Making MEFs - Mouse Embryonic Fibroblast Collection from Embryos

Note: perform procedure in cell culture hood and use aseptic technique; collection at dpc13.5

  1. Place freshly harvested embryos in 10cm cell culture dish
  2. Cover embryos in PBSA and remove placental and other maternal tissues
  3. Cut away top of head (eye and above) and eviscerate removing all innards
  4. Save head for DNA isolation for genotyping (or yolk sac)
  5. Place embryo body in separate 10cm plate
  6. Add 1mL trypsin and mince embryo with razor blade
  7. Sterilize blade between samples with Bunsen burner flame
  8. Place plate in 37°C incubator for 30-45min (tilted so trypsin covers the cells)
  9. Quench typsin activity by adding 4mL MEF media (DME w/ 10% FCS+P/S) per well
  10. Pipette 10-20x to break up tissues
  11. Transfer cellular suspension to T75 flask or 10cm plate and add 6-15mL MEF media
  12. Allow cells to grow to confluency (3-4days)
  13. Aspirate off medium and rinse with 10mL PBS
  14. Add 3mL trypsin and incubate in 37°C incubator 5-10min
  15. Quench by adding 7mL MEF media
  16. Pipette 15x to resuspend cells
  17. Transfer suspension to 2 T175 flasks and add 50mL MEF media to each flask
  18. Allow cells to grow to confluency (3-4 days)
  19. Harvest cells via trypsin and freeze down cells @ 3x106 cells/vial