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.