Canine mesenchymal stem cells isolation from bone marrow aspirates.

Bone marrow mesenchymal stem cells are an integral part of the hemopoetic microenvironment. Due to the rarity of these cells in the bone marrow and the lack of definitive markers that specifically isolate MSCs these cells are commonly isolated by adherence to plastic and consecutive passage in tissue culture. The aim of the present paper was to evaluate three bone marrow harvesting sites: the proximal humerus, proximal femur and the wing of the ilium and two isolation and cultivation media containing α-MEM and DMEM for mesenchymal cell culture in dogs. 3 ml of bone marrow were harvested from each puncture site by aspiration in flushing medium containing syringes for a total of 9 ml/dog. Two batches were established, batch number I using α-MEM cell culture medium and batch number II with DMEM culture medium. The most efficient bone marrow puncture and harvesting sites were the humeral one, followed by the femoral one. Comparing the two culture media used we have determined that α-MEM medium had a higher efficiency rate than DMEM upon cell plastic adherence, viability and proliferation capacities, detecting distinct mesenchymal cell colonies at 6 days of culture with homogenous fibroblast-like morphology and reaching 70% confluence at 7 days (passage 1). Cell viability after thawing was 70% for the first batch and 65% for the second one