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D.G. Harkin, S.L. Ainscough, Z. Barnard, Z. Upton; IGF–I Used in Conjunction With IGFBP, EGF and Vitronectin Supports ex vivo Expansion of Limbal Epithelial Cells Under Serum–Free Conditions . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1604.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the responsiveness of limbal epithelial cells to a novel combination of growth factors and matrix proteins with the view to developing a more defined culture system for ex vivo expansion.
Primary cultures of human limbal epithelial cells were initially established from donor tissue utilizing foetal bovine serum and irradiated 3T3 feeder cells (i3T3). The responsiveness of these cells was examined to varying combinations of insulin–like growth factor I (IGF–I), insulin–like growth factor binding protein 3 (IGFBP–3), epidermal growth factor (EGF) and vitronectin (VN) using trans–well migration assays and the MTT assay for metabolic activity. Optimised conditions were subsequently examined for their ability to support the establishment and serial propagation of cultures in the absence of serum and i3T3 cells. The phenotype of cultures produced was examined by immunocytochemistry for keratin 3 (mAb AE5) and p63 (mAb 4A4).
Optimal responses for cells derived from established cultures were observed in the presence of IGF+EGF (for MTT assay) or IGF–I+IGFBP–3+EGF+VN (for migration assay). Responses in both instances were typically greater than those observed towards serum–supplemented growth medium. A combined treatment of IGF–I+IGFBP(3 or 5)+EGF+VN was subsequently found to support the establishment and serial propagation of cultures in the absence of serum, but i3T3 were still required. Positive staining for keratin 3 and p63 confirmed the presence of immature limbal–corneal epithelial cells.
This study demonstrates the potential of a novel combination of growth factors (IGF–I, EGF) and matrix proteins (VN, IGFBP) for ex vivo expansion of limbal epithelial cells under serum–free conditions.
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