Ex-vivo human anterior segment culture: a suitable model for glaucoma research




Patel, Pinkal
Kasetti, Ramesh
Zode, Gulab


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Purpose: The available human models to study the trabecular meshwork (TM) pathology in glaucoma are very limited. The well-established ex-vivo human anterior segment perfusion system requires intact eyes and cost intensive. Here we tested the feasibility of ex-vivo human anterior segment culture to use as a model for glaucoma research. Methods: Human rejected corneas with intact TM ring were obtained from the Lions Eye Institute (Tampla, Florida) in accordance with Declaration of Helsinki guidelines for research involving human tissue. The anterior segments were dissected into 4 equal quadrants and each quadrant was cultured in a 24 well plate using DMEM media added with 10% FBS and 1% Pen-strep. The anterior segment quadrants either treated with 100nM dexamethasone or 5ng per ml recombinant Tgfβ2 (in 0.5% FBS containing media) with appropriate vehicles or transduced with Adenoviral mediated wild type or mutant myocilin for up to 7 days. The spent media was collected for western blot analysis and cultured tissues were fixed and paraffin sections were utilized for immnostaining. Results: The TM morphology is well preserved in human rejected corneas before and after 7 days of culture, observed in H&E staining. The dexamethasone, Tgfβ2 and mutant myocilin induced glaucomatous changes like increased extracellular matrix proteins, increased ER stress were observed in the TM of dexamethasone, Tgfβ2 and mutant myocilin treated tissue sections compared to their respective controls. The western blot analysis of spent media clearly showed an increase of myocilin and fibronectin levels in dexamethasone treated samples whereas an increase in fibronectin observed in Tgfβ2 treated samples. As expected mutant myocilin secretion is hampered in Ad5-mutant-myocilin transduced quadrant tissue samples compared to that of Ad5-wildtype-myocilin. The tissue disintegration rate in an ex-vivo culture was monitored by counting tunnel positive cells at the TM region at different time points. Conclusion: The ex-vivo human anterior segment culture is an effective model to study the TM pathology in glaucoma research. Moreover this model is cost-effective and the rejected corneas available with ease.