Bone marrow-derived mesenchymal stem cells (BM-MSCs) are isolated from bone marrow, harvested from the posterior iliac crest, under moderate sedation and local anesthesia. Preclinical validation studies have demonstrated the reproducible generation of >200 million mesenchymal stem cells from aspirated bone marrow, within 21-28 days of culture.
Mononuclear cell fraction is isolated using a density gradient with Lymphocyte Separation Medium (specific gravity 1.077). The low-density cells will be washed with Plasma-Lyte A containing 1% HSA. The washed cells will be sampled, and viable cell counts performed. The bone marrow cells are seeded in 225 cm2 tissue culture flasks in α minimum essential medium containing FBS (20%). After 14 days of culture, P0 cells are harvested by trypsin treatment and expanded into 30 flasks. These flasks are incubated for an additional 7 days and the MSCs are harvested by trypsin treatment (P1). The P1 cells are washed, and viability counts performed. The MSCs are resuspended in cryoprotectant consisting of Pentaspan (10% pentastarch in 0.9% NaCl) supplemented with 2% HSA and 5% dimethyl sulfoxide. The cells will be frozen and stored in a liquid nitrogen freezer. Upon request, the cells will be thawed in a 37°C water bath. In a biosafety cabinet, the cell suspension will be transferred to conical tubes and slowly diluted with a PBS buffer supplemented with HSA (1%). The suspension will be centrifuged, and the cell pellet will be resuspended in the dilution buffer. Viability counts will be performed, and the cells will be delivered to the catheterization laboratory.