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.

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: To evaluate the use of RESEP device (ALCHIMIA S.r.l.), intended for antibiotic removal from liquid samples, on tissue homogenates.

Material and method
RESEP treatment of tissues:
– Homogenization: 1 gr in 10 ml of BASE solution with IKA ULTRA TURRAX homogenizer
– 2 passages in RESEP, 20 min/each at RT RESEP was evaluated for the following parameters:
1. Bacterial retention: sterile human fresh aorta fragments were homogenized, contaminated with P.aeruginosa (3 doses) and treated with RESEP.
2. Interference with bacterial growth: sterile human fresh aorta and pericardium fragments were contaminated with P.aeruginosa and S.aureus (3 doses).
3. Antibiotic removal ability: decontamination (BASE 128_125ml/g: 14 h, 37°C) washing (BASE 6,5 ml/g. 2×5 mln, 1x6h, 4°C) and homogenization procedures were applied on sterile porcine cardiovascular tissues. Microbial strains were spiked on RESEP treated/untreated homogenates.
All the homogenates were tested on agar plates for microbal quantification and in BactAlert.

Results: Two sequential passages of the homogenates on RESEP only slightly reduced P.aeruginosa load tested on agar plates. Samples were positive in BacAlert.
P.aeruginosa and S.aures in RESEP treated and untreated homogenates showed similar growth capability on agar plates and were positive in BacAlert.
RESEP-treated homogenates showed a slightly better bacterial recovery than RESEP-untreated samples both in agar plates and in BactAlert.

Conclusions: RESEP device is compatible with homogenized tissues ensuring a good bacterial recovery and absence of interference with bacterial growth. Preliminary data suggest also the capability of RESEP to remove antibiotic in tissue homogenates.

Purpose: Establish a method to decontaminate, wash and freeze cardiovascular tissues.

Methods: Samples sterile porcine cardiovascular tissues, decontaminationantibioticsolutionBASE128_125ml/g 14h 37°C, washing: BASE 6.5 ml/g. 2×5 min. 1x6h. 4°C, RESEP treatment of hormogenates (antibiotic removal device): 2×20 min. RT.

Evaluation of:
1. Decontamination efficiency: samples were immersed in contaminated solutions (103-108 cfu/ml: 10 bacteria and 2 fungi strains), decontaminated, washed and homogenised. Homogenates (RESEP treated/untreated) were tested for contamination on agar plates and BactAlert.
2. Capability of washing procedure to remove antibiobiotic: decontamination and washing procedure was applied. 12 microbial strains were spiked on RESEP treated/untreated homogenates, tested on agar plates and BactAlert.
3. Freezing method: several profiles were tested on aortic, pulmonary and pericardium specimens.

Results:
1. Decontamination resulted in 6 Log 10 reduction for 5 strains (P.aeruginosa, B.atrophaeus, S.marcescens, P.acnes, E.coli), 5 Log 10 for 4 strains (S.epidermidis, K.pneumoniae, C.sporogenes, B.fragilis), 4 Log 10 for S.pyogenes, 1.0 Log 10 for C.albicans and A.brasiliensis.
2. Microbial growth was demonstrated on:
– RESEP-treated tissues: 12/12 strains (agar plates and BactAlert)
– RESEP-untreated tissues: 11/12 strains on agar plates (at less extent for 4 strains), 10/12 strains on BactAlert.
3. A freezing profile was defined leading to optimal freezing curve (-1°C/min to -40°C, -3°C/min to -120°C).

Conclusions: Decontamination was effective (4-6 Log 10) for 10/12 strains. The extensive washing procedure, able to remove antibiotic from the solutions and tissues. In combination with the RESEP ensures a reliable sterility assay. An optimal freezing profile was set-up.

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.).

