This work presents a bottom-up approach to the engineering of local magnetic forces for control of neuronal organization. Neuron-like cells loaded with magnetic nanoparticles (MNPs) are plated atop and controlled by a micro-patterned platform with perpendicular magnetization. Also described are magnetic characterization, MNP cellular uptake, cell viability, and statistical analysis.
The ability to direct neurons into organized neural networks has great implications for regenerative medicine, tissue engineering, and bio-interfacing. Many studies have aimed at directing neurons using chemical and topographical cues. However, reports of organizational control on a micron-scale over large areas are scarce. Here, an effective method has been described for placing neurons in preset sites and guiding neuronal outgrowth with micron-scale resolution, using magnetic platforms embedded with micro-patterned, magnetic elements. It has been demonstrated that loading neurons with magnetic nanoparticles (MNPs) converts them into sensitive magnetic units that can be influenced by magnetic gradients. Following this approach, a unique magnetic platform has been fabricated on which PC12 cells, a common neuron-like model, were plated and loaded with superparamagnetic nanoparticles. Thin films of ferromagnetic (FM) multilayers with stable perpendicular magnetization were deposited to provide effective attraction forces toward the magnetic patterns. These MNP-loaded PC12 cells, plated and differentiated atop the magnetic platforms, were preferentially attached to the magnetic patterns, and the neurite outgrowth was well aligned with the pattern shape, forming oriented networks. Quantitative characterization methods of the magnetic properties, cellular MNP uptake, cell viability, and statistical analysis of the results are presented. This approach enables the control of neural network formation and improves neuron-to-electrode interface through the manipulation of magnetic forces, which can be an effective tool for in vitro studies of networks and may offer novel therapeutic biointerfacing directions.
Micropatterning of neurons holds great potential for tissue regeneration1,2,3,4,5 and the development of neuro-electronic devices6,7,8. However, the micron-scaled positioning of neurons at high spatial resolution, as in biological tissues, poses a significant challenge. Forming predesigned structures at this scale requires the guidance of nerve cell processes by locally controlling soma motility and axonal outgrowth. Previous studies have suggested the use of chemical and physical cues9,10,11,12 for guiding neuronal growth. Here, a novel approach focuses on controlling cell positioning by magnetic field gradients13,14,15,16,17, turning cells loaded with MNPs into magnetic-sensitive units, which can be remotely manipulated.
Kunze et al., who characterized the force needed to induce cellular responses using magnetic chip- and MNP-loaded cells, proved that early axonal elongation can be triggered by mechanical tension inside cells18. Tay et al. confirmed that micro-fabricated substrates with enhanced magnetic field gradients allow for wireless stimulation of neural circuits dosed with MNPs using calcium indicator dyes19. Moreover, Tseng et al. coalesced nanoparticles inside cells, resulting in localized nanoparticle-mediated forces that approached cellular tension20. This led to the fabrication of defined patterns of micromagnetic substrates that helped to study cellular response to mechanical forces. Cellular tension arising from the application of localized nanoparticle-mediated forces was achieved by coalescing nanoparticles within cells20. A complementary metal oxide semiconductor (CMOS)-microfluidic hybrid system was developed by Lee et al. who embedded an array of micro-electromagnets in the CMOS chip to control the motion of individual cells tagged with magnetic beads21.
Alon et al. used micro-scale, pre-programmed, magnetic pads as magnetic "hot spots" to locate cells22. Specific activity could also be stimulated within cells using micro-patterned magnetic arrays to localize nanoparticles at specific subcellular locations23. Cellular MNP uptake has been successfully demonstrated in leech, rat, and mouse primary neurons24,25,26. Here, this has been demonstrated on a rat PC12 pheochromocytoma cell line, which has been previously reported to show high uptake of MNPs27. In recent years, there have been various medical applications of MNPs, including drug delivery and thermotherapy in cancer treatments28,29,30,31. Specifically, studies deal with the application of MNPs and neuron networks32,33,34,35. However, the magnetic organization of neurons using MNPs at a single-cell level deserves further investigation.
