Human corneal stromal stem cells exhibit survival capacity following isolation from stored organ-culture corneas.

Abstract

To assess the suitability of human donor corneas maintained in long-term organ culture for the isolation and expansion of viable and functional corneal stromal stem cells (CSSCs). These cells display properties similar to mesenchymal stem cells and demonstrate the ability to reproduce an organized matrix in vitro. Therefore, CSSCs have great potential for the development of cell-based therapies for corneal blindness or stromal tissue bioengineering. Human donor corneas that had been stored either in organ-culture medium (OC) up to 4 weeks (n = 3) or in Optisol medium (OS) up to 6 days (n = 3) were used for isolation of CSSCs and maintained in culture until passage 4. Cell phenotype of isolated CSSCs was assessed with light microscopy and immunocytochemistry (PAX6, CD73, and CD90). PAX6 protein expression was further confirmed with immunoblot analysis. A comparison of CSSCs isolated from corneas stored under OC and OS conditions revealed no obvious differences in their morphology. Immunocytochemistry revealed CSSCs from both OC and OS corneas maintained positive staining for PAX6 and mesenchymal stem cell markers CD73 and CD90. Immunoblotting confirmed protein expression of PAX6 in cells from both tissue types. Human CSSCs exhibit survival capacity by retaining their phenotype following isolation from long storage, OC corneas. This advantageous property enables the retrieval of CSSCs from OC corneas that are more abundantly available for research than OS-stored corneas. Organ-culture corneas are also often discarded for retrieval of other cell types, such as corneal epithelial and endothelial cells, which require high tissue quality for their preservation.