Mesenchymal Stem Cells Labeled With Superparamagenetic Iron Oxide Homing To Tumor Metastasis And In Vivo Visualization By 7.0T Magnetic Resonance Imaging

Part 1:The Isolation and Cultivation of MSCs from SD Rats and Labeling the Obtained MSCs with SPIOObjectiveTo obtain and expand bone marrow cells, and evaluate the feasibility of labeling MSCs with superparamegnetic iron oxide(SPIO) using liposome as transfection agent, and the iron content in per cell and cell viability was also detected after MSCs were labeled with different SPIO concentration.Methods1) Tibias and femurs of SD rats were flushed to isolate MSCs and MSCs were expanded in vitro. MSCs were incubated with SPIO for 6 hours to label MSCs, and liposome was used as transfection agent in the process;2) Transmission electron microscopy and prussion blue staining were used to detect iron within cells;3) Cell viability at different SPIO concentration (20 ug/ml,40 ug/ml, 80 ug/ml,120 ug/ml,160 ug/ml) was determined by CCK-8 kit;4) The iron content in per cell was determined by atomic absorption spectrometry(AAS).Results1) Prussion blue staining and electron microscopic imagings showed internalized SPIO nanoparticles in vitro cultured SPIO-MSCs;2) There was no significant difference found in SPIO-labeled MSCs at different SPIO concentrations through comparing OD values of different groups;3) When the SPIO concentration in cultured medium was 20 ug/ml, 40 ug/ml,80 ug/ml,120 ug/ml,160 ug/ml, the iron content of labeled MSCs was 3.8 pg/cell,6.6 pg/cell,13.7 pg/cell,18.5 pg/cell,23.7 pg/cell;4) The iron content of SPIO-labeled MSCs was 21.3 pg/cell,10.7 pg/cell,4.2 pg/cell,1.9 pg/cell,0.9 pg/cell,0.5 pg/cell at 1 day,3 days,5 days,7days,9 days,11 days after the MSCs were labeled at the SPIO concentration of 160 ug/ml.Conclusions1) MSCs can be successfully labeled through incubating SPIO with MSCs for 4 hours using liposome as transfection agent;2) SPIO-MSCs viability is not affected by SPIO;3) The iron content of SPIO-MSCs can be increased when the SPIO concentration in cultured medium is increased, and the iron content of SPIO-MSCs is decreased as the cells is splitting.Part 2:Application of 3.0T Magnetic Resonance Imaging in Motoring A Liver Hematogenous Metastasis Model Established by C6 Cells InjectionObjectiveTo establish liver hematogenous metastasis model in nude mice through injection of C6 glioma cells to hepatic portal vein and investigate the feasibility and accuracy of 3.0T magnetic resonance imaging(MRI) in detection of the liver metastasis model.MethodsHepatic portal veins of 36 nude mice were exposed by surgical operation. Then cultured C6 glioma cells were injected into hepatic portal veins of the 36 nude mice. Every time 12 nude mice were randomly sampled for magnetic resonance scanning at 7 days,14 days and 21 days. After scanning, these mice were sacrificed. All livers of these mice were collected for pathological examination, and immunohistochemical staining was used to assay the expression of S-100beta.ResultsC6 cells injected through portal vein successfully formed multifocal metastases in livers of nude mice. Hematoxylin-eosin staining results first showed the formation of metastases at 7 days after cells delivery. Morphologic changes and 3.0T MRI signal changes were observed at 14 days after cells injection. Pathological findings and MRI characteristics were more obvious at 21 days after the injection. Immunohistochemical staining of S-100beta revealed positive results in tissue slices. Metastases manifested high signal in MRI T2WI characteristics.3.0T MRI obtained more details about metastatic nodules and also showed edema around tumors.ConclusionsThe liver metastasis model can be successfully established by portal vein injection of C6 cells with high success rate and good repeatability. A clinical 3.0T MRI scanner can be used to detect formation and transform of liver metastases in this animal model. And T2WI is more sensitive than T1WI in metastasis detection.Part 3:MR imaging of SPIO-labeled MSCs migration towards liver metastasisObjectiveTo explore whether MSCs can be a potential vehicle for delivery of SPIO to tumor metastasis, and the SPIO gathering at tumor sites can be detected by 7.0T MRI, we established a liver hematogenous metastasis model in nude mice and labeled MSCs with SPIO in vitro in previous studies. In this part, we test this hypothesis.Methods1) 6 mouse model of liver metastasis were randomly divided into 2 groups, and then unlabeled-MSC(1×106 cell per mouse) and SPIO-labeled MSCs (1×106 cell per mouse) were injected into model mouse of two groups separately through tail veins;2) The 6 mice underwent 7.0T magnetic resonance scanning for livers to obtain T2WI and T2* value 9 days after injection of cells;3) Liver tissues from the 6 model mice were performed with frozen section, and prussian blue staining was used to confirm the SPIO-labeled MSCs.Results1) T2WI and T2* value(T2* relaxation time) of normal liver tissue in the unlabeled-MSCs group had no significant difference compared to the SPIO-MSCs group(P>0.05), and intension of T2WI and T2* value of tumor metastases in the SPIO-MSCs group was significantly decreased compared to the unlabeled-MSCs group(P<0.05);2) The result of prussion blue staining in the frozen section revealed the blue particles around tumor metastases.ConclusionsMSCs can be used as a potential vehicle for delivery of SPIO to tumor metastasis, and the SPIO gathering at the tumor sites can also be detected by 7.0T MR scanning causing the T2WI signal intension and T2* value changes.