In this work, a bottom-up approach has been described to engineer local magnetic forces via predesigned platforms for controlling neuronal arrangement. The fabrication of micron-scale patterns of FM multilayers has been presented. This unique, FM multilayered structure creates stable perpendicular magnetization that results in effective attraction forces toward all the magnetic patterns. Via incubation, MNPs were loaded into PC12 cells, transforming them into magnetic sensitive units. MNP-loaded cells, plated and differentiated atop the magnetic platforms, were preferentially attached to the magnetic patterns, and the neurite outgrowth was well-aligned with the pattern shape, forming oriented networks. Several methods have been described to characterize the magnetic properties of the FM multilayers and the MNPs, and techniques for cellular MNP uptake and cell viability assays have also been presented. Additionally, morphometric parameters of neuronal growth and statistical analysis of the results are detailed.
NOTE: Perform all biological reactions in a biosafety cabinet.
1. Magnetic platform fabrication
2. Characterization of magnetic device via transport measurements
3. Characterization of MNPs and magnetic multilayers by magnetometry measurements
4. Collagen-coating protocol
5. Cellular MNP uptake and viability
6. Characterization of MNP concentration inside the cells using inductively coupled plasma (ICP)
7. Cell differentiation and growth on magnetic platform
8. MNP-loaded cell staining
9. Measurements and statistical analysis
Magnetic platforms with different geometric shapes were fabricated (Figure 1A). Magnetic patterns were deposited by sputtering: 14 multilayers of Co80Fe20 and Pd, 0.2 nm and 1 nm, respectively. Electron microscopy revealed the total height of the magnetic patterns to be ~18 nm (Figure 1B). This unique FM multilayer deposition creates a stable platform with perpendicular magnetization anisotropy (PMA) relative to the substrate plane that enables the attraction of the MNP-loaded cells toward the entire magnetic pattern and not only to the edges22,37. The parameters of the FM multilayer structure were characterized by magneto-transport measurements for which a cross-shaped device of FM multilayers was fabricated (Figure 1C inset), and the magnetization perpendicular to the substrate was measured via the anomalous hall effect (AHE)38, where the AHE resistance is proportional to the perpendicular magnetization. The AHE vs magnetic field measurement showed a hysteresis loop indicative of PMA ferromagnets (Figure 1C). The remnant magnetization of the FM multilayers (magnetic moment at zero field) was identical to the magnetization saturation (MS) at high fields. In addition, the coercive field of the FM multilayers was ~500 Oe, reaching saturation at 1,200 Oe, which enabled easy magnetization of the device and ensured stability against the influence of unintentional magnetic fields. The MS value of the multilayers was measured using a magnetometer (Figure 1D), as described in section 3.1. The MS was 270 emu/cm3, which is at par with previous measurements of similar structures.
Fluorescent iron oxide (γ-Fe2O3) MNPs were prepared according to a previous publication39. The MNPs were synthesized by nucleation, including the covalent conjugation of six-layered thin films of iron oxide to rhodamine isothiocyanate and coating with human serum albumin. The dry diameter size of the MNPs was ~15 nm with zeta potential of -45, according to the transmission electron microscopic measurement. The magnetometric measurements of the MNPs (Figure 2A) show that the magnetization curve had no hysteresis, indicating superparamagnetic behavior of the MNPs, a low saturation field of 500 Oe, and relatively high MS of 22 emu/g. To control cell localization using magnetic patterns, PC12 cells were incubated in medium mixed with iron oxide fluorescent MNPs for 24 h, transforming them into magnetic units. The MNP concentration in the medium can be varied; it was 0.25 mg/mL in the plating experiments. Confocal microscopic images were taken after DAPI staining (Figure 2B). The MNPs were internalized into the PC12 cells' soma, but not into the nuclei, which was reflected by the dark shadow in the center. The results show that there was no red florescence in the nuclei, which indicates that the MNPs were not internalized into the nuclei or had attached to the outer surface of the cells. Using ICP measurements, it was possible to quantify the amounts of MNPs internalized into the PC12 cells. The iron concentration inside the cells increased with the increase in MNP concentration in the medium (Figure 2C).
