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Labeling of mesenchymal stem cells for MRI with single-cell sensitivity.

Ariza de Schellenberger A, Kratz H, Farr TD, Löwa N, Hauptmann R, Wagner S, Taupitz M, Schnorr J, Schellenberger EA - Int J Nanomedicine (2016)

Bottom Line: Attempts should be made to achieve better cell separation for homogeneous NP loading and to thus improve NP-uptake-dependent biocompatibility studies and cell detection by MRI and future MPI.Additionally, using a 7 T MR imager equipped with a cryocoil resulted in approximately two times higher detection.In conclusion, we established labeling conditions for new high-relaxivity MCP, VSOP, and Resovist(®) for improved MRI of MSC with single-cell sensitivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology, Center for Stroke Research Berlin, Charité - Universitätsmedizin Berlin, Berlin, Germany.

ABSTRACT
Sensitive cell detection by magnetic resonance imaging (MRI) is an important tool for the development of cell therapies. However, clinically approved contrast agents that allow single-cell detection are currently not available. Therefore, we compared very small iron oxide nanoparticles (VSOP) and new multicore carboxymethyl dextran-coated iron oxide nanoparticles (multicore particles, MCP) designed by our department for magnetic particle imaging (MPI) with discontinued Resovist(®) regarding their suitability for detection of single mesenchymal stem cells (MSC) by MRI. We achieved an average intracellular nanoparticle (NP) load of >10 pg Fe per cell without the use of transfection agents. NP loading did not lead to significantly different results in proliferation, colony formation, and multilineage in vitro differentiation assays in comparison to controls. MRI allowed single-cell detection using VSOP, MCP, and Resovist(®) in conjunction with high-resolution T2*-weighted imaging at 7 T with postprocessing of phase images in agarose cell phantoms and in vivo after delivery of 2,000 NP-labeled MSC into mouse brains via the left carotid artery. With optimized labeling conditions, a detection rate of ~45% was achieved; however, the experiments were limited by nonhomogeneous NP loading of the MSC population. Attempts should be made to achieve better cell separation for homogeneous NP loading and to thus improve NP-uptake-dependent biocompatibility studies and cell detection by MRI and future MPI. Additionally, using a 7 T MR imager equipped with a cryocoil resulted in approximately two times higher detection. In conclusion, we established labeling conditions for new high-relaxivity MCP, VSOP, and Resovist(®) for improved MRI of MSC with single-cell sensitivity.

No MeSH data available.


Related in: MedlinePlus

Increasing the NP labeling concentrations was sufficient to increase cellular NP uptake, allowing us to avoid use of protamine sulfate.Notes: Cells were loaded at 0.2 mM with Resovist® or MCP in combination with 12 µg/mL PS or VSOP alone (open symbols) and cells loaded at 2 mM without PS for all three NPs (filled symbols). Iron quantification after 24-hour incubation and ECM disruption indicates that Resovist®-PS complexes have better uptake by MSC than MCP-PS. VSOP show poor uptake at low (0.2 mM) loading concentration. However, 10× higher loading concentration (2 mM) in absence of PS was sufficient to significantly increase cellular uptake of all NPs. (mean; error bars, ± SD).Abbreviations: ECM, extracellular matrix; MCP, multicore carboxy-methyl-dextran- coated iron oxide nanoparticle; NP, nanoparticle; PS, protamine sulfate; VSOP, very small iron oxide nanoparticle.
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f2-ijn-11-1517: Increasing the NP labeling concentrations was sufficient to increase cellular NP uptake, allowing us to avoid use of protamine sulfate.Notes: Cells were loaded at 0.2 mM with Resovist® or MCP in combination with 12 µg/mL PS or VSOP alone (open symbols) and cells loaded at 2 mM without PS for all three NPs (filled symbols). Iron quantification after 24-hour incubation and ECM disruption indicates that Resovist®-PS complexes have better uptake by MSC than MCP-PS. VSOP show poor uptake at low (0.2 mM) loading concentration. However, 10× higher loading concentration (2 mM) in absence of PS was sufficient to significantly increase cellular uptake of all NPs. (mean; error bars, ± SD).Abbreviations: ECM, extracellular matrix; MCP, multicore carboxy-methyl-dextran- coated iron oxide nanoparticle; NP, nanoparticle; PS, protamine sulfate; VSOP, very small iron oxide nanoparticle.

Mentions: Cells incubated with NP for 4 hours and 24 hours showed substantial extracellular iron clusters, mostly attached to the ECM. Clustering was observed irrespective of the particle type, the incubation concentration, or the use of PS as TA. Figure 1 shows representative micrographs of cells labeled with Resovist®. For meaningful quantification of NP uptake into cells, the extracellular NP clusters were significantly reduced by ECM disruption with trypsin and cell passage (Figure 1C and D). Following ECM disruption, the quantified averaged NP iron per cell was also reduced but now represented the real intracellular NP uptake (Figure 1E). Comparison of intracellular NP between cells incubated with 0.2 mM Resovist® or MCP, alone or with PS, showed that the conditions used with PS were ideal to increase intracellular uptake of MCP and Resovist® by a factor of ~2.5 and ~1.5, respectively (data not shown). However, increasing the NP loading concentration to 2 mM was sufficient to avoid the use of PS and resulted in significantly increased intracellular NP uptake for both MCP and Resovist® (three-fold and 1.5-fold, respectively) and even more significantly for smaller NPs such as VSOP, for which a ten-fold uptake was observed (Figure 2).


