Magnetic Labeling Of Rat Bone Marrow Mesenchymal Stem Cells And In Vivo MR Imaging For Tracking The Labeled Cells Transplanted Into Liver

Part IIsolation, cultivation and biologic characteristics of rat bone morrow-derived mesenchymal stem cellsObjective To establish a method for isolation and in vitro proliferation of mesenchymal stem cells (MSCs) from rat bone marrow, and to explore the biologic characteristics of MSCs, preparing for the application of MSCs in the following study. Methods The bone marrow cells were collected from the bilateral thighbones and tibias of Wistar rats. After centrifugation at 1500 r/min for 5 minutes, the cell pellet was resuspended in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum. The cultures were maintained at 37° C in air of 5% CO₂. Cells were cultured and purified by using plastic adhesion method, with an initial media changing 3 days after inoculation and then media changing every 2 days. The morphology of MSCs at different time was observed with a phase-contrast microscopy. For the 1, 3, 5 passage MSCs, the growth curves were drawn and the adhesive rates were measured. All data were expressed as X|-±S. An analysis of variance was used to test the difference of the cell numbers or adhesion rates among three passages. Results (1) The primitive MSCs grow slowly with some colonies adhered to the culture plastics. The distribution was heterrogenous, with shuttle-shaped, triangle-shaped, or star-shaped morphology. Thereafter, the colonies of MSCs gradually fused to each other, and the morphology tended to be heamogenous. By 12-14 day, the cells had conflenced to about 100 per cent. Compared with the primary MSCs, the passage MSCs proliferated rapidly, with a more hemogenous morphology and distribution. (2) The growth curve showed that the passage cells started to proliferate rapidly by the 3rd day after passaging. The time for cell duplication was 28.1 hours in mean, and the passaging individual was about 4 to 6 days. The statistic analysis implied no significance between the cell numbers at different times. (3) For passage MSCs, the ability of adhesion to the plastics enhanced significantly, with an adhesive rate of approximately 60% by 4 h, and above 90% by 10 h. There was no statistically significance between the adhesion rate for the P₁, P₃, and P₅ cells. Conclusion (1) MSCs can be isolated and purified by plastic adhesion method. (2) DMEM/F12 supplemented With 10% fetal bovine serum suits to in vitro cultivation and expansion of MSCs. (3) MSCs have a powerful capacity to proliferate and to adhere to the plastics, and have a homogenous morphology, with a fibroblast-like shape. Passaging does not affect the growth and proliferation characteristics of cells. MSCs can be used as an idial cell source for the following study.Part II Magnetic labeling of rat bone marrow mesenchymal stem cells with superparamagnetic iron oxides nanoparticlesObjective To explore the feasibility of magnetic labeling of mesenchymal stem cells (MSCs) with superparamagnetic iron oxides nanoparticles, and to raise the magnetic labeling efficiency. Methods Feridex was mixed with poly-left-lysine (PLL) to obtain a complex of feridex-PLL, in which the ultimate concentration of feridex and PLL was 250 ug/ml and 7.5 ug/ml respectively. The culture media containing feridex-PLL of different concentration were added to the 4^th passage MSCs such that the ultimate concentration of feridex was 150 ug/ml, 100 ug/ml, 50 ug/ml, 25 ug/ml, 10 ug/ml, and 5 ug/ml respectively. After incubation overnight, a Prussian blue staining for demonstrating intracytoplastic nanoparticles, atomic absorption spectrophotometry for iron content, and trypan blue exclusion test for cell viability were performed at 24 h, 1w, 2 w, 3 w, 4 w after labeling respectively. Data were expressed as the mean plus or minus standard deviation (X|- ±S) and an analysis of variance was used to determine the significant differences. Results Numerous intracytoplastic iron particles were stained with Prussian blue in the feridex-PLL lebeled cells, with the labeling efficiency about 100 per cent. A limited staining was found in the control cells labeled with feridex alone. The iron content for feridex-PLL labeling group was higher than for the control group (p<0.05). In the feridex-PLL groups, the 25ug/ml group had an obviously higher iron content than the 10ug/ml or below groups (P<0.05) , but not lower than the 50ug/ml or above groups. With division of stem cells, the stained particles were seen decreased gradually. Trypan blue exclusion test showed that the viability of the cells labeled with 100 ug/ml or below concentration were not significantly impaired at different time points compared with that of nonlabeled cells. Conclusion (1) It is feasible and practical to lebel MSCs with feridex-PLL complex. (2) The iron concentration of 25 ug/ml, not only efficient, but also safe, can be considered an optimal dose for cell labeling. (3) With division of the labeled cells, the intracyte iron can be transferred to the next passage cells. Part IIIIn vitro MR imaging of magnetically labeled rat mesenchymal stem cellsObjective To investigate the