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
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	<li>Lithography
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		<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>16% Paraformaldehyde (formaldehyde) aqueous solution</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>15710</td>
			<td></td>
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			<td>6-well cell culture plate</td>
			<td>FALCON</td>
			<td>353846</td>
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			<td>96-well cell culture plate</td>
			<td>SPL life sciences</td>
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			<td>Biological Industries</td>
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			<td>AZ 1514H photoresist</td>
			<td>MicroChemicals GmbH</td>
			<td></td>
			<td></td>
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			<td>AZ 351 B developer</td>
			<td>MicroChemicals GmbH</td>
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			<td>Biological Industries</td>
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			<td>Cell and Tissue cultur flask</td>
			<td>Biofil</td>
			<td>TCF002250</td>
			<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>
			<td>627-160</td>
			<td>35 mm</td>
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			<td>Cell Proliferation Kit (XTT-based)</td>
			<td>Biological Industries</td>
			<td>20-300-1000</td>
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			<td>Centrifuge tube</td>
			<td>Biofil</td>
			<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>99.95%</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>
			<td></td>
			<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>
			<td></td>
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			<td>DAPI fluoromount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-20</td>
			<td></td>
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			<td>Disposable needle</td>
			<td>KDL</td>
			<td></td>
			<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>BioTek synergy 4 hybrid microplate reader</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>
			<td></td>
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			<td>Fresh murine &#946;-NGF</td>
			<td>Peprotech</td>
			<td>450-34</td>
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		<tr>
			<td>GMW C-frame electromagnet .</td>
			<td>Buckley systems LTD</td>
			<td></td>
			<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>
			<td></td>
			<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>
		</tr>
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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>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Biological Industries</td>
			<td>03-020-1B</td>
			<td></td>
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			<td>Light microscope</td>
			<td>Leica</td>
			<td></td>
			<td>DMIL LED</td>
		</tr>
		<tr>
			<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>
			<td></td>
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			<td>Nitric acid 70%</td>
			<td>Sigma-Aldrich</td>
			<td>438073</td>
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			<td>Normal donkey serum (NDS)</td>
			<td>Sigma</td>
			<td>D9663</td>
			<td></td>
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		<tr>
			<td>PBS 10x</td>
			<td>hylabs</td>
			<td>BP507/1LD</td>
			<td></td>
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		<tr>
			<td>PC12 cell line</td>
			<td>ATCC</td>
			<td>CRL-1721</td>
			<td></td>
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		<tr>
			<td>Pd sputter target</td>
			<td>ACI Alloys</td>
			<td></td>
			<td>99.95%</td>
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			<td>Penicillin-streptomycin nystatin solution</td>
			<td>Biological Industries</td>
			<td>03-032-1B</td>
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			<td>PrestoBlue cell viability reagent</td>
			<td>Molecular probes</td>
			<td>A-13261</td>
			<td>resazurin-based</td>
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			<td>Rabbit antibody to &#945;-tubulin</td>
			<td>Santa Cruz Biotechnology, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RF magnetron sputtering system</td>
			<td>Orion AJA Int.</td>
			<td></td>
			<td>Orion 8</td>
		</tr>
		<tr>
			<td>RPMI 1640 with l-glutamine</td>
			<td>Biological Industries</td>
			<td>01-100-1A</td>
			<td></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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		<tr>
			<td>SQUID magnetometer</td>
			<td>Quantum Design, CA</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Triton X-100</td>
			<td>CHEM-IMPEX INTERNATIONAL</td>
			<td>1279</td>
			<td>non-ionic surfactant</td>
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		<tr>
			<td>XTT cell viability reagent</td>
			<td></td>
			<td></td>
			<td></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. ...

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