Chitosan-Gelatin Biopolymers as Carrier Substrata for Limbal Epithelial Stem Cells

Abstract

Purpose: The aim of this work was to evaluate different semi-synthetic biopolymers based on chitosan (CH) and gelatin (G) as potential in vitro carrier substrata for human cells derived from the limbal niche, the so-called limbal epithelial stem cells (LESC).

Methods: Semi-synthetic biopolymers were obtained using 85% deacetylated CH and different concentrations of pig skin G (0%, 20%, 50% and 80%). Glutaraldehyde (0.50% wt) was added as crosslinker. Human corneal epithelial cells (HCE) were cultured onto the different membranes for several days. Cell adhesion, viability and proliferative capacity were analyzed using Calcein/EthDIII kit and Alamar blue test, respectively. LESC derived from human limbal explants were expanded using a culture medium lacking components of animal origin. Tissue culture plastic was used as control substratum. The expression of specific corneal epithelial cell markers (K3, K12, E-caherin, desmoplakin, and zonula occludens (ZO)-1) and LESC markers (K15 and ABCG2) was evaluated by real time RT-PCR and immunoflurescence microscopy.

Results: None of the polymers tested were cytotoxic and HCE cell proliferation was higher when CH formulations were crosslinked with G. Expression levels of specific corneal epithelial marker mRNAs KRT3, KRT12, E-caherin, desmoplakin, and ZO-1, as well as proteins K3 and K12 were better in HCE cells grown on CH-G 20:80 membranes. Because of the sustained expression levels of these markers by cells grown on that polymer, and because of the easy handling of this substratum, CH-G 20:80 was chosen for the subsequent expansion of human LESC. Cells derived from limbal explants were successfully expanded on CH-G 20:80 membranes. The expression levels found for the LESC markers K15 and ABCG2 in limbal cells expanded on CH-G 20:80 membranes were similar to or higher than those found in limbal cells grown onto the control substratum.

Conclusions: The successful growth of cells with a stem cell-like phenotype derived from limbal explants was achieved on a semi-synthetic substratum in a medium free of non-human animal components. CH-G 20:80 membranes were suitable for the expansion and maintenance of these LESC. These results strongly support the use of polymers as alternative substrates for the transplantation of cultivated limbal cells onto the corneal surface.

Keywords: 482 cornea: epithelium • 687 regeneration