Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum

Adult mesenchymal stem cells (MSCs) are considered as valuable mediators for tissue regeneration and cellular therapy. This study was performed to develop conditions for regularly propagating a clinical quantity of >2 x 10(8) MSCs without animal serum from small bone marrow (BM) aspiration volumes within short time. We established optimized culture conditions with pooled human platelet lysate (pHPL) replacing fetal bovine serum (FBS) for MSC propagation. MSC quality, identity, purity, and function were assessed accordingly. Biologic safety was determined by bacterial/fungal/mycoplasma/endotoxin testing and genomic stability by array comparative genomic hybridization (CGH). We demonstrate that unmanipulated BM can be used to efficiently initiate MSC cultures without the need for cell separation. Just diluting 1.5-5 mL heparinized BM per 500 mL minimum essential medium supplemented with L-glutamine, heparin, and 10% pHPL sufficiently supported the safe propagation of 7.8 +/- 1.5 x 10(8) MSCs within a single 11- to 16-day primary culture under defined conditions. This procedure also resulted in sustained MSC colony recovery. MSC purity, immune phenotype, and in vitro differentiation potential fully matched current criteria. Despite high proliferation rate, MSCs showed genomic stability in array CGH. This easy single-phase culture procedure can build the basis for standardized manufacturing of MSC-based therapeutics under animal serum-free conditions for dose-escalated cellular therapy and tissue engineering.