Corneal Fibroblast Isolation and Myofibroblast Generation

Donor human corneas were procured from an eye bank and were used in the study in accordance with the Declaration of Helsinki for the use of human tissue. Primary human corneal fibroblasts were generated from donor human corneas according to a method described previously.^{41 (link)} Briefly, the cornea was washed with cell culture medium, and the epithelium and endothelium were removed by gentle scraping with a scalpel blade. Corneal stroma was cut into small pieces and incubated in a humidified CO₂ incubator at 37°C in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum to obtain human corneal fibroblasts. Seventy percent confluent cultures of human corneal fibroblasts (passages 1–3) were used for experiments. For myofibroblast generation, cultures were exposed to TGF-β1 (1 ng/mL) for 7 days under serum-free conditions. TSA was used at 50 to 500 nM concentrations. TGF-β1 was purchased from R&D Systems (Minneapolis, MN), and TSA was purchased from Sigma-Aldrich (St Louis, MO).