eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Material Setup
<ol>
	<li>Incubate chick eggs in a commercially available tabletop egg incubator at 37 &#176;C. Incubate eggs for 7 days for E7 embryos.</li>
	<li>Prepare multi-electrode arrays (MEAs). Sterilize the required number of MEAs by soaking them in 70% ethanol for 3 to 4 h and then rinsing them three times in approximately 10 mL of sterile distilled water in the biosafety level-II (BSLII) hood. 
	NOTE: The MEAs contain 64 nanoporous platinum electrodes arranged in an 8 x 8 grid. The electrode diameter is 30 &#181;m, and the spacing between the electrodes is 200 &#181;m. Do not use denatured ethanol to sterilize the MEAs, as this will lead to corrosion.</li>
	<li>Place the MEAs inside a sterile container with a lid (to maintain sterile conditions) and move to a tabletop incubator. Bake for at least 6 hours at 55 &#176;C. This step is critical for proper sterilization, and the temperature should not exceed 60 &#176;C. To maintain sterility, move the dish containing MEAs to the BSLII hood for storage until further use. 
	NOTE: We use an oven-safe glass baking dish with a plastic lid and surface sterilize it with 70% ethanol and place it in the BSLII hood for drying.</li>
	<li>Aliquot the extracellular matrix (ECM) solution. Thaw the vial at 4 &#176;C overnight and freeze 100 &#181;L aliquots at -20 &#176;C. 
	NOTE: ECM is shipped frozen. It should not be brought to room temperature until ready to coat as it polymerizes at room temperature and is impossible to coat once polymerized. ECM without growth factors is preferred to prevent additional effects due to unknown factors.</li>
	<li>Sterilize all dissection instruments (Dumont #5 forceps, curved forceps, small scissors, and spring scissors) and self-sealing material-coated dissection dishes with 70% ethanol and let them dry in the dissection hood.</li>
</ol>
2. Chick Embryonic Neuron Culture and Network Activity
<ol>
	<li>On the day of plating, remove a vial of ECM from the -20 &#176;C freezer, spray with 70% ethanol, and place it on ice. Dilute to 25% by adding 300 &#181;L cold neurobasal medium inside the BSLII hood.</li>
	<li>Using a P200 pipetteman add 100 &#181;L of 25% ECM to the center of the MEA taking care not to touch the electrodes and remove immediately leaving a thin film on the surface. Cover the MEA and place in the CO2 incubator (37&#176; C and 5% CO2) until ready to plate the neurons.</li>
	<li>Sterilize the outer shell of an E7 egg (embryonic day 7, incubated at 37 &#176;C for 7 days) with 70% ethanol. Decapitate the embryo into a Sylgard bottom dissection dish containing cold sterile Hank&#39;s Balanced Salts Solution (HBSS) without calcium. Cut around the eyes and remove the eyeballs. Using DuMont #5 fine forceps and spring scissors make an incision on the ventral side and remove the outer layers of skin to expose the forebrain and optic tectum. Peel and remove the pial membrane carefully. Transfer the forebrain into another petri dish and cut it into small pieces of about 2 mm with spring scissors. 
	NOTE: There should not be any blood vessels attached to the forebrain after removal of the pial membrane. If available, it is preferable to perform the dissection in a sterile dissection hood, although we have obtained fairly good results when dissection is performed outside a hood as long as the instruments and dissection area are thoroughly sterilized with 70% ethanol.</li>
	<li>Using a sterile transfer pipette, collect the pieces of forebrain into a 15 mL centrifuge tube and remove as much of the HBSS as possible after letting them sink to the bottom of the centrifuge tube.</li>
	<li>Add 1 mL of 0.05% trypsin/ethylenediamine tetraacetic acid (Trypsin/EDTA) (pre-warmed to 37 &#176;C) and incubate at 37 &#176;C for 15 minutes.</li>
	<li>Using a Pasteur pipette, carefully remove trypsin without disturbing the pieces of tissue and add 1 mL of neurobasal medium. Let the pieces of tissue sink to the bottom and remove the medium. Repeat this wash once more. This step is to wash off the trypsin/EDTA.</li>
	<li>Add 2 mL of neurobasal medium and start triturating. Trituration involves taking a sterile fire-polished Pasteur pipette and gently passing the tissue through it several times until no more pieces are seen. If any pieces remain after repeated passes, just leave them to settle to the bottom. 
	NOTE: Avoid frothing while triturating - if froth does form, stop and remove by aspiration.</li>
	<li>Dilute the re-suspended cells 1:10 with neurobasal medium and count viable cells using Trypan Blue dye and a hemocytometer. Mix 50 &#181;L of the diluted cells with 50 &#181;L of Trypan Blue solution in a 0.5 mL centrifuge tube. Add 10 &#181;L to the hemacytometer after placing on the coverslip and count the bright clear cells (blue cells are dead and should not be counted). 
	NOTE: Typical cell numbers from a single E7 optic tectum range between 1 and 5 x 107 cells/mL.</li>
	<li>Plate the dissociated cells on ECM-coated MEAs at a density of 2,200 cells/square mm. For our MEA system, this translates to approximately 130,000 cells per MEA. Add the neurobasal medium to bring the volume in the MEA to 1 mL and place MEAs in the CO2 incubator overnight for cell attachment and neurite extension (Figure 1). 
	NOTE: For MEAs of different areas and electrode numbers, different cell densities could be tried - in general, higher cell density results in greater network activity, but also leads to shorter-lived cultures.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#5 foreceps</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Axion Muse MEA</td>
			<td>Axion Biosystems</td>
			<td>M64-GL1-30Pt200</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Fine Science Tools</td>
			<td>11272-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Fisher</td>
			<td>14170112</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Fisher</td>
			<td>02-671-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced, Phenol Red-Free</td>
			<td>BD Biosciences</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>BrainBits</td>
			<td>Nb4-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes</td>
			<td>Fisher</td>
			<td>13-678-20A</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15514-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard bottom dissection dishes</td>
			<td>Living Systems Instrumentaion</td>
			<td>DD-90-S-BLK-3PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue dye</td>
			<td>Fisher</td>
			<td>15-250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Fisher</td>
			<td>15400054</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating and culturing chick embryonic neurons on a multi-electrode array

