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The aim of this work was to evaluate semi-synthetic biopolymers based on chitosan (CH) and gelatin (G) as potential in vitro carrier substrata for human limbal epithelial cells (hLECs). To that end, human corneal epithelial cells (HCE) were cultured onto different CH–G membranes. None of the polymers were cytotoxic and cell proliferation was higher when CH was functionalized with G. Expression levels of corneal epithelial markers (K3, K12, E-caherin, desmoplakin, and zonula occludens (ZO)-1) were better maintained in HCE cells grown on CH–G 20:80 membranes than other proportions. Consequently, CH–G 20:80 was chosen for the subsequent expansion of hLECs. Cells derived from limbal explants were successfully expanded on CH–G 20:80 membranes using a culture medium lacking components of non-human animal origin. The expression levels found for corneal (K3 and K12) and limbal …