Primary culture of human trabecular meshwork cells

Primary HTM cell lines were generated from single residual cornea rims of patients after surgical corneal transplants at the UNC Eye Clinic. The TM was isolated from surrounding tissue under a dissecting microscope by making incisions both anterior and posterior to the meshwork and removing it using forceps. The tissue was cut into small pieces, carefully attached to the bottom of a 2% porcine gelatin-coated (Sigma/Aldrich, St. Louis, MO, USA, cat #G2500) 35 mm dish, and coverslipped with a drop of MEM Richter’s Modification medium (Modified IMEM, Gibco/ThermoFisher, Waltham, MA, USA cat # A1048901) supplemented with 20% fetal bovine serum (FBS, Gibco/ThermoFisher cat #10437-028), 50 µg/ml gentamicin (Gibco/Life Technologies/ThermoFisher cat #15750-060). Cells from these specimens were not treated with enzymes, and were allowed to grow from the explant for 4 weeks changing the media every other day; upon confluency, cells were harvested and stored in liquid nitrogen. When reconstituted, these primary nontransformed cells are grown in IMEM, 10% FBS, gentamicin (complete medium) and subsist for five to six passages. All cells were used at passages 3–5. These outflow pathway cultures comprise all cell types involved in maintaining resistance to flow. That includes cells from the three distinct regions of the TM plus cells lining the Schlemm’s canal. Because most of the cells in these cultures come from the TM, they are commonly referred to as “TM cells”. The cells used in this study were from a 70-year-old Caucasian male (HTM-210, Ind #1), a 62-year-old Caucasian female (HTM-213, Ind #2), a 90-year-old Caucasian male (HTM-195, Ind #3), and a 57-year-old Caucasian female (HTM-216, Ind #4). Each of these individual cell lines was characterized by morphology and by the specific induction of myocilin (MYOC) by dexamethasone (D-8893 Sigma/Aldrich) treatment [71, 72].