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Copyright 2003 Cascade Biologics, Inc. All rights reserved.
PRIMARY ISOLATION AND SERIAL PROPAGATION OF NORMAL HUMAN
KERATINOCYTES IN AN ANIMAL PRODUCT-FREE ENVIRONMENT
Paul W. Cook, Ann K. Shipley, Michelle Van Kleeck, Christine Parrish, Erin Tucker and Shiwei Li.
Cascade Biologics, Inc.
1341 SW Custer Drive, Portland, OR USA 97219
(503) 292-9521, (503) 292-0566 (FAX)
ABSTRACT: Existing culture media for the isolation and serial propagation of human keratinocytes are
supplemented with animal-derived materials that can include bovine serum albumin (BSA), transferrin,
insulin, serum (FBS), and/or bovine pituitary gland extracts (BPE). Additionally, xenotrophic feeder layer
cells are sometimes utilized to promote the growth of these cells. Disadvantages of utilizing animal-
derived supplements include expense, and potential contamination with infectious agents such as
mycoplasma, viruses and prions (BSE), which can pose a health risk to both workers exposed to the
contaminated supplements, and patients treated with contaminated therapeutic agents. We have recently
discovered that certain plant-derived extracts can act as replacements for animal-derived products in
supporting the primary isolation and serial propagation of both neonatal and adult human epidermal
keratinocytes in an animal product -free (APF) environment. Application of a defined Coating Matrix to the
plastic substrate enhances the plating and proliferation of keratinocytes under these APF conditions.
Utilizing this APF system, human keratinocytes can be propagated for up to 55 cumulative population
doublings after the primary culture, and have initial growth rates of up to 1.2 population doublings per day.
Human keratinocytes cultured in the APF system appear to be free of contaminating dermal fibroblasts
and melanocytes, possess normal morphology, and express both epidermis-specific cytokeratins,
calcium-inducible involucrin, and the p63 gene product. Our results demonstrate the first APF primary