Abstract
Purpose :
To use a 3D corneal organotypic model to study which cell types, either alone or in combination, contribute to the assembly of the epithelial basement membrane (EBM).
Methods :
A 3D corneal organotypic model was established by culturing the rabbit corneal epithelial cells with either corneal fibroblasts or myofibroblasts embedded in collagen type I for 18 or 30 days. The myofibroblasts were derived either from bone marrow or differentiated from corneal fibroblasts. Fresh rabbit corneas had overnight enzymatic digestion to collect the keratocytes/corneal fibroblasts. The fibroblast were differentiated into myofibroblasts by incubating in TGFb1 (20 ng/ml) and mature myofibroblasts were confirmed with ICC for markers alpha-SMA, vimentin, desmin and vinculin. Immunohistochemistry for laminin alpha-5, laminin beta-3, perlecan, nidogen-1 and collagen IV was performed on cryofixed sections to detect EBM generation and IHC staining for vimentin and alpha-SMA was used to differentiate the fibroblasts and myofibroblasts. Each experiment was repeated at least 3 times.
Results :
Expression and localization of EBM components laminin alpha 5, laminin beta 3, perlecan, nidogen 1 and collagen IV at the interface of the epithelial cells and corneal fibroblasts confirmed generation of a normally-assembled EBM in 3D organotypic cultures of epithelial cells and corneal fibroblasts. The presence of vimentin+, SMA– cells in the organotypic culture of corneal fibroblasts with epithelial cells revealed that corneal fibroblasts retained their phenotype after 18 days of culture. Epithelial cells or corneal fibroblasts alone in culture did not produce EBM. EBM was not observed in 3D organotypic cultures of myofibroblasts (either cornea- or bone marrow-derived) with epithelial cells, even with a long term (30 days) organotypic culture. However, a thickened EBM was observed in epithelial cell-corneal fibroblast organotypic cultures when incubation was continued for 30 days.
Conclusions :
Corneal EBM assembly is mediated by the coordinated action of epithelial cells and corneal fibroblasts—with both cell types producing EBM component proteins. This in vitro organotypic model can be used to further elucidate EBM assembly in the cornea and to study other functions such as regeneration of epithelial barrier function after injury.
This is a 2021 ARVO Annual Meeting abstract.