AlloStem® is comprised of adipose-derived mesenchymal stem cells (MSCs) combined with a partially demineralized cancellous bone matrix (DCBM). DCBM constitutes an effective substrate for selection and retention of MSCs derived from the stromal vascular fraction (SVF) of adipose tissue. The bone graft contains three key elements for bone regeneration: an osteoconductive, natural scaffold, growth factors mediating osteoinductive signals and osteogenic stem cells.
Recovered adipose tissue (2000 ml) was washed in PBS and digested with collagenase type I. After centrifugation at 500xg at room temperature, mature adipocytes in the supernatant were discarded and the pellet was identified as the SVF. The cells were seeded in a culture flask, at a density of 3x105 cells/cm2, to establish the primary culture.
Cancellous bone matrices were recovered from human donors and sectioned into the desired shape (e.g. cubes). Matrices were then placed in a demineralization solution and rinsed until the pH was neutral. The matrices were then stored in DMEM/F12 supplemented with FBS (10% (v/v)). Demineralized bone cubes were seeded with 1x106 cells from the SVF and then incubated at 37°C with 5% CO2 and 100% humidity for 36 hours. Grafts were then washed thoroughly with PBS to remove nonadherent cells. AlloStem® allografts were placed into freezing medium containing FCS (90%) and DMSO (10%) and frozen at - 80C.