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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation and Coating of the Multicompartment Chips 
<ol>
	<li>In a bio-safety cabinet, place the chip into a Petri dish or other suitable sterile container.</li>
	<li>Add 100 &#181;L of pre-coating solution to the upper left well of the chip and allow it to flow through the main channel into the adjoining well. 
	NOTE: The pre-coating solution is used to pre-coat the microfluidic channels to eliminate the potential for trapping air bubbles within the chip.</li>
	<li>Fill the lower left well with 100 &#181;L of pre-coating solution. Wait 5 min to allow the solution to flow through the microgrooves.</li>
	<li>Add 100 &#181;L of pre-coating solution to the upper right well and allow it to flow through the main channel into the adjoining well. Fill the lower right well with 100 &#181;L of pre-coating solution.</li>
	<li>Aspirate the solution from each well. Aspirate away from the main channels to avoid removing liquid from the main channels (Figure 1A). Immediately add 150 &#181;L of phosphate-buffered saline (PBS) to the upper left well. Wait 1.5 min. 
	CAUTION: Do not aspirate all liquid from the enclosed main channels.</li>
	<li>Add 150 &#181;L PBS to the lower left well. Wait 5 min to allow liquids to flow through the microgrooves. Add 150 &#181;L PBS to the upper right well. Add 150 &#181;L PBS to the lower right well. Wait 10 min.</li>
	<li>Repeat steps 1.5-1.6 for a second PBS wash.</li>
	<li>Check the chip under a tissue culture microscope for bubbles in the main channels. If bubbles are present, perform the procedures below. If no bubbles are present, skip to step 1.9.
	<ol>
		<li>Aspirate PBS from the wells, angling the pipet tip away from the channel opening (Figure 1A).</li>
		<li>Dispense 100 &#181;L of pre-coating solution into the upper well, angling the tip of the pipet near the channel opening (Figure 1B). The bubbles should move through the channel into the lower well. Wait 1.5 min.</li>
		<li>Repeat steps 1.3-1.8.</li>
	</ol>
	</li>
	<li>Aspirate PBS from the wells, angling the pipet tip away from the channel opening (Figure 1A).</li>
	<li>Add 100 &#181;L of 0.5 mg/mL poly d-lysine (PDL) to the upper left well of the chip. Wait 1.5 min. Fill the lower left well with 100 &#181;L of PDL.</li>
	<li>Add 100 &#181;L of PDL to the upper right well of the chip. Wait 1.5 min. Add 100 &#181;L to the lower right well.</li>
	<li>Close the petri dish and place the chip in an incubator at 37 &#176;C for 1 h.</li>
	<li>Repeat PBS wash steps 1.5-1.6 twice to remove excess PDL.</li>
	<li>Aspirate the PBS from the device.</li>
	<li>Immediately add 100 &#181;L of cell culture media to the upper left well of the chip. Wait 1.5 min. Add media to the lower left well. Add media to the upper right well. Wait 1.5 min. Add 100 &#181;L medium to the lower right well of the chip.</li>
	<li>Place the chip in the 37&#176;C incubator until ready to plate cells.</li>
</ol>
2. Seeding Neurons into the Multicompartment Chips 
<ol>
	<li>Prepare cell suspension of dissociated rat hippocampal neurons according to established protocols to yield a density of ~12 &#215; 106 cells/ mL. 
	NOTE: Use of cell suspension densities between 3 and 12 &#215; 106 cells/mL is possible. If a lower density is used, the volume of cell suspension to be added to the chip may be increased. The procedure described below is applicable for murine dissociated cortical or hippocampal neurons. Optimal cell densities for other neuron types may vary.</li>
	<li>Remove the majority of media in each well of the chip, leaving approximately 5 &#181;L in each well. Aspirate away from the main channels to avoid removing liquid from the main channels (Figure 1A). 
	CAUTION: Do not aspirate liquid from the enclosed main channels. Air bubbles may become trapped in the chip if fluid is aspirated from the main channels.</li>
	<li>Load 5 &#181;L of cell suspension in the upper right well and another 5 &#181;L of cell suspension in the lower right well (~120,000 cells total). Load the cells by dispensing them close to the main channel (Figure 1B). Check under a microscope to ensure the neurons are in the main channel. Wait for 5 min to allow the cells to attach. 
	NOTE: Neurons can be loaded into either compartment. For explanation purposes, the somatic compartment is the main channel on the right side, but either compartment can be used as the somatic compartment. Use of lower cell densities down to 60,000 cells per chip is possible. Up to 10 &#181;L of cell suspension may be added to each well of the somatic compartment in combination with a cell suspension with fewer cells than described above.</li>
	<li>Add approximately 150 &#181;L of neuronal culture media to each of the upper and lower right wells, and then add 150 &#181;L of media to each upper and lower left well. Place the chip into the humidified tray in a 5% CO2 37 &#176;C incubator.</li>
	<li>After 24 h, perform a media change by removing media from the wells. Make sure the main channel remains filled. Add 150 &#181;L of media to each top well, then fill the bottom wells.</li>
	<li>Place the chip back in the incubator for the desired number of days. 
	NOTE: Monitor the media every few days to ensure it remains light pink. If the media is yellowish, replace 50% of it with fresh media. If the fluid level is low, ensure adequate humidity and appropriate secondary containment of the chips to prevent evaporation. Minimizing or eliminating media changes is possible using secondary containment and/or covering the dish containing the chip with polytetrafluoroethylene (PTFE)-FEP film.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>XonaChip</td>
			<td>Xona Microfluidics LLC</td>
			<td>XC150</td>
			<td>150 &#181;m length microgroove barrier</td>
		</tr>
		<tr>
			<td></td>
			<td>Xona Microfluidics LLC</td>
			<td>XC450</td>
			<td>450 &#181;m length microgroove barrier&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td>Xona Microfluidics LLC</td>
			<td>XC900</td>
			<td>900 &#181;m length microgroove barrier</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>XonaPDL</td>
			<td>Xona Microfluidics, LLC</td>
			<td>XonaPDL</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuronal culture media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobassal medium</td>
			<td>ThermoFisher Scientific</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Plus Supplement (50x)&#160;</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>A3582801</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>ThermoFisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100x)</td>
			<td>ThermoFisher Scientific</td>
			<td>15240112</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorinated ethylene propylene film</td>
			<td>American Durafilm&#160;</td>
			<td>50A&#160;</td>
			<td>0.5 mil thickness</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>PBS (10x)&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>QVC0508</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator, 5% CO2 37 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

