Fabrication of Magnetic Platforms for Micron-Scale Organization of Interconnected Neurons

Summary

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.

Abstract

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.

Introduction

Micropatterning of neurons holds great potential for tissue regeneration^1,2,3,4,5 and the development of neuro-electronic devices^6,7,8. 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 cues^9,10,11,12 for guiding neuronal growth. Here, a novel approach focuses on controlling cell positioning by magnetic field gradients^{13,14,15,16,17}, 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 neurons^24,25,26. 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 treatments^28,29,30,31. Specifically, studies deal with the application of MNPs and neuron networks^32,33,34,35. 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.

Protocol

NOTE: Perform all biological reactions in a biosafety cabinet.

1. Magnetic platform fabrication

1. Lithography
   1. Cut glass slides into 2 x 2 cm² using a scriber pen. Clean the glass slides in acetone and then isopropanol for 5 min each in an ultra-sonication bath. Dry with ultra-high purity (UHP) nitrogen.
   2. Coat the glass with photoresist using spin-coating at 6,000 rpm for 60 s, to attain 1.5 µm thickness, and bake at 100 °C for 60 s. Expose the sample to a light source, using an appropriate wavelength for photoresist, with a desired pattern, using a photomask or mask-less lithography.
   3. Develop for 40 s in a developer, diluted in distilled water (DW) according to the manufacturer's instructions; wash in DW for 45 s, and dry with UHP nitrogen gas. Inspect the pattern under an optical microscope.
2. Sputter deposition
   1. Insert the sample into the main chamber of the deposition system and wait for base pressure (~5 × 10^-8 Torr). Open the gas flow; set the argon flow for standard sputtering (28 sccm [standard cubic cm per min) herein. Ignite the sputter targets, then set the sputter pressure to 3 mTorr.
   2. Increase the power on each target until the desired rate is achieved.
      ​NOTE: Pd Rate: 0.62 A/s = 1.0 nm in 16 s; Co₈₀Fe₂₀ Rate: 0.32 A/s, 0.2 nm in 6.25 s.
   3. Turn on rotation. Deposit the FM multilayer, alternating between the Co₈₀Fe₂₀ and Pd targets, by opening and closing the target shutters, respectively. Deposit 14 bilayers of Co₈₀Fe₂₀ (0.2 nm)/Pd (1.0 nm), and finish with an additional 2 nm Pd capping layer.
   4. Lift-off: Soak the sample in acetone for 30 min, and rinse with isopropanol. Then, dry with UHP nitrogen, and keep the sample in a clean and dry environment until use.

2. Characterization of magnetic device via transport measurements

1. Use a Si substrate or glass slide with a cross-shaped magnetic bar of 100 µm width, deposited with FM multilayers (see Figure 1C inset). Attach the sample to the holder using double-sided tape.
2. Using a wire bonder, bond 4 wires to the sample, one on each leg of the cross electrode. Set the sample-holder and sample inside the transport measurement system with a magnetic field so that the magnetic field is perpendicular to the sample. Perform measurements at room temperature.
3. Perform transverse voltage (V_T) measurement of the device; follow the markings in Figure 1C (inset): apply a current of 1 mA between contacts 1 and 3; measure the V_T between contacts 2 and 4; then, apply a current between 2 and 4, and measure the voltage between 1 and 3. Finally, calculate the difference between the voltages of both the measurements and divide by 2 to obtain V_T. Use a switching system to automatically change between the two measurement configurations.
4. Sweep the magnetic field between 0.4 T to -0.4 T in steps of 5 mT and measure the V_T as a function of the field. Plot the transverse resistance (V_T/I) vs. the magnetic field to determine the anomalous Hall signal, which is proportional to the perpendicular magnetization in the film.

