Date Published: March 1, 2017
Publisher: Public Library of Science
Author(s): Laura García-Posadas, Laura Soriano-Romaní, Antonio López-García, Yolanda Diebold, Masaya Yamamoto.
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.
Inflammatory ocular surface diseases are very prevalent among the global population. Patients demand more efficacious, new treatments for their diseases and, at the same time, governments and pharmaceutical companies are concerned about the cost of the research needed to develop new drugs . The increasing use of three-dimensional models has shown their utility in decreasing research costs by providing more reliable results and reducing the use of animals in research .
While there is a large amount of ongoing conjunctival research, there is a need to develop new methodologies that can reduce the reliance on animal use and accurately simulate the in vivo physiology of this tissue. In this study we engineered biocompatible scaffolds that contained primary cultures of fibroblasts and epithelial cells derived from human conjunctivas. These constructs maintained the conjunctival phenotype and retained biological function. In addition, we presented a proof of concept for its use to study ocular surface inflammation. We exposed the engineered conjunctiva to different conditions that mimic dry eye disease and allergic conjunctivitis, developing in this way a new three-dimensional in vitro model that could be used to test novel drugs against those prevalent diseases.