PURPOSE
To simulate pars plana vitrectomy in porcine eyes ex-vivo using intraoperative devices and to evaluate viability of retinal cells.

METHODS
25 enucleated porcine eyes were divided in following groups Group A) No surgery control: Group B) Sham surgery; Group C) Cytotoxic control; Group D) Surgery with residues; Group E) Surgery with minimal residues. The retina was extracted from each eye bulb and the cell viability was determined by MTT assay. The in vitro cytotoxicity of each used compounds was tested on ARPE-19 cells.

RESULTS
No cytotoxicity was detected in retinal samples in groups A, B and E. Samples from eye bulbs that had undergone surgery with minimal removal of residues (group D) and cytotoxic controls (group C) showed high retinal cytotoxicity. The simulation of vitrectomy indicated that the combined use of compounds does not affect retinal cells viability if all the compounds are properly removed, whereas the cytotoxicity detected in group D may suggest that the presence and accumulation of the residues of the compounds used intraoperatively could negatively impact retinal viability.

CONCLUSIONS
The present study demonstrate the crucial role of an optimal removal of the intraoperative devices used in eye surgery to ensure safety to the patient.

Purpose
To extract crystalline proteins from porcine eye lenses and to evaluate their relation with oxidative stress.

Methods
10 lenses were extracted from porcine eyes. The lenses were washed with phosphate-buffered saline (PBS) and homogenized in extraction buffer (20 mM Tris HCl, 5 mM EDTA, 2.6 ml/g of lenses) using Polytron homogenizer. The water insoluble residues were eliminated from the homogenate by subsequent centrifugations at 12000 rpm for 10 minutes. Crystalline proteins concentration of the extract was assessed with Bradford assay using Bradford reagent (Sigma Aldrich) and HPLC using Jupiter 5 μm C18 300 Å column.
The reactive oxygen species (ROS) probe Dihydrorhodamine-123 (DHR123, Sigma Aldrich) was added to the lens homogenate solution containing 3 mg/ml of crystalline proteins and irradiated by UV light (254 nm) for 0, 5, 10, 15 minutes. Irradiated samples were analyzed both by HPLC for protein characterization
and fluorimetry recording emission fluorescence spectra from 510 to 700 nm, using a Perkin Elmer luminescence spectrophotometer with excitation at 505 nm to determine ROS production.

Results
HPLC analysis showed the presence of a specific peak pattern of crystalline proteins in the porcine eye lens extract corresponding to 21% of α, 66% of β, and 13% of γ crystalline proteins. The concentration of each crystalline protein decreased after 15 minutes of UV irradiation. The emission fluorescence spectra showed a peak at 527 nm corresponding to the presence of Rhodamine 123, as a result of the oxidation of DHR123 probe induced by the presence of ROS.

Conclusions
α, β and γ crystalline proteins were extracted from porcine eye lens and quantified. UV irradiation of crystalline proteins solution induced the protein degradation that could be related to ROS production.
Additional studies are necessary to evaluate the oxidative stress mechanism that induce crystalline proteins degradation.

Purpose
To develop a quantitative method to assess the toxicity of intraocular endotamponades using human retina ex-vivo model. The study aimed at determination of retina viability, positive and negative controls, method accuracy and repeatability.

Methods
Human donor eye globes for research use were transported to FBOV and stored in DMEM/F-12 medium at 4°C for a maximum of 48 hours. 2-mm and 3-mm retina samples were evaluated for viability using TOX-1 In Vitro Toxicology Assay Kit (Sigma-Aldrich, Italy) immediately (T0) and 24 h, 48 h, 72 h and 7 days after retina extraction. Sodium Dodecyl Sulfate (SDS) concentrations of 0.25 mg/ml, 0.50 mg/ml and 1 mg/ml were tested as positive (cytotoxic) controls. DMEM/F-12 medium was used as a negative control. The toxicity of perfluorooctane samples (PFO) and the same PFO containing toxic residues (1H-PFO and PFOA, positive controls) was assessed in a comparison between donor retina ex vivo model and cytotoxicity test in vitro model using ARPE-19 cell line.

Results
Retina extracted within 24 and 48 hours post mortem showed optimal viability at T0. Incubation of the samples with 0.25, 0.50 mg/ml and 1 mg/ml SDS (cytotoxic control) induced 75.3 ± 4 %, 98.6 ± 2 % and 98.5 ± 2 % mortality at T0, respectively. In average 53 ± 2 samples with 3-mm diameter were prepared from one donor retina. 3-mm sample size was suitable to allow good method repeatability. Both donor retina ex vivo and cell line models showed comparable cytotoxic results; not cytotoxic samples resulted in similar % of cell viability corresponding to 92 ± 3 % and 97 ± 1 % for donor retina ex vivo and cell line models respectively. Cytotoxic samples induced higher mortality in donor retina ex vivo model compared to the cytotoxicity test in vitro in cell line model.

Conclusion
A quantitative method to assess the toxicity of intraocular liquid devices in human retina ex-vivo model was standardized with high sensibility and repeatability.

Background: Mesenchymal stromal cells (MSCs) are an Advanced Therapy Medicinal Product (ATMP) that has been extensively used as second-line treatment for acute Graft-versus-host disease (GvHD) after hematopoietic stem cell transplanation.
We currently isolate and expand MSCs starting from umbical cord fragments (UC) collected after caesarian sections. To ensure final sterility of the cells, UC are decontaminated immediately after collection in the operationg room by submerging them for 24 hours at 4°C in BASE.128 (AL.CHI.MI.A. S.r.l.), an antibiotic solution containing vancomycin, gentamicin, cefotaxime, and amphotericin B deoxycolate. Cefotaxime, a cephalosporin, is a beta-lactamic potentially inducing IgE-mediated reactions in humans. For this reasons, the quantification of residues of cefotaxime in the final product is a requirement related to the MSCs product safety.