The viability of MNP-loaded PC12 cells for different concentrations of MNPs was evaluated using XTT- and resazurin-based assays. Figure 3A shows PC12 cells after MNP treatment, growing and differentiating atop a collagen-coated plastic dish. To examine the impact of internalized MNPs on differentiation, Sholl morphological measurement was performed. No significant difference was observed in cell morphology between the MNP-loaded cells and control cells (t-test, p > 0.05, n = 3) (Figure 3B). The metabolic activity of the PC12 cells was measured using XTT- and resazurin-based assays after cell incubation with different MNP concentrations. The results were normalized to the control measurement of PC12 cells without MNPs. These concentrations of MNPs showed no significant cytotoxicity toward the cells, evident in the lack of significant differences in cell viability for any of the preparations (t-test, p > 0.05, n = 3) (Figure 3C,D). The effects of MNPs on cell plating and development were determined by comparing MNP-loaded cells to non-loaded cells, plated and grown on identical magnetic substrates. Cells were seeded and left to adhere to the substrate. Every 2 days, cells were treated with fresh medium and NGF as described in section 7. Figure 4 shows PC12 cells with and without MNP treatment, growing and differentiating on a magnetic substrate with 20 µm wide stripes and 100 µm spacing. After 3 days of growth, the cells were immunostained, DAPI-stained, and images were taken.
The magnetized cells were found to attach to the magnetic patterns and grow branches according to the patterns, while cells without MNP treatment grew with no affinity to the magnetic devices (Figure 4A,B). Figure 4C presents the positioning of cells and network formation on a substrate with hexagonal geometry of a side length of 200 µm and line width of 50 µm. Cells were imaged after 6 days. The magnetized cells were located on the magnetic pattern, with similar affinity to the striped substrates. Cell positioning was quantified by counting the cell bodies located on the magnetic patterns or attached to them. The relative proportion of cells was calculated from the total cell population. This was done for cells with and without MNP treatment. The effective area of the magnetic response was calculated by adding the cells' diameter (~10 µm for PC12 cells) to both edges of the magnetic pattern. For the magnetic stripe patterns, the effective magnetic area ratio was 33%, which corresponded to the probability of the cells landing randomly on the magnetic stripes. The results showed that 75% of MNP-loaded cell bodies were in contact with the magnetic stripes, whereas only 35% of the un-magnetized cells were located on the stripes (in statistical agreement with an unbiased distribution) (Figure 4D). Statistical analysis indicated that the measurement was not derived from arbitrary cell landing (one-sample z-test, p < 10-6, n = 430).
In contrast, the statistical analysis of the magnetic hexagons revealed that 92% of the MNP-loaded cells' soma were attached to the magnetic patterns, compared to 38% for cells without MNP treatment (Figure 4E). The effective area ratio of the hexagons was 32% of the substrate. Statistical analysis for the hexagons also indicated that the measurement was not derived from arbitrary cell landing (one-sample z-test, p < 10-6, n = 370). The results revealed a clear preference of the MNP-loaded cells for the magnetic pattern, while cells without MNPs adhered randomly to the entire substrate. In addition to the cell-positioning effect, these magnetic platforms were found to also control the directionality of the growing neurites. Figure 5A shows MNP-loaded cells with neurites, aligning according to the stripes' orientation. In contrast, the control measurement of cells without MNPs showed neurite growth across the platform regardless of the magnetic patterns.
To evaluate the magnetic effect on neuronal growth directionality, the angle between the neurites and the magnetic stripes was measured. The data revealed that 80% of the neurites of MNP-loaded cells exhibited correlation with the magnetic stripes' orientation, within Δθ < 15° relative to the stripes' direction. However, only 32% of the neurites of cells without MNPs developed in that range. Cells without MNP treatment showed no correlation with the magnetic stripes and grew according to a uniform angle distribution (Figure 5B). Statistical analysis of the distribution of Δθ revealed that it was not normal or uniform (Chi-square test, p < 0.001). The effect of the hexagonal geometry on neurite growth was demonstrated as well. Figure 5C shows fluorescence images of neural network development of magnetized and un-magnetized PC12 cells atop a magnetic pattern of hexagons and large circles between the edges. The side length of the hexagon was 200 µm and the lines width was 10 µm; the circle diameter was 30 µm. The cell somas showed high affinity for the circles and developed a well-oriented neural network along the hexagonal outlines. A zoom-in image demonstrates cells attached to a magnetic circle and neurites growing along those outlines (Figure 5D).
