In order to prepare hBM-MSCs, bone marrow aspirate was harvested from the iliac crest of a normal donor who was negative for HIV, HBV and HCV laboratory tests, after informed consent was obtained. Then, hBM-MSCs were cultured and isolated as previously reported.26 (link)
Mononuclear cells were isolated by gradient centrifugation at 2500 rpm for 30 minutes on Ficoll-PaqueTM Plus (Amersham Pharmacia Biotech, Uppsala, Sweden). Mononuclear cells were then plated at a concentration of 10-30 × 103 cells/cm2 in Dulbecco’s Modified Eagle Medium (DMEM) containing 20% (v/v) of FBS. Non-adherent cells were removed 2 days later, and fresh medium was added. When the cultures reached 80-100% confluence, hBM-MSCs were trypsinized and subcultured. The purity of hBM-MSC suspensions was evaluated by flow cytometry using the following monoclonal antibodies: CD105-PE, CD45-FITC and CD90-PerCP, along with corresponding isotype controls (BD Biosciences, San Jose, CA, USA). Finally, the suspensions were analyzed utilizing a BD FACS CantoTM flow cytometry System (BD Biosciences, CA).