eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Dissection of Embryos and Dissociation of Tissue
<ol>
	<li>House adult female rats (200-250 g) at a constant temperature (22 &#177; 1 &#176;C) and relative humidity (50%) under a regular light&#8211;dark schedule (light-on 7 AM&#8211;7 PM) in the animal facility. Ensure that food and water are freely available.</li>
	<li>Anesthetize a pregnant female rat after 18 days of development (E18) using 3% isoflurane. Then, sacrifice the animal by cervical dislocation.</li>
	<li>Remove hippocampi from each rat embryo and place them into ice cold Hank&#39;s balanced salt solution without Ca2+ and Mg2+. At day 18 of development, hippocampus and cortex tissues are very soft and do not need to be cut into small pieces. Further details can be found.</li>
	<li>Dissociate the tissue in 0.125% of Trypsin/Hank&#39;s solution containing 0.05% of DNAse for 18-20 min at 37 &#176;C.</li>
	<li>Remove the supernatant solution with a Pasteur pipette and stop the enzymatic digestion by adding medium with 10% fetal bovine serum (FBS) for 5 min.</li>
	<li>Remove the medium with FBS and wash once with the medium with its supplement, 1% L-glutamine, gentamicin 10 &#181;g/ml. Remove again the medium with a Pasteur pipette and refill it again with a small amount (500 &#181;l) of growth medium with its supplement, 1% L-glutamine, gentamicin 10 &#181;g/ml.</li>
	<li>Dissociate the tissue pellet mechanically with a narrow Pasteur pipette until a milky suspension of cells is apparent. It is not necessary to centrifuge the cell suspension.</li>
	<li>Dilute the small volume of cell suspension with the growth medium to obtain a final volume of 2.0 ml. Count the obtained cellular concentration with a hemocytometer chamber. Dilute this concentration at 1:5 in order to obtain the desired cell concentration of 600-700 cells/&#181;l.</li>
</ol>
2. Cell Plating
<ol>
	<li>Plate cells at a density of about 2,000 cells/mm2 onto the active area of the MEA, defined by the PDMS constraint to create a 2D neuronal network. 
	NOTE: Each hippocampus contains about 5 x 105 cells, (1 x 106 for a single embryo). Dissect 6 embryos to get a total amount of 6 x 106 cells in 2 ml, and an estimated concentration of 3,000 cells/&#181;l. Dilute this concentration at 1:5 in order to obtain the desired cell concentration and plate about 600-700 cells/&#181;l. If the MEA area delimited by the PDMS constraint is about 7.065 mm2, and the total number of plated cells 600 cells/&#181;l x 24 &#181;l = 14,400 cells, the final density of 2,038 cells/mm2.</li>
	<li>Place the MEA devices into the incubator with humidified CO2 atmosphere (5%) at 37 &#176;C.</li>
	<li>Distribute 160 &#181;l of the suspension with a cell concentration of 600-700 cells/&#181;l (about 100,000 cells) onto the surface of the microbeads monolayer positioned inside the multiwell plates to complete the preliminary step for constructing three-dimensional culture. Put the multiwell plates in the incubator with humidified CO2 atmosphere (5%) at 37 &#176;C.</li>
</ol>
3. 3D Neuronal Network Construction
<ol>
	<li>6-8 hr after the plating, transfer the suspension (microbeads with neurons) from the multiwell plates very carefully inside the area delimited by the PDMS constraint by using a pipette set for a volume of about 30-40 &#181;l. After each transfer, wait for about half a minute, to allow the microbeads to self-assemble in a hexagonal compact structure.</li>
	<li>Once all the layers are deposited and spontaneously assembled, refill with a large drop of about 300 &#181;l of medium the top of the area delimited by the PDMS constraint.</li>
	<li>Put the 3D structure coupled to the MEA in incubator (T = 37.0 &#176;C, CO2 = 5%) for 48 hr before adding a final volume (about 1 ml) of growth medium culture with its supplement. 
	NOTE: Consider the total number of beads on the multiwell plates with membrane insert (30,000) and the number of beads on a single layer onto the MEA device (6,000). The resulting 3D structure is composed of 5 layers of microbeads and cells. Considering that the 3D neuronal network is not geometrically perfect, the resulting 3D structure could comprise 5-8 layers.</li>
	<li>The day after plating, carefully add the final volume of the medium (about 900 &#181;l) inside the MEA ring. Maintain the 3D cultures in a humidified CO2 atmosphere (5%) at 37 &#176;C for 4-5 weeks. Replace half of the medium once a week.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>0.05 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407</td>
			<td>0.05 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25050-014</td>
			<td>0.125% diluted 1:2 in HBSS w\o calcium and magnesium ions</td>
		</tr>
		<tr>
			<td>DNAase</td>
			<td>Sigma-Aldrich</td>
			<td>D5025</td>
			<td>0.05% diluted in Hanks solution</td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco Invitrogen</td>
			<td>21103049</td>
			<td>Culture medium</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco Invitrogen</td>
			<td>17504044</td>
			<td>2% medium supplent</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F-2442</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Glutamax-I</td>
			<td>Gibco</td>
			<td>35050038</td>
			<td>0.5 mM</td>
		</tr>
		<tr>
			<td>gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G.1272</td>
			<td>5mg/liter</td>
		</tr>
		<tr>
			<td>Poly-Dimethyl-Siloxane (PDMS)</td>
			<td>Corning Sigma</td>
			<td>481939</td>
			<td>Curing agent and the polymer</td>
		</tr>
		<tr>
			<td>Micro-Electrode Arrays</td>
			<td>Multi Channel Systems (MCS)</td>
			<td>60MEA200/30-Ti-pr</td>
			<td>MEA with: Electrode grid 8x8; Electrode spacing and diameter 200 and 30 &#181;m, respectively; plastic ring without thread</td>
		</tr>
		<tr>
			<td>Microbead</td>
			<td>Distrilab-Duke Scientific</td>
			<td>9040</td>
			<td>1gr Glass Part.Size Stds 40 &#181;m</td>
		</tr>
		<tr>
			<td>Transwell</td>
			<td>Costar Sigma</td>
			<td>CLS 3413</td>
			<td>Multiwell plates with membrane insert (6.5 mm diameter porous 0.4 &#181;m)</td>
		</tr>
		<tr>
			<td>HBSS w\o calcium and magnesium ions</td>
			<td>Gibco Invitrogen</td>
			<td>14175-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Buffer Solution</td>
			<td>Sigma</td>
			<td>H8264</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat</td>
			<td>Sprague Dawley</td>
			<td>Wistar Rat</td>
			<td></td>
		</tr>
	</tbody>
</table>

