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To investigate the possible toxic effect of air exposure for an in vitro model of primary human corneal endothelial cells (HCECs).Primary HCECs were isolated from donor corneal rings and cultivated at 37°C in 5% CO2 and 95% humidified air. Six groups of HCEC cultures were set up, and 4 samples were enclosed in each group: group 1 consisted of samples in which HCECs were exposed to air for 30 minutes. Group 2 consisted of HCECs exposed to air for 1 hour, group 3 for 3 hours, group 4 for 6 hours, group 5 for 12 hours, and group 6 for 24 hours.Three hours after exposure, the morphology of the cells was still intact; however, a few cells within the monolayer appeared enlarged and exhibited characteristics of more senescent cells. Six hours after exposure to air, the endothelial cells started losing their typical hexagonal morphology and appeared enlarged and compromised. Viability was superior to 95% in groups 1 to 3, whereas for groups 4, 5, and 6 was 71%, 22.4%, and 6.3%, respectively.The present study illustrates that the toxic effect of air exposure for the studied in vitro model of primary human-cultured corneal endothelial cells is not significant for the period of 3 hours, whereas after 6 hours it starts to induce major apoptotic mechanisms, leading to reduced viability until the period of 24 hours where the percentage of living cells is drastically decreased.