Name: fabrication of magnetic platforms for micron-scale organization of interconnected neurons
Uploaded: 2021-07-14T14:00:00
Description: Watch this Scientific Journal Video about Fabrication of Magnetic Platforms for Micron-Scale Organization of Interconnected Neurons at JoVE.com

This research was supported by the Ministry of Science &#38; Technology, Israel, and by the Israeli Science Foundation (569/16).

NOTE: Perform all biological reactions in a biosafety cabinet.
1. Magnetic platform fabrication
<ol>
	<li>Lithography
	<ol>
		<li>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.</li>
		<li>Coat the glass with photoresist using spin-coating at 6,000 rpm for 60 s, to attain 1.5 &#181;m thickness, and bake at 100 &#176;C for 60 s. Expose t...

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, b...

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 &#34;hot spots&#34; 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.

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			<td>AZ 1514H photoresist</td>
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			<td>AZ 351 B developer</td>
			<td>MicroChemicals GmbH</td>
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			<td>Cell and Tissue cultur flask</td>
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			<td>75.0 cm^2 250 mL Vent cap, Non-treated</td>
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			<td>Cell culture dish</td>
			<td>Greiner Bio-One</td>
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			<td>Centrifuge tube</td>
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			<td>CFT021500</td>
			<td>50 mL</td>
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			<td>Co80Fe20 at% sputter target</td>
			<td>ACI Alloys</td>
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			<td>Collagen type I</td>
			<td>Corning Inc.</td>
			<td>354236</td>
			<td>Rat Tail, concentration range 3-4 mg/mL</td>
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			<td>Confocal microscope</td>
			<td>Leica</td>
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			<td>TCS SP5</td>
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			<td>Cy2-conjugated AffiniPure Donkey Anti-rabbit secondary antibody</td>
			<td>Jackson ImmunoResearch Laboratories, Inc.</td>
			<td>711-165-152</td>
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			<td>DAPI fluoromount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-20</td>
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			<td>Disposable needle</td>
			<td>KDL</td>
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			<td>23 G</td>
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			<td>Disposable&#160; syringe</td>
			<td>Medispo</td>
			<td>1160227640</td>
			<td>10 mL</td>
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			<td>Donor horse serum</td>
			<td>Biological Industries</td>
			<td>04-124-1A</td>
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			<td>ELISA reader</td>
			<td>Merk Millipore</td>
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			<td>Ethanol 70%</td>
			<td>ROMICAL LTD</td>
			<td>19-009102-80</td>
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			<td>Ethanol absolute (Dehydrated)</td>
			<td>Biolab-chemicals</td>
			<td>52505</td>
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			<td>Fetal bovine serum (FBS)</td>
			<td>Biological Industries</td>
			<td>04-127-1A</td>
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			<td>Fresh murine &#946;-NGF</td>
			<td>Peprotech</td>
			<td>450-34</td>
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			<td>GMW C-frame electromagnet .</td>
			<td>Buckley systems LTD</td>
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			<td>3470, 45 mm</td>
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			<td>Hydrochloric acid 32%</td>
			<td>DAEJUNG CHEMICAL &#38; METALS</td>
			<td>4170-4100</td>
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			<td>ImageJ</td>
			<td>US National Institutes of Health, Bethesda</td>
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			<td>NeuronJ plugin</td>
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			<td>Inductively coupled plasma (ICP)</td>
			<td>Ametek Spectro</td>
			<td></td>
			<td>SPECTRO ARCOS ICP-OES, FHX22 MultiView plasma</td>
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			<td>Keithley source-measure</td>
			<td>Keithley</td>
			<td></td>
			<td>2400</td>
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			<td>Keithley switching system</td>
			<td>Keithley</td>
			<td></td>
			<td>3700</td>
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			<td>L-glutamine</td>
			<td>Biological Industries</td>
			<td>03-020-1B</td>
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			<td>Light microscope</td>
			<td>Leica</td>
			<td></td>
			<td>DMIL LED</td>
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			<td>Maskless photolithography</td>
			<td>Heidelberg Inst.</td>
			<td></td>
			<td>MLA150</td>
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			<td>Microscope Slides</td>
			<td>BAR-NAOR</td>
			<td>BN1042000C</td>
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			<td>Nitric acid 70%</td>
			<td>Sigma-Aldrich</td>
			<td>438073</td>
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			<td>Sigma</td>
			<td>D9663</td>
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			<td>PBS 10x</td>
			<td>hylabs</td>
			<td>BP507/1LD</td>
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			<td>PC12 cell line</td>
			<td>ATCC</td>
			<td>CRL-1721</td>
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			<td>Pd sputter target</td>
			<td>ACI Alloys</td>
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			<td>99.95%</td>
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			<td>resazurin-based</td>
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			<td>Rabbit antibody to &#945;-tubulin</td>
			<td>Santa Cruz Biotechnology, Inc.</td>
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			<td>RF magnetron sputtering system</td>
			<td>Orion AJA Int.</td>
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			<td>Orion 8</td>
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			<td>RPMI 1640 with l-glutamine</td>
			<td>Biological Industries</td>
			<td>01-100-1A</td>
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			<td>Sonication bath</td>
			<td>KUDOS</td>
			<td>SK3210HP</td>
			<td>Frequency: 53 kHz. Ultrasonic power: 135 W</td>
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			<td>SQUID magnetometer</td>
			<td>Quantum Design, CA</td>
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			<td>Triton X-100</td>
			<td>CHEM-IMPEX INTERNATIONAL</td>
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			<td>non-ionic surfactant</td>
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			<td>XTT cell viability reagent</td>
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fabrication of magnetic platforms for micron-scale organization of interconnected neurons