Figure 1: Characterization of the magnetic devices. (A) Optical microscopic images of magnetic devices with various geometric shapes. Scale bar = 200 µm. (B) Scanning electron microscopic image of Co80Fe20/Pd multilayers and a schematic of the multilayers. The total height of the magnetic patterns is 18 nm. Scale bar = 100 nm. (C) Anomalous Hall effect measurement of the magnetic device showing the coercive and remnant magnetic fields of the FM. Inset: image of device with marked electrodes. (D) Magnetometry of multilayer device shows the magnetization saturation value calculated per volume. This figure has been modified from Marcus et al.37. Abbreviations: AHE = Anomalous Hall effect; FM = ferromagnetic; B = magnetic field. Please click here to view a larger version of this figure.
Figure 2: PC12 cell uptake of MNPs. (A) Magnetometric measurement of MNPs at room temperature. (B) Confocal microscopy image of MNP uptake by PC12 cells. Nuclei stained with DAPI; MNPs labeled with rhodamine enter the cells. Scale bar = 10 µm (C) ICP measurement of the internalized iron oxide MNPs (pg) by PC12 cells after 24 h of incubation with several MNP concentrations. This figure has been modified from Marcus et al.37. Abbreviations: MNPs = magnetic nanoparticles; DAPI = 4′,6-diamidino-2-phenylindole; ICP = inductively coupled plasma. Please click here to view a larger version of this figure.
Figure 3: MNP-loaded PC12 cell viability. (A) Confocal microscopic images of differentiated PC12 cells incubated with MNPs. Arrows show differentiated neurites with internalized MNPs. Scale bars = 50 nm. (B) Sholl analysis of neurite outgrowth of PC12 cells after 3 days of differentiation. (C) XTT viability assay of PC12 cells treated with increasing concentrations of MNPs after 24 h of incubation. Measurements are normalized to control. (D) Resazurin-based viability assay of PC12 cells treated with increasing concentrations of MNPs after 24 h of incubation. Measurements are normalized to control. There is no statistical significance in both analyses. This figure has been modified from Marcus et al.37. Abbreviations: MNPs = magnetic nanoparticles; XTT = 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide. Please click here to view a larger version of this figure.
Figure 4: Cell localization atop magnetic devices. (A) Fluorescent images of PC12 cells loaded with MNPs growing on the magnetic stripes: (i) α-tubulin labeling, (ii) DAPI staining, and (iii) merged image. Scale bar = 100 µm. (B) Fluorescent images of PC12 cells without MNP treatment, growing on top of the magnetic stripes as in (A). (C) Fluorescence images of PC12 cells, with and without MNPs, on the hexagonal magnetic pattern. Scale bar = 200 µm. (D) Percentage of cell bodies, with and without MNPs, located on the magnetic stripes. Error bars represent standard deviation. The dotted line represents the probability of cells landing on the magnetic stripes. (E) Percentage of cell bodies, with and without MNPs, located on the magnetic hexagons. Error bars represent standard deviation. The dotted line represents the probability of cells landing on the magnetic patterns for a random distribution. There is no statistical significance in both analyses. This figure has been modified from Marcus et al.37. Abbreviations: MNPs = magnetic nanoparticles; DAPI = 4′,6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Figure 5: Neuronal network directionality. (A) Confocal microscopic images of α-tubulin-labeled PC12 cells with MNP treatment (left) and without MNP treatment (right), growing atop magnetic stripes. The stripes are marked. (B) Polar histograms present the neurite directionality effect on PC12 cells (left) with MNP treatment and (right) without MNP treatment. The deviation of the orientation angle is defined as the difference in the angle between the neurites and the magnetic stripe orientation (15° bins). (C) Fluorescence images of PC12 cells grown atop a magnetic hexagonal pattern, (left) with MNP treatment and (right) without MNP treatment. Scale bar = 200 µm. (D) Zoom-in image of a magnetized cell developing neurites according to the magnetic pattern. Scale bar = 30 µm. This figure has been modified from Marcus et al.37. Abbreviation: MNPs = magnetic nanoparticles. Please click here to view a larger version of this figure.
