191 EPIDERMAL GROWTH FACTOR (EGF) EXTENDS LIFE SPAN OF NORMAL HUMAN FIBROBLASTS IN CULTURE

Abstract

Normal human fibroblast cultures derived from newborn foreskin (HF) have a limited in vitro life span of 50 ± 10 cumulative population doublings (CPD). Adding EGF (25 ng/ml) to cultures at different stages of in vitro aging increased the survival of individual strains by as much as 25% (10-15 CPD). In early single passages, EGF produced a nearly 2 fold greater cell density over control cultures. With increasing CPD, HF progressively showed decreased EGF sensitivity as reflected by decreased stimulation of cells to initiate DNA synthesis and decline in saturation density of EGF treated cultures. Cell density was similar in control and EGF cultures during the final 4-6 CPD. HF exposed to EGF from early stages of explant outgrowth showed no greater CPD than those exposed to EGF at later stages. Intermittent or discontinuous EGF exposure from early passages produced maximum CPD. Removal of EGF from cultures after early continuous exposure did not abolish the expanded culture survival. After control HF reached about 80% of expected CPD, exposure to EGF did not significantly extend life span of the cultures. Colony size distribution was determined during early, middle and late passages of strains with/without EGF exposure. In each strain regardless of CPD, EGF significantly increased the size distribution curve of derived colonies. Thus, EGF enhanced the survival of cells in subculture and prolonged their proliferative activity significantly beyond control cultures.