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Attachment and growth of human keratinocytes in a serum‐free environment
Author(s) -
Gilchrest Barbara A.,
Calhoun Jean K.,
Maciag Thomas
Publication year - 1982
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041120207
Subject(s) - keratinocyte , epidermal growth factor , incubation , fibronectin , in vitro , population , cell growth , cell culture , andrology , growth factor , doubling time , chemistry , biology , microbiology and biotechnology , extracellular matrix , biochemistry , medicine , genetics , receptor , environmental health
Using a serum‐free system, we have investigated the influence of human fibronectin (HFN) and selected growth factors (GF) on the attachment and growth of normal human keratinocytes in vitro. Single‐cell suspensions of keratinocytes from near‐confluent primary plates, plated on 5–10 μg/cm 2 HFN, showed approximately 30–40% attachment after 2–24 hours of incubation at 37°C, compared with 4–6% attachment on uncoated platic plates. Percentage of attached cells was independent of seed density, tissue donor age, in vitro culture age, or medium composition, while subsequent cellular proliferation was strongly dependent on these factors. Keratinocytes grown on an adequate HFN matrix in a previously described hormone‐supplemented medium (Maciag et al., 1981a) achieved four to eight population doublings over 7–12 days at densities ≥ 10 4 cell/cm 2 . Removal of most GF individually from the medium had little or no effect on growth, while removal of epidermal growth factor (EGF) alone reduced growth by 30–35% and removal of bovine brain extract (BE) alone reduced growth by approximately 90%. Conversely, EGF alone in basal medium supported approximately 10% control growth, BE alone supported 30–40% control growth, and the combination of EGF and BE approximately 70%. In addition to its major effect on proliferation in this system, BE was necessary to preserve normal keratinocyte morphology and protein production. These findings expand earlier observations that HFN facilitates keratinocyte attachment in vitro and that a brain‐derived extract can exert a major positive influence on cultured keratinocytes.