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.

growth-differentiation-magnetic-nano-particle-loaded-neurons-on

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

EoE Sub Articles

eoe sub articles

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.

13 Aufrufe. Wachstum und Differenzierung von magnetischen, mit Nanopartikeln beladenen Neuronen auf magnetischen Plattformen

Serie: Wachstum und Differenzierung von magnetischen, mit Nanopartikeln beladenen Neuronen auf magnetischen Plattformen - Experiment

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described.
Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the St&#246;ber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.

This work describes protocols for the preparation of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine using a succinyl moiety as a linker. A deep structural characterization description and a capture bacteria assay using Y. enterocolitica for all the intermediate nanoparticles and the final conjugate are also described in detail.

Synthese funktionalisierter magnetischer Nanopartikel, deren Konjugation mit dem Siderophor Feroxamin und deren Bewertung für den Bakteriennachweis

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with ...

Forschung

JoVE Journal

Chemie

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

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.
Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.&#34;

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

Ein Neurit Engrowth Assay und Neurotoxizität Satonie-Bewertung mit menschlichen neuronalen Vorläufer Cell-Derived Neuronen

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides ...

Entwicklungsbiologie

In Vitro and In Vivo Delivery of Magnetic Nanoparticle Hyperthermia Using a Custom-Built Delivery System

Hyperthermia has long been used in the treatment of cancer. Techniques have varied from the intra-tumoral insertion of hot iron rods, to systemically delivered tumor antibody-targeted magnetic nanoparticles, at temperatures from 39 &#730;C (fever-level) to 1,000 &#730;C (electrocautery) and treatment times from seconds to hours. The temperature-time relationship (thermal dose) dictates the effect with high thermal doses resulting in the tissue ablation and lower thermal doses resulting in sublethal effects such as increased blood flow, accumulation of drugs and immune stimulation. One of the most promising current medical therapies is magnetic nanoparticle hyperthermia&#160; (mNPH). This technique involves activating magnetic nanoparticles, that can be delivered systemically or intratumorally, with a non-invasive, non-toxic alternating magnetic field. The size, construct and association of the magnetic nanoparticles and the frequency and field strength of the magnetic field are major heating determinants. We have developed sophisticated instrumentation and techniques for delivering reproducible magnetic nanoparticle hyperthermia in large and small animal models and cultured cells. This approach, using continuous, real time temperature monitoring in multiple locations, allows for the delivery of well-defined thermal doses to the target tissue (tumor) or cells while limiting non-target tissue heating. Precise control and monitoring of temperature, in multiple sites, and use of the industry standard algorithm (cumulative equivalent minutes at 43 &#730;C /CEM43), allows for an accurate determination and quantification of thermal dose. Our system, which allows for a wide variety of temperatures, thermal doses, and biological effects, was developed through a combination of commercial acquisitions and inhouse engineering and biology developments. This system has been optimized in a manner that allows for the rapid conversion between ex vivo, in vitro, and in vivo techniques. The goal of this protocol is to demonstrate how to design, develop and implement an effective technique and system for delivering reproducible and accurate magnetic nanoparticle therapy (mNP) hyperthermia.

This protocol presents techniques and methodology necessary for the accurate delivery of magnetic nanoparticle hyperthermia using a sophisticated delivery and monitoring system.

In-vitro- und In-vivo-Verabreichung von magnetischer Nanopartikel-Hyperthermie mit einem maßgeschneiderten Verabreichungssystem

Hyperthermia has long been used in the treatment of cancer. Techniques have varied from the intra-tumoral insertion of hot iron rods, to systemically ...

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

Transkript

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