3. Characterization of MNPs and magnetic multilayers by magnetometry measurements

1. Magnetometric measurement for FM multilayers
   1. Deposit the FM multilayer on the Si substrate (see section 1.2). Cut the sample into 6 squares of 4 x 4 mm² size. Stack the samples one on top of the other and arrange them in the capsule perpendicular to the direction of the magnetic field (see Figure 1D inset).
   2. Insert the capsule into the magnetometer and measure the magnetization at room temperature. Sweep the magnetic field between -0.4 T and 0.4 T.
   3. Calculate the total volume of the magnetic material, considering the thickness of the magnetic layer, the size of the samples, and the number of substrates. Divide the magnetization by the total volume of the magnetic material.
   4. Plot the magnetization (per unit volume) vs. the magnetic field. Subtract the diamagnetic background of the substrate from the high magnetic field response and extrapolate the saturation magnetization of the FM from the graph.
2. Magnetometric measurement for MNPs
   1. Insert a designated mass of MNPs into a synthetic polymer capsule. Consider a larger volume if measuring small magnetization saturation values.
   2. If the MNPs are suspended in a solvent, dry the MNPs by leaving the capsule open overnight. Insert the capsule into the magnetometer and measure the magnetization at room temperature. Sweep the magnetic field between -0.2 T and 0.2 T.
   3. Calculate the total mass of the MNPs by multiplying the designated volume by the particle concentration. Normalize the results to 1 g.
   4. Plot the normalized magnetization (per gram) vs. the magnetic field. Extrapolate the magnetization saturation of the MNPs from the graph.

4. Collagen-coating protocol

1. Coating plastic dishes
   1. Prepare 0.01 M HCl by adding 490 µL of HCl to 500 mL of autoclaved double-distilled water (DDW).
      NOTE: Perform this step only in the chemical hood.
   2. Dilute collagen type 1 (solution from rat tail) 1:60-1:80 in 0.01 M HCl to obtain the final working concentration of 50 µg/mL. Place 1.5 mL of the diluted solution in a 35 mm culture dish. Leave the dish in the hood for 1 h, covered.
   3. Remove the solution, and wash 3x in sterile 1x phosphate-buffered saline (PBS). The dish is ready for cell seeding.
2. Coating glass slides
   1. Dilute collagen type 1 (solution from rat tail) 1:50 in 30% v/v ethanol. For coating a 35 mm dish, add 20 µL of collagen to 1 mL of 30% ethanol.
   2. Cover the dish with the solution, and wait until all the solution evaporates, leaving the dish uncovered for a few hours. Wash 3x in sterile 1x PBS; the glass slide is ready for cell seeding.

5. Cellular MNP uptake and viability

1. Cellular MNP uptake
   1. Prepare basic growth medium for PC12 cell culture by adding 10% horse serum (HS), 5% fetal bovine serum (FBS), 1% L-glutamine, 1% penicillin/streptomycin, and 0.2% amphotericin to Roswell Park Medical Institute (RPMI) medium, and filter using a 0.22 µm nylon filter.
   2. Add 1% horse serum (HS), 1% L-glutamine, 1% penicillin/streptomycin, and 0.2% amphotericin to RPMI medium to prepare PC12 differentiation medium, and filter using a 0.22 µm nylon filter.
   3. Grow cells in a non-treated culture flask with 10 mL of basic growth medium; add 10 mL of basic growth medium to the flask every 2-3 days, and sub-culture the cells after 8 days.
   4. For cellular uptake, centrifuge the cell suspension in a centrifuge tube for 8 min at 200 × g and room temperature, and discard the supernatant.
   5. Resuspend the cells in 3 mL of fresh basic growth medium. Again, centrifuge the cell suspension for 5 min at 200 × g and room temperature, and discard the supernatant. Resuspend the cells in 3 mL of fresh differentiation medium.
   6. Aspirate the cells 10x using a syringe and a needle to break up cell clusters. Count the cells using a hemocytometer, and seed 10⁶ cells in a regular uncoated 35 mm dish.
   7. Add to the dish the calculated volume of MNP suspension and volume of differentiation medium to achieve the desired MNP concentration and total volume. Mix the cells, MNPs, and differentiation medium; incubate the dish in a 5% CO2 humidified incubator at 37 °C for 24 h.
   8. Centrifuge the cell suspension for 5 min at 200 × g at room temperature, and discard the supernatant. Resuspend the cells in 1 mL of fresh differentiation medium, and count the cells using a hemocytometer.
2. MNP-loaded cell differentiation
   1. Perform uptake protocol (section 5.1). Seed 8 × 10⁴ MNP-loaded cells on a 35 mm, collagen type l-coated dish in the presence of differentiation medium (see collagen coating protocol in section 4.1). After 24 h, add 1:100 fresh murine beta-nerve growth factor (β-NGF) (final concentration 50 ng/mL).
   2. Renew the differentiation medium and add fresh murine β-NGF every 2 days. Image the cells every 2 days using optical microscopy. After network formation (6-8 days for PC12 cells), image the cells using confocal microscopy, and observe the fluorescence of the particles.
3. Viability assay for MNP-loaded cells: 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) cell viability test.
   1. Prepare the basic growth medium according to step 5.1.1. Cultivate the PC12 cells with MNPs at different concentrations (0.1 mg/mL, 0.25 mg/mL, and 0.5 mg/mL in basic growth medium) and also without MNPs for the control in triplicate in a flat 96-well plate (total volume of 100 µL/well). Incubate the cells for 24 h in a 5% CO₂, humidified incubator at 37 °C.
   2. Prepare blank wells containing medium without cells for background correction. Thaw the XTT reagent solution, and the reaction solution containing N-methyl dibenzopyrazine methyl sulfate) in a 37 °C bath immediately prior to use. Swirl gently until clear solutions are obtained.
   3. For one 96-well plate, mix 0.1 mL of activation solution with 5 mL of XTT reagent. Add 50 µL of the reaction solution to each well, slightly shake the plate for an even distribution of the dye in the wells, and then incubate the plate in an incubator for 5 h.
   4. Measure the absorbance of the sample against the blank wells using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm. Measure the reference absorbance using a wavelength of 630 nm and subtract it from the 450 nm measurement.
   5. As slight spontaneous absorbance occurs in the culture medium incubated with the XTT reagent at 450 nm, subtract the average absorbance of the blank wells from that of the other wells. Subtract signal values from parallel samples of MNPs at the same tested concentrations as the cell samples.
4. Viability assay for MNP-loaded cells: resazurin-based cell viability test
   1. Prepare basic growth medium according to step 5.1.1. Cultivate the PC12 cells with MNPs at different concentrations (0.1 mg/mL, 0.25 mg/mL, and 0.5 mg/mL in basic growth medium) and without MNPs as control in triplicate in a flat 96-well plate for 24 h. Incubate the cells for 24 h in a 5% CO₂ incubator at 37 °C. Prepare blank wells containing medium without cells.
   2. Wash the cells with 1x PBS. Add the resazurin-based reagent (10% w/v) to the medium and incubate for 2 h in a 37 °C incubator.
   3. Place 150 µL aliquots of the samples in the ELISA reader, and measure the absorbance at an excitation wavelength of 560 nm and emission wavelength of 590 nm. Subtract the signal values from the parallel samples of MNPs at the same tested concentrations as the cell samples.

