Limbus processing procedures

Limbal biopsy (2 × 2 mm²) was performed on the healthy eyes of adult patients in the period from July 2019 to May 2022. The biopsy of the superior limbus region (corneoscleral tissues of limbal rings) was taken aseptically in the operating room with local anesthesia at the Department of Ophthalmology, University Hospital Center Sestre Milosrdnice, Zagreb, Croatia. Transport of the biopsy specimen from the operating room to the Tissue and Cell Bank was carried out using a 4 mL sterile tube (Techno Plastic Products AG, Trasadingen, Switzerland) with 2.5 mL growth medium (GM) (Dulbecco's Modified Eagle Medium (DMEM) and Ham’s F12 media (2:1 mixture) containing heat-inactivated fetal bovine serum (FBS) (10%) (Gibco, Thermo Fisher Scientific), with added growth supplements: 4 mM L-glutamine (Gibco, Thermo Fisher Scientific), 0.19 mM adenine (Sigma‒Aldrich, Saint Louis, USA), 5 µg/mL insulin (Sigma‒Aldrich, Saint Louis, USA), 0.1 nM cholera toxin (List Biological Laboratories, Campbell, California, USA), 1.36 ng/mL triiodothyronine (2 nM) (Sigma‒Aldrich, Saint Louis, USA), 0.4 μg/mL hydrocortisone (Sigma‒Aldrich, Saint Louis, USA), 5 μg/mL apotransferrin (Sigma‒Aldrich, Saint Louis, USA), 10 μg/mL human epidermal growth factor (EGF) (Sigma‒Aldrich, Saint Louis, USA) and 1% penicillin‒streptomycin-amphotericin B (100 lU/mL) (Gibco, Thermo Fisher Scientific).