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D. R. Overby, Y. Lei, C. R. Ethier; Puromycin-Based Selection of Porcine Aqueous Plexus Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3215.
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© ARVO (1962-2015); The Authors (2016-present)
Endothelial cells from Schlemm’s canal are important for influencing IOP, but in vitro studies are limited by sparse availability of human tissue from mostly elderly donors. Here, we explored porcine eyes as a source of aqueous plexus (AP) cells (AP is the porcine equivalent of Schlemm’s canal). Cells were differentially selected using puromycin, a toxin often used to select brain microvascular endothelial cells based upon expression of P-glycoprotein (Pgp), a multi-drug resistance efflux pump.
Trabecular meshwork containing AP was dissected and pooled from fresh porcine eyes and digested in collagenase I (1 mg/mL) for 1 hr at 37°C. Liberated cells were twice washed in culture medium, filtered through a cell strainer, and cultured for 8 days in a gelatin-coated plastic flask. For selection, cells were exposed to 4 µg/mL puromycin for 2 days in culture medium, with fetal bovine serum replaced by platelet-poor plasma-derived serum to support endothelial cell growth. Cells were fixed and immuno-stained for Pgp, ICAM II, von Willebrand factor (vWF), VE-cadherin and alpha-smooth muscle actin (α-SMA) using standard techniques. Six separate isolations were done in this study, each pooling tissue from 6 eyes.
Histology of the limbus showed that our dissection was limited to the trabecular meshwork region, including the AP, without extending into the iris or cornea. Prior to puromycin treatment, cells appeared heterogeneous, with polygonal and fusiform shapes, suggestive of a mixed population. Over 90% of the cells were removed by puromycin, leaving a population that appeared uniformly cobblestone-like when grown to confluence. Puromycin-selected cells stained positively for ICAM-II, vWF and VE-cadherin, but negatively for α-SMA. Pgp was expressed at very low levels in untreated cells, but was strongly upregulated in surviving cells following puromycin treatment.
Based upon marker expression and morphology, puromycin-selected cells from porcine trabecular meshwork appear consistent with Schlemm’s canal (AP) endothelial cells. This supports using porcine eyes as a plentiful alternative to human eyes for cell sourcing for in vitro outflow studies related to glaucoma.
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