Long-standing cultures from human cornea limbal and stromal grafts form 3D structures ex vivo - potential implications for future transplantation

Abstract

Purpose: Long-standing cultivation and characterizion of human cornea limbal epithelial- and stromal stem cells (LESCs and CSSCs), respectively, into 3D tissue grafts was developed for future tissue engineering and clinical applications.

Methods: The limbal and stromal tissue explants were obtained from cadavers according to the guidelines of the Helsinki Declaration, and cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for markers of stemness (p63α, ABCG2, CK19 and Vimentin in case of LESCs), proliferation (Ki-67), limbal epithelial- (CK 8/18) and differentiated cornea epithelial- cells (CK3 and 12), and collagen I, IV and V.

Results: Morphological and immunostaining analysis revealed that both cells types when cultured long-term can form stratified 3D tissue layers with a distinct extracellular matrix deposition and organization. The LESCs showed robust expression of p63α, ABCG2 and Vim, low level expression of CK3/12 and a subpopulation of proliferative cells - a clear advantage for their use in future transplantation. Both the LESCs and the CSSCs produced distinct and organized collagen structure.

Conclusions: Overall, we provide a model for generating highly pluripotent, long-standing 3D cultures from LESCs and CSSCs which can be used for further research purposes or transplantation