Rat mesenchymal stem cell culture
StemPro® Rat Alk Phos Expressing MSCs were purchased from ThermoFisher Scientific (cat. no. R7789120) and cultured in α-Minimum Essential Medium, with nucleosides and GlutaMAX™ (ThermoFisher Scientific, cat. no. 32571), supplemented with 10% fetal bovine serum (ThermoFisher Scientific, cat. no. 10270) and 1% penicillin-streptomycin solution 100× (Euroclone, cat. no. ECB3001D). MSCs were isolated from bone marrow of transgenic Fischer 344 rats expressing the human placental alkaline phosphatase (hPAP) gene. The medium was changed every third day and MSCs were maintained at 37 °C, 5% CO2.
SPIO labelling and 19F labelling
MSCs (passage 5) were treated with trypsin-EDTA (cat. no. Euroclone, ECB3052D) and centrifuged at 300 g for 5 min. MSCs count (expressed as number of cells/mL) and cell viability (calculated as number of viable cells/number of dead cells + number of viable cells and expressed as %) were evaluated with the trypan blue exclusion method. 2 × 106 viable MSCs were seeded in a tissue culture flask (75 cm2) for labelling with Molday ION Rhodamine-B (MIRB, BioPal Inc, Worcester, MA, USA), SPIO nanoparticles conjugated with Rhodamine B, that can be visualised by fluorescent imaging. MIRB has a colloidal size of 35 nm, a zeta potential of ~ +31 mV and an iron concentration of 2 mg/mL. MIRB was added to MSC culture at a concentration of 50 μg/mL in 6 mL culture medium for 24 h at 37 °C. 2 × 106 MSCs were labelled with Cell Sense (CS-ATM DM Green), a PFC emulsion conjugated with a green fluorescent dye, commercially obtained from Celsense Inc. (Pittsburgh, PA, USA). Cell Sense has a total fluorine content of 120 mg/mL. MSCs were incubated with Cell Sense at a concentration of 10 mg/mL in 6 mL of culture medium for 24 h at 37 °C. Unlabelled control MSCs (2 × 106 cells) were incubated in 6 mL of culture medium for 24 h at 37 °C. After incubation, the culture medium was aspirated and all the MSCs cultures were washed twice with phosphate-buffered saline (PBS) to remove extracellular labelling agents. MIRB-MSCs, Cell Sense-MSCs and control MSCs were then treated with trypsin-EDTA and centrifuged at 300 g for 5 min; cell count and cell viability were evaluated with the trypan blue exclusion method. 104 labelled cells (MIRB-MSCs and Cell Sense-MSCs) were resuspended in culture medium and seeded on a chamber slide. The culture medium was aspirated, cells were washed twice with PBS and fixed with 4% paraformaldehyde (PFA) for 15 min and stored at 4 °C. Cells were permeabilised with PBS-Triton 0,1% for 10 min and nuclei were stained with DAPI (Thermo Fisher Scientific, cat. no. D1306).
Images were acquired by confocal microscopy (Eclipse TE2000-E microscope equipped with EZ-C1 scan-head; Nikon) using a 20× (NA 0.85) objective to evaluate labelling efficiency; structured illumination microscopy (SIM) with 100× Apo-TIRF (NA 1.49) objective (Nikon) was used to assess MIRB and Cell Sense cytoplasmic distribution. SIM achieves a lateral resolution of 100–130 nm [14].
The number of labelled MSCs (visualised by green fluorescence for Cell Sense-labelled MSCs and by red fluorescence for MIRB-labelled MSCs) and the number of analysed cells (number of cell nuclei, blue stained by DAPI) were evaluated using the ImageJ Software Cell-Counter plug-in. The percentage of MIRB-labelled and Cell Sense-labelled MSCs was then calculated.
Cell phantom preparation
Labelled MSCs were fixed in 4% paraformaldehyde and divided into four samples containing increasing numbers of cells (0.125 × 106, 0.25 × 106, 0.5 × 106, 1 × 106). MSC samples were prepared by centrifugation at 300 g for 5 min and each microcentrifugation tube was filled up with low-melting 1% agarose (Bio-Rad, 162–0017). Cell phantoms of unlabelled cells and a positive control with MIRB and Cell Sense at the same concentrations used for cell labelling were prepared.
