A new method for selection of angular aqueous plexus cells from porcine eyes: a model for Schlemm's canal endothelium.

Published

Journal Article

The authors sought to develop a technique for isolating and culturing angular aqueous plexus (AAP) cells from more plentiful porcine eyes. AAP is an analogue of Schlemm's canal.Cells were differentially selected with puromycin, a toxin often used to select brain microvascular endothelial cells based on the expression of P-glycoprotein (P-gp), a multidrug resistance efflux pump. Trabecular meshwork containing AAP was dissected and pooled from fresh porcine eyes, digested in collagenase I, washed, filtered, and cultured for 8 days in a gelatin-coated plastic flask. Cells were then selected by exposure to 4 μg/mL puromycin for 2 days in the culture medium. Cells were fixed and immunostained for P-gp, ICAM II, von Willebrand factor (vWF), VE-cadherin, and α-smooth muscle actin (α-SMA).Histology of the limbus showed that the dissection was limited to the trabecular meshwork region, including the AAP. Before puromycin treatment, cells appeared heterogeneous and polygonal, suggestive of a mixed population. More than 90% of the cells were removed by puromycin, leaving a population that appeared uniformly cobblestone-like when grown to confluence and that was contact inhibited. Puromycin-selected cells stained positively for the endothelial markers ICAM II, vWF, and VE-cadherin but negatively for α-SMA, consistent with staining patterns in whole tissue.Based on marker expression, morphology, and behavior in culture, puromycin-selected cells from porcine outflow tissues are AAP endothelial cells. Thus, porcine eyes can provide a plentiful alternative cell source for studying Schlemm's canal biology related to ocular hypertension.

Full Text

Duke Authors

Cited Authors

  • Lei, Y; Overby, DR; Read, AT; Stamer, WD; Ethier, CR

Published Date

  • November 2010

Published In

Volume / Issue

  • 51 / 11

Start / End Page

  • 5744 - 5750

PubMed ID

  • 20554623

Pubmed Central ID

  • 20554623

Electronic International Standard Serial Number (EISSN)

  • 1552-5783

International Standard Serial Number (ISSN)

  • 0146-0404

Digital Object Identifier (DOI)

  • 10.1167/iovs.10-5703

Language

  • eng