In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds

Abstract

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.

Investigation of Cancer Cell Migration and Proliferation on Synthetic Extracellular Matrix Peptide Hydrogels

“…Of course, steps have to be taken to investigate the influence of the density of each peptide and their combination, to introduce other ECM molecules such as hyaluronic acid and proteoglycan signaling, and to elaborate their densities, to shape the matrix creating tissue-like structures like capillaries and nerves with the respective topography and biochemistry. Taking into account that PEG-CLP hydrogel is shape-retaining and can be precisely molded at a fine microscale level (Haagdorens et al, 2019), such organ-mimicking micro-shaping might be introduced for further microenvironmental tuning.…”

“…Further, 12% (w/w) PEG-peptide aqueous solution was crosslinked with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) at RT ( Haagdorens et al, 2019 ). Due to the hydrophobic nature of the CLP-IKVAV peptide, 10% (w/w) PEG-CLP-IKVAV solution was used.…”

Selecting Appropriate Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Studies in Isolated and Cultured Ocular Surface Epithelia

“…Limbal biopsies were isolated and processed according to the protocol described by Haagdorens et al . 85 . In brief, biopsies were taken from the superior and inferior keratolimbal region (Fig.…”

New Technologies in Clinical Trials in Corneal Diseases and Limbal Stem Cell Deficiency: Review from the European Vision Institute Special Interest Focus Group Meeting

“…Therefore, there remains a need for optimized carrier membranes that combine the advantages of fibrin and HAM, while tackling its shortcomings. A very promising approach is the application of collagen hydrogels as a carrier for LESCs in CLET as collagen is characterized by inherent biocompatibility and cost-effectiveness [188]. After all, collagen is the most abundant protein of the human body, and more than 90% of the human cornea consists of collagen type I. Collagen hydrogels have already been applied successfully in corneal tissue engineering [189-191].…”