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Making Mouse Embryonic Fibroblasts (MEFs)

Method

  1. Dissect E12.5-14.5 mouse embryos into a 10-cm dish with PBS. To ensure a purer fibroblastic population, remove the brain and limbs, and scoop out the internal organs (You can use some of this for genotyping.).
  2. Place each individual embryo in a well of a 6-well plate in PBS and transfer plate to tissue culture hood. Transfer to new sterile 6-well plate with 1 ml trypsin per well.
  3. Mince embryos up with a sterile razor blade (ethanol and flamed) into very small pieces, with a consistency similar to sludge. Then use 1-ml pipette to help homogenize the embryos further. Place in 37°C incubator for 10 min.
  4. Remove from incubator and once again use 1-ml pipette to pipet up and down to further breakup the cells. There will be some viscous material on the bottom of the plates. Immediately add 2 ml MEF media to each well.
  5. Split volume between 3 10-cm dishes which have been gelatinized with 0.1% gelatin and incubate at 37°C. Cells should attach within next two days. Change media once the cells have attached.
  6. Aspirate remaining cartilaginous chunks (very obvious when you change media).
  7. Grow to confluency, then freeze two 10-cm dishes as p0 and continue to grow the other plate, splitting 1:10.

Gelatinizing plates

  1. Make 0.1% gelatin solution (bovine skin type I collagen) by dissolving 0.5 g collagen into 500 ml PBS and autoclaving.
  2. Cover surface of tissue culture dish with gelatin and let sit at RT 1-2 hours.
  3. Pipet off excess solution and let dry min 15 minutes. These dishes can now be stored at 4°C indefinitely.

Solutions

MEF Media

  • 440 ml DMEM
  • 50 ml FBS (heat-inactivated)
  • 5 ml Pen/Strep
  • 5 ml L-glutamine
  • Filter sterilize

b-Mercaptoethanol (b-ME)

  • 72 µl 14M b-ME
  • 100 ml 1X PBS

© 2014 The University of Texas MD Anderson Cancer Center