6. Characterization of MNP concentration inside the cells using inductively coupled plasma (ICP)

1. Prepare basic growth medium according to step 5.1.1. Cultivate the PC12 cells with MNPs at different concentrations (0.1 mg/mL, 0.25 mg/mL, and 0.5 mg/mL in basic growth medium) and without MNPs as control in triplicate in a flat 96-well plate (total volume of 100 µL/well). Incubate in a 5% CO₂, humidified incubator at 37 °C for 24 h.
2. Transfer the suspension to a centrifuge tube (from each well separately), centrifuge cells at 200 × g for 5 min at room temperature and discard the supernatant. Resuspend the cells in 1 mL of fresh differentiation medium, and count the cells using a hemocytometer.
3. Lyse the cells by treatment with 100 µL of 70% nitric acid to each well separately for at least 15 min. Add 5 mL of DDW to the lysed cells and filter the solutions.
4. Measure the iron concentration using ICP and use the cell count to record Fe concentration per cell.

7. Cell differentiation and growth on magnetic platform

1. Clean the patterned substrate with 70% v/v/ ethanol and place the substrate in a 35 mm culture dish in the hood. Place a large magnet (~1500 Oe) below the patterned substrate for 1 min and remove the magnet by first moving the dish up and away from the magnet, and then take the magnet out of the hood. Turn on the ultraviolet light for 15 min.
2. Coat the substrate with collagen type 1 according to section 4.2. Suspend the cells after cellular MNP uptake (section 5.1), seed 10⁵ cells in a 35 mm culture dish, and add 2 mL of differentiation medium. Incubate the culture in a 5% CO₂, humidified incubator at 37 °C.
3. After 24 h, add 1:100 fresh murine β-NGF (final concentration of 50 ng/mL). Renew the differentiation medium and add fresh murine β-NGF every 2 days. Image the cells every 2 days using light microscopy, and after network formation, perform immunostaining on the cells (section 8.1).

