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Karen Yud Torrejon, Adam Goodcoff, Cula Dautriche, Magnus Bergkvist, Yubing Xie, Susan Sharfstein, John Danias; Bioengineered 3D HTM is physiologically responsive to glucocorticoid. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5654.
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© ARVO (1962-2015); The Authors (2016-present)
To study extracellular matrix (ECM) deposition of a bioengineered 3D human trabecular meshwork (HTM) and test whether the glucocorticoid prednisolone acetate (PA) affects flow through the "artificial TM".
We previously reported the establishment of an “Artificial HTM” on microfabricated, well-defined, porous SU-8 scaffolds, demonstrating in vivo-like phenotype of human HTM cells (e.g., morphological characteristics, HTM marker expression) and responding to a pharmacological agent, latruncunlin-B, in a similar fashion to the one seen in vivo. In the present work, further biological characterization of this system was performed. ECM deposition of collagen type IV (Col.IV), fibronectin (FN) and laminin was confirmed by immunocytochemistry and confocal microscopy. Additionally, the bioengineered HTM treated with PA (3nM to 30µM) for 3-5 days was assayed for cellular expression and secretion of myocilin, FN and Col.IV. In parallel, the responsiveness of this “Artificial HTM” to PA under flow was investigated using a stand-alone perfusion chamber with a pressure sensing system. Before and after 4-day PA treatment at 300nM, the pressure was monitored continuously and the response to flow was recorded. The outflow facility of the bioengineered HTM was determined in a similar way to ex vivo organ cultures and in vivo studies: Outflow Facility = Δ(flow rate)/Δ(pressure); this was done for both treated and untreated samples. The perfused HTM cells were subsequently fixed and stained using phalloidin to reveal F-actin cytoskeleton organization. Scanning electron microscopy was used to reveal the topography of the PA-treated bioengineered tissue.
Primary HTM cells grown on porous SU-8 scaffolds exhibited enhanced ECM deposition that was enriched in Col.IV and FN, creating a 3D HTM construct over 45µm thick. The “Artificial HTM” showed dose-dependent PA response, with increased myocilin and Col.IV expression and secretion after 3-day treatment with 300nM PA compared to untreated controls. The outflow facility decreased 2-fold (from 4.7±0.4 to 2.42±0.48 μl/min/mmHg) after 300nM PA treatment. Additionally, PA-perfused HTM cells presented cross-linked actin networks that are distinct from those treated without flow.
A bioengineered in-vitro HTM showed PA responsiveness, confirming the applicability of this system in TM physiology studies and for high-throughput screening of agents affecting IOP.
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