CESC Culture and Treatment

Mouse corneal epithelial stem/progenitor cell line (TKE2, Public Health England, 11033107, http://www.phe‐culturecollections.org.uk/) was presented by Dr. Tetsuya Kawakita of Keio University (Tokyo, Japan). The cells were cultured in a keratinocyte serum‐free medium (KSFM, Gibco‐Invitrogen, Carlsbad, CA) supplemented with 5 ng/ml human recombinant epithelial growth factor, 50 μg/ml bovine pituitary extract (BPE) at 37°C and 5% CO₂ ³⁹. The cell line has been characterized in our group ⁴⁰, ⁴¹. TKE2 cells were treated with 1, 5, 10, and 20 ng/ml of IL‐1β (Millipore, Temecula, CA), TNF‐α (R&D, Minneapolis, MN), or varying osmolarities (340, 360, 400, or 450 mosm) achieved by the addition of sodium chloride in a BPE‐free KSFM medium. In some experiments, TKE2 cells were treated with 10 ng/ml of IL‐1β or TNF‐α combined with hyperosmotic stress (340, 360, or 400 mosm).

The rabbits used for the primary corneal limbal epithelial cells culture was purchased from Qingdao Kangda Rabbit Industry Development Co., Ltd. Three rabbits were used in this assay. Rabbit corneal limbal epithelial cells were cultured as below procedure: the corneal limbal tissues from New Zealand rabbits were treated with 2.4 U/ml Dispase (Roche, Indi‐anapolis, IN) in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, CA) overnight at 4°C. The limbal epithelium was detached under a dissecting microscope and digested with 0.25% trypsin/0.02% EDTA for 15 minutes at 37°C. The acquired rabbit primary limbal epithelial cells were suspended in DMEM/F‐12 medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY), Insulin‐Transferrin‐Selenium Supplement (ITS, Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1 nM cholera toxin (Sigma, St. Louis, MO), 2 nM 3,305‐Triiodo‐l‐thyronine Sodium salt (Sigma), 0.4 ng/ml hydrocortisone succinate (Wako, Osaka, Japan), 2 mM L‐Glutamine (Invitrogen), penicillinestreptomycin (Hyclone, Logan, UT), and 10 ng/ml recombinant human epidermal growth factor (R&D Systems). NIH‐3 T3 cells were treated with 4 mg/ml mitomycin C (Haiz‐heng, Taizhou, China) for 2 hours at 37°C and reinoculated at a density of 3 × 10⁵ cells in six‐well plate as feeder layers. The rabbit primary limbal epithelial cells were seeded on NIH‐3T3 feeder surface. Rabbit corneal limbal epithelial cells were treated with 10 ng/ml of IL‐1β, TNF‐α, or hyperosmotic stress at 400 mosm. All of the animal experiments were conducted in accordance with the Animal Care and Use Committee guidelines of the Shan‐dong Eye Institute and the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.