Objective: This validation study investigates the treatment of cornea organ culture medium (Modified Eagle Medium, Biochrom GmbH, Berlin, Germany) with RESEP, a new medical device for antibiotics removal, before microbiological testing with BACTEC^TM blood culture bottles.

Methods and analysis: 10–100 colony forming units of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtillis, Aspergillus brasiliensis, Clostridium sporogenes, Enterobacter cloacae and Staphylococcus epidermidis were inoculated in 9mL of cornea organ culture medium.
In group A, the medium was withdrawn with RESEP and treated for 20 min at room temperature, and then inoculated in BACTEC Plus Aerobic/F/Anaerobic/F blood culture bottles.
In group B, the medium, spiked by the inoculation of microorganism, was injected directly. For each strain, a growth control was performed, by direct inoculation of the microorganisms in BACTEC^TM vials (positive control). All samples were incubated in the automated BACTEC^TM blood culture system at 36°C ±1°C for maximum of 14 days or until a positive reading. The elimination of antibiotics from the medium by RESEP was determined by high-performance liquid chromatography.

Results: After 20 min of RESEP treatment, 100% (n=9) of streptomycin, 100% (n=9) of amphotericin B and 99.7% (n=9) of penicillin G were eliminated.
In group A , all microorganisms were detected within 3 days of incubation with a sensitivity of 100% (n=99) and showed no significant delay compared with the positive controls.
In group B, the overall sensitivity was 67.9% (n=96) with a significant delay until detection of microbial growth for all tested microorganisms except for A. brasiliensis.

Conclusion: The use of RESEP to eliminate the antibiotics from cornea organ culture medium increases the sensitivity of the microbiological testing with BACTEC^TM Plus blood culture bottles significantly and fulfils the requirements of the European Pharmacopoeia method suitability test.

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.

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.

Methods: BALB3T3 and ARPE19 cell lines were cultured in 96-well plates for direct contact cytotoxicity test of 22%, 59%, and 83% contact areas and 2.5, 12, and 24 h contact times. Cell morphology was graded by light microscopy. Cell viability was quantified by MTT assay in ARPE19 cells and neutral red uptake viability assay for BALB3T3. 1-H perfluorooctane (1H PFO) and purified perfluoro-n-octane (PFO) were used as positive and negative controls, respectively.

Results: Qualitative evaluation showed that positive control induced the presence of severe reactivity zones, resulting in 2.2 to 3.0 grading score, and cytotoxicity according to ISO 10993-5 in all tested conditions. Quantitative evaluation of 1H PFO applied on 22%, 59%, and 83% contact areas corresponded to 14–26%, 84–96%, and 86–99% cell mortality ranges, respectively. No cytotoxicity according to ISO 10993-5 was detected in tested cell lines, when 1H PFO was applied on a 22% area at the tested time intervals. The negative control was not cytotoxic with both approaches in all tested conditions.

Conclusions: Direct contact cytotoxicity test according to ISO 10993-5 of PFCLs was validated quantitatively and qualitatively, using ARPE19 and BALB 3T3 cell lines and covering 59% of the cell surface areas for 24 h of contact time. Test conditions using a smaller contact area could not be validated.

Purpose: To compare the protective properties and ease of manipulation during cataract surgery of corneal coating with a gel (eyeDRO; AL.CHI.MI.A. S.R.L, Italy) and corneal irrigation with balanced salt solution (BSS).

Methods: We analyzed the data of 51 patients receiving either eyeDRO or BSS during routine cataract surgery performed within a 20-day period in 2016. The selected parameters were intraoperative clarity and ease of manipulation; postoperative epithelial integrity; and patient discomfort.

Results: Compared with BSS irrigation, eyeDRO coating significantly increased intraoperative clarity and ease of manipulation (P < 0.01). Single application was required in eyeDRO-treated eyes, whereas BSS was applied 5.3 ± 0.4 times on average (P < 0.01). Two hours postoperatively, a normal epithelium was observed in 90.0% and 60.0% of eyeDRO-coated and BSS-irrigated eyes, respectively; punctate epithelial damage was observed in 9.7% and 40.0% (P < 0.05) of eyeDRO-coated and BSS-irrigated eyes, respectively; eye irritation and foreign body sensation were experienced by 13.0% and 37.0% of eyeDRO-treated patients and by 65.0% and 100% of BSS-treated patients, respectively (P < 0.01). Twenty-four hours postoperatively, 80.0% of BSS-treated patients versus 19.0% of eyeDRO-treated patients still experienced foreign body sensation (P < 0.01).

Conclusions: eyeDRO coating was shown to be a safer and more effective option than BSS irrigation in cataract surgery because single application provided optimal hydration and intraoperative clarity during the entire surgery, better preserved the corneal epithelium, and offered postoperative comfort to the patient.

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.