Purpose. To investigate the efficacy of recombinant human collagen type I (RHC I) and collagen-like peptide (CLP) hydrogels as alternative carrier substrates for the cultivation of limbal epithelial stem cells (LESC) under xeno-free culture conditions. Methods. Human LESC were cultivated on seven different collagen-derived hydrogels: (1) unmodified RHC I, (2) fibronectin-patterned RHC I, (3) carbodiimide-crosslinked CLP (CLP-12 EDC), (4) DMTMM- (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium-) crosslinked CLP (CLP-12), (5) fibronectin-patterned CLP-12, (6) “3D limbal niche-mimicking” CLP-12, and (7) DMTMM-crosslinked CLP made from higher CLP concentration solution. Cell proliferation, cell morphology, and expression of LESC markers were analyzed. All data were compared to cultures on human amniotic membrane (HAM). Results. Human LESC were successfully cultivated on six out of seven hydrogel formulations, with primary cell cultures on CLP-12 EDC being deemed unsuccessful since the area of outgrowth did not meet quality standards (i.e., inconsistence in outgrowth and confluence) after 14 days of culture. Upon confluence, primary LESC showed high expression of the stem cell marker ΔNp63, proliferation marker cytokeratin (KRT) 14, adhesion markers integrin-β4 and E-cadherin, and LESC-specific extracellular matrix proteins laminin-α1, and collagen type IV. Cells showed low expression of differentiation markers KRT3 and desmoglein 3 (DSG3). Significantly higher gene expression of KRT3 was observed for cells cultured on CLP hydrogels compared to RHC I and HAM. Surface patterning of hydrogels influenced the pattern of proliferation but had no significant effect on the phenotype or genotype of cultures. Overall, the performance of RHC I and DMTMM-crosslinked CLP hydrogels was equivalent to that of HAM. Conclusion. RHC I and DMTMM-crosslinked CLP hydrogels, irrespective of surface modification, support successful cultivation of primary human LESC using a xeno-free cultivation protocol. The regenerated epithelium maintained similar characteristics to HAM-based cultures.

A methodology was developed and optimised for the preparation of a new drug delivery system (DDS) with sustained release properties to allow ocular protein delivery and to limit destructive production steps during manufacturing. Elevated temperatures, shear forces and an oxidative environment should be avoided in order to prevent denaturation or oxidation of proteins. An aqueous HPMC solution was prepared using heat and casted into small semi-rod-shaped PVC blisters. The polymer solution was allowed to cool down and was partially dehydrated at room temperature. A drug solution containing glycerol, drug and water was subsequently added to rehydrate the partially dehydrated polymer matrix at a temperature of 2°C. Several parameters of the production process were varied to determine their influence on the release kinetics from HPMC inserts from three different molecules of different molecular weight. This production method was further optimised in order to shorten the rehydration time from weeks to days, while eliminating heat and shear forces on the selected drug molecules sodium fluorescein, lysozyme and albumin. Slow release kinetics were achieved for sodium fluorescein and lysozyme as model drug molecules. The higher molecular weight of albumin prevented a good penetration into the insert during the rehydration process resulting in predominantly burst release. The biocompatibility of a viscous HPMC solution was evaluated on SV40-human corneal epithelial cells with PrestoBlue and no cytotoxic effects were observed.