A NEW HUMAN PLATELET LYSATE FOR MESENCHYMAL STEM CELL PRODUCTION COMPLIANT WITH GOOD MANUFACTURING PRACTICE CONDITIONS

Mesenchymal stem cells (MSCs) are classified as Advanced Therapy Medicinal Products, a new category of medicines produced in good manufacturing practice (GMP) conditions for clinical use. We isolated MSCs from bone marrow (BM), using human platelet lysate (HPL) as an alternative to foetal bovine serum. Our aim was to compare the standard HPL-E, obtained by freezing and thawing cycles of platelets (PLTs), and HPL-S, obtained via a new method that consist in treating PLT pools with Ca-Gluconate to form a gel clot mechanically squeezed to release growth factors. HPL-S resulted without PLTs and fibrinogen, but the quantity of total proteins and growth factors obtained was comparable to that of HPL-E. HPL-S, avoiding the addiction of heparin in the cell culture, resulted more advantageous for MSC production in GMP conditions. We isolated MSCs from 7 BM samples testing HPL-S vs HPL-E. We observed that MSCs cultivated in HPL-E generated a higher number of colony forming unit-fibroblasts at the seeding density of 10.000 cells/cm2 but not at 100.000 cells/cm2 in comparison with HPL-S and showed a cumulative population doubling higher than that of MSCs in HPL-S in the earlier passages. At the 4th passage we observed an inverted trend of the cell growth in MSCs cultivated in HPL-S and a maintenance of stem cell markers during the expansion. Immunophenotypic analysis showed a significant lower expression of HLA-DR (1.30 %) in MSCs cultivated in HPL-S compared to HPL-E (14.10 %) showing an immune-privilege. All data demonstrated that HPL-S is an effective alternative for MSC production compliant with GMP conditions.