8. MNP-loaded cell staining

1. Tubulin immunostaining
   1. Prepare 4% paraformaldehyde (PFA) solution by mixing 10 mL of 16% w/v PFA solution, 4 mL of 10x PBS, and 26 mL of DDW. Prepare 50 mL of 1% PBT by adding 500 µL of a non-ionic surfactant to 50 mL of 1x PBS. Prepare 50 mL of 0.5% PBT by mixing 25 mL of 1% PBT with 25 mL of 1x PBS. Prepare blocking solution by mixing 1% bovine serum albumin and 1% normal donkey serum in 0.25% PBT.
      ​NOTE: Use PFA only inside the chemical hood.
   2. Remove the supernatant medium from the cells. Fix the MNP-loaded cells in 4% PFA for 15 min at room temperature inside a chemical hood. Wash the MNP-loaded cells 3x in 1x PBS, 5 min each wash, inside a chemical hood.
   3. Permeabilize the MNP-loaded cells with 0.5% PBT for 10 min. Incubate the MNP-loaded cells first in blocking solution for 45 min at room temperature and then with rabbit anti- α-tubulin antibody in blocking solution overnight at 4 °C. Wash MNP-loaded cells 3x in 1x PBS, 5 min each wash.
   4. Incubate the MNP-loaded cells with Cy2-conjugated donkey anti-rabbit secondary antibody for 45 min in darkness and at room temperature. Wash the MNP-loaded cells 3x in 1x PBS, 5 min each wash.
   5. Perform confocal imaging. For tubulin, use an excitation wavelength of 492 nm and an emission wavelength of 510 nm. For the MNPs (rhodamine), use an excitation wavelength of 578 nm and an emission wavelength of 613 nm.
2. Nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI)
   1. Wash the MNP-loaded cells 3x in 1x PBS, 5 min each wash. Remove excess liquid around the sample, add 1 drop (~50 µL) of mounting medium containing DAPI to cover an area of 22 mm x 22 mm, and incubate for 5 min in darkness and at room temperature.
   2. Wash the MNP-loaded cells 3x in 1x PBS, 5 min each wash. Perform confocal imaging. For DAPI, use an excitation wavelength of 358 nm and an emission wavelength of 461 nm. For the MNPs (rhodamine), use an excitation wavelength of 578 nm and an emission wavelength of 613 nm.

9. Measurements and statistical analysis

1. Morphometric analysis of MNP-loaded cell differentiation
   1. To measure the number of intersections at various distances from the cell body, acquire phase images of cultured cells up to 3 days after treatment with NGF.
      ​NOTE: If done later, the cells may develop networks, preventing single-cell resolution measurements.
      1. Open the images in the image processing program, ImageJ, and use the NeuronJ plug-in, which enables a semi-automatic neurite tracing and length measurement³⁶. Using the neurite tracer plug-in, trace the neurites and convert the data to binary images. Define the center of the soma.
      2. Perform Sholl analysis, available in the NeuronJ plug-in. Define the maximal radius. Repeat the experiment three times. Analyze more than 100 cells in each experiment.
2. Cell localization analysis
   1. To determine the percentage of cells localized on the magnetic area after 3 days of incubation, acquire confocal microscopic images of cells with and without MNP uptake. Use DAPI staining (section 8.2).
   2. Manually count the cells atop or partially atop the pattern (touching cells) and the cells that are not. Repeat for three experiments. Analyze more than 400 cells with MNP and without uptake.
   3. Calculate the relative proportion of the cells that are atop the magnetic patterns out of the total number of cells, with and without MNPs. Additionally, calculate the percentage of the magnetic pattern's effective area by adding the cell body diameter to the pattern width to determine the random probability of cells landing on a magnetic pattern.
   4. Perform a single sample Z-test to analyze whether the cell distribution is a result of isotropic cell landing, or if there is a preferred bias to the magnetic pattern.
3. Growth directionality analysis
   1. To quantify the effect on neurite outgrowth directionality, acquire confocal microscopic images of the cells with and without MNP treatment after 8 days of incubation. Perform immuno-staining (section 8.1).
   2. Using ImageJ software, measure the angle between the cell neurite and the magnetic stripes in both conditions.
      ​NOTE: Analyze only neurites that originate from somas located on the magnetic stripes.
   3. Plot the distribution of the neurites' angles relative to the direction of the stripes (Δθ). Perform a Chi-squared test of the distribution of Δθ to demonstrate that the distribution is not normal or uniform.

Representative Results

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 edges^22,37. 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 (M_S) 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 M_S value of the multilayers was measured using a magnetometer (Figure 1D), as described in section 3.1. The M_S 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 M_S 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.

Discussion

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 outgrowth^40,41, 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 stimulation^42,43.

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 magnetization^44,45 easy axis and attraction to different edges of the electrodes^22,37. 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 endocytosis^47,48.

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.

Materials

Name	Company	Catalog Number	Comments
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