Establishing Primary Corneal Epithelial Cultures

All work with human tissue was done in accordance with the tenets of Helsinki. Donor human corneoscleral rims with no history of corneal diseases that were unsuitable for transplant were used to establish primary epithelial cultures as previously described [9] (link), [38] (link). Human cadaver corneas were graciously donated by the Lions Eye Bank of Wisconsin, Madison or the Missouri Lions Eye Bank (Columbia, MO). These tissue samples obtained were not human subjects research and as such approval from committee or kin were not necessary. Briefly, sclera and limbal regions of the cornea were trimmed and the tissue was immersed in dispase (1.2 U/ml, Boehringer, Mannheim, Germany) at 37°C for 4 h. Corneal epithelial cells were removed by gently rubbing the anterior surface with a sterile pipette tip. Cell suspensions were pooled, centrifuged and re-suspended in EpiLife medium (Life Technologies, Carlsbad, CA) supplemented with EpiLife defined growth supplement (EDGS; Life Technologies) and 1% penicillin/streptomycin (Life Technologies) and used between passages 2 and 3. Experiments were repeated with cells isolated from three different donors.
Immortalized human corneal epithelial cells (hTCEpi; [44] (link)), kindly provided by Dr James V Jester (UC Irvine), were maintained in EpiLife medium as above and were used between passages 40–60.