Characterization of corneal endothelial cells cultured to the third passage using refined dual media culture system. (A) Quantitative RT-PCR of mRNA extracted from confluent cultures of human CEnCs grown using regular dual medium (green), human CEnCs(GMP) propagated using the refined dual medium (blue), and expanded human corneal fibroblast (red) were performed. Relative quantification values of each genes were calculated with GAPDH set as the internal control, and respective gene expression of COL8A2, SLC4A11, GPC4, CD200, ALDH3A1, LUM were calculated relative to human CEnCs from the regular culture. It should be noted that human CEnCs used for the gene expression study were non-donor matched but were both at the third passage. Primary human CEnCs(GMP) were cultured to the third passaged and characterized for their expression of (B) Na+K+ATPase, (C) ZO-1, and cell surface markers (D) CD166; TAG-1A3 and (E) PRDX-6; TAG-2A12 by immunocytochemistry. Scale bar: 100 µm. (F) Representative karyograms of chromosomal spreads of CEnCs(GMP) propagated to the third passage from a male donor (46, XY; left) and a female donor (46, XX; right).