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.

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) relativ...

Watch this Scientific Journal Video about Fabrication of Magnetic Platforms for Micron-Scale Organization of Interconnected Neurons at JoVE.com

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

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. ...

Our approach makes it possible to control neural network formation using magnetic manipulations. This is an effective tool for in vitro studies of networks and offers novel therapeutic direction for biointerfacing devices. With this technique we can control both cell's location and growth at the micron scale, allowing flexible design of the magnetic pattern and thus the network organization.An efficient and non-toxic magnetic nanoparticle steriloptic is crucial. To succeed, appropriate magnetic nanoparticles are needed. The important features are the size, coating, and situation magnetization value.Demonstrating the procedures will be Reut Plen and Dafna Levenberg, master students for my lab. To begin, cut glass slides into two by two centimeter pieces using a scriber pen. Clean the glass slides in acetone and then isopropanol for five minutes each in an ultrasonication bath.Then dry them with ultra high purity nitrogen. Coat the glass with photoresist using spin coating at 6, 000 RPM for 60 seconds to attain 1.5 micrometer thickness and bake 100 degrees Celsius for 60 seconds. Expose the sample to a lightsource using a photomask or maskless lithography to achieve the desired pattern.Develop the sample for 40 seconds in a developer diluted in distilled water according to the manufacturer's instructions. Then wash it in water for 45 seconds and dry it with UHP nitrogen gas. Inspect the pattern under an optical microscope.Insert the sample into the load lock chamber of the deposition system and wait for base pressure. Transfer the sample in the main chamber and set the sample at the appropriate height for deposition. Increase the power on each target until the desired rate is achieved.Turn on rotation, then deposit the ferromagnetic multilayer, alternating between the cobalt, iron, and palladium targets by opening and closing the target shutters respectively. Deposit 14 bilayers of cobalt iron palladium and finish with an additional palladium capping layer. After deposition is complete, unload the sample from the deposition system.Soak the sample in acetone for 30 minutes and rinse it with isopropanol. Then dry it with UHP nitrogen and examine it under the microscope. Keep it in a clean and dry environment until use.Use a glass slide with a cross shaped magnetic bar of 100 micrometer width deposited with ferromagnetic multilayers. Attach the sample to the holder using double sided tape. Use a wire bonder to bond four 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 transverse voltage measurements as described in the text manuscript. Sweep the magnetic field and measure the transverse voltage as a function of the field.Plot the transverse resistance as a function of the magnetic field to determine the anomalous Hall signal, which is proportional to the perpendicular magnetization in the film. Prepare basic growth medium and differentiation medium for PC12 cell culture as described in the text manuscript. Grow cells in a non-treated culture flask with 10 milliliters of basic growth medium, adding 10 milliliters of medium to the flask every two to three days.Subculture the cells after eight days. For cellular uptake, centrifuge the cell suspension for eight minutes at 200 G and room temperature, then discard the supernatant. Resuspend the cells in three milliliters of fresh basic growth medium.Centrifuge the cells again for five minutes, then discard the supernatant and resuspend the cells in three milliliters of fresh differentiation