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NOTE:&#160;Figure 1A, B&#160;shows a schematic of the pre-assembled cyclic olefin copolymer (COC) chip, including the locations of the main channels or compartments, wells, and microgrooves. The compartmentalized chip can establish distinct fluidic microenvironments within a compartment as demonstrated by the isolation of food coloring dye (Figure 1C).&#160;
1. Coating of multi-compartment chips for hSC-neurons
NOTE:&#160;Figure 2A&#160;shows an overview of the coating procedures.
<ol>
	<li>Dissolve poly-L-ornithine in cell culture-grade distilled water to make 600 &#181;L of solution per chip of a 20 &#181;g/mL working solution.</li>
	<li>Aspirate the remaining phosphate-buffered saline (PBS) left after preparing the chips from the wells. When aspirating, ensure that the pipet tip is away from the channel opening. 
	NOTE: The microfluidic channels must remain filled for the entirety of this procedure. Do not aspirate liquid from the channels/compartments, only the wells.</li>
	<li>Load 150 &#181;L of poly-L-ornithine working solution in the upper right well. Wait for 90 seconds or until the liquid begins to fill the lower right well. Add 150 &#181;L of media to the lower right well and wait for 5 min to let the liquid pass through the microgrooves.</li>
	<li>Add 150 &#181;L of media to the upper left well. Wait 90 s and then add 150 &#181;L to the lower left well. Wrap the holder of the chip(s) with parafilm and place at 4 &#176;C overnight. 
	NOTE: Alternatively, place the chip and holder at 37 &#176;C for 1 h.</li>
	<li>Aspirate the media from the wells, ensuring that the pipet tip is placed away from the channel opening. Add 150 &#181;L of sterile water to the upper right well. Wait for 90 s and then add 150 &#181;L to the lower right well. Wait for 5 min to let the liquid pass through the microgrooves.</li>
	<li>Add 150 &#181;L of sterile water to the upper left well, wait for 90 s, and then add another 150 &#181;L to the lower left well.</li>
	<li>Repeat steps 1.5-1.6.</li>
	<li>Thaw laminin slowly at 2 &#176;C to 8 &#176;C and prepare a 10 &#181;g/mL working solution.</li>
	<li>Load 150 &#181;L of the laminin working solution to the upper right well. Wait for 90 seconds or until the liquid begins to fill the lower right well. Pipet 150 &#181;L to the lower right well. Wait for 5 min to let the laminin go through the microgrooves.</li>
	<li>Add 150 &#181;L of laminin to the upper left well. Wait 90 s and then add 150 &#181;L to the lower left well. Incubate the chip within its holder for 2 h at 37 &#176;C.</li>
	<li>Rinse the chip with PBS (without Ca2+&#160;and Mg2+). Load 150 &#181;L of PBS to the upper right well. Wait for 90 seconds or until the liquid begins to fill the lower right well. Add 150 &#181;L of PBS to the lower right well and wait for 5 min to let the PBS go through the microgrooves.</li>
	<li>Pipet 150 &#181;L of PBS to the upper left well. After 90 s pipet 150 &#181;L to the lower left well.</li>
	<li>Aspirate PBS from the chip and then rinse the chip with NSC media&#160;(see&#160;Table of Materials). Load 150 &#181;L of media to the upper right well. After 90 s or when liquid passes through the right channel into the lower right well, add 150 &#181;L to the lower right well. Wait for 5 min to let the media flow through the microgrooves.</li>
	<li>Add 150 &#181;L of media to the upper left well and another 150 &#181;L to the lower left well. Incubate the chip in a 37 &#176;C incubator with 5% CO2&#160;to pre-condition the chip until ready to seed NSCs. 
	NOTE: Chips can be pre-conditioned overnight if needed.</li>
</ol>
2. Seeding NSCs into the multicompartment chip
NOTE: This protocol used commercially purchased NSCs, unlike our previous publication which began with human embryonic stem cells. In this protocol neural stem cells are plated directly into the plastic chips where they differentiate into neurons. The cells can be allowed to proliferate as NSCs (without differentiation) for up to 2 passages and stored in vials in liquid N2&#160;for further use. This protocol uses NSC vials stored in liquid N2&#160;after passage 1 or 2.&#160;Figure 2B&#160;shows an overview of the coating procedures.
<ol>
