In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds
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 di...
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Wiley
2019-01-01
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| Series: | Stem Cells International |
| Online Access: | http://dx.doi.org/10.1155/2019/7867613 |
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| author | Michel Haagdorens Vytautas Cėpla Eline Melsbach Laura Koivusalo Heli Skottman May Griffith Ramūnas Valiokas Nadia Zakaria Isabel Pintelon Marie-José Tassignon |
| author_facet | Michel Haagdorens Vytautas Cėpla Eline Melsbach Laura Koivusalo Heli Skottman May Griffith Ramūnas Valiokas Nadia Zakaria Isabel Pintelon Marie-José Tassignon |
| author_sort | Michel Haagdorens |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-b75633d2da1a4deab1e57add717ff582 |
| institution | Kabale University |
| issn | 1687-966X 1687-9678 |
| language | English |
| publishDate | 2019-01-01 |
| publisher | Wiley |
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| spelling | doaj-art-b75633d2da1a4deab1e57add717ff5822025-02-03T01:28:47ZengWileyStem Cells International1687-966X1687-96782019-01-01201910.1155/2019/78676137867613In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen ScaffoldsMichel Haagdorens0Vytautas Cėpla1Eline Melsbach2Laura Koivusalo3Heli Skottman4May Griffith5Ramūnas Valiokas6Nadia Zakaria7Isabel Pintelon8Marie-José Tassignon9Faculty of Medicine and Health Sciences, Department of Ophthalmology, Visual Optics and Visual Rehabilitation, University of Antwerp, Campus Drie Eiken, T building, T4-Ophthalmology, Universiteitsplein 1, 2610 Antwerp, BelgiumDepartment of Nanoengineering, Center for Physical Sciences and Technology, Savanorių 231, 02300 Vilnius, LithuaniaDepartment of Ophthalmology, Antwerp University Hospital, Wilrijkstraat 10, 2650 Antwerp, BelgiumFaculty of Medicine and Health Technology, Tampere University, Arvo Ylpön katu 34, 33014, FinlandFaculty of Medicine and Health Technology, Tampere University, Arvo Ylpön katu 34, 33014, FinlandMaisonneuve-Rosemont Hospital Research Centre and Department of Ophthalmology, University of Montreal, Montreal, QC, H1T 4B3, CanadaDepartment of Nanoengineering, Center for Physical Sciences and Technology, Savanorių 231, 02300 Vilnius, LithuaniaFaculty of Medicine and Health Sciences, Department of Ophthalmology, Visual Optics and Visual Rehabilitation, University of Antwerp, Campus Drie Eiken, T building, T4-Ophthalmology, Universiteitsplein 1, 2610 Antwerp, BelgiumLaboratory of Cell Biology and Histology, Antwerp University, Campus Drie Eiken, T building, T1-Veterinary Sciences, Universiteitsplein 1, 2610 Antwerp, BelgiumFaculty of Medicine and Health Sciences, Department of Ophthalmology, Visual Optics and Visual Rehabilitation, University of Antwerp, Campus Drie Eiken, T building, T4-Ophthalmology, Universiteitsplein 1, 2610 Antwerp, BelgiumPurpose. 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.http://dx.doi.org/10.1155/2019/7867613 |
| spellingShingle | Michel Haagdorens Vytautas Cėpla Eline Melsbach Laura Koivusalo Heli Skottman May Griffith Ramūnas Valiokas Nadia Zakaria Isabel Pintelon Marie-José Tassignon In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds Stem Cells International |
| title | In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds |
| title_full | In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds |
| title_fullStr | In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds |
| title_full_unstemmed | In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds |
| title_short | In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds |
| title_sort | in vitro cultivation of limbal epithelial stem cells on surface modified crosslinked collagen scaffolds |
| url | http://dx.doi.org/10.1155/2019/7867613 |
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