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 the sample to a light source, using an appropriate wavelength for photoresist, with a desired pattern, using a photomask or mask-less lithography.</li>
		<li>Develop for 40 s in a developer, diluted in distilled water (DW) according to the manufacturer&#39;s instructions; wash in DW for 45 s, and dry with UHP nitrogen gas. Inspect the pattern under an optical microscope.</li>
	</ol>
	</li>
	<li>Sputter deposition
	<ol>
		<li>Insert the sample into the main chamber of the deposition system and wait for base pressure (~5 &#215; 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.</li>
		<li>Increase the power on each target until the desired rate is achieved. 
		&#8203;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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Characterization of magnetic device via transport measurements
<ol>
	<li>Use a Si substrate or glass slide with a cross-shaped magnetic bar of 100 &#181;m width, deposited with FM multilayers (see Figure 1C inset). Attach the sample to the holder using double-sided tape.</li>
	<li>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.</li>
	<li>Perform transverse voltage (VT) measurement of the device; follow the markings in Figure 1C (inset): apply a current of 1 mA between contacts 1 and 3; measure the VT 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 VT. Use a switching system to automatically change between the two measurement configurations.</li>
	<li>Sweep the magnetic field between 0.4 T to -0.4 T in steps of 5 mT and measure the VT as a function of the field. Plot the transverse resistance (VT/I) vs. the magnetic field to determine the anomalous Hall signal, which is proportional to the perpendicular magnetization in the film.</li>
</ol>
3. Characterization of MNPs and magnetic multilayers by magnetometry measurements
<ol>
	<li>Magnetometric measurement for FM multilayers
	<ol>
		<li>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).</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Magnetometric measurement for MNPs
	<ol>
		<li>Insert a designated mass of MNPs into a synthetic polymer capsule. Consider a larger volume if measuring small magnetization saturation values.</li>
		<li>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.</li>
		<li>Calculate the total mass of the MNPs by multiplying the designated volume by the particle concentration. Normalize the results to 1 g.</li>
		<li>Plot the normalized magnetization (per gram) vs. the magnetic field. Extrapolate the magnetization saturation of the MNPs from the graph.</li>
	</ol>
	</li>
</ol>
4. Collagen-coating protocol
<ol>
	<li>Coating plastic dishes
	<ol>
		<li>Prepare 0.01 M HCl by adding 490 &#181;L of HCl to 500 mL of autoclaved double-distilled water (DDW). 
		NOTE: Perform this step only in the chemical hood.</li>
		<li>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 &#181;g/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.</li>
		<li>Remove the solution, and wash 3x in sterile 1x phosphate-buffered saline (PBS). The dish is ready for cell seeding.</li>
	</ol>
	</li>
	<li>Coating glass slides
	<ol>
		<li>Dilute collagen type 1 (solution from rat tail) 1:50 in 30% v/v ethanol. For coating a 35 mm dish, add 20 &#181;L of collagen to 1 mL of 30% ethanol.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
5. Cellular MNP uptake and viability
<ol>
	<li>Cellular MNP uptake
	<ol>
		<li>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 &#181;m nylon filter.</li>
		<li>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 &#181;m nylon filter.</li>
		<li>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.</li>
		<li>For cellular uptake, centrifuge the cell suspension in a centrifuge tube for 8 min at 200 &#215; g and room temperature, and discard the supernatant.</li>
		<li>Resuspend the cells in 3 mL of fresh basic growth medium. Again, centrifuge the cell suspension for 5 min at 200 &#215; g and room temperature, and discard the supernatant. Resuspend the cells in 3 mL of fresh differentiation medium.</li>
		<li>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.</li>
		<li>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 &#176;C for 24 h.</li>
		<li>Centrifuge the cell suspension for 5 min at 200 &#215; 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.</li>
	</ol>
	</li>
	<li>MNP-loaded cell differentiation
	<ol>
