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: 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.).

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).

Abstract: We investigated the presence of antibiotics in cryopreserved cardiovascular tissues and cryopreservation media, after tissue decontamination with antibiotic cocktails, and the impact of antibiotic residues on standard tissue bank microbiological analyses. Sixteen cardiovascular tissues were decontaminated with bank-prepared cocktails and cryopreserved by two different tissue banks according to their standard operating procedures. Before and after decontamination, samples underwent microbiological analysis by standard tissue bank methods. Cryopreserved samples were tested again with and without the removal of antibiotic residues using a RESEP tube, after thawing. Presence of antibiotics in tissue homogenates and processing liquids was determined by a modified agar diffusion test. All cryopreserved tissue homogenates and cryopreservation media induced important inhibition zones on both Staphylococcus aureus- and Pseudomonas aeruginosa-seeded plates, immediately after thawing and at the end of the sterility test. The RESEP tube treatment markedly reduced or totally eliminated the antimicrobial activity of tested tissues and media. Based on standard tissue bank analysis, 50% of tissues were found positive for bacteria and/or fungi, before decontamination and 2 out of 16 tested samples (13%) still contained microorganisms after decontamination. After thawing, none of the 16 cryopreserved samples resulted positive with direct inoculum method. When the same samples were tested after removal of antibiotic residues, 8 out of 16 (50%) were contaminated. Antibiotic residues present in tissue allografts and processing liquids after decontamination may mask microbial contamination during microbiological analysis performed with standard tissue bank methods, thus resulting in false negatives.

Abstract: Residual antibiotic concentrations may induce bacteriostasis of microorganisms eventually present on tissue samples and lead to false negative results during microbiological analysis of the sample.
The aim of the study was to validate RESEP for removal of antibiotic residues from solid and liquid samples before microbiological analysis.
The amount of antibiotics in 3-5 ml samples of four bank-prepared antibiotic cocktails, BASE.128 (AL.CHI.MI.A. Srl), cardiovascular, skin, amniotic membrane and corneal tissue samples was determined by HPLC and agar diffusion analyses, before and after treatment with RESEP. Interference of the device with bacterial growth was evaluated performing the microbiological recovery test with inoculants containing 1-10, 10-100 and 100-1000 CFU of European Pharmacopoeia (EP) reference strains (S. Aureus, P. Aeruginosa, C. Albicans, B. Subtilis, A. Niger and C. Sporogenes). Additional tests of the device were performed using different fluids and tissue samples provided by five different tissue banks.
Agar diffusion test and HPLC showed important antibiotic residues in liquid and tissue samples not treated with the RESEP; the amount of antibiotic residues varied depending on the antibiotic cocktail. Complete antibiotic removal from liquid and tissue homogenates decontaminated with BASE.128 was observed after RESEP treatment. Removal of antibiotics from bank-prepared antibiotic cocktails varied from 94-99%.
For EP reference strains, growth was observed for all the tested inoculants.
RESEP treated samples showed significantly higher bacterial recovery as compared to tissues analysed with traditional microbiological methods.
In conclusion, RESEP was validated for the removal of antibiotic residues from both liquid and tissue samples, thus allowing  detection of microbial growth.
The device resulted to be harmless for EP reference strains and showed higher sensibility as compared to currently used microbiological methods in tissue banks.

Abstract: Our previous studies indicated that the use of antibiotic cocktails during tissue processing can lead to an antibiotic carry-over effect, which in turn can generate false negative results in microbiological analysis.

Purpose: To validate a single-use device for elimination of antibiotics from liquid samples undergoing microbiological analysis to prevent interference with microbial growth.

Methods: The efficacy of the ResEP device was tested on 5 ml of BASE.128 (AL.CHI.MI.A.S.r.l.) and determined by HPLC after 5, 10, 20, 30 min. The recovery of S. Aureus, P. Aeruginosa, C. Albicans, B. Subtilis, A. Niger and C. Sporogenes was determined by dilution plating of inoculants containing 1-10, 10-100 and 100-1000 CFU, 20 min after treatment.

The device was further tested by three cardiovascular and skin Italian banks. Standard bacteriological tests were performed on transport, rinsing and cryopreservation liquids (BACT-ALERT Biomerieux or thioglycollate/TSB media) of twenty tissue samples decontaminated with BASE.128; additional bacteriological tests were performed on liquid samples using ResEP device, after antibiotic removal. The presence of antibiotic residues in all samples undergoing the microbiological analyses was evaluated by HPLC.

Results: The HPLC analysis of the samples treated with ResEP device showed complete antibiotic removal from the BASE.128 within 20 min of treatment. A total bacterial recovery was obtained for all investigated inoculants.
HPLC analysis on processing liquids showed that important antibiotic residues were present in rinsing and cryopreservation liquids, after decontamination of skin and cardiovascular tissues. The ResEP device removed efficiently antibiotic residues from all liquid samples prior to microbiological analysis allowing the detection of 10 % of false negative results.

Conclusion: The ResEP device was validated for easy and quick removal of antibiotic residues from liquid samples undergoing microbiological analysis, showing complete microbial recovery and high specificity for BASE.128 medium and allowing the detection of false negative results in microbiological analyses.

Abstract: Each tissue bank validates its own method for microbiological analysis of tissues, which is essential to determine whether they can be released for transplantation.
The aim of the study was to compare the results of microbiological analyses of tissues intended for transplantation obtained by using different microbiological testing methods.
Human cardiovascular tissues, skin and corneas were retrieved and processed by five different tissue banks. Tissues were decontaminated at 4°C for 24h/72h (cardiovascular) or at 22°C for 90 min. (skin) either with bank prepared solutions or BASE.128 (AL.CHI.MI.A. Srl., Italy) and then cryopreserved in RPMI 1640 with the addition of 10% DMSO. Corneas were processed and stored under organ culture conditions. Microbiological analysis of tissues and processing liquids were performed by tissue banks according to their standard procedures, using BacT/ALERT, BACTEC and direct inoculation of culture media (Thioglycollate/TSB); samples were tested in parallel at AL.CHI.MI.A. with the sterility test according to European Pharmacopoeia (EP), with the removal of antibiotic residues with RESEP.
All bacteriological analyses of decontaminated cardiovascular tissues performed with direct inoculum method resulted negative. 3% of liquid and tissue samples were found positive with the BACTEC method. The percentages of positive liquid and tissue samples with RESEP were 16% and 25%, respectively.
The bacteriological analysis of decontaminated skin samples were negative with both BACTEC and direct inoculum method. 33% of liquid samples and 50% of tissue samples were found positive using RESEP.
Corneas processing liquids resulted negative with BACTEC and direct inoculum method. Instead, 59% of the samples resulted positive with RESEP and sterility test according to EP.
Different results were obtained depending on the used method to perform microbiological analyses after tissue decontamination. A validation of the microbiological method, including accurate removal of possible antibiotic residues in decontaminated tissue and processing liquid is necessary to ensure the safety of tissue allografts.