Cell uptake, differentiation and cytotoxicity. uptake, differentiation and cytotoxicity. Uptake of SPIONs was evaluated in a murine mesenchymal stem/stromal cell line (MSC D1, ATCC CRL-12424). Cells (MSCs) were grown in Dulbecco's Modified Eagle's Medium (DMEM, Sigma) supplemented with 10 % foetal calf serum (FCS, Gibco) and kept at 37 °C in a 104 humidified atmosphere with 5 % CO . In a typical labelling experiment, 5 × cells 2 were seeded in each well of a 24-well plate and, after 24h, the culture medium was replaced with 400 μL of fresh medium containing the SPIONs. Labelling was carried out for 24 hours, after which the cells were carefully washed with phosphate-buffered saline (PBS) to remove excess nanoparticles and then used for downstream measurements. For intracellular iron quantification, cells were trypsinised, counted, 104 and 5 × cells taken for measurement using a modified ferrozine based method as described.16 previously A typical standard curve obtained via this method if shown in Figure S11. For Prussian blue staining cells were fixed with 4% formaldehyde, stained with an iron stain kit (Sigma) according to the manufacturer's instructions and imaged with a Leica DM IL inverted microscope coupled to a DFC420C camera. Differentiation potential was assessed by inducing the cell’s differentiation immediately after labelling with SPIONs. For that purpose, the medium containing the SPIONs was replaced with medium containing adipogenic or osteogenic supplementation. Adipogenic supplementation consisted of 100 nM Dexamethasone, 10 mM β-glycerophosphate disodium salt hydrate and 77 µM 2-Phospho-L-ascorbic acid trisodium salt (all from Sigma), whereas osteogenic supplementation consisted of 100 nM Dexamethasone, 155 µM 2-phospho-L-ascorbic acid trisodium salt, 50 µM indomethacin and 175 nM bovine pancreas insulin (all from Sigma). Differentiation was induced for a total of 7 d with periodic medium changes. Effective differentiation was evaluated by fixation and histochemical staining with Oil Red O (0,5% in isopropanol, Sigma) Alizarin Red S (2%, pH 4.3, Sigma). Viability was measured via the quantification of ATP (CellTiter-Glo® Luminescent Cell Viability Assay, (104) Promega). Cells were seeded in each well of a 96-well plate and labelled as above using 100 µL of medium per well. After labelling, the SPION containing medium was replaced with 50 µL of fresh medium and then 25 µL of the ATP reagent was added to each well and the plate was mixed in an orbital shaker. After 10 minutes the contents of the plate were transferred to white, opaque, 96-well plates and the luminescence measured with a plate reader (Fluostar Omega, BMG Labtech). MRI. MR data was acquired with a Bruker 9.4 T Avance III HD instrument (Bruker, Coventry, UK) using a 40 mm transmit/receive quadrature volume coil. To obtain the relaxivity of the particles, those were embedded in 1 % low-melting temperature agarose (Sigma). For measurements of relaxivities in cells, cells were labelled as above, trypsinised, fixed and then embedded in agarose. In each experiment, the intracellular concentration of iron was calculated and the number of cells to obtain a given molar concentration of iron calculated. As particle distribution affects relaxivity, cells were labelled at an exposure dose that yielded comparable intracellular mL-1 mL-1 concentrations (50 µg for particles of 6.7 nm, 3 µg for particles of 8.4 nm mL-1 and 2.5 µg for particles of 9.9 nm) thus requiring equivalent number of cells. Under these labelling conditions cell uptake is ~ 4 pg[Fe]/cell requiring e.g. ~ 2.8 × 105 cells suspended in 100 µL to obtain 0.25 mM of Fe. The T relaxation times of the 2 samples was obtained using a Multi Slice Multi Echo (MSME) spin echo sequence with 25 echoes, an inter-echo time of 8 ms and repetition time of 3577 ms from which relaxation maps were generated with Paravision 6.0 (Bruker). For T * relaxation maps, 2 a multi gradient echo (MGE) sequence with 8 echoes, an inter-echo time of 5.5 ms and a TR of 800 ms was used. For imaging, cells were trypsinised, fixed, resuspended in 40 μL of 1% low-melting temperature agarose and loaded into 100 μL polypropylene tubes between two layers of agarose containing no cells. The tubes were then mounted