Abstract
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.
Highlights
There is no accepted alternative regulatory method to eliminate the use of animals in ocular irritation testing for the risk assessment of chemicals, pharmaceuticals and cosmetics
Aiming at the establishment of a multilayered cornea model to replace animal experiments in eye irritation testing, our work focuses on the optimisation of an existing full-thickness-model of the human cornea constructed from three SV40-immortalised human cell lines (Engelke et al, 2004; Zorn-Kruppa et al, 2005) with regard to standardised serum-free culture conditions for all the three different cell types of this tissue engineered cornea
Murine monoclonal anti-α-smooth-muscle-specific actin (α-SMA), anti-mouse IgG-FITC secondary antibody, human transforming growth factor β1 (TGFβ1), aprotinin, bovine serum albumin (BSA), bromophenol blue, collagenase from Clostridium histolyticum, type I collagen from rat tail, deoxycholic acid, dimethyl sulphoxide (DMSO), dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), gelatine from cold water fish skin, glycerol, goat serum, HEPES, isopropanol, leupeptin, MowiolTM, 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT), NaHCO3, NaOH, Nonidet P-40, pepstatin, phenylmethanesulfonyl fluoride (PMSF), sodium azide, sodium chloride, sodium dodecyl sulphate (SDS), sodium fluoride, calcium chloride, sodium orthovanadate, Tris, Tris/HCl, Triton X-100 and Tween 20 were purchased from SIGMA-Aldrich (Deisenhofen, Germany)
Summary
There is no accepted alternative regulatory method to eliminate the use of animals in ocular irritation testing for the risk assessment of chemicals, pharmaceuticals and cosmetics. The gap between mild and moderate eye irritation potential has to be closed by new alternative methods and the call for a mechanistically based method that considers the depth of the corneal injury and covers the whole severity range of toxic reactions, demands an implementation of corneal structures, such as stroma and endothelium, in an organotypic in vitro model of the human cornea (Maurer et al, 2002; Maurer et al, 2001; Jester et al, 2001; Jester, 2006). Most ocular irritants cause progressive injury to the cornea with stromal injury occurring beneath the areas of epithelial denudation Clear exceptions to this rule are some chemicals (Maurer et al, 2001) which are more harmful to the stroma than to the epithelium. Assays using only stratified epithelium are restricted to the detection of those materials causing only slight irritation, whereas the inclusion of stroma and stromal keratocytes broadens the range of potential irritation that can be detected
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