Fibroblasts and Preparation of Mouse Embryonic Feeder Layer Cells

Anna E. Michalska1

1 Monash University, Victoria, Australia
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1C.3
DOI:  10.1002/9780470151808.sc01c03s3
Online Posting Date:  October, 2007
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Abstract

To realize their potentials, embryonic stem (ES) cells must be maintained in optimal culture conditions that preserve their pluripotency and self‐renewal capacity. Mouse embryonic fibroblasts (MEFs) are used to prepare a feeder cell layer that supports the growth of ES cells and the quality of feeders is crucial for the maintenance of undifferentiated ES cells in prolonged culture. The protocols provided in this unit describe aspects of isolation and expansion of MEFs and maintenance of established feeder cells. Preparation of mitotically inactivated feeder cell layer (treatment with mitomycin C or γ‐irradiation) is also described. In addition, a method for counting cell numbers and a simple method for detection of mycoplasma contamination by in situ DNA staining are also provided. Methodology described has been tested in a real laboratory environment and provides detailed information regarding resource and time requirements as well as critical parameters and troubleshooting. Curr. Protoc. Stem Cell Biol. 3:1C.3.1‐1C.3.17. © 2007 by John Wiley & Sons, Inc.

Keywords: embryonic stem (ES) cells; culture; mouse embryonic fibroblasts (MEFs); isolation and expansion; feeder cell layer; MEFs freezing and thawing; mycoplasma detection; protocols and methods

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation and Propagation of Mouse Embryonic Fibroblasts
  • Support Protocol 1: Freezing MEFs
  • Support Protocol 2: Thawing MEFs
  • Support Protocol 3: Mycoplasma Testing
  • Basic Protocol 2: Preparation of Mouse Embryonic Feeder Cell Layers by Mitomycin C Treatment
  • Alternate Protocol 1: Preparation of Mouse Embryonic Feeder Cell Layers by γ‐Irradiation
  • Support Protocol 4: Counting Number of Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Isolation and Propagation of Mouse Embryonic Fibroblasts

  Materials
  • 12.5 to 14.5 days post‐coitum (dpc) pregnant female mouse
  • 70% (v/v) ethanol (see recipe)
  • Phosphate‐buffered saline, calcium and magnesium free (CMF‐PBS; see recipe), ice cold and room temperature
  • 0.25% (w/v) trypsin/EDTA solution for fetal tissue digestion (see recipe)
  • MEF culture medium (see recipe)
  • 0.05% (w/v) trypsin/EDTA solution for MEFs passaging (see recipe)
  • Pair of scissors and forceps (do not have to be sterile)
  • Sterile instruments (iris scissors, 2 pairs of watchmaker forceps no. 5, scalpel blade)
  • 10‐cm bacteriological petri dishes
  • 5‐ and 10‐ml plastic pipets
  • 50‐ml centrifuge tubes
  • 37°C water bath
  • 37°C, 5% CO 2 incubator
  • 75‐cm2 tissue culture flasks
  • Inverted microscope
  • Additional reagents and equipment for euthanasia by cervical dislocation (Donovan and Brown, )

Support Protocol 1: Freezing MEFs

  Materials
  • 75‐cm2 flask(s) of 80% to 90% confluent MEFs ( protocol 1)
  • Freezing solution (see recipe)
  • Liquid nitrogen
  • 1‐ml cryovials
  • 15‐ml centrifuge tubes
  • Container with ice
  • Freezing container (e.g., Mr. Frosty or Styrofoam box; Nalgene)
  • Additional reagents and equipment for removing cells from the flasks ( protocol 1)

Support Protocol 2: Thawing MEFs

  Materials
  • MEF culture medium (see recipe)
  • Cryovials containing frozen MEFs ( protocol 2)
  • 70% (v/v) ethanol (see recipe)
  • 15‐ml centrifuge tubes
  • 37°C water bath
  • 75‐cm2 tissue culture flasks

Support Protocol 3: Mycoplasma Testing

  Materials
  • Cultures of MEFs
  • MEF culture medium (see recipe) with and without antibiotics
  • Fixative solution (see recipe)
  • Phosphate‐buffered saline, calcium and magnesium free (CMF‐PBS; see recipe)
  • Hoechst 33258 dye solution (see recipe)
  • 6‐cm tissue culture petri dishes
  • Cover slips
  • UV fluoresence microscope

Basic Protocol 2: Preparation of Mouse Embryonic Feeder Cell Layers by Mitomycin C Treatment

  Materials
  • 75‐cm2 flask(s) of 80% to 90% confluent MEFs ( protocol 1)
  • Mitomycin C (MMC) solution (see recipe)
  • MEF culture medium (see recipe)
  • 0.1% (w/v) gelatin solution (see recipe)
  • Phosphate‐buffered saline, calcium and magnesium free (CMF‐PBS; see recipe)
  • 0.05% (w/v) trypsin/EDTA solution (see recipe)
  • 37°C, 5% CO 2 incubator
  • Appropriate tissue culture plates, dishes, or flasks for growing ES cells
  • Inverted microscope
  • 10‐ml plastic pipets
  • 15‐ml centrifuge tubes
  • 37°C, 5% CO 2 incubator

Alternate Protocol 1: Preparation of Mouse Embryonic Feeder Cell Layers by γ‐Irradiation

  Materials
  • 75‐cm2 flask(s) of 80% to 90% confluent MEFs ( protocol 1)
  • 0.1% (w/v) gelatin solution (see recipe)
  • MEF culture medium (see recipe)
  • Appropriate tissue culture plates/dishes/flasks for growing ES cells
  • 50‐ml centrifuge tubes
  • Controlled cesium source for γ‐irradiation
  • Additional reagents and equipment for detaching MEFs ( protocol 1)

Support Protocol 4: Counting Number of Cells

  Materials
  • 0.1‐ to 0.2‐ml‐size aliquot of MEFs
  • 0.4% (w/v) trypan blue solution (see recipe)
  • Hemacytometer and coverslip
  • Piston‐driven air displacement pipet (e.g., Gilson pipet)
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Figures

Videos

Literature Cited

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Key References
   Lanza, R., ed. 2004. Handbook of Stem Cells, Vol. 1: Embryonic Stem Cells (R. Lanza, J. Gearhart, B. Hogan, D. Melton, R. Pedersen, J. Thomson, and M. West, eds.) pp. 15‐26. Elsevier Academic Press, Amsterdam.
  Collection of articles by world's experts in the field of embryonic stem cell research, providing background information and methods on various aspects of ES manipulation.
   Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. 2002. Manipulating the Mouse Embryo: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Press, New York.
  A comprehensive manual describing all aspects of early mouse embryo culture and manipulation including isolation and propagation of mouse embryonic stem cells.
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