medium. Aspirate the cells 10 times using a syringe and needle to break up the cell clusters, then count the cells using a hemocytometer and seed one million cells in a regular, uncoated 35 millimeter dish. Add the calculated volume of MNP suspension and differentiation medium to the dish to achieve the desired MNP concentration.Mix the cells, MNPs, and differentiation medium, then incubate the dish in a 5%carbon dioxide humidified incubator at 37 degrees Celsius for 24 hours. Clean the patterned substrate with 70%ethanol and place it in a 35 millimeter culture dish in the hood. Place a large magnet below the patterned substrate for one minute, then remove it by moving the dish up and away and taking the magnet out of the hood.Turn on the ultraviolet light for 15 minutes. To prepare a collagen coated glass substrate, dilute collagen type one at a one to 50 ratio in 30%ethanol. Cover the glass with the solution.Leave the dish uncovered in the hood for four hours until all the solution evaporates, then wash it three times in sterile PBS. Remove the cells from the incubator, centrifuge the cell suspension for five minutes at 200 G and discard the supernatant. Resuspend the cells in one milliliter of fresh differentiation medium and count the cells using a hemocytometer.Seed 10 to the five cells atop the substrate in a 35 millimeter culture dish and add two milliliters of differentiation medium. Incubate the culture in a 5%carbon dioxide humidified incubator at 37 degrees Celsius. After 24 hours, add one to 100 fresh murine beta-NGF to the cells.Renew the differentiation medium and add fresh beta-NGF every two days. Image the cells every two days using light microscopy and perform immunostaining on the cells after network formation. Magnetic platforms with different geometric shapes were fabricated and fluorescent iron oxide MNPs were synthesized by nucleation.Magnetometric measurements of the MNPs show that the magnetization curve had no hysteresis, a low saturation field, and relatively high magnetization saturation. PC12 cells were incubated in medium mixed with the iron oxide fluorescent MNPs, transforming them into magnetic units. The MNPs were internalized into the cells'soma, but not in the nuclei.The iron concentration inside the cells increased with the increase in MNP concentration in the medium. The viability of MNP loaded PC12 cells for different concentrations of MNPs was evaluated by comparing morphological parameters of MNP loaded cells using Shoal analysis, showing no difference from control cells. XTT and Resazurin based assays confirmed that the evaluated concentrations of MNPs showed no significant cytotoxicity toward the cells.PC12 cells with and without MNP treatment were grown and differentiated on a magnetic substrate. 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. Positioning of cells on a substrate with hexagonal geometry is shown here.75%of MNP loaded cell bodies were in contact with the magnetic stripes, whereas only 35%of the unmagnetized cells were located on the stripes. In addition to the cell positioning effect, these magnetic platforms were found to control the directionality of the growing neurites. To evaluate the magnetic effect on neuronal growth directionality, the angle between the neurites and the magnetic stripes was measured, showing significant correlation with the magnetic stripes'orientation.This procedure is easily adapted for directing active compounds, like drugs, after conjugating them to magnetic nanoparticles. The can be translated to high throughput drug screening assays or local treatment within tissues. This novel approach opens new possibilities for functionalizing other substrates or devices by adding magnetic attractive structures.Currently, we are developing improved biocompatible neuron chip interfaces.

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