possibility of in vitro detection of magnetically labeled MSCs with a clinical 1.5 T MR, and to find an optimal sequence and parameter for MR imaging of labeled cells, preparing for the following in vivo study. Methods (1) 1×10³, 1×10⁴, and 1×10⁵ cells were respectively suspended in 0.5 ml gelatin (10%) for MR imaging. (2) 1×10⁵ cells were collected at 24 h, 1 w, 2 w, 3 w, 4 w after labeling respectively. (3) MR imaging of cell suspensions in gelatin was performed at a 1.5 T MR imaging system with a head coil, by using a SE sequence (T1WI: TR/TE, 500 msec/15 msec; T2W1: TR/TE, 4000 msec/100 msec)and a GRE sequence (T2*WI: TR/TE, 300 msec/20 msec, flip angle, 15°). All images were obtained with a section thickness of 3 mm, a matrix size of 256×160, and a 6～8 cm × 6～8 cm field of view. The signal changing was analyzed according to the flowing equation: ΔSI= (( SI_l-SI_u) /SI_u)× 100% , where ΔSI was signal changing rate, SI_l was signal intensity of labeled cells, and SIu signal intensity of unlabeled cells. (4) The iron content of the cells at different time was assayed with an atomic absorption spectrophotometer. (5) All data were expressed as X ±S. An analysis of variance was used to determine the differences of signal intensity changing for three sequences, and a linear regression analysis was applied to test the relationship between the iron contents and T2*WI signal changing rates. Results (1) The signal intensity changing rate of 1×10³, 1×10⁴, 1×10⁵ labeled cells was (-67.93±2.94) % , (-78.77±1.37) % , (-88.73±1.12) % respectively on T2WI images, and (-72.50±5.96)%,(- 82.90±2.72)%, (-92.60±1.01)% respectively on T2*WI images, both were higher than that of (-4.93±1.99) % , (-9.90±1.95)% and (-5.87±3.05) respectively on T1WI images(p<0.05). (2) At 24 h, 1 w,2w,3 w,4 w after labeling, the T2* signal intensity decreasing rate of 1×10⁵ labeled cells was (3.37±1.12)%, (65.52±0.92)%, (30.63±1.27)%, (13.63±0.96)%, (5.60±0.10) % respectively, very liniealy correlated with the iron content of 1.87±0.35 ug, 0.81±0.09 ug, 0.40±0.02 ug, 0.19±0.02 ug, 0.08±0.01 ug (r=0.94). Conclusion (1) The MSCs labeled with feridex-PLL complex can give rise to a obvious T2 signal decreasing, which can be detected by a clinical 1.5 T MR imaging system. (2) GRE T2*WI is the most sensitive sequence to detect the signal intensity changing of labeled cells. (3) The T2 signal intensity changing correlated with the cell number. With the cell number increasing, the signal decreases more obviously. (4) The degree of T2 signal intensity decreasing fades with the cells proliferating.Part IVIn vivo MR imaging of magnetically labeled mesenchymal stem cellstransplanted into rat liverObjective To evaluate in vivo magnetic resonance imaging with a clinical 1.5 T MR imaging system for depiction and trafficking of the magnetically labeled MSCs transplanted into the liver through portal vein injection. Methods To establish the acute liver necrosis, 2 % carbon tetrachloride was administrated orally to 30 Wistar rats, 20 of which were included in the experimental group, and 10 in the control group. MSCs were simultaneously labeled with feridex-PLL and DAPI. The cells transplantation was performed by injection of 1×10⁶ duplicaly labeled MSCs in 0.5 ml DMEM medium via portal vein, and the control group received an injection of the same quantity of unlabeled cells via the same vascular. MR imaging was performed with a clinical 1.5 T MR imager immediately before and at 1 h, 3 d, 7 d, 14 d respectively after transplantation. MR imaging was performed with GRE T2*WI (TR/TE, 300 msec/20 msec, flip angle, 15°). The signal changing was expressed as signal to noise ratio (SNR). After MR examination, the animals were sacrificed, and the liver, kidney, lung and muscle were prepared for fluorescence observation and Prussian staining. All data were expressed as X|-±S, and an analysis of variance was applied to determine the differences of SNR at different time. Results The SNR for liver was 1.10±0.26 at 1 hour, 8.18±1.55 at 3 day, 11.08±1.30 at 7 day, and 14.15±1.02 at 14 day after transplantation. Within 7 days after transplantation of labeled cells, the SNRs for liver were obviously lower than that before transplantation (P<0.05). For the kidney, muscle in experimental group, as well as for the liver, kidney, and muscle in control group, there was no significant difference between the SNRs after and before transplantation. For experimental group, abundance of blue fluorescent particles under the fluorescence microscopy was detected, which faded over time. Prussian staining showed the distribution of iron particles at different time matched to that found under the fluorescent microscopy. Both the iron staining and the fluorescence displaying had a good correspondence with the SNR changing on MR images at different time. Conclusion (1) 1.5 T clinical MR can be used to detect in vivo the magnetic labeled MSCs, which give rise to an obvious signal changing. The cells can be tracked for up to 7 days. (2) The MR signal changing seems to fade over time. (3) The degree of MR signal changing has a good correspondence with both the iron staining and the fluorescence displaying.