Tissue-cultured C3H/Hej (H-2
k), BALB/c (H-2
d), and C57BL/6J (H-2
b) corneal epithelial and endothelial cells were used as target cells for complement-mediated cytotoxicity assays, rather than the usual lymphoid cells, because corneal cells are the relevant target cells in vivo during corneal allograft rejection. Furthermore, it has been shown that the corneal epithelial and stromal cell layers express MHC class I antigens, with little to no expression on the corneal endothelial cell layer,
22 23 whereas lymphoid cells express high levels of MHC class I antigens. Cell cultures were established as described previously.
24 Briefly, cultures were established from freshly dissected corneal explants
25 26 and propagated in minimum essential medium (MEM; BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated fetal bovine serum (FBS; HyClone Laboratories, Logan, UT). Once primary cultures were established, the cells were immortalized with human papilloma virus genes
E6 and
E7, using the disabled recombinant retroviral vector pLXSN16E6/
E7.
27 The transformed corneal cells proliferate indefinitely, maintaining their original morphologic characteristics and expressing the same histocompatibility antigens as their nontransformed counterparts.
28 Cell lines were maintained in complete MEM containing 10% heat-inactivated FBS, 2 mM
l-glutamine, 1 mM sodium pyruvate, 2 mM MEM vitamins, and 1% penicillin-streptomycin-fungizone solution (all from BioWhittaker).