Publication protocol
Human Limbal Epithelial Cell Isolation and Cultivation
Cornea-limbal rings were harvested from five healthy donors just after corneal transplantation, informed consent was sought, and the sample harvesting protocol was approved by the Institutional Review Board (IRB) of Jilin University. Fresh cornea-limbal rings were treated with 0.25% Dispase II at 4°C overnight, and epithelial layer was scrubbed from the underlying stroma tissue and treated with 0.05% trypsin-0.02% EDTA at 37°C for 15 minutes. Trypsin activity was neutralized by 10% FBS and dissociated limbal epithelial cells were collected and centrifuged at 1,500 rpm for 5 minutes. Epithelial cell viability was determined by trypan blue excluding staining and cell number was counted using hemocytometer.
Mouse 3T3 fibroblasts were maintained in Dulbecco's Modified Eagle's Medium (DMEM, high glucose) supplemented with 10% FBS, L-glutamine (2 mM), and penicillin-streptomycin (50 IU/mL) and cultured with 5% CO2 and humidified atmosphere. 3T3 cells were subcultured every 6 days when reaching 80–90% confluence. 3T3 cells were serially maintained, and only cells before passage 20 were used for preparation of feeder layer. To prepare feeder layer, confluent 3T3 cells were treated with mitomycin C (10 μg/mL) for 2 hours at 37°C, washed with PBS twice, and treated with 0.05% trypsin for 5 minutes at 37°C. 3T3 fibroblasts were then collected and plated at a density of 30,000 cells/cm2 one day before seeding epithelial cells.
Human limbal epithelial cells were cultivated on 3T3 feeder layer using either FAD medium or serum replacement (SR) medium. The FAD medium is a mixture of DMEM and Ham's F-12 medium (1 : 1) containing 10% fetal bovine serum, L-glutamine (2 mM) and penicillin-streptomycin (50 IU/mL), epidermal growth factor (10 ng/mL), insulin (5 μg/mL), adenine (0.18 mM), hydrocortisone (0.4 μg/mL), cholera toxin (0.1 nM), and triiodothyronine (2 nM). The SR medium is a mixture of DMEM and Ham's F-12 medium (1 : 1) containing 10% KnockOut SR serum replacement, L-glutamine (2 mM) and penicillin-streptomycin (50 IU/mL), epidermal growth factor (10 ng/mL), insulin (5 μg/mL), adenine (0.18 mM), hydrocortisone (0.4 μg/mL), cholera toxin (0.1 nM), triiodothyronine (2 nM), transferrin (5 μg/mL), and selenium (5 ng/mL). Limbal epithelial cells were seeded onto 3T3 feeder layer at a density of 6,000 cells/cm2 and cultured with 5% CO2 and humidified atmosphere. The FAD medium was changed every 3 days while the SR medium was changed every 2 days.
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