This study aimed to identify the risk factors for endothelial cell density (ECD) loss after Descemet membrane endothelial keratoplasty (DMEK) and analyse whether donor tissues from cold versus organ culture differ in terms of ECD loss after DMEK. Consecutive DMEK cases from a prospective database for Fuchs' endothelial corneal dystrophy were retrospectively analysed between 2011 and 2016 at the University of Cologne, and the possible risk factors for ECD loss, including patient-related factors, type of tamponade (air or 20% sulphur hexafluoride gas), type of surgery (triple DMEK or DMEK alone), re-bubbling, immune rejection, and donor-related factors were determined. Eight hundred and forty-one eyes were selected. There was no significant difference in the best-corrected visual acuity (logarithm of the minimal angle of resolution) and corneal thickness (P = 0.540 and P = 0.667) between groups. Immune reactions were more common in cold cultures (P = 0.019), but ECD loss (1 year after DMEK) was greater in organ cultures (38.3 ± 0.8%) than in cold cultures (34.7 ± 1.4%) (P = 0.022). Only re-bubbling was significantly associated with ECD loss (P < 0.001). Re-bubbling was found to be a key factor for ECD loss at 1 year after DMEK.