SV40-transformed human corneal keratocytes: optimisation of serum-free culture conditions.

Aiming at the replacement of animal experiments in eye irritation testing, we have established a multilay ered cornea model comprising the co-culture of all three corneal cell types. It was the objective of this study to optimise serum-free culture conditions to preserve both growth and phenotype of an SV40-immortalised human corneal keratocyte cell line (HCK). Our results revealed that HCK continue to proliferate in both monolayer cultures as well as after seeding in a collagen matrix and resemble primary corneal keratocytes in morphology and functional characteristics under defined serum-free conditions. Furthermore, HCK were shown to transform into activated corneal fibroblast phenotypes in response to serum and TGF(beta)1. In summary, HCK cells mimic their in vivo (primary) precursors, both in sustaining the quiescent keratocyte phenotype (serum-starved conditions) and in responding to growth factor stimulation. Hence, this cell line may provide a useful tool to study the toxicity and wound healing response of corneal keratocytes in vitro.