Establishing Orbital Fibroblast Cultures from Graves' Ophthalmopathy Patients

Orbital fibroblast cultures were established from surgical waste of four patients with GO during decompression surgery and from apparently normal orbital tissues in three age-matched patients undergoing surgery for noninflammatory conditions. All were not smokers or ex-smokers. All GO patients achieved stable euthyroidism for at least 6 months before surgery and were in the inactive stage of GO. All patients did not undergo corticosteroid treatment for at least 1 month before surgery. The study was performed according to the tenets of the Declaration of Helsinki and these activities have been approved by the Institutional Review Board of Taipei Veterans General Hospital. Briefly, the orbital tissues were minced aseptically in phosphate-buffered saline (PBS), and then incubated with a sterile solution containing 0.5% collagenase and dispase (Sigma-Aldrich Chemical Co., St. Louis, MO) for 24 h at 37 °C in a humidified chamber filled with 5% CO₂. The digested orbital tissues were pelleted by centrifugation at 1,000× g, and then resuspended in DMEM containing 10% fetal bovine serum (FBS) and antibiotics (Biological Industries, Kibbutz Beit Haemek, Israel), which was composed of 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate, respectively. [8 (link),9 (link)]. Cultured orbital fibroblasts were used between the 3rd and 5th passages and the cultures at the same passage number were used for the same set of experiments.