Full thickness 3D in vitro conjunctiva model enables goblet cell differentiation

In vitro culture and generation of highly specialized goblet cells is still a major challenge in conjunctival 3D in vitro equivalents. A model comprising all physiological factors, including mucus-secreting goblet cells has the potential to act as a new platform for studies on conjunctival diseases. We isolated primary conjunctival epithelial cells and fibroblasts from human biopsies. 3D models were generated from either epithelial layers or a combination of those with a connective tissue equivalent. Epithelial models were investigated for marker expression and barrier function. Full-thickness models were analyzed for goblet cell morphology and marker expression via immunofluorescence and quantitative real-time PCR. Simple epithelial models cultured at the air-liquid interface showed stratified multi-layer epithelia with pathologic keratinization and without goblet cell formation. The combination with a connective tissue equivalent to generate a full-thickness model led to the formation of a non-keratinized stratified multi-layer epithelium and induced goblet cell differentiation. In our model, a high resemblance to natural conjunctiva was achieved by the combination of conjunctival epithelial cells with fibroblasts embedded in a collagen-hydrogel as connective tissue equivalent. In the future, our conjunctival in vitro equivalent enables the investigation of goblet cell differentiation, conjunctival pathologies as well as drug testing.