The representative results demonstrate the effectiveness of the presented methodology for controlling and organizing neuronal network formation at the micron-scale. The MNP-loaded PC12 cells remained viable and were transformed into magnetic sensitive units that were attracted by the magnetic forces from the FM electrodes to specific sites. This is best demonstrated in Figure 5C, where the cells preferentially adhered to the larger vertices of the hexagons and not the thin lines. Moreover, branching of the cells also developed favorably following the magnetic patterns. All control experiments demonstrated unambiguously that the magnetic forces directed the localization of the cell bodies and outgrowths. Although it was demonstrated that topographic cues can be used for directing neurite outgrowth40,41, this is not the case here, as cells without MNPs showed no response to the shape of the pattern.
A bottom-up approach was employed to engineer the local magnetic forces, using standard photolithography and sputtering depositions that are available in many research facilities, facilitating the adoption of these techniques by many researchers. The bottom-up approach allows freedom in the design of complex patterns and shapes according to the researchers' needs, with micron-scale resolution for centimeter length areas. Although the results were demonstrated on glass slides, in principle, it is possible to prepare the devices atop other biocompatible materials that are suitable for in vivo therapeutic applications such as flexible electrode arrays for neuronal recording and stimulation42,43.
These unique PMA platforms, attained by multilayer deposition, produce a strong magnetic field along the entire magnetic pattern and not only on the edges, as observed earlier22. Additionally, FMs were designed with a large remnant magnetization saturation, i.e., even when the external magnetic field is removed, the electrodes remain fully magnetized and keep attracting the cells without the need of an external magnet. However, an external magnetic force can assist in fully magnetizing the MNPs in the cells, thus increasing the force of attraction and efficiency during plating. An important consideration in FM design was the number of repetitions. While more repetition will increase the total magnetic moment, which is favorable, adding many layers will also increase layer intermixing, causing less stable PMA and finally result in an in-plane magnetization44,45 easy axis and attraction to different edges of the electrodes22,37. Therefore, it is necessary to optimize the FM number and composition of the multilayers to ensure stable PMA with maximal magnetic fields.
The MNPs presented in the representative results showed almost no toxicity toward the PC12 cells at the tested concentrations, nor did they affect cellular behavior, despite being able to enter the cells and having a relatively high magnetic moment. The attraction force on a cell depends on the number of MNPs in the cell and the magnetization of each MNP. Ideally, both should be high; however, there may be a tradeoff between the two. With some commercial MNPs, cell viability was good, but the magnetic moment was too weak. For other particles fabricated in the laboratory, the magnetic moment was high, but the MNPs tended to aggregate and cell viability was low. Thus, it is important to test cell viability and characterize their magnetization when choosing MNPs. The MNPs used here are also fluorescent, which makes it easy to track their location in the cells. The results show neurites developing according to the shape of the magnetic pattern, and the fluorescence indicates the presence of MNPs along the neurites.
The internalization mechanism of MNPs into cells has been previously investigated46. Cellular uptake of MNPs occurs via endocytosis according to their size, shape, and surface chemistry. Previous studies examined the uptake of different types of MNPs into neurons24; cellular uptake of coated MNPs was better than the cellular uptake of uncoated MNPs. As shown in Figure 3A,B, MNPs were internalized into the cytoplasm, but remained outside the nuclei and were transferred to the neurites during their development. Additionally, MNPs conjugated to NGF that activated the NGF signaling pathway were also internalized into cells via endocytosis47,48.
To conclude, this paper presents an effective toolbox of magnetic manipulation for research requiring biological element organization. The use of magnetic forces enables the positioning of cells, directing neurite growth. This method enables the design of platforms with complex geometric shapes. The forces of magnetic attraction can be engineered to manipulate the neural network formation remotely by changing the magnetic force landscape over time. This entire methodology can be easily extended to control other factors or chemicals that can be coupled to the MNPs and bring them to predesigned points of interest, all with micron-scale resolution.
