The Purification and Culture of Canine Bone Marrow Mesenchymal Stem Cells in vitro with Nyco- prep~(? 1. 068

Objective The present study was aimed to investigate the appropriate and reliable methods for isolation, culture-expansion of canine bone marrow mesenchymal stem cells (MSCs). Methods The bone marrow mononuclear cells were isolated from 15 -20 ml canine bone marrow aspirates, by using density gradient centrifugation with 1.068 g/ml or 1.073 g/ml separating mediums. All cells were cultured at 37℃ under5% CO2 atmosphere in DMEM supplemented with 15% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 25 μg/ml amphotericin B. The medium was replaced every 2 or 3 days. The MSCs were identified by special cellular surface antigens and pluripotent differentiation potential. Results With the density gradient centrifugation method, the mononuclear cells were successfully isolated, and the Nycoprep 1. 068 group could obtain more purified monocytes. The adherent cells proliferated quickly at a doubling time of 24 hr. When the cells were confluent, the morphology of adherent cells was spindle-shaped, small and arranged like pectinate. The characteristics of proliferation and morphology did not change after 8 passages, and there was no evidence of spontaneous differentiation. The expanded adherent cells were positive for SH2, and negative for CD45 surface antigen. Under the induction of chemicals, the MSCs differentiated into cardiomyocytes, adipocytes and osteocytes. Conclusions The mesenchymal stem cells from canine bone marrow could be isolated, cultured and expanded in vitro through the density gradient centrifugation with Nycoprep?1.068 and adherent cell cultivation. These cells showed the characteristics of mesenchymal stem cells, with self-replenishing ability and pluripotent differentiation potential.