		<li>Perform uptake protocol (section 5.1). Seed 8 &#215; 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 (&#946;-NGF) (final concentration 50 ng/mL).</li>
		<li>Renew the differentiation medium and add fresh murine &#946;-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.</li>
	</ol>
	</li>
	<li>Viability assay for MNP-loaded cells: 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) cell viability test.
	<ol>
		<li>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 &#181;L/well). Incubate the cells for 24 h in a 5% CO2, humidified incubator at 37 &#176;C.</li>
		<li>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 &#176;C bath immediately prior to use. Swirl gently until clear solutions are obtained.</li>
		<li>For one 96-well plate, mix 0.1 mL of activation solution with 5 mL of XTT reagent. Add 50 &#181;L 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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Viability assay for MNP-loaded cells: resazurin-based cell viability test
	<ol>
		<li>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 &#176;C. Prepare blank wells containing medium without cells.</li>
		<li>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 &#176;C incubator.</li>
		<li>Place 150 &#181;L 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.</li>
	</ol>
	</li>
</ol>
6. Characterization of MNP concentration inside the cells using inductively coupled plasma (ICP)
<ol>
	<li>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 &#181;L/well). Incubate in a 5% CO2, humidified incubator at 37 &#176;C for 24 h.</li>
	<li>Transfer the suspension to a centrifuge tube (from each well separately), centrifuge cells at 200 &#215; 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.</li>
	<li>Lyse the cells by treatment with 100 &#181;L 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.</li>
	<li>Measure the iron concentration using ICP and use the cell count to record Fe concentration per cell.</li>
</ol>
7. Cell differentiation and growth on magnetic platform
<ol>
	<li>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.</li>
	<li>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 &#176;C.</li>
	<li>After 24 h, add 1:100 fresh murine &#946;-NGF (final concentration of 50 ng/mL). Renew the differentiation medium and add fresh murine &#946;-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).</li>
</ol>
8. MNP-loaded cell staining
<ol>
	<li>Tubulin immunostaining
	<ol>
		<li>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 &#181;L 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. 
		&#8203;NOTE: Use PFA only inside the chemical hood.</li>
		<li>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.</li>
		<li>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- &#945;-tubulin antibody in blocking solution overnight at 4 &#176;C. Wash MNP-loaded cells 3x in 1x PBS, 5 min each wash.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Nuclear staining with 4&#8242;,6-diamidino-2-phenylindole (DAPI)
	<ol>
		<li>Wash the MNP-loaded cells 3x in 1x PBS, 5 min each wash. Remove excess liquid around the sample, add 1 drop (~50 &#181;L) 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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
9. Measurements and statistical analysis
<ol>
	<li>Morphometric analysis of MNP-loaded cell differentiation
	<ol>
		<li>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. 
		&#8203;NOTE: If done later, the cells may develop networks, preventing single-cell resolution measurements.
		<ol>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cell localization analysis
	<ol>
		<li>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).</li>
		<li>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.</li>
		<li>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&#39;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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Growth directionality analysis
	<ol>
		<li>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).</li>
		<li>Using ImageJ software, measure the angle between the cell neurite and the magnetic stripes in both conditions. 
		&#8203;NOTE: Analyze only neurites that originate from somas located on the magnetic stripes.</li>
		<li>Plot the distribution of the neurites&#39; angles relative to the direction of the stripes (&#916;&#952;). Perform a Chi-squared test of the distribution of &#916;&#952; to demonstrate that the distribution is not normal or uniform.</li>
	</ol>
	</li>
</ol>

