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.

Background: The endothelial cell mortality of the donor corneas is determined qualitatively by the eye bank technicians using the trypan blue staining of the endothelium and visual evaluation of the mortality zones. The present study aimed at developing a quantitative method to determine the endothelial cell mortality based on the trypan blue staining and Image Analysis Using ImageJ software.

Method: Porcine eyes were retrieved at local abattoir, transported to our labs, the cornea was dissected and endothelial cell mortality was immediately assessed. Evaluation of corneas was repeated in five different experiments each including 10 corneas. Endothelial cell mortality was determined by staining the endothelium with trypan blue (TB-S Alchimia S.r.l.) and acquiring stereomicroscopy pictures with 10x magnification. The pictures were analyzed with the Fiji ImageJ software, adopting a customized macro sequence to count blue pixels, corresponding to dead cells, in the central 8-mm diameter area. Surface irregularity of the corneal tissue was compensated during macro sequence optimization phase. Mean percentage of cell mortality was determined for each group of tissues. The normal distribution of percentages of cell mortality within groups was evaluated with Shapiro-Wilk Normality Test and differences between groups was verified with ANOVA one-way analysis of variance.

Results: Customized macro sequence of Fiji ImageJ software provided an accurate and repeatable quantification of the mortality area of corneal endothelium despite the irregular surface of the corneal tissue. The mean endothelial cell mortality was 4.50 % ± 0.94%; the low standard error indicated the repeatability of the method. The distribution of values within groups resulted to be normal according to Shapiro-Wilk Normality Test (p>0.05) and no significant differences were observed between groups based on ANOVA test (p>0.05).

Conclusion: The present study allowed the development of a semi-automatized quantitative method for the measurement of corneal endothelial cell mortality using image analysis with Fiji ImageJ customized macro sequence with optimal method sensibility and repeatability. Additional method optimization will be investigated in order to implement the endothelial cell mortality evaluation within the daily routine of the eye banks.

Background: This study aimed at assessing the stability of new cold storage medium with antimycotic tablet, Kerasave (AL.CHI.MI.A. S.r.l.), in terms of shelf-life and stability under the conditions of use.

Methods: The Kerasave medium shelf-life was determined by monitoring the antimicrobials concentration by HPLC, the tablet integrity and dissolution time, and the medium transparency, pH, and osmolality  after storage at room temperature and at 4°C for 2 years.

Additionally, corneal cold storage was mimicked by using porcine corneas and monitoring the medium pH, osmolality and concentration of antimicrobial and antifungal agents after 14 days of corneal storage. The same media parameters were measured without adding the tissue.

Results:  The Kerasave parameters, including tablet integrity and dissolution time, medium transparency, pH and osmolality remained unchanged after 2 years of storage at room temperature and at 4°C. The antimicrobial concentrations remained within the acceptability range of values for 18 months and 24 months, when media were stored at room temperature and at 4°C, respectively.

Chemical-physical analysis of the media, after mimicking the corneal cold storage, indicated that the pH and osmolality remained unchanged while the antimycotic concentration decreased of approximately 10% after 14 days at 4°C.

In the absence of the cornea, all the measured parameters remained unchanged at 4°C for 14 days.

Conclusions: The stability studies allowed to assign a shelf-life of 2 years to the new cold storage medium with antimycotic tablet, Kerasave, when stored at 4°C. The medium exhibited excellent stability under the conditions of use.

Background: This study aimed at assessing the Candida spp. killing efficacy of the new cornea cold storage medium with antimycotic tablet, Kerasave (AL.CHI.MI.A. S.r.l.).

Material and methods: Kerasave antimycotic activity was determined by in vitro time-kill studies. In order to simulate tissue contamination, sterile porcine corneas were immersed in inoculum solutions respectively containing 10⁵ cfu/ml of 5 Candida spp. clinical isolates (C. albicans ATCC 10231, C. albicans ATCC 90028, C. albicans ATCC MYA-2876, C. tropicalis ATCC 750 and C. parapsilosis ATCC 90018) for 5 h at room temperature. The killing rate at 4°C was monitored by determination of residual contamination in tissue homogenates and storage media after 5 and 10 days of incubation. The samples were treated with RESEP for elimination of antimicrobials before plating by spread-plate technique. Killing rate of the media was evaluated at additional 1, 2 and 3 days time points for C. albicans ATCC 10231, C. albicans ATCC 90028 and C. tropicalis ATCC 750.

Results:  In vitro time-kill studies on cornea homogenates showed at least 3 log10  decrease after 5 days incubation at 4°C and 4 to 6 log10 reduction for all Candida strains was achieved within 10 days of incubation at 4°C.

The media showed almost maximal elimination of the contamination corresponding to 3.1 to 5.6 Log 10 already after 5 days of incubation at 4°C for all tested strains.

Conclusions: The new cold storage medium with antimycotic tablet, Kerasave, exhibited an excellent killing rate of all tested Candida clinical isolates in corneal tissue already after 5 days of tissue cold storage.

Methods: Serial dilutions of perfluorooctanoid acid, 1H,1H,7H-dodecafluoro-1- heptanol, 1H,1H,1H,2H,2H-Perfluorooctane, 1H perfluorooctan; ethylbenzene, paraxylene, perfluotobutyfurane, and hexafluoro-1,2,3,4-tetrachlorobutane were tested by direct contact cytotoxicity test using BALB3T3 and ARPE19 cell lines, after application on 59% of the area for 24 h.

