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siran wang, Rachel Gomes, Chiara E Ghezzi, Kenneth Kenyon, James L Funderburgh, David L Kaplan; In vitro study of 3D tissue model of innervated corneal epithelium and stroma. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5310.
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© ARVO (1962-2015); The Authors (2016-present)
In vitro tissue models are powerful tools to study physiological and pathological processes. Current cornea tissue models lack innervation and the dynamic conditions mimicking in vivo parameters. Thus, we designed a silk protein biomaterial-based in vitro tissue model that includes human corneal epithelium (hCECs), stromal (hCSCs) and neurons (hNCs) cultured in a bioreactor to assess corneal ECM formation, mimics of physiological tear washing and the application of ocular pressure (IOP) to study impact on cellular phenotypes in static and dynamic environments.
The in vitro corneal tissue model was formed by co-culturing hCECs, hCSCs and hNCs in silk scaffolds to mimic corneal architecture. A bioreactor composed of an artificial anterior chamber creating ocular pressure (10-20mmHg), and an artificial tear outlet on the lid with 15-30 drops /min washing rate was used to house the 3D corneal tissue systems and sustain the culture for 2 months. Live and dead assay, immunohistochemistry, RT-PCR, and mechanical properties were investigated to assess cellular phenotype, ECM formation, tight junction (TJ) formation, and neuronal function. Data were compared between innervated and non-innervated, static and dynamic cultivated tissue systems using statistical analysis
Significantly higher TJ formation and keratocytic ECM protein expression were observed in the innervated models compared to the non-innervated controls. During dynamic cultivation, the scaffold gained curvature and increased stiffness when compared to static cultivation conditions. Tear washing increased the cell layers and tight junction formation in the epithelium, while the IOP significantly increased the synthesis of ECM protein in the stroma.
This innervated corneal epithelium and stroma tissue model cultivated in a dynamic environment provides a useful system for studying long term interactions between neuronal innervation and corneal tissue, and the impact of IOP and tear washing on corneal tissue. Future applications include utility in drug screening and artificial cornea development for clinical translation.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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