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.
Highlights
Located at the corneoscleral limbus, limbal epithelial stem cells (LESC) play a pivotal role in rejuvenating the corneal epithelium and keeping the cornea healthy, transparent, and avascular [1, 2]
Human LESC were cultivated on seven different collagen-derived hydrogels: (1) unmodified recombinant human collagen type I (RHC I), (2) fibronectin-patterned RHC I, (3) carbodiimide-crosslinked collagen-like peptide (CLP) (CLP-12 ethylcarbodiimide hydrochloride (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
Optimum cutting temperature (OCT) formulation was purchased from Sakura Finetek Europe (Zoeterwoude, the Netherlands); nitrocellulose paper and filter sterilizers were from Merck Millipore (Darmstadt, Germany); polydimethylsiloxane (PDMS) was from Dow Corning (Midland, MI, USA); balanced salt solution (BSS) was from Alcon (Fort Worth, TX, USA); CnT-prime medium (CnT-PR) was from CELLnTEC (Bern, Switzerland); phosphate-buffered saline (PBS)/glycerol Citifluor was from Citifluor Ltd. (London, UK); and RNeasy Mini Kit was from QIAGEN (Hilden, Germany)
Summary
Located at the corneoscleral limbus, limbal epithelial stem cells (LESC) play a pivotal role in rejuvenating the corneal epithelium and keeping the cornea healthy, transparent, and avascular [1, 2]. In vitro processing remains labor intensive, costly, and challenging [9] These limitations hamper the application of HAM in ocular tissue engineering. In 2015, Holoclar® (Chiesi, Italy), a technique in which limbal cells are expanded on fibrin scaffolds, was conditionally approved for release in Europe as the first commercially available stem cell therapy for LSCD. Use of this medicinal product is restricted to autologous stem cell transplantation in unilateral cases after chemical or thermal burn. A safe and standardized therapy that targets all LSCD patients has yet to be developed
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