Optimizing of bone marrow- and adipose tissue-derivedmesenchymal stromal cells manufacturing for clinical trialsaccording to GMP guidelines

Mesenchymal stromal cells (MSCs) are nonhematopoietic multipotent cells displaying immunomodulatory, pro-angiogenic and reparative properties that were demonstrated both in animal and human models and has generated markedly increasing interest in a wide variety of biomedical disciplines. The International Society for Cellular Therapy (ISCT) proposed minimal criteria to define human MSCs: MSCs must be plastic-adherent, have a phenotype of CD105+, CD73+, CD90+, CD45-, CD34-, CD14-, HLA-DR-, and have the capacity to undergo in vitro differentiation to osteoblasts, adipocytes, and chondrocytes. We optimized techniques for isolation of MSCs from bone marrow and adipose tissue, their expansion in humanized serum-free culture system, cryopreservation, transport, quality control and potency testing and established protocol for their manufacturing in validated GMP aseptic conditions in Clean Rooms. It was necessary to establish humanized serum free cell culture system without risk of ineligible immune reaction and transmission of zoonoses. The best expansion potential was achieved in medium DMEM supplemented with 5% platelet lysate made from trombocyte apheresis. Controls of the production as contamination control and visual control of morphology and confluence are carried out during the cell expansion; cell number and viability are monitored at every cell harvest. To prevent the decrease of MSCs quality and proliferation rate, not more than 4 passages are performed, however, MSCs are tested for genetic stability in third and sixth passage by karyotyping and CGH. Release criteria take into account the effectiveness and safety of the product: cell number according to indication, 80% viability and immunophenotype fulfilling ISCT criteria and excluding impurities. MSCs from bone marrow and adipose tissue had similar morphology, immunophenotype and differentiation potential. MSCs from adipose tissue displayed higher proliferation rate and lower senescence ratio. Genetic stability was preserved even in higher passages. MSCs are resident in the source tissues in small numbers of nucleated cells, but can be readily expanded in vitro to provide sufficient number of GMP grade cells for local and systemic cellular therapy (10-50 x 106 cells for local and 1-5 x 106 per kg for systemic administration). The methods used in the manufacturing protocol will be validated and the clinical protocol will be approved by Czech State Institute for Drug Control. Supported by the Ministry of Education of the Czech Republic, NPVII-2B06058, NPVII-2B08066 and endowment fund “Jistota“.