MSCs were expanded and labelled in culture medium at physiological pH. Before MRI/MRS analysis, cells were fixed in 4% PFA solution pH 7.4 and washed in PBS pH 7.4; therefore, the pH was always maintained at physiological level.
MRI/MRS characteristics and features
The acquisitions were performed on a horizontal 7T MRI scanner (Bruker BioSpec 70/30, Ettlingen, Germany) equipped with a gradient system reaching a maximum amplitude of 440 mT/m. Both MRI for MIRB-labelled cells and MRS for Cell Sense-labelled cells were acquired by a double nuclei (1H/19F) volume resonator with an inner diameter of 72 mm.
Since contrast agents relaxivity depends on extrinsic factors such as applied field and temperature, all MRI acquisition were performed at the magnet bore temperature, strictly maintained constant at 18 °C.
T1, T2 and T2* MRI maps were acquired with the same geometry (one 1-mm coronal slice including the cells pellet) on MIRB-labelled cells, the MIRB phantom and cell negative control. T2-weighted images with 0.7-mm slices in axial, sagittal and coronal geometries (with respect to the magnet reference frame) were also acquired to perform relaxometry maps reference images by visualising the hypo-intensity corresponding to the labelled cells pellets.
T1 mapping was based on a rapid acquisition with relaxation enhancement (RARE) sequence with a RARE factor of 2; 28 images, with a different repetition time (TR) in the range of 25–15,000 ms, were acquired; echo time (TE) was set to 4.6 ms, in-plane resolution was 313 × 313 μm2, the slice thickness was 1 mm, the number of excitations was 2; the total acquisition time was 14 min. A region of interest (ROI) was then selected corresponding to the sample signal and signal intensity (SI) plotted versus the image TR. The curve was fitted by the function
$$ S I= A+ C\left(1- \exp -\frac{ T R}{T_1}\right) $$
where A is the absolute bias and C the maximal SI.
T2 mapping was based on a multi-slice multi-echo (MSME) sequence with a series of 200 echo images a with different TE (range 5–937.5 ms); TR was set to 5000 ms, in-plane resolution was 313 × 313 μm2, the slice thickness was 1 mm, the number of excitations was 28; the total acquisition time was 13 h. A ROI was then selected corresponding to the sample signal and SI was plotted versus the image TE. The curve was fitted by the function
$$ S I= A+ C\left( \exp -\frac{ T E}{T_2}\right) $$
where A is the absolute bias and C the maximal SI.
T2* mapping is based on a multiple gradient echo (MGE) sequence with a series of 500 echo images with a different TE (range of 2–807 ms); TR is set to 5000 ms, in-plane resolution was 313 × 313 μm2, the slice thickness was 1 mm, the number of excitations was 5; the total acquisition time was 50 min. A ROI was selected corresponding to the sample signal and its SI was plotted versus the image TE. The fitting was performed by the T2 maps analysis function.
All curves fittings were performed by the built-in least squares minimisation Levenberg-Marquardt routines of OriginLab (Northampton, MA, USA, http://www.originlab.com/) software. Reported errors are the fit’s standard deviations.
A T2-weighted RARE sequence was performed with the following parameters: Hermite pulse centred at 300 MHz with 2 kHz bandwidth, TR = 3100 ms, TE = 54 ms, flip angle (FA) = 90°, slice thickness = 0.7 mm, in-plane resolution = 156 μm × 156 μm2. The total acquisition time was 1 min 36 s.
Non-localised single-pulse 19F-MRS sequences (Block pulse centered at 209 MHz with 14 kHz bandwidth, FA 90°, TR 20 s, 100 excitations, total acquisition time 33 min) were acquired on Cell Sense-labelled cells, Cell Sense phantom and cell negative control to ensure the detectability of each sample. For each sample, two acquisition sessions were performed one week apart.
