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.