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Cell labeling with magnetic nanoparticles: Opportunity for magnetic cell imaging and cell manipulation
© Kolosnjaj-Tabi et al.; licensee BioMed Central Ltd. 2013
Published: 10 December 2013
This tutorial describes a method of controlled cell labeling with citrate-coated ultra small superparamagnetic iron oxide nanoparticles. This method may provide basically all kinds of cells with sufficient magnetization to allow cell detection by high-resolution magnetic resonance imaging (MRI) and to enable potential magnetic manipulation. In order to efficiently exploit labeled cells, quantify the magnetic load and deliver or follow-up magnetic cells, we herein describe the main requirements that should be applied during the labeling procedure. Moreover we present some recommendations for cell detection and quantification by MRI and detail magnetic guiding on some real-case studies in vitro and in vivo.
Magnetic labeling provides living cells with new features, which allow cell magnetic resonance imaging (MRI), enable distal cell manipulation applicable to tissue-engineering techniques, or could be even used for magnetically assisted cell delivery to target organs in vivo. Among magnetic nanoparticles, superparamagnetic iron oxide nanoparticles have an extensively documented background about particle synthesis and surface modification. Moreover, if properly used (i.e. when well dispersed), such particles do not alter viability, function, proliferation or differentiation of cells. In order to efficiently and safely label different cell types, including stem cells, this tutorial presents a well-established method of controlled cell labeling with citrate-coated ultra small superparamagnetic iron oxide nanoparticles (herein referred to as magnetic nanoparticles - MNP). In addition, we also provide a method of detection and quantification of single cells with high resolution MRI and describe the basis of cell sorting and magnetic manipulation for engineering and therapeutic purposes.
Cell labeling with magnetic nanoparticles
Different strategies can be applied in order to endow cells with sufficient magnetization to be detectable by MRI and/or to be manipulated by an external magnetic field. The handiest way is the co-incubation of cells with magnetic nanoparticles, where the particles are generally internalized through the spontaneous endocytosis pathway  or phagocytosis . However cellular uptake may strongly depend on nanoparticle properties, especially on surface functionalization . While dextran-coated nanoparticles show very poor uptake due to steric repulsions between particles and cell membrane, the best strategy to facilitate endocytosis of nanoparticles is to favor a specific binding or non-specific adsorption to the cell membrane. This can be achieved by linking biological effectors on nanoparticles such as antibodies, transferrin or HIV-Tat peptide that target specific receptors on plasma membrane . The use of cationic transfection agents that form highly charged complexes with nanoparticles is also efficient to trigger cellular uptake, but usually requires long incubation times (>6 hours) . Moreover the aggregation state of nanoparticles in the formed complexes cannot be controlled.
The importance of nanoparticle stability in cell labeling medium
As the cells react in a different manner depending on whether the nanoparticles remain dispersed in suspension or become aggregated, the stability of MNPs is a key issue to achieve an efficient and controllable magnetic labeling. Moreover, cell toxicity might arise from MNPs aggregates, whereas the same MNPs would have no deleterious effect when correctly dispersed. In addition, the surface properties of nanoparticles can be changed upon dynamic adsorption of the proteins and macromolecules encountered in the biological medium. Therefore what the cell perceives is not the original nanoparticle designed by a chemist, but a modified heterogeneous surface reconfigured by the biological milieu [6, 7]. Both the physical state (aggregated versus isolated nanoparticles) and the biological identity of particles (comprising the adsorbed proteins) dictate the uptake by different cell types and the in vivo biodistribution of nanoparticles.
Practical aspects of cell labeling
After a short incubation time (typically less than one hour, compared to several hours of cell labeling with other types of magnetic nanoparticles), cells are rinsed with the citrate-enriched, serum-free medium and left for particle chase in the standard cell medium at 37°C. Once the chase period is over, cells appearance should be attentively examined. The main qualitative check points are summarized in Figure 3.
