Mesenchymal stromal cell labeling by new uncoated superparamagnetic maghemite nanoparticles in comparison with commercial Resovist--an initial in vitro study

Abstract

Objective: Cell therapies have emerged as a promising approach in medicine. The basis of each therapy is the injection of 1-100×10(6) cells with regenerative potential into some part of the body. Mesenchymal stromal cells (MSCs) are the most used cell type in the cell therapy nowadays, but no gold standard for the labeling of the MSCs for magnetic resonance imaging (MRI) is available yet. This work evaluates our newly synthesized uncoated superparamagnetic maghemite nanoparticles (surface-active maghemite nanoparticles - SAMNs) as an MRI contrast intracellular probe usable in a clinical 1.5 T MRI system.

Methods: MSCs from rat and human donors were isolated, and then incubated at different concentrations (10-200 μg/mL) of SAMN maghemite nanoparticles for 48 hours. Viability, proliferation, and nanoparticle uptake efficiency were tested (using fluorescence microscopy, xCELLigence analysis, atomic absorption spectroscopy, and advanced microscopy techniques). Migration capacity, cluster of differentiation markers, effect of nanoparticles on long-term viability, contrast properties in MRI, and cocultivation of labeled cells with myocytes were also studied.

Results: SAMNs do not affect MSC viability if the concentration does not exceed 100 μg ferumoxide/mL, and this concentration does not alter their cell phenotype and long-term proliferation profile. After 48 hours of incubation, MSCs labeled with SAMNs show more than double the amount of iron per cell compared to Resovist-labeled cells, which correlates well with the better contrast properties of the SAMN cell sample in T2-weighted MRI. SAMN-labeled MSCs display strong adherence and excellent elasticity in a beating myocyte culture for a minimum of 7 days.

Conclusion: Detailed in vitro tests and phantom tests on ex vivo tissue show that the new SAMNs are efficient MRI contrast agent probes with exclusive intracellular uptake and high biological safety.

Keywords: magnetic resonance imaging; mesenchymal stromal cells; stem cell labeling; stem cell tracking; superparamagnetic iron oxide nanoparticles.

Figure 1

Comparison of MRI contrast effect of SAMNs and Resovist. Notes: (A) Tubes with 10, 20, 30, 40, 80 μg ferumoxides/mL and their illustrative MRI T2 weighted image. All data are summarized into signal intensity curve of SAMNs (blue) and Resovist (violet). (B and C) Images of negative contrast of SAMN-labeled compared to Resovist-labeled MSCs (50, 100, 250, 500×10³ cells per mL of agar) and their signal intensity curves measured by fast-recovery fast spin echo (FRFSE) or gradient echo (GRE), respectively (average ± SD). Plastic tubes diameter =10 mm, water used as a background. The central photo between Figure B and C shows hMSC labeled with SAMNs after trypsinization and resuspension in fresh liquid agar. (E) Photo of freshly extracted heart with inserted tubes containing 50 and 500×10³ of SAMN (left couple of tubes) and Resovist-labeled cells (right couple). (D and F) Representative scans with hypointense lesions of SAMN- and Resovist-labeled cells in the heart tissue scanned in FRFSE and GRE sequences, respectively. Abbreviations: MRI, magnetic resonance imaging; SAMNs, surface-active maghemite nanoparticles; MSC, mesenchymal stromal cell; RES, Resovist; SD, standard deviation.

Figure 2

Transmission electron microscopy image of surface-active maghemite nanoparticles.

Figure 3

Comparison of viability and proliferation of SAMNs and Resovist-labeled cells. Notes: (A) Human mesenchymal stromal cell (hMSC) viability after 48 hours incubation in 50 μg and 100 μg ferumoxides/mL, measured by two independent methods (gray bars – fluorescence microscopy calcein assay; green bars – flow cytometer propidium iodide assay). (B) hMSC growth curve measured by microscopy assay (blue bars – growth curve of control hMSC without nanoparticle labeling; violet bars – growth curve of hMSCs incubated for 48 hours with 50 μg of SAMNs/mL). (C) Illustrative hMSC growth curves measured by the xCELLigence automatic system (cell sample from donor 1), cells were incubated without nanoparticles or with addition of 50 μg/mL of SAMNs or Resovist. (D) Summarized data from xCELLigence for all cell donors. E–H denote analogous results for rat mesenchymal stromal cells (rMSCs). Error bars represent standard deviations. Statistically significant differences between mean value of the sample and mean value of its control are highlighted by a star and P-value by a number. Abbreviations: incub, incubation; h, hours; CTRL, control; SAMNs, surface-active maghemite nanoparticles; SAMN50, surface-active maghemite nanoparticles 50 μg/mL; RES50, Resovist 50 μg/mL; RES 100, Resovist 100 μg/mL; SAMN100, surface-active maghemite nanoparticles 100 μg/mL.

Figure 4

Light microscopy images. Notes: (A) MSCs (top line – hMSC; bottom line – rMSC), (left) control nonlabeled cells, (middle) SAMN-labeled cells, and (right) Resovist-labeled cells. Incubation 48 hours, 50 μg ferumoxide/mL; objective 20×. (B) SAMN-labeled MSCs after Prussian blue staining showing the presence of iron (blue spots) in the cell cytoplasm. Red spots depict the nuclei of cells. (C) Representative image of fully labeled hMSC with SAMNs. Objective 40×. Abbreviations: MSCs, mesenchymal stromal cells; hMSC, human mesenchymal stromal cell; rMSC, rat mesenchymal stromal cell; SAMN, surface-active maghemite nanoparticle.

Figure 5

Optical images of MSCs labeled with SAMNs. Notes: (A) Immediately after trypsinization and injection into myocardial culture (highlighted by red arrows). (B) After 24 hours of cocultivation with myocytes (highlighted by blue border). The flattened shape of MSCs is clearly visible. (C) MSCs in myocardial culture after 7 days. Abbreviations: MSC, mesenchymal stromal cell; SAMNs, surface-active maghemite nanoparticles.