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Isolation of Primary Human Keratinocytes from Foreskin   

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Keratinocytes are the primary constituents of human skin, the functional barrier between our bodies and the external environment. The balance between keratinocyte differentiation and self-renewal is crucial to skin homeostasis. Primary keratinocyte culture serves as a tractable model for understanding human epithelial cell differentiation as well as self-renewal.

Materials and Reagents

  1. Foreskin
  2. 0.05% Trypsin (Life Technologies, InvitrogenTM, catalog number: 25300-054 )
  3. 2x dispase (Life Technologies, InvitrogenTM, catalog number: 17105-041 )
  4. DMEM (Life Technologies, InvitrogenTM, catalog number: 11965-092 )
  5. Fetal bovine serum (FBS) (Life Technologies, InvitrogenTM, catalog number: 16000-044 )
  6. Defined keratinocyte serum-free medium (Life Technologies, InvitrogenTM, catalog number: 10744-019 )
  7. 154 medium (Life Technologies, InvitrogenTM, catalog number: M-154-500 )
  8. Penicillin and streptomycin (Life Technologies, InvitrogenTM, catalog number: 10378-016 )
  9. Phosphate buffered saline (PBS)
  10. DMEM cultural medium (see Recipes)
  11. 50: 50 medium (see Recipes)


  1. Centrifuges
  2. Incubator (37 °C and 5% CO2)
  3. Sterile petridish
  4. Cell culture hood
  5. Cell culture dishes
  6. Scissors
  7. Forceps
  8. 50 ml falcon tube
  9. Disposable sterile cell culture strainer


  1. In a sterile petridish, remove vascular and adipose tissue from foreskin using scissors and forceps.
    Note: All manipulations should be done with sterile instruments and in the hood.
  2. Incubate prepared foreskin with 10 ml of 1x dispase for 12-18 h at +4 °C. 2x dispase stock should be diluted in PBS, containing penicillin (500 units/ml) and streptomycin (50 μg/ml).
  3. Peel off the epidermis and place into 2-3 ml of 0.05% trypsin. Incubate at 37 °C for 15 min in a 50 ml falcon tube.
  4. Quench trypsin by adding 2 volumes of DMEM, containing 10% FBS. Invert falcon tube several times to detach keratinocytes from the dermis.
  5. Pass solution through a sterile sieve and centrifuge filtered solution at 1,000 rpm and 37 °C to pellet cells.
  6. Aspirate medium, gently resuspend cells in 30 ml of 50:50 medium supplemented with penicillin and streptomycin and plate on 3, 10 cm dishes.
  7. Incubate at 37 °C and 5% CO2 for several days until colonies grow out. Change the medium every 2 days.


  1. DMEM cultural medium
    DMEM supplemented with 10% FBS.
  2. 50:50 medium
    1:1 mixture of defined KSF medium and 154 medium


  1. Rheinwald, J. G. and Green, H. (1975). Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6(3): 331-343.
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Reuter, J. (2011). Isolation of Primary Human Keratinocytes from Foreskin. Bio-101: e165. DOI: 10.21769/BioProtoc.165.

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