Preservation and Expansion of the Primate Keratocyte Phenotype by Downregulating TGF-β Signaling in a Low-Calcium, Serum-Free Medium

METHODS. Rhesus monkey keratocytes were isolated from central corneal buttons by collagenase digestion for 16 hours, seeded on plastic in Dulbecco’s modified Eagle’s medium (DMEM) containing insulin-transferrin-sodium selenite (ITS) supplement (DMEM/ITS) or 10% fetal bovine serum (DMEM/ 10% FBS), or in a defined keratinocyte serum-free medium (KSFM). After confluence, cells in KSFM were continuously subcultured at a 1-to-3 split. Cellular proliferation was analyzed by immunostaining for Ki67 and the MTT assay. The cellular phenotype was determined by immunostaining for aldehyde dehydrogenase (ALDH), keratocan, and CD34 and by the expression of keratocan promoter-driven enhanced cyan fluorescent protein (ECFP). The stability of the keratocyte phenotype was examined by switching KSFM to DMEM/ITS and DMEM/ 10% FBS. TGFsignaling was monitored by measuring the promoter activity of TGF1, 2, and RII after transient adenoviral transfection, and cytolocalization of Smad2 and Smad4.