Human Trabecular Meshwork Cell Isolation and Culture

Ocular anterior segments from human donors 1 to 3 were obtained from the Iowa Lions Eye Bank (Iowa City, IA), and those from donors 4, 5, and 6 were obtained from Beijing Tongren Eye Hospital (Beijing, China). The protocol for collecting human tissues was approved by the institutional review boards of the University of Iowa Institutional and Beijing Tongren Eye Hospital. After dissection, the TM was visible as a brown circle in the irideocorneal angle and isolated by using a 0.5-mm curette. After rinsing with Dulbecco's modified eagle medium (DMEM; Sigma-Aldrich, St. Louis, MO), the tissue was digested in DMEM containing 4 mg/mL collagenase A (Sigma-Aldrich) and 4 mg/mL human serum albumin (Sigma-Aldrich) at 37°C for 2 hours. After centrifugation (1000 rpm for 3 minutes), the pellets were resuspended in TM cell growth medium comprised of Gibco Medium 199E containing 20% Gibco FBS, 90 µg/mL porcine heparin (Sigma-Aldrich), 20 U/mL endothelial cell growth supplement (Sigma-Aldrich), and 1.7 mM l-glutamine (Sigma-Aldrich). The digested tissue was seeded onto 1% gelatin precoated six-well plates (Thermo Fisher Scientific, Waltham, MA) and cultured in the incubator with 5% CO₂ at 37°C. As in our previous studies,8 cells at passages five to eight with robust expression of TM biomarkers and the capacity for dexamethasone (DEX)-induced myocilin (MYOC) secretion were used in this study.