compartmentalized primary murine neuron culture using plastic microfluidic chips

Source: Nagendran, T., et al. <a href="https://app.jove.com/v/58421">Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons.</a> J. Vis. Exp. (2018).
This video demonstrates the utilization of a pre-assembled plastic microfluidic chip for the compartmentalized culture of primary rat hippocampal neurons. The neurons are plated into one of the microfluidic compartments, where they attach and grow, extending their axons through the chip&#39;s microgrooves into the adjacent compartment. The narrow microgrooves prevent neuronal cell bodies from entering, thereby enabling physical separation between the somatic and axonal compartments.

<img alt="Figure 1" src="/files/ftp_upload/22365/22365_Figure1.jpg" /> 
Figure 1. Pipetting techniques are needed when using plastic multicompartment chips. (A) When adding and aspirating media for washes, the pipet tip should be angled away from the entrance of the main channel, as shown. (B) When loading neurons or performing axotomy, the pipet tip should be angled towards the entrance of the main channel.

Compartmentalized Primary Murine Neuron Culture Using Plastic Microfluidic Chips

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Take a plastic microfluidic chip comprising somatic reservoirs and compartments, as well as axonal reservoirs and compartments, connected by microgrooves.
Add a pre-coating solution to saturate the compartments and grooves, preventing air bubble entrapment.
Aspirate the solution, leaving the main compartments filled. Wash with buffer.
Coat the wells with a synthetic polymer that facilitates neuronal adhesion. Wash with buffer and add culture media to prevent the chip from drying.
To seed a suspension of rat hippocampal neurons, aspirate the media.
Load the suspension into the somatic reservoirs. The neurons enter and attach to the somatic compartment.
Add neuronal culture media to the wells and incubate. Growth factors present in the media facilitate neuronal growth.
Gradually, the neurons extend their axons, which grow into the axonal compartment through the microgrooves.
The narrowness of the grooves prevents the entry of neuronal cell bodies, ensuring the physical separation of somatic and axonal regions.

compartmentalized-primary-murine-neuron-culture-using-plastic

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

75 Views. Źródło: Nagendran, T., et al. Użycie wstępnie zmontowanych plastikowych chipów mikroprzepływowych do podziału pierwotnych neuronów mysich. J. Vis. Exp. (2018). Ten film demonstruje wykorzystanie wstępnie zmontowanego plastikowego chipa mikroprzepływowego do podzielonej na przedziały hodowli pierwotnych neuronów hipokampa szczura. Neurony są umieszczane w jednym z przedziałów mikroprzepływowych, gdzie przyczepiają się i rosną, przedłużając swoje aksony przez mikrorowki chi...

Video: Podzielona na przedziały pierwotna hodowla neuronów mysich przy użyciu plastikowych chipów mikroprzepływowych - Concept

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

JoVE Journal

Bioengineering

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a pre-assembled multi-compartment chip made in a cyclic olefin copolymer (COC) to compartmentalize neurons differentiated from human stem cells. The footprint of these COC chips are the same as a standard microscope slide and are equally compatible with high resolution microscopy. Neurons are differentiated from human neural stem cells (NSCs) into glutamatergic neurons within the chip and maintained for 5 weeks, allowing sufficient time for these neurons to develop synapses and dendritic spines. Further, we demonstrate multiple common experimental procedures using these multi-compartment chips, including viral labeling, establishing microenvironments, axotomy, and immunocytochemistry.

This protocol demonstrates the use of compartmentalized microfluidic chips, injection molded in a cyclic olefin copolymer to cultured neurons differentiated from human stem cells. These chips are preassembled and easier-to-use than traditional compartmentalized poly(dimethylsiloxane) devices. Multiple common experimental paradigms are described here, including viral labeling, fluidic isolation, axotomy, and immunostaining.

Compartmentalized Primary Murine Neuron Culture Using Plastic Microfluidic Chips

Transkrypcja

Compartmentalized Primary Murine Neuron Culture Using Plastic Microfluidic Chips