The authors declare no competing financial interests.

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&#39; 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.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16% Paraformaldehyde (formaldehyde) aqueous solution</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plate</td>
			<td>FALCON</td>
			<td>353846</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well cell culture plate</td>
			<td>SPL life sciences</td>
			<td>30096</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B solution</td>
			<td>Biological Industries</td>
			<td>03-028-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>AZ 1514H photoresist</td>
			<td>MicroChemicals GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AZ 351 B developer</td>
			<td>MicroChemicals GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Biological Industries</td>
			<td>03-010-1B</td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>Greiner Bio-One</td>
			<td>627-160</td>
			<td>35 mm</td>
		</tr>
		<tr>
			<td>Cell Proliferation Kit (XTT-based)</td>
			<td>Biological Industries</td>
			<td>20-300-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>Biofil</td>
			<td>CFT021500</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Co80Fe20 at% sputter target</td>
			<td>ACI Alloys</td>
			<td></td>
			<td>99.95%</td>
		</tr>
		<tr>
			<td>Collagen type I</td>
			<td>Corning Inc.</td>
			<td>354236</td>
			<td>Rat Tail, concentration range 3-4 mg/mL</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td></td>
			<td>TCS SP5</td>
		</tr>
		<tr>
			<td>Cy2-conjugated AffiniPure Donkey Anti-rabbit secondary antibody</td>
			<td>Jackson ImmunoResearch Laboratories, Inc.</td>
			<td>711-165-152</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI fluoromount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable needle</td>
			<td>KDL</td>
			<td></td>
			<td>23 G</td>
		</tr>
		<tr>
			<td>Disposable&#160; syringe</td>
			<td>Medispo</td>
			<td>1160227640</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Donor horse serum</td>
			<td>Biological Industries</td>
			<td>04-124-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA reader</td>
			<td>Merk Millipore</td>
			<td></td>
			<td>BioTek synergy 4 hybrid microplate reader</td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>ROMICAL LTD</td>
			<td>19-009102-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute (Dehydrated)</td>
			<td>Biolab-chemicals</td>
			<td>52505</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Biological Industries</td>
			<td>04-127-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fresh murine &#946;-NGF</td>
			<td>Peprotech</td>
			<td>450-34</td>
			<td></td>
		</tr>
		<tr>
			<td>GMW C-frame electromagnet .</td>
			<td>Buckley systems LTD</td>
			<td></td>
			<td>3470, 45 mm</td>
		</tr>
		<tr>
			<td>Hydrochloric acid 32%</td>
			<td>DAEJUNG CHEMICAL &#38; METALS</td>
			<td>4170-4100</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>US National Institutes of Health, Bethesda</td>
			<td></td>
			<td>NeuronJ plugin</td>
		</tr>
		<tr>
			<td>Inductively coupled plasma (ICP)</td>
			<td>Ametek Spectro</td>
			<td></td>
			<td>SPECTRO ARCOS ICP-OES, FHX22 MultiView plasma</td>
		</tr>
		<tr>
			<td>Keithley source-measure</td>
			<td>Keithley</td>
			<td></td>
			<td>2400</td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>BAR-NAOR</td>
			<td>BN1042000C</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitric acid 70%</td>
			<td>Sigma-Aldrich</td>
			<td>438073</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal donkey serum (NDS)</td>
			<td>Sigma</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 10x</td>
			<td>hylabs</td>
			<td>BP507/1LD</td>
			<td></td>
		</tr>
		<tr>
			<td>PC12 cell line</td>
			<td>ATCC</td>
			<td>CRL-1721</td>
			<td></td>
		</tr>
		<tr>
			<td>Pd sputter target</td>
			<td>ACI Alloys</td>
			<td></td>
			<td>99.95%</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin nystatin solution</td>
			<td>Biological Industries</td>
			<td>03-032-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>PrestoBlue cell viability reagent</td>
			<td>Molecular probes</td>
			<td>A-13261</td>
			<td>resazurin-based</td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Sonication bath</td>
			<td>KUDOS</td>
			<td>SK3210HP</td>
			<td>Frequency: 53 kHz. Ultrasonic power: 135 W</td>
		</tr>
		<tr>
			<td>SQUID magnetometer</td>
			<td>Quantum Design, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>CHEM-IMPEX INTERNATIONAL</td>
			<td>1279</td>
			<td>non-ionic surfactant</td>
		</tr>
		<tr>
			<td>XTT cell viability reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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) 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 (&#947;-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&#39; 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 &#62; 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 &#62; 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 &#181;m wide stripes and 100 &#181;m 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 &#181;m and line width of 50 &#181;m. 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&#39; diameter (~10 &#181;m 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 &#60; 10-6, n = 430).
In contrast, the statistical analysis of the magnetic hexagons revealed that 92% of the MNP-loaded cells&#39; 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 &#60; 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&#39; 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&#39; orientation, within &#916;&#952; &#60; 15&#176; relative to the stripes&#39; 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 &#916;&#952; revealed that it was not normal or uniform (Chi-square test, p &#60; 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 &#181;m and the lines width was 10 &#181;m; the circle diameter was 30 &#181;m. 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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62013/62013fig01.jpg" /> 
Figure 1: Characterization of the magnetic devices. (A) Optical microscopic images of magnetic devices with various geometric shapes. Scale bar = 200 &#181;m. (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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62013/62013fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62013/62013fig02.jpg" /> 
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 &#181;m (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&#8242;,6-diamidino-2-phenylindole; ICP = inductively coupled plasma. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62013/62013fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62013/62013fig3v2.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62013/62013fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62013/62013fig04.jpg" /> 
Figure 4: Cell localization atop magnetic devices. (A) Fluorescent images of PC12 cells loaded with MNPs growing on the magnetic stripes: (i) &#945;-tubulin labeling, (ii) DAPI staining, and (iii) merged image. Scale bar = 100 &#181;m. (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 &#181;m. (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&#8242;,6-diamidino-2-phenylindole. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62013/62013fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62013/62013fig05.jpg" /> 
Figure 5: Neuronal network directionality. (A) Confocal microscopic images of &#945;-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&#176; 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 &#181;m. (D) Zoom-in image of a magnetized cell developing neurites according to the magnetic pattern. Scale bar = 30 &#181;m. This figure has been modified from Marcus et al.37. Abbreviation: MNPs = magnetic nanoparticles.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62013/62013fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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. ...

fabrication-magnetic-platforms-for-micron-scale-organization

Nanyang Technological University

Research

JoVE Journal

Bioengineering

3.9K Views.  Bar-Ilan University. 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.

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

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment2.
Coagulation Factor VIII (FVIII)3 is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A&#160;-&#160;a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting4. Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete5. Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa6,7.
In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.&#160;The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.

We present a combination of Cryo-electron microscopy, lipid nanotechnology, and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory&#160;to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...