The remote actuation of cellular processes such as migration or neuronal outgrowth is a challenge for future therapeutic applications in regenerative medicine. Among the different methods that have been proposed, the use of magnetic nanoparticles appears to be promising, since magnetic fields can act at a distance without interactions with the surrounding biological system. To control biological processes at a subcellular spatial resolution, magnetic nanoparticles can be used either to induce biochemical reactions locally or to apply forces on different elements of the cell. Here, we show that cell migration and neurite outgrowth can be directed by the forces produced by a switchable parallelized array of micro-magnetic pillars, following the passive uptake of nanoparticles. Using live cell imaging, we first demonstrate that adherent cell migration can be biased toward magnetic pillars and that cells can be reversibly trapped onto these pillars. Second, using differentiated neuronal cells we were able to induce events of neurite outgrowth in the direction of the pillars without impending cell viability. Our results show that the range of forces applied needs to be adapted precisely to the cellular process under consideration. We propose that cellular actuation is the result of the force on the plasma membrane caused by magnetically filled endo-compartments, which exert a pulling force on the cell periphery.

Wnt signaling is a key developmental pathway that regulates dopaminergic progenitor cell proliferation and differentiation during neuronal development. This makes Wnt signaling an important therapeutic target for neurodegenerative conditions such as Parkinson's disease. Wnt signaling can be modulated using peptides such as UM206, which bind to the Wnt receptor Frizzled. Previous work has demonstrated remote activation of the Wnt pathway through Frizzled using peptide‐functionalized magnetic nanoparticles (MNPs) with magnetic field stimulation. Using this technology, Wnt signaling is remotely activated in the neuronal cell line SH‐SY5Y, and the phenotypic response to stimulation is assessed. Results indicate β‐catenin translocalization and activation of TCF/LEF responsive transcription in response to MNP and magnetic fields, which result in dopaminergic marker expression when synergistically combined with differentiation factors retinoic acid and the phorbol ester phorbol 12‐myristate 13‐acetate. This approach is translated into ex vivo postnatal rat brain slices modeling the developing nigrostriatal pathway. Dopaminergic marker expression is maintained in MNP‐labeled SH‐SY5Y cells after injection and magnetic stimulation. These results demonstrate the translational value of remote control of signal transduction for controlling neuronal precursor cell behavior and highlight the potential applications for controlled cell differentiation as part of cell therapies for neurodegenerative disease.

The ability to control the growth and orientation of neurites over long distances has significant implications for regenerative therapies and the development of physiologically relevant brain tissue models. In this study, the forces generated on magnetic nanoparticles internalised within intracellular endosomes are used to direct the orientation of neuronal outgrowth in cell cultures. Following differentiation, neurite orientation was observed after 3 days application of magnetic forces to human neuroblastoma (SH-SY5Y) cells, and after 4 days application to rat cortical primary neurons. The direction of neurite outgrowth was quantified using a 2D Fourier transform analysis, showing agreement with the derived magnetic force vectors. Orientation control was found to be effective over areas >1cm² using modest forces of ∼10 fN per endosome, apparently limited only by the local confluence of the cells. A bioinformatics analysis of protein expression in cells exposed to magnetic forces revealed changes to cell signaling and metabolic pathways resulting in enhanced carbohydrate metabolism, as well as the perturbation of processes related to cellular organisation and proliferation. Additionally, in cell culture regions where the measured force vectors converged, large (∼100 µm) SH-SY5Y neuroclusters loaded with nanoparticles were found, connected by unusually thick linear neurite fibres. This could suggest a magnetically driven enhancement of neurocluster growth, with the clusters themselves contributing to the local forces that direct outgrowth. Such structures, which have not been previously observed, could provide new insights into the development and possible enhancement of neural circuitry. STATEMENT OF SIGNIFICANCE: A magnetic force approach for directing outgrowth in neuronal cells over macroscopic areas is successfully demonstrated. Cells were incubated with magnetic nanoparticles which were sequestered into intracellular compartments. Permanent magnet arrays created local intracellular magnetic force vectors mediated via the internalized nanoparticles, which were found to precisely guide neurite orientation. Analysis of cellular protein expression suggested the mechanism for directed growth involved specific cell signaling and metabolic pathways. In addition, highly unusual straight and thick neural fibers were observed that connected large 'magnetic' spherical cell clusters. The results reported will advance nanotechnology and cell therapy for neuro-regeneration where magnetic forces could help to reconnect damaged neurons, or even build artificial neuronal architectures.

The remote actuation of cellular processes such as migration or neuronal outgrowth is a challenge for future therapeutic applications in regenerative medicine. Among the different methods that have been proposed, the use of magnetic nanoparticles appears to be promising since magnetic fields can act at a distance without interactions with the surrounding biological system. To control biological processes at a subcellular spatial resolution, magnetic nanoparticles can be used either to induce biochemical reactions locally or to apply forces on different elements of the cell. Here, we show that cell migration and neurite outgrowth can be directed by the forces produced by a switchable parallelized array of micro-magnetic pillars, following passive uptake of nanoparticles. Using live cell imaging, we first demonstrate that adherent cell migration can be biased toward magnetic pillars and that cells can be reversibly trapped onto these pillars. Second, using differentiated neuronal cells we were able to induce events of neurite outgrowth in the direction of the pillars without impending cell viability. Our results show that the range of forces applied needs to be adapted precisely to the cellular process under consideration. We propose that cellular actuation is the result of the force on the plasma membrane caused by magnetically filled endo-compartments, which exert a pulling force on the cell periphery.