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To assess the endothelial cell density before and after the preparation of precut posterior corneal lamellae for Descemet membrane (DM) endothelial keratoplasty with a stromal rim with the aim of standardizing this new technique and establishing the parameters of corneas intended for manual preparation under the conditions of an ocular tissue bank.Two groups of corneoscleral buttons were used to prepare lamellae consisting of a central zone of endothelium–DM surrounded by a supporting peripheral stromal rim. Group 1 contained 12 corneas with a live endothelial cell density (LECD) of 2500 cells per square millimeter or more. Group 2 contained 10 corneas with a density of 2500 cells per square millimeter or less. The preparation was performed manually using an artificial anterior chamber. The endothelial cell density (ECD) and the percentage of dead cells were assessed before and immediately after preparation and after 48 hours of organ culture storage at 31°C.Immediately after preparation, on average, 4.9% and 5.2% of dead cells were found in group 1 and group 2, respectively. After 48 hours of storage, the percentage decreased significantly in both the groups. There was no significant decrease in the ECD 48 hours after preparation in group 1; however, there was a significant decrease in group 2. The amount of tissue wasted during preparation was 24%.The decrease in ECD is dependent on the initial values: the cell loss is less in corneas with higher original densities. We suggest that the minimal acceptable LECD of lamellae should be 2500 cells per square millimeter.