Aims: To evaluate the efficacy of tissue decontamination and quantification of cefotaxime residues during the different steps of the UC-MSCs production process in the GMP facility of the Advanced Cellular Therapy Laboratory (LTCA), Hematology Unit, Vicenza Hospital, Italy.

Methods: In the operating room UC (n=3) were submerged in BASE.128 immediately after caesarian section. After 24h at 4°C UC were introduced in the clean room, transferred into 50-ml tubes and washed twice with DPBS at room temperature in order to remove debris and red blood cells.
Subsequently, UC were minced in fragments of about 1-2 mm, seeded in reclosable lid TPP flasks in medium containing human-derived platelet lysate and incubated at 37°C, 5% CO2. Medium was changed and cells detached and replated periodically until passage 3 in Corning Hyperflasks. After 26 days in culture, cells were detached, counted, collected in bags and cryopreserved in liquid nitrogen vapor phase.
Samples of processing liquids, UC fragments and cells were collected during UC-MSCs production steps, frozen and sent to Alchilife S.r.l. laboratory for analyses. Cefotaxime residues were determined using HPLC Dionex Ultimate 3000, with Ultra C8 3µm 150 x 2.1 mm column (Restek Superchrom) in BASE.128 solution, after UC decontamination, DPBS washing solution, UC homogenates after washing steps and in the expanded MSCs with and without the thawing solution (UC-MSC final product). UHPLC analyses were repeated in triplicate and means and standard errors were calculated.

Results: No decontaminations were detected in all UC samples processed after 24 hours in BASE.128 at 4°C. At the end of the production process UC-MSC were sterile, mycoplasma and endotoxin free. HPLC analysis showed the presence of 17.308±0.303 µg/ml of cefotaxime in BASE.128 solution after UC decontamination.
Traces of cefotaxime corresponding to 0.105±0.016 µg/ml were detected in DPBS washing solution. Importantly, no cefotaxime residues were detected in UC homogenates after the washing steps as well as in the final MSCs product.

Conclusion: The present study showed the efficacy of the decontamination strategy in BASE.128 solution and the complete absence of cefotaxime residues in both UC tissue after washing and in the final UC-MSC product demonstrating the efficacy of the washing strategy during production process thus ensuring the absence of allergenic residues potentially harmful for the transplant recipient.

Background: Advancements in cellular and molecular biology led to the development of the so-called Advanced Therapy Medicinal Products (ATMP) namely gene therapy, somatic cell therapy and tissue engineering products.
ATMPs have been classified as medicines after the entry in force of the EU regulation 1394/2007 and must be prepared according to Good Manufacturing Practice (GMP) and be produced in authorized facilities.
Graft-versus-host disease (GvHD) is a severe complication in the setting of allogeneic hematopoietic stem cell transplantation (HSCT). Mesenchchymal stromal cells (MSCs) have been extensively used as second-line treatment for acute GvHD. Umbical cord derived (UC) MSCs display  low immunogenicity, which makes them suitable for an allogeneic use.

Aims: Here we describe the production process of UC-derived MSC (UC-MSC) in the LTCA GMP facility (Vicenza, Italy) starting from the cord collection to ATMP release.

Methods/Materials: UC fragments are collected from caesarian sections and immediately submerged in transport and decontamination solution (BASE.128, AL.CHI.MI.A. S.R.L., Italy) containing a cocktail of four antibiotics. Harvested tissues are transferred to the Transfusion Medicine where it is anonymized and screened for viruses and T. pallidum. UC is then transferred to LTCA and after 24h in BASE.128 introduced in clean room. UC is minced in small fragments and seeded on flasks. Cells are expanded in Corning HYPERFLASKS in medium containing human derived platelet lysate. At day 7 media is changed and at days 13, 19 and 23 cells are detached and replated. At day 26 cells are harvested, counted, collected in bags and cryopreserved in liquid nitrogen vapor phase.
UC_MSC are released once sterility, mycoplasma, endotoxins, cell count, phenotype, karyotype, cell viability and impurities have been determined following compendial methods.

Results: At the end of the expansion, MSCs resulted to be sterile, endotoxin and Mycoplasma-free. Detection was performed as prescribed in EU Pharmacopeia. Expanded cells expressed MSC markers and were able to differentiate into mesodermal tissues and to exert immunomodulatory activity on activated lymphocytes. Starting from 10cm of UC we have produced a mean of 255×106 cells at P3 with a cell viability of ≥80%.

Conclusion: One of the critical steps in ATMP production is to ensure product sterility since cells are not terminally sterilized. The UC collection during caesarian section is crucial and the reduction of bioburden depends on operator training and decontamination strategy used. In our experience none of the UC collected was contaminated by bacteria once treated with BASE.128 and no residual cefotaxime was found in the final product.
Academia plays a pivotal role in the development of ATMPs but the translation of preclinical research into GMP procedures has been facilitated from research centers such as our Transnational Research Laboratory. In our center we have both the availability of tissues and cells thanks to donors and to the connection to the hospital and the knowledge of aseptic tissue manipulation, thus filling the gap from bench to bedside.

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.

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.