Karyotype changes in cultured human corneal endothelial cells

PURPOSE To examine karyotype changes in cultured human corneal endothelial cells (HCECs). METHODS HCECs with Descemet's membrane were removed from 20 donors of various ages (range, 2-77 years; average, 43.7+/-26.4 years) and cultured on dishes coated with extracellular matrix produced by bovine corneal endothelial cells (BCECs). Karyotype changes were examined by G-band karyotyping of HCECs at the third passage from 12 donors and the fifth passage from 16 donors. The number of chromosomes was analyzed in 40-50 cells from the third and fifth passages of each HCEC preparation. A detailed karyotype analysis of 10-16 cells from the third and fifth passages of each HCEC preparation was performed. The frequency of aneuploid cells per case (the number of abnormal cells divided by the total number of cells examined at metaphase) was tested for correlation with age by Spearman's correlation analysis. RESULTS At the third passage, five cases (41.7%) showed an almost normal karyotype, and five cases (41.7%) showed sex chromosome loss. One case (8.3%) showed chromosome 21 trisomy. At the fifth passage, five cases (31.3%) showed an almost normal karyotype, and four cases (25%) showed sex chromosome loss. Three cases (18.8%) showed chromosome 8 trisomy, and one case (6.3%) showed chromosome 21 trisomy. Donor age and the frequency of aneuploidy had a statistically significant correlation at the fifth passage (R=0.653, p=0.042). CONCLUSIONS Donor age and the frequency of aneuploidy have a positive correlation in cultured HCECs at the fifth passage. Therefore, HCECs for clinical therapies should be obtained from donors as young as possible. Karyotyping cultured HCECs is crucial before clinical application.