Cell Culture Information for Keratinocytes

Keratinocyte Cell Culture Protocol:

Keratinocyte cells should be cultured in a 50%-50% mixture of DMEM and Ham’s F12. Use a 1:1 ratio of the two medias. The mixture should be modified by addition of 10% FBS. Make sure that F12 contains 1.2 g/L sodium bicarbonate, 2.5 mM L-glutamine, 15 mM HEPES and 0.5 mM sodium pyruvate supplemented with 400 ng/ml hydrocortisone. Incubate cells at 37.0°C in humidified cell culture incubator. Cell cultures will double approximately every 30 hours. This will vary depending on the keratinocyte cells source and culturing conditions.


Split the cells 1:4 to 1:8 once just confluent, and renew the medium ever 2 to 3 days.

  1. Remove medium
  2. Rinse cells with 1xPBS or solution containing 0.05% Trypsin and 0.03% EDTA
  3. Add an additional 1-2 mL of Trypsin-EDTA and allow cells to disperse
  4. If cells are not dispersing, place cells in incubator for 3-5 minutes to encourage rounding up
  5. Centrifuge cells at 125 x g for 5-10 minutes
  6. Remove supernatant (Trypsin-EDTA)
  7. Add fresh culture medium
  8. Split and dispense cells into new cell culture flasks

Keratinocyte cryopreservation:

Freeze cells in conditioned growth medium supplemented with 5-10% (v/v) DMSO by freezing at 1 degree (C) per minute, and store in the liquid nitrogen vapor phase or at -135 °C.


Keratinocyte Stable Cell Line Generation Services

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Keratinocyte (ATCC)

Keratinocyte Transfection Resources:

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