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

被引：28

作者：

Haagdorens, Michel ^{[1
,2
]}

Cepla, Vytautas ^{[3
,4
]}

Melsbach, Eline ^{[2
,5
]}

Koivusalo, Laura ^{[6
]}

Skottman, Heli ^{[6
]}

Griffith, May ^{[7
,8
]}

Valiokas, Ramunas ^{[3
,4
]}

Zakaria, Nadia ^{[1
,2
,5
]}

Pintelon, Isabel ^{[9
]}

Tassignon, Marie-Jose ^{[1
,2
]}

机构：

[1] Univ Antwerp, Fac Med & Hlth Sci, Dept Ophthalmol Visual Opt & Visual Rehabil, Campus Drie Eiken,T Bldg,T4 Ophthalmol,Univ Pl 1, B-2610 Antwerp, Belgium

[2] Antwerp Univ Hosp, Dept Ophthalmol, Wilrijkstr 10, B-2650 Antwerp, Belgium

[3] Ctr Phys Sci & Technol, Dept Nanoengn, Savanoriu 231, LT-02300 Vilnius, Lithuania

[4] Ferentis UAB, Savanoriu 235, LT-02300 Vilnius, Lithuania

[5] Antwerp Univ Hosp, Ctr Cell Therapy & Regenerat Med, CCRG Oogheelkunde, Wilrijkstr 10, B-2650 Edegem, Belgium

[6] Tampere Univ, Fac Med & Hlth Technol, Arvo Ylpon Katu 34, Tampere 33014, Finland

[7] Univ Montreal, Maisonneuve Rosemont Hosp, Res Ctr, Montreal, PQ H1T 4B3, Canada

[8] Univ Montreal, Dept Ophthalmol, Montreal, PQ H1T 4B3, Canada

[9] Antwerp Univ, Lab Cell Biol & Histol, Campus Drie Eiken,T Bldg,T1 Vet Sci,Univ Pl 1, B-2610 Antwerp, Belgium

来源：

STEM CELLS INTERNATIONAL | 2019年 / 2019卷

关键词：

AMNIOTIC MEMBRANE; CORNEAL; RECONSTRUCTION; EXPRESSION; GEL;

D O I：

10.1155/2019/7867613

中图分类号：

Q813 [细胞工程];

学科分类号：

摘要：

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 Np63, proliferation marker cytokeratin (KRT) 14, adhesion markers integrin-4 and E-cadherin, and LESC-specific extracellular matrix proteins laminin-1, 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.

引用

页数：17

共 82 条

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