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.

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.

Objective: This study aimed at validating the method for sterility testing of the corneal culture medium, TISSUE-C, and the transport/deswelling medium, CARRY-C, according to the method suitability test, as defined by the European Pharmacopoeia (EP), using RESEP, which is a new medical device for removal of antimicrobial agents and an automated culture system.

Methods and analysis: 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 micro-organisms in the culture broths. Microbial growth was read by an automated light scattering culture system within 48 hours.

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 micro-organism, 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.