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


METHODS
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).


RESULTS
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.


CONCLUSIONS
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.
Lei
Yuan
Y
Department of Bioengineering, Imperial College London, London, United Kingdom.
Overby
Darryl R
DR
Read
A Thomas
AT
Stamer
W Daniel
WD
Ethier
C Ross
CR
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001082:Aqueous Humor / Q000166:cytology*; D015415:Biomarkers / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods*; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D008954:Models, Biological; D011691:Puromycin / Q000494:pharmacology; D013552:Swine; D014129:Trabecular Meshwork / Q000166:cytology* / Q000187:drug effects; D014841:von Willebrand Factor / Q000378:metabolism; D000099152:Cadherin 5
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000199:Actins; D015703:Antigens, CD; D015415:Biomarkers; D015820:Cadherins; D015815:Cell Adhesion Molecules; D011691:Puromycin; D014841:von Willebrand Factor; D000099152:Cadherin 5