| PART ONE:Labeling of rat bone marrow mesenchymal stem cells with superparamagnetic iron oxideObjectives:To label rat bone marrow mesenchymal stem cells (MSCs) with superparamagnetic iron oxide nanoparticles (SPIO) and DiR, and to determine the appropriate labeling method of stem cells labeledwith SPIO and DiR.Methods:Rat mesenchymal stem cells were isolated, culture expanded3passages, and identified by Fluorescence-activated Cell Sorting (FACS) in vitro. MSCs were magnetically labeled with different concentration of SPIO in culture medium for24h, so as to determine the appropriate labeling methods according to both cellular iron incorporation and cell toxity.Then, MSCs were labeled with5μg/ml of DiR for20min. Labeling effciency was assessed by fluorescence microscope.Results:Labeling MSCs with25μg/ml of SPIO in culture medium for24hours is appropriate. Intracytoplasmic particles stained with Prussian blue stain were detected for all cells. The containing iron dense granular vesicles in cytoplasm were observed by electron microscopy and the ultrastructure of MSCs had not to be destroyed. Labeling MSCs with5μg/ml of DiR and4’-6-diamidino-2phenylindole (DAPI), fluorescence microscope showed that the labeling effciency of DiR was closed to100%.Conclusions:Rat bone marrow mesenchymal stem cells can be efficiently and safely labeled with SPIO and DiR.PART TWO:The characteristics of permanent magnet and magnetic responsiveness of magnetically labeled MSCs in vitro.Objectives:To clarify characteristics of the cylinder permanent magnet,and to determine magnetic responsiveness of static and flowing magnetically labeled MSCs in vitro.Methods:The magnetic characteristics were analysed by the magnetic field physics. Magnetically labeled MSCs in culture flasks were exposure to a0.21Tesla (T) magnetic field for24h. The magnetic responsiveness of static MSCs was observed by magnetic resonance imaging and inverted phase contrast microscope. In order to simulate blood flow state,1×106magnetic MSCs in turn flowed through a quartz tube served as a model of blood vessels in the0.15T,0.3T and0.6T magnetic field. The capture efficiency (CE) were respectively calculated to evaluate the magnetic responsiveness of The flowing MSCs. The0.6T magnet was placed tightly at the mid-segment of the tube,and cell suspension circulated at flows of4mm/s,20mm/s,100mm/s and500mm/s to mimic circulatory conditions in animals and human beings,observing the effects of the fluid velocity on accumulation of magnetic MSCs.Results:According to the physics analysis, the magnetic field of cylindrical permanent magnet symmetrically distribute along Z axis. When the radius is constant, The magnetic flux density reduce dramatically with the increase of Z axis. When the Z axis is constant, the magnetic flux density escalate significantly from center to peripheral.The static magnetically labeled MSCs in the magnetic field redistributed to form brown ring. Magnetic resonance ring detected the ring signal deletion.The0.15T,0.3T and0.6T magnets showed44.67%±5.5%,68%±3%and80.3%±1.5%of the capture efficiencies respectively (all p<0.01). The capture efficiency (CE) was85.0%±3.6%,68.7%±4.7%,11.3%±4.5%and1.6%±0.75%when the flow velocity was4,20,100and500mm/s, respectively (all P<0.01).The capture efficiency was negatively related to the average velocity (R=-0.770,P=0.003).Conclusions:The cylinder permanent magnet exhibit the important characteristics,which are the horizontal axis polarization and the vertical axis attenuation.The magnetically labeled MSCs have a strong ability to magnetically response to the magnetic field,which can be effectively attracted. The capture efficiency was positively related to the magnetic flux density and negatively related to the average velocity.PART THREE:Comparison of magnetic intensities for mesenchymal stem cell-targeting therapy on ischemic myocardial repair:high magnetic intensity improves cell retention but has no additional functional benefit Objectives:To investigate the effect of magnetic targeting intensities on cell transplantation, we used different magnetic targeting intensities for mesenchymal stem cells therapy in a rat model of ischemia/reperfusion.Methods:Rat mesenchymal stem cells were labeled with superparamagnetic iron oxide nanoparticles. The rats model of ischemia/reperfusion were randomized into five groups to receive, during brief aortic and pulmonary occlusion, direct intraventricular injection of PBS (n=25) or magnetically labeled MSCs, with0.15T (n=25),0.3T (n=25),0.6T (n=25) or without (n=25) magnets0-1mm above the injured myocardium during and after the injection of1×106MSCs. The cardiac retention of transplanted cells was assessed using quantitative Y chromosome-specific PCR and optical imaging. Cardiac function was measured using echocardiography, and histological analyses of infarct morphology and angiogenesis were obtained.Results:Magnetic targeting enhanced cell retention after24hours and did so in a magnetic field strength-dependent manner [(3.75%±0.44%) in0.1T group vs.(7.13%±0.28%) in0.3T group,vs.(9.95%±0.53%) in0.6T group, all P<0.001].Compared with the0T group, three magnetic targeting groups enhanced varying cell engraftment at3weeks(all P<0.001), at which time left ventricular (LV) remodeling was attenuated; the therapeutic benefit (LV ejection fraction) was also higher in the0.15T and0.3T groups. Moreover, the0.3T group demonstrated the most cell engraftment and the greatest therapeutic benefit. Interestingly, due to micro-vascular embolisms, the0.6T group failed to translate into additional therapeutic outcomes, though it had the highest cell retention.Conclusions:Magnetic targeting enhances cell retention in a magnetic field strength-dependent manner. However, too high of a magnetic intensity may increase micro-embolization and undermine the functional benefits of cell transplantation.PART FOUR:Magnetic targeting enhances retrograde cell retention in a rat model of myocardial infarctionObjectives:The aim was to investigate the effect of magnetic targeting on retrograde coronary venous cells delivery in a rat model of myocardial infarction.Methods:Rat mesenchymal stem cells were labeled with superparamagnetic iron oxide nanoparticles. In a Sprague-Dawley rat model of acute myocardial infarction, 1×106magnetic mesenchymal stem cells were transjugularly injected into the left cardiac vein, while a0.6T magnet was placed above the heart. The cardiac retention of transplanted cells was assessed using quantitative Y chromosome-specific PCR, cardiac magnetic resonance imaging and optical imaging. Cardiac function was measured using echocardiography, and histological analyses of infarct morphology and angiogenesis were obtained.Results:Twenty-four hours after cellular retrocoronary delivery, magnetic targeting significantly increased cardiac retention of transplanted cells by2.73-2.87fold (P<0.001). Histological analyses showed that more transplanted cells were distributed in the anterior wall of left ventricular. The enhanced cell engraftment persisted for at least3weeks (P<0.001), at which time left ventricular remodeling was attenuated, and functional benefit was improved.Conclusions:These results suggest that magnetic targeting offers new perspectives for the retrograde coronary venous delivery to enhance cell retention and subsequent functional benefit in heart diseases. |
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