A Porcine Anterior Segment Perfusion and Transduction Model With Direct Visualization of the Trabecular Meshwork

Abstract

PurposeTo establish a consistent and affordable, high quality porcine anterior segment perfusion and transduction model that allows direct visualization of the trabecular meshwork.MethodsPorcine anterior segments were cultured within 2 hours of death by removing lens and uvea and securing in a specially designed petri dish with a thin bottom to allow direct visualization of the trabecular meshwork with minimal distortion. Twenty-two control eyes (CO) with a constant flow rate were compared to eight gravity perfused eyes (COgr, 15 mm Hg). We established gene delivery to the TM using eGFP expressing feline immunodeficiency virus (FIV) vector GINSIN at 108 transducing units (TU) per eye (GINSIN_8, n = 8) and 107 TU (GINSIN_7, n = 8). Expression was assessed for 14 days before histology was obtained.ResultsPig eyes were a reliable source for consistent and high quality anterior segment cultures with a low failure rate of 12%. Control eyes had an intraocular pressure (IOP) of 15.8 ± 1.9 mm Hg at fixed pump perfusion with 3 μL/min compared to gravity perfused COgr with imputed 3.7 ± 1.6 μL/min. Vector GINSIN_8 eyes experienced a transient posttransduction IOP increase of 44% that resolved at 48 hours; this was not observed in GINSIN_7 eyes. Expression was higher in GINSIN_8 than in GINSIN_7 eyes. Trabecular meshwork architecture was well preserved.ConclusionsCompared with previously used human donor eyes, this inexpensive porcine anterior segment perfusion model is of sufficient, repeatable high quality to develop strategies of TM bioengineering. Trabecular meshwork could be observed directly. Despite significant anatomic differences, effects of transduction replicate the main aspects of previously explored human, feline and rodent models.

Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model

“…Ablation control eyes were transduced with 1 10 7 eGFP FIV vectors before F and S. 24 hours after × transduction, the TM cells began to express eGFP, reaching a peak intensity at 48 hours, as reported previously (Loewen et al, 2016b;Dang et al, 2016b). There were discontinuous areas of transduced TM (Fig.…”

“…Feline immunodeficiency viral vectors expressing eGFP were generated by transient cotransfection of envelope plasmid pMD.G, packaging plasmid pFP93, and gene-transfer plasmid encoding eGFP and neomycin resistance GINSIN (Saenz et al, 2007;Oatts et al, 2013;Zhang et al, 2014) using a polyethylenimine method (Loewen et al, 2016b). The vector-rich supernatant from transfected 293T cells were harvested two, four and six days after transfection and concentrated by ultracentrifugation.…”

“…To address these concerns, we developed a chemical-free, freeze-thaw method to produce a decellularized TM scaffold. Together with our anterior segment perfusion system (Loewen et al, 2016b), this scaffold model can be used for cell transplantation, allowing real-time TM visualization and IOP measurement.…”

A Porcine Ex Vivo Model of Pigmentary Glaucoma

“…In our previous work with pig eyes and the study presented here, we took advantage of the high tissue quality that is the result of only two hours from enucleation to culture, the consistency within a litter, and an outflow tract anatomy that matches several features in humans [15][16][17][18] . Notable differences are a thicker TM, Schlemm's canal-like segments instead of a mostly single lumen (angular aqueous plexus) 19 , and, in contrast to almost all other domestic animals and pets 20 , a paucity of naturally developing glaucoma or medically-induced ocular hypertension.…”

“…Notable differences are a thicker TM, Schlemm's canal-like segments instead of a mostly single lumen (angular aqueous plexus) 19 , and, in contrast to almost all other domestic animals and pets 20 , a paucity of naturally developing glaucoma or medically-induced ocular hypertension. We recently established gene transfer 17,21 , modeled segmental aqueous outflow 16,22,23 , and created a microincisional angle surgery system [24][25][26] in a pig eye model.…”