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Karen Y Torrejon, Ellen Papke, Dhruba J Bharali, Magnus Bergkvist, W Daniel Stamer, Susan T Sharfstein, Yubing Xie, Feryan Ahmed, John Danias; Human microvascular endothelial cells (HMVECs) assume Schlemm’s canal (HSC) cell characteristics after co-culture with human trabecular meshwork (HTM) cells and can be used to model conventional outflow. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3471.
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We have previously reported on the development of an artificial 3D co-culture system of HTM and HSC cells. Adapting the system for screening putative pharmacologic agents that affect outflow facility requires large numbers of HTM and HSC cells. Although HTM cells can be readily isolated from cadaveric tissue, isolation and culture of HSC cells is challenging. We explored the possibility of using HMVECs as an alternative to HSCs.
Primary HTM cells grown on well-defined scaffolds were co-cultured in a basal-to-basal orientation with either primary HSC cells or HMVECs for 21 days. Samples were then perfused at various flow rates while perfusion pressure was constantly monitored. A number of the 3D constructs were exposed to prednisolone acetate (300nM), TGFβ-2 (2.5ng/mL) and Y27632 (10µM), and the effect on perfusion pressure at the same flow rates was determined. At the conclusion of the experiment cells were fixed for morphological studies using scanning electron microscopy or used for qPCR and immunocytochemistry to determine gene and protein expression.
HMVEC/HTM cell co-cultures induced morphological changes in HMVECs by day 14 causing them to become elongated and form a thin cell layer. Moreover HMVECs expressed HSC markers (e.g. PROX-1, FOXC2) detected by qPCR and IHC. HTM cells retained HTM-marker expression during co-culture. Basal-to-apical flow led to giant vacuole-like structure formation. Co-cultures provided resistance to through flow maintaining a perfusion pressure (PP) of 7.4±1 mmHg at 2ul/min flow through an area of 78.5mm2. Under the same conditions for the HTM/HSC cell co-culture PP was 6.8±2 mmHg (p>0.05 1-way ANOVA). After exposure to prednisolone or TGFβ-2, both HTM/HSC and HTM/HMVEC constructs responded by increasing extracellular matrix (ECM), myocilin & VE-cadherin protein expression; and increasing resistance to through flow (9.5±0.9 and 8.7±1 mmHg, p>0.05 1-way ANOVA). Exposure to Y27632 resulted in decreased ECM deposition and lower resistance (5.1±0.9 and 4.6±1 mmHg, p>0.05 1-way ANOVA).
HTM/HMVEC 3D outflow co-culture system induces HMVECs to express HSC markers and behave similarly to HSCs. This model may allow higher throughput screening of agents that affect conventional outflow tract and provide insight into HSC development.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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