The effect of culture medium and carrier on explant culture of human limbal epithelium: a comparison of ultrastructure, keratin profile and gene expression

Abstract

PurposeTo examine the effect of medium and substrate on morphology, on expression of selected keratins and on global gene expression in ex vivo engineered corneo‐limbal grafts.MethodsLimbal biopsies retrieved from corneo‐scleral rings of cadaveric donors were placed on human amniotic membranes or on plastic inserts and cultured for 3 weeks in parallel in either a complex medium (COM) or in medium containing human serum (HM) as single growth promoting supplement at 37°C and 5% CO2. Culture medium was changed every 2–3 days. Grafts were examined using light microscopy (LM) and transmission electron microscopy (TEM) and immunohistochemistry (IHC) (for 8 keratins), microarray and qRT‐PCR were performed.ResultsLM and TEM examination revealed similar multilayered stratified epithelium with cuboidal basal cells and flattened superficial cells with microplicae, attached to one another by desmosomes. In explants cultured on HAM, hemidesmosome‐like junctions were observed between the expanded cells and the underlying HAM. Using IHC, expression of keratins was found to be similar in grafts engineered in the different conditions. We observed that cells cultured in the same medium (HS or COM) have more similarities in gene expression than cells cultured on same scaffold (PL or HAM).ConclusionsAll explants have similar morphology and keratin expression. The presence of hemidesmosome‐like junctions between the expanded cells and underlying HAM on TEM may indicate better attachment of the expanded cells to the carrier surface as compared to the cells cultured on PL. Gene expression was more similar in cells cultured in the same medium (HS or COM) as compared to cells cultured on the same scaffold (PL or HAM), suggesting that choice of media is of greater significance for explant cell characteristics than choice of carrier.