Isolation and Characterization of Primary Human Trabecular Meshwork Cells

Primary normal human trabecular meshwork (HTM) cells were harvested from donor eyes (n = 4) obtained from the Liverpool Research Eye Bank and approved by the local ethics review board (RETH000833). Samples were handled in accordance with the Declaration of Helsinki. Eyes were obtained from the Royal Liverpool University Hospital Mortuary and medical history was unknown; although no donor had previous ocular surgery or a known diagnosis of glaucoma. Donor information is listed in Supplemental Figure S1. Donor eyes were excluded if the maximum post-mortem time exceeded 48 h and HTM cells were isolated using the blunt dissection method as reported previously [24 (link)]. Cells were maintained in Dulbecco’s Modified Eagle Media (DMEM)-low glucose (Sigma, Gillingham, UK) supplemented with 10% fetal calf serum (Biosera, Heathfield, UK), 2 mM L-glutamine (Sigma, Gillingham, UK), Pen/Step (Sigma, Gillingham, UK), and 2.5 µg/mL Fungizone (amphotericin B, Sigma, Gillingham, UK). Samples were incubated at 37 °C (5% CO₂ and 95% humidity). HTM characterisation was carried out as previously described [24 (link)] and included upregulated myocilin protein expression in response to dexamethasone treatment (polyclonal rabbit anti-myocilin primary antibody was a kind gift from Dr. W. Daniel Stamer) as previously described by our group [25 (link)].