	<li>Thaw NSCs according to the manufacturer&#39;s instructions. 
	NOTE: This procedure applies to H9-derived NSCs. Other cell lines will require cell density optimization.</li>
	<li>Count the cell concentration using a hemocytometer and adjust using NSC media to get a concentration of 7 x 106&#160;cells per mL.</li>
	<li>Aspirate media from each well of the chip. Avoid aspirating media from the main channels.</li>
	<li>Pipet 5 &#181;L of the cell solution to the upper right well, followed by 5 &#181;L to the lower right well (70,000 cells total). Ensure that the cells are pipetted towards the main channel openings. Use a microscope to check that cells have entered the channel. Wait 5 min for cells to adhere to the bottom of the chip. 
	NOTE: Either or both compartments can be used to load cells.</li>
	<li>Pipet ~150 &#181;L of NSC media to the upper right well and then 150 &#181;L to the lower right well. Repeat on the left side. Incubate at 5% CO2, 37 &#176;C within a suitable humidified container.</li>
	<li>After 48 hours, aspirate NSC media from the wells and replace it with neural differentiation media by adding 150 &#181;L to each top well and each bottom well.</li>
	<li>Culture neurons within a 5% CO2, 37 &#176;C incubator within a suitable humidified container.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>complete neural stem cell media:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>REC HU EGF 
			10 UG BIOSOURCE (TM)</td>
			<td>ThermoFisher Scientific</td>
			<td>PHG0314</td>
			<td>20ng/mL</td>
		</tr>
		<tr>
			<td>REC HU FGF BASIC 
			10 UG BIOSOURCE (TM)</td>
			<td>ThermoFisher Scientific</td>
			<td>PHG0024</td>
			<td>20ng/mL</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>35050061</td>
			<td>2mM</td>
		</tr>
		<tr>
			<td>KnockOut DMEM/F-12</td>
			<td>ThermoFisher Scientific</td>
			<td>12660012</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Neural Supplement</td>
			<td>ThermoFisher Scientific</td>
			<td>A1050801</td>
			<td>2%</td>
		</tr>
		<tr>
			<td>Gibco DPBS without Calcium and Magnesium</td>
			<td>ThermoFisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>GIBCO HUMAN NSC (H9) KIT COMBO KIT</td>
			<td>Gibco</td>
			<td>N7800200</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Laminin</td>
			<td>ThermoFisher Scientific</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur pipettes</td>
			<td>Sigma-Aldrich</td>
			<td>CLS7095D5X SIGMA</td>
			<td>5.75 in length</td>
		</tr>
		<tr>
			<td>H9-DERIVED HU NEURAL STEM CELL 
			1E6 CELLS/VIAL; 1 ML</td>
			<td>ThermoFisher Scientific</td>
			<td>510088</td>
			<td></td>
		</tr>
		<tr>
			<td>hibernate-E Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>A1247601</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator, 5% CO2 37 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty</td>
			<td>ThermoFisher Scientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neural differentiation media</td>
			<td></td>
			<td></td>
			<td>Per 100 mL.</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100x)</td>
			<td>ThermoFisher Scientific</td>
			<td>15240112</td>
			<td>1mL (100X)</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma Aldrich</td>
			<td>A8960</td>
			<td>200mM</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>ThermoFisher Scientific</td>
			<td>PHC7074</td>
			<td>40 ng/mL</td>
		</tr>
		<tr>
			<td>Gibco B27 Plus Supplement (50X)</td>
			<td>FisherScientific</td>
			<td>A3582801</td>
			<td>2mL (50X)</td>
		</tr>
		<tr>
			<td>Gibco CultureOne Supplement (100X)</td>
			<td>FisherScientific</td>
			<td>A3320201</td>
			<td>1mL (100X)</td>
		</tr>
		<tr>
			<td>Gibco Neurobasal Plus Medium</td>
			<td>FisherScientific</td>
			<td>A3582901</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr>
			<td>XC pre-coat</td>
			<td>Xona Microfluidics, LLC</td>
			<td>XC Pre-Coat</td>
			<td>included with XonaChips</td>
		</tr>
		<tr>
			<td>XonaChip</td>
			<td>Xona Microfluidics, LLC</td>
			<td>XC450</td>
			<td>450 &#181;m length microgroove barrier</td>
		</tr>
		<tr>
			<td>Humidifier Tray</td>
			<td>Xona Microfluidics, LLC</td>
			<td>humidifier tray</td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiation of neural stem cells to neurons using a compartmentalized microfluidic device

