Study In Vitro Of Human Bone Marrow-derived Mesenchymal Stem Cells Labeled With 5-Bromodeoxyuridine

AimTo establish the methods of culture,expansion and identification of adult bone marrow mesenchymal stem cells(MSCs).To explore the best time and best dose for labeling the MSCs with bromodeoxyuridine(BrdU)in vitro,so as to attain the best tracing guide line of applying MSCs to future research.Methods10mL bone marrow was collected from the healthy volunteer.mononuclear cell were obtained by density gradient centrifugation with Percoll solution.,MSCs Were cultured purified,and passaged by sticking plastic bottle repeatedly,with inverted phase contrast microscope observe MSCs shape.Flow cytometry(FCM) Identificate CD44,CD71,CD34,HLA-DR.Chose passage one,three,five of the MSCs,traced them with BrdU(10μmol/L).chose passage three of the MSCs, traced them with BrdU in different dose(5,10 and 15μmol/L)and time(12,24, 48,72 and 96 h),and then Immunocytochemistry was performed to detect the expression of BrdU,so as to know whether our labeling was successful and calculate the labeling index(LI).To observe the morphous of labeled cells after serial passage. ResultsThe Human marrow MSCs harvested were homogenous population and exhibited a spindle-shaped fibroblastic morphology.Flow cytometry showed that MSCs expressed CD44 and CD71 but not expressed CD34 and HLA-DR.Most MSCs labeled by BrdU.The effect of MSCs labeled with BrdU was steady among passage cells.Incubation of the MSCs with BrdU 10μmol/L and 72h appeared to achieve the highest LI,both of which exceeded 90%.BrdU has little effect on morphous and proliferation of labeled cells after serial passage.ConclusionThe combination of density gradient centrifugation and adherent culture and digestion control is an ideal method for culture and purify MSCs in vitro;BrdU labeling provides a feasible means for a dynamic observation of the survival, growth and differentiation of the implanted MSCs.the incubation of which with 10μmol/L BrdU for 72h is the optimal dosage and timing.