Source: Sanchez, K. R., et al., <a href="https://app.jove.com/v/56300">Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays.</a> J. Vis. Exp.(2018)
This video demonstrates a method to isolate neurons from the forebrain of an early-stage chick embryo and culture them on a microelectrode array to form a neuronal network.

<img alt="Figure 1" src="/files/ftp_upload/22624/22624_Figure1.jpg" /> 
Figure 1: Schematic of the protocol depicting the steps from dissection of chick forebrain to dissociation, plating, and recording of network activity.

Isolating and Culturing Chick Embryonic Neurons on a Multi-Electrode Array

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

Take a fertilized chicken egg containing an early-stage embryo.
Sterilize the shell and carefully open it.
Remove the embryo. Dissect and transfer the head to a culture plate containing a buffer. Remove the eyeball.&#160;
Make an incision and remove the skin layers, exposing the forebrain and midbrain.
Remove the inner membranous layer and blood vessels. Dissect and transfer the forebrain to a culture plate with a buffer.
Cut the tissue into smaller fragments and transfer them to a conical tube. Once the fragments settle, remove the buffer.
Add a proteolytic enzyme solution. Incubate to digest the tissue&#39;s extracellular matrix and loosen neurons.
Remove enzyme-rich media, wash with neurobasal media, removing any remaining enzymes.
Add neurobasal media. Mechanically dissociate the tissue to obtain a neuronal suspension.
Seed these neurons on a multi-electrode array system coated with an extracellular matrix. Add neurobasal media and incubate.
Neurons attach and extend projections, forming a neuronal network.

isolating-culturing-chick-embryonic-neurons-on-multi-electrode

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

35 Views. Źródło: Sanchez, K. R., et al., Ocena wpływu związków zaburzających funkcjonowanie układu hormonalnego na rozwój funkcji sieci neuronowej kręgowców przy użyciu układów wieloelektrodowych. J. Vis. Exp.(2018) Ten film demonstruje metodę izolowania neuronów z przodomózgowia zarodka pisklęcia we wczesnym stadium i hodowli ich na matrycy mikroelektrod w celu utworzenia sieci neuronalnej. 

Video: Izolowanie i hodowla neuronów embrionalnych piskląt na matrycy wieloelektrodowej - Concept

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

JoVE Journal

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...

Developmental Biology

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

Isolating and Culturing Chick Embryonic Neurons on a Multi-Electrode Array

Transkrypcja

Isolating and Culturing Chick Embryonic Neurons on a Multi-Electrode Array