Source: Paranjape, S. R., et al. <a href="https://app.jove.com/v/59250">Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips.</a> J. Vis. Exp. (2019)
The video demonstrates the differentiation of neural stem cells (NSCs) using a microfluidic device comprising a somatic and an axonal compartment separated by a microgroove barrier. NSCs are introduced into the somatic compartment and are allowed to adhere. The NSCs differentiate into neurons inside the somatic compartment and extend axons through the microgroove barrier to the axonal compartment.

<img alt="Figure 1" src="/files/ftp_upload/22433/22433_Figure1.jpg" /> 
Figure 1: The pre-assembled COC, multicompartment microfluidic chip.&#160;(A) A drawing of the chip identifying the upper and lower wells. The size of the chip is 75 mm x 25 mm, the size of standard microscope slides. (B) A zoomed-in region showing the channels and microgrooves separating the channels. (C) This photograph illustrates the creation...

Differentiation of Neural Stem Cells to Neurons Using a Compartmentalized Microfluidic Device

NOTE:&#160;Figure 1A, B&#160;shows a schematic of the pre-assembled cyclic olefin copolymer (COC) chip, including the locations of the main channels or ...

Take a microfluidic device consisting of reservoir wells connected to two compartments, a somatic and an axonal, separated by a microgroove barrier.
The compartments and microgrooves are coated with a cell culture substrate to facilitate cell adhesion.
Add a neural stem cell medium to each well and incubate to precondition the device for cell culture.
Discard the media. Add neural stem cells, or NSCs, to the wells of the somatic compartment and allow the cells to adhere.
Add the NSC medium to each well and incubate in a humidified chamber to prevent media evaporation. During incubation, the NSCs proliferate.
Replace the NSC medium with a neural differentiation medium and incubate.
The small molecules in the medium induce the differentiation of NSCs into neurons within the somatic compartment, with their axons extending through the microgrooves into the axonal compartment.
The differentiated neurons with extended axons are ready for further assays.

differentiation-neural-stem-cells-to-neurons-using-compartmentalized

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

35 vistas. Fuente: Paranjape, S. R., et al. Compartimentación de neuronas derivadas de células madre humanas dentro de chips microfluídicos de plástico preensamblados. J. Vis. Exp. (2019) El video demuestra la diferenciación de las células madre neurales (NSCs) utilizando un dispositivo microfluídico compuesto por un compartimento somático y otro axonal separados por una barrera de microsurcos. Las NSC se introducen en el compartimento somático y se les permite adherirse. Las NSC se diferencian e...

Video: Diferenciación de células madre neurales en neuronas mediante un dispositivo microfluídico compartimentado - Concepto

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

Diferenciación celular precursora neuronal inducida por campo eléctrico en dispositivos microfluídicos

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...

Investigación

JoVE Journal

Bioingeniería

Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons

Microfabricated methods to compartmentalize neurons have become essential tools for many neuroscientists. This protocol describes the use of a commercially available pre-assembled plastic chip for compartmentalizing cultured primary rat hippocampal neurons. These plastic chips, contained within the footprint of a standard microscope slide, are compatible with high-resolution, live, and fluorescence imaging. This protocol demonstrates how to retrograde label neurons via isolated axons using a modified rabies virus encoding a fluorescent protein, create isolated microenvironments within one compartment, and perform axotomy and immunocytochemistry on-chip. Neurons are cultured for &gt;3 weeks within the plastic chips, illustrating the compatibility of these chips for long-term neuronal cultures.

This protocol describes the use of plastic chips to culture and compartmentalize primary murine neurons. These chips are preassembled, user-friendly, and compatible with high-resolution, live, and fluorescence imaging. This protocol describes how to plate rat hippocampal neurons within these chips and perform fluidic isolation, axotomy and immunostaining.

Uso de Chips microfluídicos plástico premontados para compartimentar primarias neuronas murinas

Microfabricated methods to compartmentalize neurons have become essential tools for many neuroscientists. This protocol describes the use of a ...

Neurociencias

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle contraction. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), NMJs degenerate, resulting in muscle atrophy and progressive paralysis. The underlying mechanism of NMJ degeneration is unknown, largely due to the lack of translatable research models. This study aimed to create a versatile and reproducible in vitro model of a human motor unit with functional NMJs. Therefore, human induced pluripotent stem cell (hiPSC)-derived motor neurons and human primary mesoangioblast (MAB)-derived myotubes were co-cultured in commercially available microfluidic devices. The use of fluidically isolated micro-compartments allows for the maintenance of cell-specific microenvironments while permitting cell-to-cell contact through microgrooves. By applying a chemotactic and volumetric gradient, the growth of motor neuron-neurites through the microgrooves promoting myotube interaction and the formation of NMJs were stimulated. These NMJs were identified immunocytochemically through co-localization of motor neuron presynaptic marker synaptophysin (SYP) and postsynaptic acetylcholine receptor (AChR) marker &#945;-bungarotoxin (Btx) on myotubes and characterized morphologically using scanning electron microscopy (SEM). The functionality of the NMJs was confirmed by measuring calcium responses in myotubes upon depolarization of the motor neurons. The motor unit generated using standard microfluidic devices and stem cell technology can aid future research focusing on NMJs in health and disease.

We describe a method to generate human motor units in commercially available microfluidic devices by co-culturing human induced pluripotent stem cell-derived motor neurons with human primary mesoangioblast-derived myotubes resulting in the formation of functionally active neuromuscular junctions.

Generación de Unidades Motoras Humanas con Uniones Neuromusculares Funcionales en Dispositivos Microfluídicos

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle ...

Differentiation of Neural Stem Cells to Neurons Using a Compartmentalized Microfluidic Device

Transcripción

Differentiation of Neural Stem Cells to Neurons Using a Compartmentalized Microfluidic Device