Mechanistic aspects in cell labeling with MNPs
Intracellular storage of internalized particles
Intralysosomal sequestration of MNPs has the advantage to protect the cell from the release of any free toxic iron species in the cytoplasm. Moreover the lysosomes are used by cells to metabolize MNPs and to degrade them at long term [10, 11]. Likewise the in vivo biotransformation of MNPs occurs intracellularly within the lysosomes, and the iron, coming from the degradation of MNPs, is locally transferred and stored within the ferritin, the iron storage protein [12, 13].
Impact of magnetic nanoparticles on cell viability
To date different cell types have been labeled with MNPs (immune cells, endothelial cells, cancer cells, primary culture or established cell lines and progenitors cells, to mention just a few) and detrimental effects on cell proliferation and cell functions at short and long terms, in vitro or in vivo, were not observed . The labeling of stem cells is more tricky as these cells should conserve their self-renewal and multipotency after internalization of MNPs . Human neural precursor cells were also efficiently labeled without impairment of their differentiation capacity [17, 18]. However in some studies using transfection agents for cell labeling, controversial effects were observed on the multilineage differentiation capacity of mesenchymal stem cells. The chondrogenesis (i.e. the capacity to differentiate in cells of cartilage) was partially inhibited in one study , but not in others [14, 20–22], whereas adipogenesis and osteogenesis were not impaired. On the contrary, while labeling cells with citrate-coated MNP, we could modulate the amount and the physical state of nanoparticles interacting with cells and could conclude that only high dose of MNPs or an aggregated state, could have adverse effects on cell differentiation (chondrogenesis)  (Figure 6). Labeling conditions with perfectly stable MNP is thus recommended for use in cell therapy assays.
Fate of the particles in a living cell
During the division process, the cell shares the magnetic endosomes between its two daughter cells. The iron load is thus reduced by a factor of two at each division. In normal conditions, there is no exocytosis of MNPs. However, under stress conditions, some magnetically labeled cells can release nanoparticle-loaded microvesicles in the extracellular medium [23, 24]. These cell-released vesicles can transfer nanoparticles to other naïve cells , especially macrophages . This process, if confirmed in vivo, could participate to a horizontal intercellular transfer of nanoparticles, challenging to some extent the initial specificity of cell labeling [26, 27].
Quantification of iron load
Cell responsiveness to the magnetic forces
As lysosomes in labeled cells concentrate several millions of MNPs, a labeled cell becomes responsive to an inhomogeneous magnetic field, generated, for example, by a permanent magnet. In a non uniform magnetic field B, defined by an unidirectional magnetic field gradient gradB, a labeled cell experiences a magnetic force M(B)gradB, where M(B) is the magnetic moment of the cell in the field B (equal to the magnetic moment of one MNP multiplied by the number of MNPs per cell). Typically a permanent magnet generates a magnetic field gradient of 10-50 T/m over a distance of approximately 1 cm. The corresponding force experienced by the cell (with an average iron load of 10 pg) may vary from 1 pN to a few nN . For cells in suspension, the magnetic force is balanced by the viscous force 6πηRV, where η is the viscosity of the medium, R the cell radius and V the cell velocity. In a set-up with calibrated B and gradB (18 T/m), it is easy to deduce iron load from the determination of V and R for each cell by video-microscopy (Figure 7 top). From this experiment we can thus determine the distribution of MNP uptake in a cell population (Figure 7 middle). If the cells have not been labeled in the appropriate way (and are consequently covered with particle aggregates and cellular debris), magnetophoresis will not reflect the cell velocity that is due to intracellular iron, but will indicate the velocity that is due to internalized and membrane-attached nanoparticles. Besides, as we can see on Figure 7 (bottom), chains of aggregates that are not attached to cell membranes also migrate towards the magnet. In contrast to other global dosage of iron load in cell pellet, single cell magnetophoresis allows to visualize potential artifact linked to nanoparticle aggregation. The control of nanoparticle stability is once again the critical point to achieve a quantifiable and reproducible magnetic labeling.
