Liver-derived mesenchymal stem cells (MSCs) are obtained from the washout of the graft preservation solution (perfusate) collected at the time of liver transplantation. The perfusate contains high counts of mononuclear cells that detach from the liver, and include lymphocytes, natural killer cells, antigen-presenting cells, hematopoietic stem cells and a small fraction of cells double-positive for the MSC surface markers CD90+CD105+ (0.09%) and CD90+CD166+(0.02%).
MSCs were expanded and passaged for several months under normal nonhypoxic culture conditions. Expanded cells at passages 4 to 9 revealed a surface marker profile typical for MSCs. Functional analysis of the liver-derived MSCs demonstrated a multilineage potential with a capacity for adipogenic and osteogenic differentiation that was similar to the capacity of BM-MSCs.
End-stage liver disease tissue samples were obtained from the explanted livers of liver transplant recipients.
Liver graft preservation fluids (perfusates) were collected from human liver grafts at the time of transplantatio. Mononuclear cells from perfusates were isolated within 12 hours by density gradient centrifugation using Ficoll Paque Plus and cultured for the presence of MSCs. Cells were cultured in DMEM supplemented with FBS (10%), penicillin (100 IU/mL), and streptomycin (100 IU/mL).