The goal of this study was to evaluate the potential suitability of collagen Vitrigel (CV) membrane as a substrate for the separate reconstruction of the three main cellular layers of the cornea. Limbal explants, keratocytes, and endothelial cells were cultured on transparent membranes made of type I collagen. The resulting cell sheets were evaluated using RT-PCR, in addition to light and electron microscopy. Tensile testing was also performed to examine the mechanical properties of CV. Limbal explant cultures resulted in partially stratified epithelial sheets with upregulation of the putative stem cell marker p63. Keratocytes cultured in serum on CV exhibited stellate morphology along with a marked increase in expression of corneal crystallin ALDH and keratocan, (a keratan sulphate proteoglycan: KSPG), compared to identical cultures on tissue culture plastic. Endothelial cells formed dense monolayers with uniform cell size, tight intercellular junctions, and expression of voltage-dependent anion channels VDAC2 and VDAC3, chloride channel protein CLCN2, and sodium bicarbonate transporter NBC1. Epithelial and endothelial cells exhibited adhesive structures (desmosomes and hemidesmosomes) and evidence of apical specialization (microplicae), while endothelial cells also produced a Descemet's membrane-like basal lamina. CV was found to possess ultimate tensile strengths of 6.8 +/- 1.5 MPa when hydrated and 28.6 +/- 7.0 MPa when dry. Taken together, these results indicate that CV holds promise as a substrate for corneal reconstruction.