Imaging cells with magnetic resonance imaging (MRI)
Cell tracking in vivo: the advantages of MRI
One of the new emerging applications of magnetic cell labeling concerns magnetic resonance cell tracking. Magnetic resonance imaging (MRI) allows real-time whole-body examinations with excellent soft-tissue contrast and spatial resolution. Moreover, impactful development has been made on high-field MR scanners, magnetic gradient systems and radiofrequency (RF) coils . One of the new coils, such as the cryogenic probe, allows sub-milimetric resolution and gives the means to perform cellular MRI in vivo. The advantage of the cryogenic probe to improve the signal-to-noise (SNR) ratio and concomitantly improve the image resolution, has been demonstrated throughout the last decade in several studies [30, 31].
Iron oxide nanoparticles as cellular MRI contrast agents
Cell imaging in cell therapies
Quantification of punctual signal voids
Magnetic manipulation of cells: from cell sorting to magnetic targeting in tissue engineering and cell therapies
Magnetic cell sorting
Impact of the magnetic force
The effect of magnetic forces on cells will be also tightly related to the fact if the cell is suspended in a liquid or if it adheres on a substrate. While suspended cells more or less freely move when submitted to remote magnetic forces, when we try to magnetically manipulate adhering cells and the magnetic force is lower than the adhesion constraint, the cell cannot move and the magnetic force acts on MNP loaded intracellular endo-lysosomes. Such intracellular constraints can be used to deform the cell in a controlled direction and could be used, for example, to control the formation of a vascular network with magnetically labeled endothelial progenitor cells . Magnetic manipulation might allow enhanced cell seeding and engraftment in different scaffolds for tissue engineering  and may enable new perspectives for in vitro construction of organized multicellular assemblies and tissue substitutes .
Magnetic vectorization: the response to the need for localized cell delivery
Magnetic vectorization was recently evaluated for cardiac cell transplantation, where magnetically labeled endothelial progenitor cells were injected in the infarcted myocardium while a magnet was externally applied to rats in the heart zone. Magnetically assisted cell delivery resulted in an increased concentration of cells and the short-term effect on cell retention was monitored in vivo by MRI and quantified by RT-PCR . In a study evaluating the long-term engraftment, the functional benefits of magnetically assisted cell retention were also confirmed , improving cardiac ventricular function.
Practical aspects for magnetic vectorization
As we mentioned in the previous section, MRI allows in vivo follow up of magnetic cells and could be therefore used to confirm successful magnetic cell targeting. Nevertheless, in addition to this method, we should confirm that visualized spots correspond to injected cells. This might be done by immunohistological methods or flow cytometer analysis post mortem. Sometimes, especially if the cells are administered in low concentrations and systemically, the cells are difficult to find both by histology and flow cytometry. If we cannot localize the cells with these or other methods of cell detection, we should at least have a proof of an important therapeutic effect that could serve as a surrogate marker of cell delivery and local action.
Summary and conclusion
Iron oxide nanoparticles can be used for magnetic labeling of different types of cells. The labeling of living cells allows a variety of biomedical applications ranging from cell manipulation to diagnostics and regenerative medicine. This tutorial provides the basic requirements for efficient cell labeling with anionic (citrate coated) iron oxide nanoparticles and includes sections on troubleshooting to prevent the occurrence of potential cell damage during the labeling procedure. In addition, as single cells can be monitored by high resolution MRI, we provide some appreciation of cellular MRI and present an abridged method for the quantification of punctual signal voids that are generated in vitro and in vivo by labeled cells. Finally, we also assess the potential of cell manipulation that can be exploited both in vitro for tissue engineering and in vivo in cell therapies.
This article has been published as part of Journal of Nanobiotechnology Volume 11 Supplement 1, 2013: Nanophysics for Health. The full contents of the supplement are available online at http://www.jnanobiotechnology.com/supplements/11/S1. Publication charges for this tutorial were funded by the CNRS School "Nanophysics for Health", 5 - 9 November 2012, Mittelwhir, France
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