Results: Traces of paraxylene, ethylbenzene, and PFOA (≤30ppm) induced severe toxicity in both cell lines. Hexafluoro-1,2,3,4-tetrachlorobutane and H,1H,7H-Dodecafluoro-1-heptanol were cytotoxic at approximately 10000 ppm. 1H perfluorooctan was cytotoxic at approximately 60000 ppm. High concentrations of 1H,1H,1H,2H,2H-Perfluorooctane and perfluotobutyfurane were not cytotoxic.

Conclusions: Six out of eight perfluorocarbon manufacturing process residuals showed cytotoxicity in the direct contact test according to ISO 10993-5. Paraxylene, ethylbenzene, and PFOA were the most cytotoxic compounds. 1H,1H,1H,2H,2H-Perfluorooctane and perfluotobutyfurane were not cytotoxic.

Purpose: This study aimed at validating the method for sterility testing of the corneal culture medium, TISSUE-C (AL.CHI.MI.A. S.r.l.), and the transport/deswelling medium, CARRY-C (AL.CHI.MI.A. S.r.l.), according to the method suitability test, as defined by the European Pharmacopoeia (EP), using RESEP (AL.CHI.MI.A. S.r.l), which is a new medical device for removal of antimicrobial agents, and HB&L (Alifax) automated culture system.

Materials and Methods: The six EP reference strains were inoculated in TISSUE-C and CARRY-C. Half of the samples were treated with RESEP (RESEP+ group) prior to the sterility testing, whereas the remaining samples were untreated (RESEP− group). Growth controls were obtained by direct inoculation of the microorganisms in the culture broths. Microbial growth was read by HB&L automated light scattering culture system within 48 h.

Results: The use of RESEP allowed detection of microbial growth in 100% of the tested samples, with a mean time to detection (TTD) comparable with that of the growth control group. Significantly lower sensitivity (38.83% ± 20.03% for both media, p < 0.05) and TTD variability, depending on the tested microorganism, were observed in the RESEP− group. The method specificity was 100% for both groups.

Conclusion: The use of RESEP increased the sensitivity of the sterility testing method to 100% and, for the first time, allowed validation of the method for sterility testing of corneal storage media according to the EP method suitability test. This further increases the safety of the corneas intended for transplantation.

Purpose: The aim of this study was to validate the method for sterility testing of corneal storage media Tissue-C and Carry-C according to the “Method suitability test” (EP) using BACTEC (Becton Dickinson) automated system in a multicentric study.

Material & Methods: The validation study was performed at the Eye Bank of Rome and Eye Bank of Monza, Italy. Samples of organ culture medium (Tissue-C, AL.CHI.MI.A. S.r.l.), deswelling/transport medium (Carry-C, AL.CHI.MI.A. S.r.l.), and optimal growth media (growth control) were inoculated with 6 EP reference strains to obtain final microbial concentration of 10 cfu/ml, and tested at least in triplicate with BACTEC automatized system.
Method sensitivity, specificity and robustness were determined for each medium, with and without antibiotic removal from samples with RESEP (AL.CHI.MI.A. S.r.l.).

Results: Both eye banks obtained the same method sensitivity and specificity results. The method for sterility testing of Tissue-C and Carry-C samples after RESEP-treatment using BACTEC system showed 100% sensitivity and specificity. Samples treated with RESEP showed similar times to detection as compared to growth controls.

Conclusions: BACTEC system can be considered validated with 100% sensitivity and specificity, and robustness for samples of corneal storage media contaminated with 1-10 cfu/ml, and treated with RESEP.

Purpose: This study aimed at assessing the antimycotic activity of the new cold storage medium, Kerasave, and at evaluating the quality of donor corneas preserved in the medium at 4°C for 14 days in comparison with Optisol GS.

Material and Methods: Kerasave antimycotic activity was determined by in vitro time-kill studies using sterile porcine corneal tissues, contaminated with 10⁴ 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 a 3 to 4 log₁₀ reduction for both Candida strains within 10 days of incubation at 4°C.
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.

Conclusions: 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.

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.

Purpose: The aim of this study was to validate the sterility testing of tissue samples according to the “Method suitability test” defined by the European Pharmacopoeia (EP, chapter 2.6.1), using the MEB buffer (AL.CHI.MI.A. S.r.l.) for extraction of microorganisms from tissue samples and RESEP (AL.CHI.MI.A. S.r.l.) for elimination of antimicrobials before direct inoculation of growth media.

Materials and methods: Samples consisting of one gram of sterile porcine aortic valve were immersed in BASE.128 (AL.CHI.MI.A. S.r.l.) at 4°C for 24 h to simulate the tissue decontamination process with an antibiotic cocktail. The samples were then contaminated with 10-100 cfu of EP reference strains (Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtilis, Aspergillus niger, Clostridium sporogenes), and introduced in a vial containing MEB extraction buffer and stirred at room temperature for 20 min in order to extract microorganisms from the tissues. The buffer was then treated with RESEP syringe for removal of antimicrobial residues and inoculated in Tryptic Soy Broth (TSB) or Thioglycolate (TG). The turbidity of the growth media was determined visually after 5 days of incubation at 22°C (TSB) or 33°C (TG).

Results: MEB buffer extracted the whole 10-100 cfu of all tested microorganisms from aortic valve samples. All microorganisms showed growth in TG or TSB media after RESEP treatment indicating vitality and absence of BASE.128 antimicrobial residues. Turbidity of growth media was detected within 5 days after inoculation in all tested conditions.

Conclusions: The sterility test of the tissue samples, including the extraction of microbial contaminants from tissues using MEB buffer and removal of antimicrobials using RESEP, before direct inoculation, was successfully validated according to the “Method Suitability Test” of the European Pharmacopoeia (chapter 2.6.1.).