Figure 1.
Properties of contrast agents.
Schematic representation, transmission electron microscopy images and description of the contrast agents used in this study. Zeta potential (ζ) was assessed in 10 mM NaCl at a concentration of 100 µg[Fe]/ml.
Figure 2.
Intracellular iron content of murine mesenchymal stem cells as a function of labelling concentration.
Cells (5×104) were seeded in 24-well plates and labelled for 24 h by adding the contrast agents to the culture medium. Intracellular iron concentration was measured (A) quantitatively using the ferrozine assay (mean ± SD from three independent experiments) or (B) qualitatively using flow cytometry (representative data from three independent experiments). LR denotes labelling with loading reagent provided by the contrast agent manufacturer.
Table 1.
Standard set of conditions used for labelling cells.
Figure 3.
Confocal and electron microscopy images evidencing intracellular uptake and accumulation of contrast agents in lysosomes.
Cells were labelled with (A) Molday ION, (B) Feratrack or (C) Bangs Beads. Blue fluorescence corresponds to the nuclei, green to the contrast agents and red to LAMP2, scale bar represents 10 µm. Transmission electron microscopy was carried out without post-staining and electron dense (dark) areas correspond to the iron-oxide based contrast agents.
Figure 4.
Dissolution of contrast agents in an in vitro model of lysosomal microenvironment.
Contrast agents were allowed to digest in PBS containing 22(A) 7.2, (B) 5.5 or (C) 4.5. The relative dissolution is shown as a function of time for a period of 28 d. Data points represent mean ± SD from three independent experiments.
Figure 5.
The labelling efficiency, intracellular iron content and cell proliferation as a function of time.
Cells were labelled with the contrast agents for 24-well plates at a density of 5×104 cells/well. Green fluorescence of unlabelled cells (control) or cells labelled with (A) Molday ION, (B) Feratrack and (C) Bangs Beads was evaluated following labelling (day 0) and at day 3. In (D), the intracellular iron (ferrozine assay, left ordinate) and cell proliferation (trypan blue exclusion, right ordinate) was measured at 24 h intervals. Data points correspond to the mean ± SD from three independent experiments.
Figure 6.
Differentiation of cells into osteocytes and adipocytes.
Unlabelled (control) and labelled cells were induced to differentiate for 9 d with osteogenic or adipogenic media, after which cells were stained with Alizarin Red S to reveal calcium deposits or Oil Red O to reveal lipid droplets. Arrow indicates an adipocyte where the intracellular presence of labelling agent is clearly noticeable around the nucleus (zoomed view is shown in inset).
Figure 7.
Relaxation rate (ΔR2) of contrast agents (A) in solution or (B) internalised in cells.
Data points represent mean ± SD from three independent experiments and are displayed as a function of iron concentration. Lines represent the least square fit of the data points.
Table 2.
Transverse Relaxivity (r2) of contrast agents in solution or within cells. Values were obtained from the linear regression of the relaxation rate and are displayed as mean±SE from three independent measurements.
Figure 8.
Fluorescence and magnetic resonance imaging of cells implanted into the brain of a chick embryo.
Cells (5×104) expressing dTomato were labelled with (A) Molday ION, (B) Feratrack or (C) Bangs Beads and implanted into the midbrain of chick embryos at embryonic day 3. At embryonic day 5 the embryos were harvested from their eggs, imaged with a fluorescence stereomicroscope and fixed prior to MR imaging using a T2-weighed RARE sequence. Scale bars represent 1 mm. Numbers in fluorescence images indicate the position of viable (dTomato expressing) cells. The corresponding numbers in the transverse MR sections show the T2 shortening effect of the labelling agent at the same anatomical positions.