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Purpose To evaluate human DSAEK grafts during preservation in a novel corneal hypothermic storage medium under development phase XTRA·4 (AL.CHI.MI.A. SRL) at 2–8°C, in comparison to the control media Eusol-C and Optisol-GS.Methods A total of thirty-seven (n=37) human corneas unsuitable for transplantation were recovered from two eye banks (EBs) and included in this study. The corneas were stored in XTRA·4 (n=27), Eusol-C (n=5), or Optisol-GS (n=5) at 2–8°C for 3–10 days. DSAEK lamellar grafts were prepared following the internal procedures of the EBs. The grafts were evaluated immediately after the cut and during lamellar storage for up to 72–96 hours at 2–8°C. Endothelial Cell (EC) Density (ECD) was monitored using light microscopy or specular microscopy. EC mortality and morphology scores were determined after Trypan Blue staining. Graft EC viability was assessed with Calcein-AM fluorescent dye. EC morphological parameters, including CV% (coefficient of variation) and HEX% (hexagonality index), were evaluated with specular microscopy. Optical coherence tomography (OCT) was employed to measure graft thickness (GT) immediately after the cut and during storage. The transparency of grafts stored in XTRA·4 (n=22) and Eusol-C (n=5) was evaluated using a lux meter.Results All DSAEK lamellar grafts were successfully obtained after corneal storage in the tested media. Graft ECD was comparable among the groups both immediately after DSAEK graft preparation and after graft storage for up to 72–96 hours. No statistically significant differences in CV%, HEX%, EC morphology score, EC mortality, and EC viability were observed among the groups, either immediately after the cut or at the end of lamellar hypothermic storage. No statistically significant differences were observed in GT among grafts stored in different media immediately after the cut. However, the XTRA·4 medium maintained GT unchanged for up to 72–96 hours of graft storage at 2–8°C, while the Eusol-C and Optisol-GS stored grafts showed a significant increase in thickness of +57±75 µm and +56±48 µm, respectively. Graft transparency was significantly higher in XTRA·4-stored grafts (68.2±5.0%) compared to Eusol-C stored grafts (55.7±6.1%) after 72–96 hours of storage.Conclusions DSAEK grafts were successfully obtained from human corneas stored in the new XTRA·4 medium at 2–8°C for 3–10 days. The grafts preserved in XTRA·4 medium at 2–8°C showed comparable results in terms of ECD, EC morphology, and EC viability to those preserved in control media for up to 72–96 hours. However, XTRA·4 better maintained DSAEK graft thickness and transparency during storage at 2–8°C. These results provide new perspectives for achieving stable graft thickness during the transport of DSAEK grafts to operating theatres under hypothermic conditions.