Cultivation of rabbit corneal epithelial cells in serum-free medium.

PURPOSE To establish conditions for cultivation, serial growth, and normal differentiation of corneal epithelial cells in serum-free medium (SFM). METHODS Rabbit corneal epithelial cells were co-cultured with lethally treated 3T3-cell feeder layers. Instead of serum, medium was supplemented with serum albumin, hormones, and other additives. Cell growth was quantitated spectrophotometrically with a new rhodamine-B staining protocol with a sensitivity range of 5 X 10(3) to 1 x 10(5) cells/cm2. Keratin expression was analyzed by immunostaining or sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell extracts. RESULTS In SFM, without growth factors, cells grew no more than six to eight doublings, but when 10 ng/ml epidermal growth factor were added, serial transfer was possible, and epithelial cells grew to up to 18 to 20 doublings (three cell passages). Two cell colony types were seen: One type was composed of nonstratified proliferating cells, and the other of stratified cells expressing high levels of the differentiation-linked keratins K3 and K12. Confluent cultures formed a four- to five-layer stratified epithelium whose suprabasal cells were stained with anti-K12 antiserum. Acidic and basic fibroblast growth factors and epidermal growth factor reduced the expression of keratins K3 and K12. Transforming growth factor-alpha and epidermal growth factor led to the highest stimulation of cell proliferation. Limbal, peripheral, and central corneal epithelial cells showed similar clonal growth abilities, but colony size was larger for cells derived from limbal epithelium. CONCLUSIONS These SFM conditions support the serial transfer, normal differentiation, and formation of typical corneal epithelium by cultured corneal epithelial cells and are useful in studying and assaying a variety of cytokines and compounds that modulate corneal epithelial cell proliferation and differentiation.