In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds
In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds
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DOI：
10.1155/2019/7867613
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发表时间：
2019-01-01
影响因子：
4.3
通讯作者：
Tassignon, Marie-Jose
中科院分区：
文献类型：
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作者：
Haagdorens, Michel;Cepla, Vytautas;Tassignon, Marie-Jose
目的。目的探讨重组人I型胶原（RHC I）和胶原样肽（CLP）水凝胶作为载体底物在无异种培养条件下培养角膜缘上皮干细胞（LESC）的效果。方法。人类LESC在七种不同的胶原来源的水凝胶上培养：(1)未修饰的RHC I，(2)纤维连接蛋白模式的RHC I，(3)碳二酰亚胺交联CLP (CLP-12 EDC), (4) DMTMM-（4-（4,6-二甲氧基-1,3,5-三嗪-2-基）-4-甲基-morpholinium-）交联CLP (CLP-12),(5)纤维连接蛋白模式的CLP-12，(6)“3D边缘生态位模拟”CLP-12,(7)由更高浓度的CLP溶液制成的DMTMM交联CLP。分析细胞增殖、细胞形态和LESC标志物的表达情况。所有数据均与人羊膜培养（HAM）进行比较。结果。人类LESC在7种水凝胶配方中的6种上成功培养，CLP-12 EDC上的原代细胞培养被认为是不成功的，因为培养14天后，培养面积不符合质量标准（即，培养和融合不一致）。融合后，原代LESC高表达干细胞标记物Np63、增殖标记物细胞角蛋白（KRT） 14、粘附标记物整合素-4和E-cadherin、LESC特异性细胞外基质蛋白laminin-1和IV型胶原，细胞低表达分化标记物KRT3和粘粒蛋白3 （DSG3）。与RHC I和HAM相比，CLP水凝胶培养的细胞中KRT3的基因表达显著升高。水凝胶的表面图案影响细胞的增殖模式，但对培养物的表型和基因型没有显著影响。总体而言，RHC I和dmtmm交联CLP水凝胶的性能与HAM相当。结论。RHC I和dmtmm交联CLP水凝胶，无论表面修饰如何，都支持使用无xeno培养方案成功培养原代人LESC。再生上皮保持了与ham培养相似的特征。
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.