Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Schwabachanlage 6, 91054, Erlangen, Germany.
Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Freiburg, Germany.
Sci Rep. 2020 Jul 6;10(1):11074. doi: 10.1038/s41598-020-68120-0.
Limbal melanocytes, located in the basal epithelial layer of the corneoscleral limbus, represent essential components of the corneal epithelial stem cell niche, but, due to difficulties in their isolation and cultivation, their biological roles and potential for stem cell-based tissue engineering approaches have not been comprehensively studied. Here, we established a protocol for the efficient isolation and cultivation of pure populations of human limbal melanocytes, which could be expanded at high yield by using recombinant laminin (LN)-511-E8 as culture substrate. Co-cultivation of limbal melanocytes with limbal epithelial stem/progenitor cells on fibrin hydrogels pre-incubated with LN-511-E8 resulted in multilayered stratified epithelial constructs within ten days. By reproducing physiological cell-cell and cell-matrix interactions of the native niche environment, these biomimetic co-culture systems provide a promising experimental model for investigating the functional roles of melanocytes in the limbal stem cell niche and their suitability for developing advanced epithelial grafts for ocular surface surface reconstruction.
角膜缘黑色素细胞位于角巩膜缘的基底上皮层，是角膜上皮干细胞龛的重要组成部分，但由于其分离和培养困难，其生物学功能和基于干细胞的组织工程方法的潜力尚未得到全面研究。在这里，我们建立了一种有效分离和培养纯人群人角膜缘黑色素细胞的方案，该方案可通过使用重组层粘连蛋白（LN）-511-E8 作为培养底物以高产率进行扩增。将角膜缘黑色素细胞与预先用 LN-511-E8 孵育的纤维蛋白水凝胶上的角膜缘上皮干细胞/祖细胞共培养，可在十天内形成多层分层上皮结构。通过复制天然龛位环境中的生理细胞-细胞和细胞-基质相互作用，这些仿生共培养系统为研究黑色素细胞在角膜缘干细胞龛中的功能作用及其用于开发先进的眼表面重建上皮移植物的适用性提供了有前途的实验模型。