Abstract: The aim of this study was to validate the microbiological testing of cornea organ culture medium according to the European Pharmacopoeia using blood culture bottles and the automatized BACTEC system (BD) after removal of antibiotics with the new medical device RESEP (AL.CHI.MI.A. Srl, Italy).
For the validation, 10-100 CFU of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtilis, Aspergillus niger, Clostridium sporogenes, Enterobacter cloacae and Staphylococcus epidermidis were inoculated in 9 ml of cornea organ culture medium (MEM with 2% fetal calf serum and antibiotics, Biochrom). The medium was treated with RESEP for 20 min., inoculated in BACTEC blood culture bottles and incubated until a positive reading or for at least 14 days at 36 ± 1°C. Medium control samples, containing the same microorganisms, were inoculated in BACTEC vials without RESEP treatment. Microbial growth was controlled by direct  inoculation of BACTEC flasks (growth control).
The antibiotics were completely eliminated after 20 min. of treatment with RESEP at room temperature (confirmed by HPLC). In RESEP treated samples, the growth of all microorganisms was detected within 3 days of incubation in BACTEC system and showed no significant delay in comparison with the growth controls. Without the use of RESEP, several tests with Staphylococcus aureus, Enterobacter cloacae and Bacillus subtilis samples were not detected and other strains showed a prolonged detection time compared to treated RESEP samples and growth controls.
The microbiological testing of cornea organ culture medium with automatized BACTEC blood culture system could be successfully validated using RESEP for the removal of antibiotics.

Background: The aim of the study was to investigate the decontamination of human donor larynx (tissue-engineered for laryngeal replacements) with medical device BASE.128 (ALCHIMIA S.r.l) in comparison with standard decontamination procedure.

Methods: Laryngeal tissues from two donors were divided in halves and decontaminated with BASE.128 at 22°C and BASE.128 at 37°C overnight. The control halves of both tissues were disinfected with standard procedure using 20% chlorhexidine for 5 minutes at room temperature. Tissues were tested microbiologically, and any microorganism still present at the end of the process was isolated and further investigated in BASE.128 time-kill studies at 37°C.

Results: Standard chlorhexidine disinfection procedure and decontamination with BASE.128 at 22°C overnight resulted inefficient in elimination of C. albicans and S. oralis from the first donor larynx.
However, the time-kill studies showed 5-6 log elimination of both isolated strains, after treatment with BASE.128 at 37°C for 6h.
The second tissue, initially contaminated by Citrobacter koseri, was completely decontaminated by BASE.128 at 37°C overnight, while it still resulted contaminated after standard disinfection with 20% chlorhexidine.

Conclusion: An overnight decontamination with BASE.128 at 37°C was effective in decontaminating laryngeal tissue. More tissues will be tested in order to confirm the results and validate the procedure.

Background: Decontamination of umbilical cord is a critical phase before cryopreservation. We investigated the decontamination efficacy of the BASE.128 against the most frequent umbilical cord contaminants by an in vitro time-kill study.

Methods: F. magna (ATCC 29328), P. melaninogenica (ATCC 25845), and umbilical tissue clinical isolates E.coli, E. faecalis, and B. fragilis were cultivated under optimal growth conditions. Resistance to the antibiotics was determined by Kirby Bauer method. The inoculum concentrations were determined by Mc Farland method. BASE.128 and BASE as control (ALCHIMIA Srl) were inoculated with 10E5-10E6 cfu/ml of the selected microorganisms, each assessed in triplicate. All the strains were incubated at 37°C for 24 h, E. coli and E. faecalis were additionally incubated at +4°C for 48h and +22°C for 48h. Time kill of microorganisms by BASE.128 was determined at 0h, 3h, 6h, 16h, 24h (48h only for incubation at +4°C and 22°C).

Result: E. faecalis was resistant to cefuroxime, cetoconazol, clindamycin, quinupristin+dalfopristin, trimetoprim+sulfametoxazol. B. fragilis was penicillin resistant. A complete elimination of E. coli (6.56 log) was observed after 3h of incubation in BASE.128 at 37°C and 22°C and after 48h at 4°C. The initial inoculum of E. faecalis (5.7 log) was completely eliminated after 16h at 37°C, and 48h at 22°C. 5 log of F. magna and 6.6 log of P. melaninogenica were completely eliminated after 16h at 37°C. B. fragilis was reduced of 2.37 log after 16h at 37°C.

Conclusion: Decontamination with BASE.128 at 37°C for 16h allowed a complete elimination of at least 5 log of the most frequent umbilical cord contaminants (E. faecalis, E. coli, P. melaninogenica and F. magna).