Superparamagnetic Iron Oxide Labeled Mesenchymal Stem Cells And Magnetic Resonance Imaged In Vitro

OBJECTIVE: 1,To study the patterns of MR signal intensity (SI) of intracellular and extracellular superparamagnetic iron oxide (SPIO),and to determine the most optimal protocol of magnetic resonance imaging. 2,To explore the optimal factor for labeling bone mesenchymal stem cells (BMSCs) with SPIO mediated by poly-L-lysine (PLL).3,To study the changes of intracellular iron particles in BMSCs labeled with SPIO in vitro following their passaging by using magnetic resonance imaging.METHODES: 1,MR imaging was performed on the Eppendoff tubes full-filled with SPIO of different concentrations (concentrations of iron from 0μg/ml to 400μg/ml) with fast spin echo (FSE) T₁ weighted, fast spoiled gradient echo (FSPGR) T₁ weighted, FSE T₂ weighted and fast imaging employing steady-state acquisition ( FIESTA ) T₂* weighted imaging. The SI of all the tubes were measured, and the signal intensity rate (SIR) were calculated. then the Lines and Geoglyphs of the SIR were drew,and analyzed its trend of SIR at different concentrations. 2,BMSCs were incubated with SPIO–PLL complexes at the concentrations of 0μg, 4.2μg, 8.4μg, 21μg, 42μg and 84μg Fe per ml medium and with PLL at the concentrations of 0μg, 0.05μg, 0.25μg, 0.5μg, 1.0μg and 5.0μg PLL per ml medium, respectively. The concentration of BMSCs labeled was 1.0x105/ml. The labeling ratio and distribution of SPIO particles in BMSCs, and the morphology of the cells were evaluated on electron microscopy and fluorescent microscope by Prussian blue staining. Viability of the labeled BMSCs was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) assay and trypan blue dye exclusion test. Magnetic resonance imaging was performed to all of the labeled BMSCs groups. 3, Five generation rows of the BMSCs labeled were passaged, the labeling ratio and SIR on gradient-echo T₂* weighted imagings of passage 1 to 5 were measured at the concentration of 1.0x105cells/ml.CONCLUDE:1,The SI of the Eppendoff tubes became increased first and then decreased following the increasing concentrations of SPIO on T₁ weighted, while the SI became constant decreased on both T₂ and T₂* weighted images. The SI on GRE images was higher than that on FSE images (P <0.05). At the concentration of 0.5μg/ml, the SIR on FSE-T₁, FSPGR-T₁, FSE-T₂ and FIESTA-T₂* weighted images were 29.04%, 26.23%,-0.54%, and -30.01%, respectively. 2, SPIO particles were distributed in the cytoplasm and lysosome of the BMSCs. There is significant correlation between the cellular labeling ratio and the concentrations of SPIO (r=0.7636, P <0.05). BMSCs labeled with SPIO had similar viability and proliferation profiles at the concentrations from 0μg to 42μg Fe per ml medium, except at the concentration of 84μg /ml (P <0.05). The PLL can not affect the cellular viability at its concentrations from 0μg to 1.0μg PLL per ml medium (with 92.2%～96.4% of active cells), except at the concentration of 5.0μg PLL per ml medium (with 80.8% of active cells). The SIR on T₂ and T₂* weighted were greater than that on T₁ weighted images (P <0.05), especially on T₂* weighted images. On T₂* weighted images there is significant negative correlation between the SI and the concentrations of SPIO used for labeling in medium (r=-0.7401, P <0.05). 3,The labeling ratios were, respectively, 100%, 98%, 63%, 11% and 2%, and the SIR on T₂* weighted images were -49.13%, -52.55%, -39.5%, -11.65% and -1.13% at the passage 1 to 5, respectively. There is significant negative correlation between SIR and the number of iron particles in the cells(r=-0.9866, P <0.005). Both the labeling ratio and the SI became decrease following the increase of the cellular passaging.CONCLUSION: 1, The relaxation rate was different in intracellular SPIO from extracellular SPIO. The T₁ and T₂* effects were dominant in extracellular SPIO, while the T₂ and T₂* effects were dominant in intracellular SPIO and at the high concentration of SPIO. 2, T₂* weighted was the sensitive and specific sequence for detecting SPIO. 3, SPIO can label BMSCs easily and efficiently mediated by PLL without interference on the cellular viability and proliferation. 4, The iron particles in the BMSCs labeled with SPIO in vitro can be passaged to the offspring cells, and can be monitored in a certain period of time with MR imaging.