Corneal stromal cell plasticity: in vitro regulation of cell phenotype through cell-cell interactions in a 3D model
Authors
Samantha L. Wilson, Ying Yang and Alicia J. El Haj
Institution
Keele University
Country
United Kingdom
Year
2013
Journal
Tissue Engineering
Abstract
In vivo, epithelial cells are connected both anatomically and functionally with stromal keratocytes. Co-culturing aims to recapture this cellular anatomy and functionality by bringing together two or more cell types within the same culture environment. Corneal stromal cells were activated to their injury phenotype (fibroblasts) and expanded before being encapsulated in type-I collagen hydrogels constructs. Three different epithelial-stromal co-culture methods were then examined; epithelial explant; transwell; the use of conditioned media. The aim was to determine if the native, inactivated keratocyte cell phenotype could be restored in vitro. Media supplementation with transforming growth factor beta 1 (TGF-β1) was then used to determine if the inactivated stromal cells retained their plasticity in vitro and could be re-activated to the fibroblast phenotype. Finally, media supplementation with wortmannin was used to inhibit epithelial-stromal cell interactions. Two different non-destructive techniques, spherical indentation and optical coherence tomography, were used to reveal how epithelial co-culturing, TGF-β1 and wortmannin media supplementation respectively affects stromal cell behaviour and differentiation in terms of construct contraction and elastic modulus measurement. Cell viability, phenotype, morphology and protein expression was investigated to corroborate our mechanical findings. It was shown that activated stromal cells could be inactivated to a keratocyte phenotype via co-culturing and that they retained their plasticity in vitro. Activated corneal stromal cells that were fibroblastic in phenotype were successfully reverted to a non-activated keratocyte cell lineage in terms of behaviour and biological properties; and then back again via TGF-β1 media supplementation. It was then revealed that epithelial-stromal interactions can be blocked via the use of wortmannin inhibition. A greater understanding of stromal-epithelial interactions and what mediates them offers great pharmacological potential in the regulation of corneal wound healing, with the potential to treat corneal diseases and injury whereby such interactions are vital.
Keywords: Cornea, co-culture, cell differentiation, elastic modulus, contraction, 3D cell culture