This experimental study aimed to observe continuous changes in structural integrity including the morphology, quantity, and viability of the corneal endothelium preserved in hypothermic storage medium (Eusol-C) and normal temperature of organ culture storage at various durations. Five pairs of rabbit corneas and four pairs of human donor corneas were collected. One cornea from each pair was preserved in Eusol-C, while the fellow was maintained in self-prepared organ culture storage (OCS) medium. Corneas were examined prior to the storage and at day 7, 14, 21, 28, and 35,then were returned to their respective storage media after each examination. Endothelial cell parameters, including endothelial cell density (ECD) and viable endothelial cell density (VECD) with trypan-blue staining were recorded at each time point. Endothelial cell loss rate (ECLR) and viable endothelial cell loss rate (VECLR) were calculated accordingly. At the end of the storage period, all of the corneas underwent immunostaining for markers of structural integrity (ZO-1). Baseline parameters were comparable between groups. Both ECD and VECD progressively decreased in the two preservation methods throughout the storage period. For rabbit corneas, the VECD values were higher in the corneas stored in OCS (r-OC) than that in Eusol-C (r-EC) (P = 0.025, t = 3.474). The difference in ECLR was statistically significant higher in r-EC group after 21 days of preservation. While after day 28, the difference in ECD between two groups was statistically significant (P = 0.014, t = 4.189). For human donor corneas, differences in ECD, VECD, ECLR, and VECLR were all statistically significant after 35 day of preservation. For long-term corneal preservation, the organ culture method demonstrates superior performance compared with hypothermic storage, particularly in maintaining integrity of endothelial cells.