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Karen Y Torrejon, Cula N Dautriche, Andrea Unser, Mathew Kerr, Feryan Ahmed, Abbey Van Alstyne-Ponce, Dan Stamer, Susan T Sharfstein, Yubing Xie, John Danias; 3D Human Trabecular Meshwork model as a potential tool for gene screening. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3441.
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© ARVO (1962-2015); The Authors (2016-present)
Primary open angle glaucoma (POAG) is a multifactorial, polygenic disease that is dependent on intraocular pressure (IOP). IOP is regulated at the angle outflow tissues through the conventional pathway. Current understanding of the genetic basis of IOP regulation is limited due to the lack of a physiologically relevant model for gene testing. This study aims at establishing a 3D tissue model of the conventional outflow tract that enables high efficiency transfection and perfusion studies to measure gene’s effect in outflow facility.
Human trabecular meshwork (HTM) cells isolated from cadaveric donors were separately cultured to reach 70% confluence on highly porous, microfabricated scaffolds, allowing 3D cell growth (tissue constructs). HTM cells grown on glass coverslips were used as controls. Targefect-RAW reagent was used to transfect HTM cells with siRNA targetingb-actin, RhoA, and SPARC or a scramble siRNA. Gene silencing for each gene was confirmed via Immunocytochemistry and qPCR. A 24-channel microfluidic perfusion system was used to study each genes’ effect on simulated outflow facility.
3D HTM constructs exhibited enhanced gene transfection efficiency compared to 2D cultures. siRNA transfection of HTM cells caused significant downregulation of b-actin (0.23 ± 0.05 fold), RhoA (0.13 ± 0.06 fold), and SPARC (0.15 ± 0.04 fold) at the mRNA and at the protein level. Simulated outflow facility of siRhoA-tranfected constructs (0.24 ± 0.05 mL/min/mm2/mmHg) was increased compared with that of siRNA scramble control transfected ones (0.13 + 0.02mL/min/mm2/mmHg) (p<0.001, t Test, N=9). siSPARC had no effect on outflow facility (0.13 ± 0.02 versus 0.15 + 0.03 mL/min/mm2/mmHg) (N=9).
The 3D HTM constructs allow high efficiency siRNA delivery confirming feasibility of using the conductive, 3D HTM model as a platform for testing genes that regulate intraocular pressure; providing a new avenue gene screening.
This is a 2020 ARVO Annual Meeting abstract.
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