Abstract
The aim of this study was to develop a three-dimensional model of the human conjunctiva that can be used to perform physiology and pathophysiology experiments. Fibrin-based matrices (derived from human plasma or plasma cryoprecipitate) were used as scaffolds, and primary cells were obtained from conjunctival tissue. Conjunctival constructs were analyzed by immunofluorescent staining and scanning electron microscopy and cell proliferation was measured with alamarBlue® assay. After characterizing the constructs, four different experimental conditions were analyzed in cryoprecipitate matrices: controls, air-lifted cultures (to increase cell stratification), partially desiccated cultures (to mimic dry eye disease), and IL-13-treated cultures (to mimic allergy). Constructs were stained with hematoxylin/eosin to observe changes in morphology. High molecular weight glycoconjugates were identified by HPA staining. MUC5AC and IL-6 secretion was evaluated by ELISA. The fibrin-based matrices supported conjunctival cell growth. Epithelial cells grew on the surface of the scaffolds and underwent stratification that increased over time. These cells had microvilli, which suggests cell polarization and functionality. Fibroblasts were integrated in the scaffold and showed elongated shape. Compared to controls, air-lifted construct had increased epithelial stratification and upregulated MUC5AC secretion. Increased MUC5AC secretion also occurred in partially desiccated and IL-13-treated cultures. The inflammatory status of cells was evaluated by IL-6 levels which were increased in air-lifted and partially desiccated cultures, but not in IL-13-treated ones. In conclusion, we have developed a new three-dimensional model of human conjunctiva that can be used to study ocular surface inflammatory diseases.