Amphotericin b1

Human corneal limbal rims (n = 10), acquired from the Tissue and Eye Services at St. Pauls Eye Unit, Royal Liverpool University Hospital, Liverpool, were isolated from the remnants of donor corneas that had been used for penetrating keratoplasty. Excess scleral, iris tissue, and corneal endothelium were carefully removed from the corneal limbal rings, leaving a ring of approximately 3-mm diameter. The rings were dissected into ∼2-mm cuboidal segments for ex vivo culture. These segments were placed in the center of a well of a 12-well culture plate (Corning, Inc., Corning, NY, USA) and cultured at 37°C and 5% CO₂ in limbal epithelial stem cell medium consisting of equal volumes of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2.5 μg/mL amphotericin B1 (all Sigma-Aldrich), and 0.4 mg/mL hydrocortisone, 10 mg/mL insulin, 20 μg/mL tri-iodothyronine, 40 mg/mL adenine, 50 μg/mL cholera toxin, and 100 ng/mL epidermal growth factor (Lonza, Slough, UK). Medium was changed every 2 days and epithelial outgrowth monitored daily for 7 to 14 days with a phase-contrast microscope (Nikon, Surrey, UK); then explants displaying outgrowths were fixed and processed for immunohistochemistry, or embedded in OCT and frozen for immunofluorescence processing.