Human Foreskin Fibroblast (HFF) preparation

  1. Collect foreskin tissue from newborn nursery (3 or more pieces) in HFF Medium I
  2. Wash 3-4 times with 1x PBS in 50 ml conical tube, shaking vigorously (to remove blood).
  3. Put into plastic petri dish and mince with two pairs of (sterile) scissors into fine pieces of a few mm3 (easiest to do it dry).
  4. Transfer the minces tissue to a fluted flask.
  5. Add 40 ml TV into the sterile fluted flask with a stir bar.
  6. Stir at 37°C (in an incubator) for 10 minutes.
  7. Repeat step 5-6 once.
  8. Add 40 ml 37°C TV and stir at 37°C for 10 minutes.
  9. Let minced tissue settle (it shuold be getting digested) and collect the supernatant (~35 ml) by pipette into a 50 ml conical tube.
  10. Centrifuge at ~400 rcf (~1200 rpm in a swinging bucket) for 5 minutes.
  11. Carefully remove and discard the supernatant.
  12. Resuspend the pellet (cells) in 5 ml Medium II.
  13. Transfer resuspended cells into a T75 in 35 ml total volume.
  14. Repeat steps 8-13 four to five times until the tissue no longer yields a pellet of cells.
  15. Incubate at 37°C in CO2 incubator for 1-2 days.
  16. Change medium for the T75 flasks with fresh Medium II (attached cells should be visible).
  17. Change medium every third day with fresh Medium II until cells are confluent.
  18. Split the cells and remove antibiotics gradually:
    • 1st split: from 1x T75 into 1x T175, remove gentamicin and fungizone.
    • 2nd split: from 1x T175 into 2x T175
    • 3rd split: from 2x T175 into 4x T175
    • 4th split: from 4x T175 into 8x T175
  19. Freeze down the cells at passage 4 (1 vial per T175) and store in liquid nitrogen.
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