Muhammad Rizwan, Peh Gary S L, Adnan Khadijah, Law Jaslyn B K, Mehta Jodhbir S, Yim Evelyn K F
Department of Biomedical Engineering, National University of Singapore, Singapore; Institute of Materials Research and Engineering, Agency for Science, Technology and Research (A*STAR), Singapore.
Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore; Duke-NUS Graduate Medical School, Singapore.
Acta Biomater. 2015 Jun;19:138-48. doi: 10.1016/j.actbio.2015.03.016. Epub 2015 Mar 18.
One of the most common indications for corneal transplantation is corneal endothelium dysfunction, which can lead to corneal blindness. Due to a worldwide donor cornea shortage, alternative treatments are needed, but the development of new treatment strategies relies on the successful in vitro culture of primary human corneal endothelial cells (HCECs) because transformed cell lines and animal-derived corneal endothelial cells are not desirable for therapeutic applications. Primary HCECs are non-proliferative in vivo and challenging to expand in vitro while maintaining their characteristic cell morphology and critical markers. Biochemical cues such as growth factors and small molecules have been investigated to enhance the expansion of HCECs with a limited increase in proliferation. In this study, patterned tissue culture polystyrene (TCPS) was shown to significantly enhance the expansion of HCECs. The proliferation of HCECs increased up to 2.9-fold, and the expression amount and localization of cell-cell tight junction protein Zona Occludens-1 (ZO-1) was significantly enhanced when grown on 1 μm TCPS pillars. 250 nm pillars induced an optimal hexagonal morphology of HCEC cells. Furthermore, we demonstrated that the topographical effect on tight-junction expression and cell morphology could be maintained throughout each passage, and was effectively 'remembered' by the cells. Higher amount of tight-junction protein expression was maintained at cell junctions when topographic cues were removed in the successive seeding. This topographic memory suggested topography-exposed/induced cells would maintain the enhanced functional markers, which would be useful in cell-therapy based approaches to enable the in situ endothelial cell monolayer formation upon delivery. The development of patterned TCPS culture platforms could significantly benefit those researching human corneal endothelial cell cultivation for cell therapy, and tissue engineering applications.
角膜移植最常见的指征之一是角膜内皮功能障碍，这可能导致角膜盲。由于全球供体角膜短缺，需要替代治疗方法，但新治疗策略的开发依赖于原代人角膜内皮细胞（HCEC）的成功体外培养，因为转化细胞系和动物来源的角膜内皮细胞不适合用于治疗应用。原代HCEC在体内不增殖，在体外扩增并同时维持其特征性细胞形态和关键标志物具有挑战性。人们已经研究了生长因子和小分子等生化信号，以在增殖增加有限的情况下增强HCEC的扩增。在本研究中，图案化的组织培养聚苯乙烯（TCPS）被证明能显著增强HCEC的扩增。当在1μm的TCPS柱上生长时，HCEC的增殖增加了2.9倍，细胞间紧密连接蛋白闭合蛋白-1（ZO-1）的表达量和定位显著增强。250nm的柱诱导了HCEC细胞的最佳六边形形态。此外，我们证明了对紧密连接表达和细胞形态的拓扑效应在每次传代过程中都能保持，并且细胞能有效地“记住”这种效应。在连续传代中去除拓扑线索时，细胞连接处仍能维持较高水平的紧密连接蛋白表达。这种拓扑记忆表明，暴露于拓扑结构/由拓扑结构诱导的细胞将维持增强的功能标志物，这在基于细胞治疗的方法中是有用的，能够在递送时原位形成内皮细胞单层。图案化TCPS培养平台的开发可以显著造福那些研究用于细胞治疗和组织工程应用的人角膜内皮细胞培养的研究人员。
