Preparation of Human Epidermal Keratinocyte Cultures

Susan S. Yamada1

1 National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 2.6
DOI:  10.1002/0471143030.cb0206s21
Online Posting Date:  February, 2004
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Abstract

Cultured keratinocytes have been used by a number of investigators in studies investigating wound repair and carcinogenesis, and they have also proven useful as a model for differentiation. This unit describes a protocol for establishing human keratinocytes in tissue culture. Human newborn foreskins are proteolytically digested to separate the epidermis and the dermis, and keratinocytes are obtained from the epidermis.

Keywords: keratinocyte cultures; establishment of epidermal keratinocyte culture keratinocytes; human keratinocytes; murine keratinocytes; morphological features

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Human newborn foreskin (for sources, see Background Information)
  • Betadine (Purdue Frederick) or 5% (v/v) Wescodyne (STERIS Corporation)
  • HEPES‐buffered saline (see recipe)
  • 0.25% (w/v) trypsin in HEPES‐buffered saline
  • Keratinocyte primary culture medium (see recipe)
  • Hanks' balanced salt solution (HBSS; appendix 2A)
  • Keratinocyte growth medium (either Keratinocyte‐SFM from Invitrogen or KGM from Cambrex)
  • Trypsin/EDTA solution (Invitrogen) diluted 1:1 with Hanks' balanced salt solution (final 0.025% w/v trypsin, 0.26 mM EDTA)
  • 10 mg/ml soybean trypsin inhibitor (Sigma) in Hanks' balanced salt solution
  • Freezing medium: 10% (v/v) DMSO/10% (v/v) heat‐inactivated fetal bovine serum/80% (v/v) keratinocyte growth medium
  • 100‐mm sterile tissue culture dishes
  • Small curved forceps (4 pairs), sterile
  • Small sharp straight or curved scissors, sterile
  • Coarse filter such as a tea strainer or several layers of cheesecloth taped over the top of a 50‐ml centrifuge tube, sterile
  • 15‐ and 50‐ml sterile disposable centrifuge tubes
  • 75‐cm2 sterile tissue culture flasks
  • 1‐ml sterile cryotubes
  • Liquid nitrogen freezer
  • Additional reagents and equipment for cell culture (unit 1.1)
CAUTION: When working with human tissue, appropriate biosafety practices must be followed.
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Figures

Videos

Literature Cited

Literature Cited
   Boyce, S.T. and Ham, R.G. 1983. Calcium‐regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum‐free serial culture. J. Invest. Dermatol. 81:33s‐40s.
   Dlugosz, A.A., Glick, A.B., Tennenbaum, T., Weinberg, W.C., and Yuspa, S.H. 1995. Isolation and utilization of epidermal keratinocytes for oncogene research. Methods Enzymol. 254:3‐20.
   Rheinwald, J.G. and Green, H. 1975. Serial cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cells. Cell 6:331‐344.
Internet Resources
   http://www-chtn.ims.nci.nih.gov/
  Web site of the Cooperative Human Tissue Network, which is a program supported by the National Cancer Institute that can provide human tissue at nominal cost.
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