Labeling of mesenchymal stem cells for MRI with single-cell sensitivity.

Ariza de Schellenberger A, Kratz H, Farr TD, Löwa N, Hauptmann R, Wagner S, Taupitz M, Schnorr J, Schellenberger EA - Int J Nanomedicine (2016)

Increasing the NP labeling concentrations was sufficient to increase cellular NP uptake, allowing us to avoid use of protamine sulfate.Notes: Cells were loaded at 0.2 mM with Resovist® or MCP in combination with 12 µg/mL PS or VSOP alone (open symbols) and cells loaded at 2 mM without PS for all three NPs (filled symbols). Iron quantification after 24-hour incubation and ECM disruption indicates that Resovist®-PS complexes have better uptake by MSC than MCP-PS. VSOP show poor uptake at low (0.2 mM) loading concentration. However, 10× higher loading concentration (2 mM) in absence of PS was sufficient to significantly increase cellular uptake of all NPs. (mean; error bars, ± SD).Abbreviations: ECM, extracellular matrix; MCP, multicore carboxy-methyl-dextran- coated iron oxide nanoparticle; NP, nanoparticle; PS, protamine sulfate; VSOP, very small iron oxide nanoparticle.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4835118&req=5

f2-ijn-11-1517: Increasing the NP labeling concentrations was sufficient to increase cellular NP uptake, allowing us to avoid use of protamine sulfate.Notes: Cells were loaded at 0.2 mM with Resovist® or MCP in combination with 12 µg/mL PS or VSOP alone (open symbols) and cells loaded at 2 mM without PS for all three NPs (filled symbols). Iron quantification after 24-hour incubation and ECM disruption indicates that Resovist®-PS complexes have better uptake by MSC than MCP-PS. VSOP show poor uptake at low (0.2 mM) loading concentration. However, 10× higher loading concentration (2 mM) in absence of PS was sufficient to significantly increase cellular uptake of all NPs. (mean; error bars, ± SD).Abbreviations: ECM, extracellular matrix; MCP, multicore carboxy-methyl-dextran- coated iron oxide nanoparticle; NP, nanoparticle; PS, protamine sulfate; VSOP, very small iron oxide nanoparticle.
Mentions: Cells incubated with NP for 4 hours and 24 hours showed substantial extracellular iron clusters, mostly attached to the ECM. Clustering was observed irrespective of the particle type, the incubation concentration, or the use of PS as TA. Figure 1 shows representative micrographs of cells labeled with Resovist®. For meaningful quantification of NP uptake into cells, the extracellular NP clusters were significantly reduced by ECM disruption with trypsin and cell passage (Figure 1C and D). Following ECM disruption, the quantified averaged NP iron per cell was also reduced but now represented the real intracellular NP uptake (Figure 1E). Comparison of intracellular NP between cells incubated with 0.2 mM Resovist® or MCP, alone or with PS, showed that the conditions used with PS were ideal to increase intracellular uptake of MCP and Resovist® by a factor of ~2.5 and ~1.5, respectively (data not shown). However, increasing the NP loading concentration to 2 mM was sufficient to avoid the use of PS and resulted in significantly increased intracellular NP uptake for both MCP and Resovist® (three-fold and 1.5-fold, respectively) and even more significantly for smaller NPs such as VSOP, for which a ten-fold uptake was observed (Figure 2).

Bottom Line: Attempts should be made to achieve better cell separation for homogeneous NP loading and to thus improve NP-uptake-dependent biocompatibility studies and cell detection by MRI and future MPI.Additionally, using a 7 T MR imager equipped with a cryocoil resulted in approximately two times higher detection.In conclusion, we established labeling conditions for new high-relaxivity MCP, VSOP, and Resovist(®) for improved MRI of MSC with single-cell sensitivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology, Center for Stroke Research Berlin, Charité - Universitätsmedizin Berlin, Berlin, Germany.

ABSTRACT
Sensitive cell detection by magnetic resonance imaging (MRI) is an important tool for the development of cell therapies. However, clinically approved contrast agents that allow single-cell detection are currently not available. Therefore, we compared very small iron oxide nanoparticles (VSOP) and new multicore carboxymethyl dextran-coated iron oxide nanoparticles (multicore particles, MCP) designed by our department for magnetic particle imaging (MPI) with discontinued Resovist(®) regarding their suitability for detection of single mesenchymal stem cells (MSC) by MRI. We achieved an average intracellular nanoparticle (NP) load of >10 pg Fe per cell without the use of transfection agents. NP loading did not lead to significantly different results in proliferation, colony formation, and multilineage in vitro differentiation assays in comparison to controls. MRI allowed single-cell detection using VSOP, MCP, and Resovist(®) in conjunction with high-resolution T2*-weighted imaging at 7 T with postprocessing of phase images in agarose cell phantoms and in vivo after delivery of 2,000 NP-labeled MSC into mouse brains via the left carotid artery. With optimized labeling conditions, a detection rate of ~45% was achieved; however, the experiments were limited by nonhomogeneous NP loading of the MSC population. Attempts should be made to achieve better cell separation for homogeneous NP loading and to thus improve NP-uptake-dependent biocompatibility studies and cell detection by MRI and future MPI. Additionally, using a 7 T MR imager equipped with a cryocoil resulted in approximately two times higher detection. In conclusion, we established labeling conditions for new high-relaxivity MCP, VSOP, and Resovist(®) for improved MRI of MSC with single-cell sensitivity.

No MeSH data available.


Related in: MedlinePlus