The authors have nothing to disclose.
This research was supported by the Ministry of Science & Technology, Israel, and by the Israeli Science Foundation (569/16).
16% Paraformaldehyde (formaldehyde) aqueous solution | ELECTRON MICROSCOPY SCIENCES | 15710 | |
6-well cell culture plate | FALCON | 353846 | |
96-well cell culture plate | SPL life sciences | 30096 | |
Amphotericin B solution | Biological Industries | 03-028-1B | |
AZ 1514H photoresist | MicroChemicals GmbH | ||
AZ 351 B developer | MicroChemicals GmbH | ||
Bovine serum albumin (BSA) | Biological Industries | 03-010-1B | |
Cell and Tissue cultur flask | Biofil | TCF002250 | 75.0 cm^2 250 mL Vent cap, Non-treated |
Cell culture dish | Greiner Bio-One | 627-160 | 35 mm |
Cell Proliferation Kit (XTT-based) | Biological Industries | 20-300-1000 | |
Centrifuge tube | Biofil | CFT021500 | 50 mL |
Co80Fe20 at% sputter target | ACI Alloys | 99.95% | |
Collagen type I | Corning Inc. | 354236 | Rat Tail, concentration range 3-4 mg/mL |
Confocal microscope | Leica | TCS SP5 | |
Cy2-conjugated AffiniPure Donkey Anti-rabbit secondary antibody | Jackson ImmunoResearch Laboratories, Inc. | 711-165-152 | |
DAPI fluoromount-G | SouthernBiotech | 0100-20 | |
Disposable needle | KDL | 23 G | |
Disposable syringe | Medispo | 1160227640 | 10 mL |
Donor horse serum | Biological Industries | 04-124-1A | |
ELISA reader | Merk Millipore | BioTek synergy 4 hybrid microplate reader | |
Ethanol 70% | ROMICAL LTD | 19-009102-80 | |
Ethanol absolute (Dehydrated) | Biolab-chemicals | 52505 | |
Fetal bovine serum (FBS) | Biological Industries | 04-127-1A | |
Fresh murine β-NGF | Peprotech | 450-34 | |
GMW C-frame electromagnet . | Buckley systems LTD | 3470, 45 mm | |
Hydrochloric acid 32% | DAEJUNG CHEMICAL & METALS | 4170-4100 | |
ImageJ | US National Institutes of Health, Bethesda | NeuronJ plugin | |
Inductively coupled plasma (ICP) | Ametek Spectro | SPECTRO ARCOS ICP-OES, FHX22 MultiView plasma | |
Keithley source-measure | Keithley | 2400 | |
Keithley switching system | Keithley | 3700 | |
L-glutamine | Biological Industries | 03-020-1B | |
Light microscope | Leica | DMIL LED | |
Maskless photolithography | Heidelberg Inst. | MLA150 | |
Microscope Slides | BAR-NAOR | BN1042000C | |
Nitric acid 70% | Sigma-Aldrich | 438073 | |
Normal donkey serum (NDS) | Sigma | D9663 | |
PBS 10x | hylabs | BP507/1LD | |
PC12 cell line | ATCC | CRL-1721 | |
Pd sputter target | ACI Alloys | 99.95% | |
Penicillin-streptomycin nystatin solution | Biological Industries | 03-032-1B | |
PrestoBlue cell viability reagent | Molecular probes | A-13261 | resazurin-based |
Rabbit antibody to α-tubulin | Santa Cruz Biotechnology, Inc. | ||
RF magnetron sputtering system | Orion AJA Int. | Orion 8 | |
RPMI 1640 with l-glutamine | Biological Industries | 01-100-1A | |
Sonication bath | KUDOS | SK3210HP | Frequency: 53 kHz. Ultrasonic power: 135 W |
SQUID magnetometer | Quantum Design, CA | ||
Triton X-100 | CHEM-IMPEX INTERNATIONAL | 1279 | non-ionic surfactant |
XTT cell viability reagent |