PURPOSE
To compare endothelial cell density and morphometry, thickness, and transparency of corneas stored in Kerasave vs. Optisol-GS for 14 days.

METHODS
Mate corneas were recovered into Kerasave or Optisol-GS (27 pairs) and stored at 2-8°C for 14 days.
Endothelial cell density (ECD), hexagonality (HEX), and coefficient of variation (CV) were evaluated by specular microscopy, and central corneal thickness (CCT) was examined by OCT after 1, 3, 7, and 14 days of storage. Corneal transparency was scored using slit-lamp examination at days 1 and 14.

RESULTS
Average ECD, HEX, and CV for the Kerasave (2653±303, 57±4, 36±3) and Optisol-GS (2623±306, 57±5, 36±4) groups were not significantly different at day 1. There was also no difference at any other study timepoints (P>0.05 for all). ECD did not significantly change from day 1 to day 14 in either group (P>0.05), but a statistically significant change in HEX and CV was observed between day 1 and day 14 in both groups (P<0.01). Average CCT measured at day 1 for corneas stored in Kerasave was 622±49 and those stored in Optisol-GS was 580±35 (P<0.01). The difference in CCT measurements was not significant different at day 14 (674±46 v. 647±58, P>0.05). Corneal transparency was not significantly different between the two groups at day 1 or day 14.

CONCLUSION
Endothelial cell density, endothelial morphometry, and corneal transparency were not different in corneas stored in Kerasave or Optisol-GS for 14 days.
Optisol-GS was initially better at thinning the cornea than Kerasave. These results suggest that Kerasave is a viable alternative to Optisol-GS for corneal storage.

Purpose: Cold storage is the standard for corneal preservation method in the U.S., but none of the three U.S. Food and Drug Administration-approved media contains antimycotics. The aim of this study to evaluate antimycotic activity of the first cold storage medium with dissolvable tablet contained Amphotericin B (Kerasave, ALCHIMIA, S.r.l., Italy)(pending FDA approval), and compare the effect of Kerasave versus Optisol-GS on endothelial cell density and corneal transparency.

Materials: Six not-suitable-for-transplant donor corneoscleral rims prepared at Lions Eye Institute for Transplant and Research, Tampa,USA were contaminated by aqueous suspension of Candida Albicans (clinical isolate) prepared to a value of 0.52 on the McFarland Scale of optical turbidimetry and incubated in Kerasave at 4°C. Two additional donor corneoscleral rims remained unaltered in Optisol-GS as control. Kerasave antifungal efficiency was performed by creation and following assessment of Sabouraud agar plates on days two (2), six (6), ten (10), fourteen (14), and sixteen(16). Endothelial cell density and corneal transparency were measured by specular microscope, slit lamp, back light microscopy and OCT after 3, 7 and 14 days of storage at 2-8°C.

Results: Six experimental specimens grew out an average colonies as follows: showed growth too numerous to count on Day 2 (from the Day 0 plating), eleven (11) colonies on Day 6 (Day 4 plating), three (3) colonies on Day 10 (Day 8 plating), and no growth from plating on Day 12 and Day 16. No significant difference in Kerasave vs Optisol-GS corneal cell density and corneal transparency after 3,7 and 14 days of storage.

Conclusion: Addition of Amphotericin B dissolvable tablet into cornea storage media demonstrates effective consistent decrease in fungal contamination compared to control and biocompatibility with donor corneas.

Purpose: To determine the efficacy and safety of different Amphotericin-B (AmphoB) concentrations during short exposure times against Candida albicans in a cold storage media.

Method: AmphoB was added to a final concentration of 0.255 µg/mL, 1.25 µg/ml, 2.50 µg/mL and 5.0 µg/mL in a DMEM-based hypothermic storage media. AmphoB containing media and control samples (tryptic soy broth with no AmphoB) were inoculated with 10⁵ CFU/ml of C. albicans (ATCC10231) and stored at 4°C for 72 hours (triplicate cultures). The number of living microorganisms in each sample was determined initially and after 6, 24, 48, and 72 hours of storage at 4°C. AmphoB was neutralized before plating on agar plates by dilution and spread plate technique. Cell viability of cornea endothelium were also examined after 72 hours of exposure using Calcein-AM staining and FIJI segmentation.

Results: AmphoB concentrations of 1.25 µg/ml, 2.5 µg/ml and 5.0 µg/ml resulted in 0.77, 1.45 and 2.15 log10 reduction after only 6 hours of storage at 4°C, and continued to decrease to 3.63, 3.98 and 4.35 log10 reductions after 72 h (>99.9%), respectively. In contrast, AmphoB at 0.255 µg/ml showed only a 0.73 log10 decrease after 48h of incubation at 4°C. CFU counts of C. albicans were significantly higher in control samples. Endothelial cell viability was not different in donor corneas exposed to AmphoB (≤ 2.5 mg/mL) for 72h compared to non-exposed controls (P=0.52).

Conclusion: Current use of AmphoB at 0.255 mg/mL is not sufficient for C. albicans suppression. Optimal efficacy of AmphoB against C. albicans is achieved in cold storage conditions at concentrations above 1.25 µg/ml and exposure time of 24-48 hours.

Purpose: Cold corneal storage media available on the market do not contain antimycotics. A new corneal storage medium with an antimycotic tablet (Kerasave, AL.CHI.MI.A. S.r.l.) was developed to prevent fungal contamination of donor corneas intended for transplantation. The aim of this study was to assess the antimycotic activity of the new cold storage medium, Kerasave, and to evaluate the quality of donor corneas preserved in the medium at 4°C for 14 days in comparison with Optisol GS.

Method(s): Kerasave antimycotic activity was determined by in vitro time-kill studies using sterile porcine corneal tissues, contaminated with 10 cfu/ml of C. Albicans (ATCC10231 and clinical isolate). The killing rate of the microorganisms was monitored at 4°C after 5 and 10 days of incubation in Kerasave.
Kerasave performance was assessed on 16 pairs of human corneas not suitable for transplantation, procured and evaluated according to standard procedures of Monza Eye Bank, Italy. One cornea was transferred in Kerasave and the contralateral in Optisol GS. Endothelial cell density (ECD), measured by specular microscopy (Keratoanalyzer, Konan), was evaluated pre-processing, and after 7 and 14 days of storage at 4°C. Endothelial cell morphology and mortality were determined according to Stocker method, and epithelial integrity, and corneal transparency were evaluated using a Slit lamp.

Results: In vitro time-kill studies showed 3.6 log reduction for both Candida strains after 5 days of incubation at 4°C and 4 log reduction was observed for both strains after 10 days of incubation.
Kerasave -and Optisol-GS- treated tissues showed similar ECD, mortality and endothelial morphology after 7 and 14 days of cold storage. Slit lamp analysis showed comparable corneal transparency and epithelial integrity in both groups.

Conclusion: The new cold storage medium with antimycotic tablet, Kerasave, exhibited an excellent antimycotic activity and biocompatibility with donor corneas after corneal storage at 4°C for up to 14 days.