A previous 3-D raft culture originally adopted for skin regeneration
30 was modified for the regeneration of stratified corneal epithelial equivalent. Briefly, Swiss 3T3 fibroblasts were embedded in the type I collagen gel matrix (Vitrogen; Angiotech BioMaterials, Palo Alto, CA) within a 12-mm insert (Millicell; Millipore, Billerica, MA) at a density of 2 × 10
5 cells/mL, and the cytometry-sorted cells were inoculated onto the stroma equivalent at densities of 500 cells (8.3 × 10
2 cells/cm
2) or 10,000 cells per insert (1.67 × 10
4 cells/cm
2) and cultured with E-medium (DMEM/F12 [3:1 mixture], containing 10% FBS, 5 μg/mL insulin, 5 μg/mL transferrin, 400 ng/mL hydrocortisone, 10
−9 M cholera toxin, 10 U/mL penicillin, and 10 μg/mL streptomycin). After 3 weeks of submerged culture, the cells were exposed to a semidry condition by adjusting the medium level for an additional 2 weeks and were fixed with formalin. For the nude mouse transplantation, the 3-D cultures after the 3 weeks of submerged culture were implanted epithelial side up over the muscle fascia of nude mice (BALB/c Slc-nu/nu).
31 At 2 weeks after transplantation, the nude mice were euthanatized, and the tissue containing the corneal epithelial equivalents was excised. After fixation in formalin, paraffin-embedded sections (4 μm) were deparaffinized and hydrated, and the staining was performed as previously described.
32 The antibodies against p63 (Oncogene, Boston, MA), keratin 3/12 (Chemicon, Temecula, CA), and keratin 1/10 (Biomeda Corp., Foster City, CA) were applied according to the recommended titer supplied by the manufacturer.