growth and differentiation of magnetic nano particle-loaded neurons on magnetic platforms

Growth and Differentiation of Magnetic Nano Particle-Loaded Neurons on Magnetic Platforms

Seed 1 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 I at a 1-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 5 minutes at 200 g and discard the supernatant. Resuspend the cells in 1 milliliter of fresh differentiation medium, and count the cells using a hemocytometer.
Seed 105 cells atop the substrate in a 35-millimeter culture dish, and add 2 milliliters of differentiation medium. Incubate the culture in a 5% carbon dioxide humidified incubator at 37 degrees Celsius. After 24 hours, add 1 to 100 fresh Murine beta-NGF to the cells. Renew the differentiation medium and add fresh beta-NGF every two days.

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Universidad San Sebastian

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JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

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1. Cellular magnetic nanoparticle (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>
2. 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 Oersted) 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 by diluting 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>
	<li>Suspend the cells after cellular MNP uptake, 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.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<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>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>Centrifuge tube</td>
			<td>Biofil</td>
			<td>CFT021500</td>
			<td>50 mL</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>Disposable needle</td>
			<td>KDL</td>
			<td></td>
			<td>23 G</td>
		</tr>
		<tr>
			<td>Disposable 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>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>L-glutamine</td>
			<td>Biological Industries</td>
			<td>03-020-1B</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>Penicillin-streptomycin nystatin solution</td>
			<td>Biological Industries</td>
			<td>03-032-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 with l-glutamine</td>
			<td>Biological Industries</td>
			<td>01-100-1A</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Indech, G., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/62013">Fabrication of Magnetic Platforms for Micron-Scale Organization of Interconnected Neurons.</a> J. Vis. Exp. (2021).
This video demonstrates the uptake of fluorescent magnetic nanoparticles (MNPs) by the neuronal cells via endocytosis. Growing these MNP-loaded neurons on the magnetically patterned substrate enables the attachment of the cells. The addition of a growth factor containing medium aids in the growth of neuron extensions, while the magnetic field of the substrate directs the orientation of the extension.

1. Cellular magnetic nanoparticle (MNP) uptake

	Prepare basic growth medium for PC12 cell culture by adding 10% horse serum (HS), 5% fetal bovine serum ...

Begin by adding neuronal cells to a suitable medium in a culture dish.
Add fluorescent magnetic nanoparticles, or MNPs, containing a magnetic core tagged with a fluorescent dye.
During incubation, the cell takes up the MNP through endocytosis and becomes magnetized.
Place a clean magnetized patterned substrate in a culture dish. This glass substrate has embedded geometrical patterns with magnetic properties.
Sterilize the substrate by exposing it to ultraviolet light.
Coat the substrate with collagen to promote cell attachment.
Add the MNP-loaded cells to the substrate and incubate.
The external magnetic field of the substrate attracts the MNP-loaded cells, enabling their attachment.
Add a medium containing a specific growth factor at regular intervals.
The growth factor assists cells in forming extensions, while the magnetic pattern of the substrate directs the orientation of these extensions.

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Video: Рост и дифференцировка нейронов, нагруженных магнитными наночастицами, на магнитных платформах - Experiment

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JoVE Journal

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Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect.
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Growth and Differentiation of Magnetic Nano Particle-Loaded Neurons on Magnetic Platforms

ТРАНСКРИПТ

Growth and Differentiation of Magnetic Nano Particle-Loaded Neurons on Magnetic Platforms