building three-dimensional neuronal networks coupled to micro-electrode arrays

Source: Tedesco, M., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/53080">Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model.</a> J. Vis. Exp. (2015)
This video demonstrates the development of a 3D neuronal culture coupled to a planar microelectrode array with hippocampal neurons. The isolated neurons form a 2D network on the microelectrode array&#39;s active area and adhere to glass microbeads in a multi-well plate with a membrane insert. Upon transferring these neuron-coated microbeads to the electrode, they organize into an interconnected 3D neural network.

Building Three-Dimensional Neuronal Networks Coupled to Micro-Electrode Arrays

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Dissection of Embryos and ...

Take a suspension of rat embryo hippocampal neurons.
Transfer the neurons onto the adhesion factor-coated active area of a microelectrode array, or MEA. This active area is confined by a polymer structure.
Incubate the MEA. Neurons adhere to the coated MEA active area, forming a 2D neuronal network.
Additionally, transfer the neurons onto an adhesion factor-coated glass microbead monolayer in membrane inserts within a multi-well plate.
Incubate for the neurons to attach to the adhesion factor-coated beads.
Transfer the microbeads with adherent neurons to the confined MEA area containing the neuronal network.
Microbeads self-assemble to form a hexagonal structure.
Repeat the microbead transfer on the MEA several times, building layers that self-assemble, forming a packed 3D structure.
Add media to the MEA confined active area and incubate.
Neurons grow, forming an interconnected 3D neuronal network.

building-three-dimensional-neuronal-networks-coupled-to-micro

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

30 Görüntüleme. Kaynak: Tedesco, M., et al., 3D Mühendislik Nöronal Kültürlerin Mikro-Elektrot Dizilerine Arayüzlenmesi: Yenilikçi Bir İn Vitro Deneysel Model. J. Vis. Deneyim (2015) Bu video, hipokampal nöronlara sahip düzlemsel bir mikroelektrot dizisine bağlı bir 3D nöronal kültürün gelişimini göstermektedir. İzole edilmiş nöronlar, mikroelektrot dizisinin aktif alanı üzerinde 2 boyutlu bir ağ oluşturur ve bir zar eki olan çok kuyulu bir plakada cam mikro boncuklara yapışır. Bu n...

Video: Mikro elektrot dizilerine bağlı üç boyutlu nöronal ağlar oluşturma - Kavram

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

Sinaptik Microcircuit modelleme 3D Cocultures Astrocytes ve Neurons insan Pluripotent kök hücre ile

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

JoVE Journal

Gelişim Biyolojisi

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural underpinnings of brain disease and injury. As electrodes are miniaturized to the scale of individual cells, a corresponding rise in the interface impedance limits the quality of recorded signals. Additionally, conventional electrode materials are stiff, resulting in a significant mechanical mismatch between the electrode and the surrounding brain tissue, which elicits an inflammatory response that eventually leads to a degradation of the device performance. To address these challenges, we have developed a process to fabricate flexible microelectrodes based on Ti3C2 MXene, a recently discovered nanomaterial that possesses remarkably high volumetric capacitance, electrical conductivity, surface functionality, and processability in aqueous dispersions. Flexible arrays of Ti3C2 MXene microelectrodes have remarkably low impedance due to the high conductivity and high specific surface area of the Ti3C2 MXene films, and they have proven to be exquisitely sensitive for recording neuronal activity. In this protocol, we describe a novel method for micropatterning Ti3C2 MXene into microelectrode arrays on flexible polymeric substrates and outline their use for in vivo micro-electrocorticography recording. This method can easily be extended to create MXene electrode arrays of arbitrary size or geometry for a range of other applications in bioelectronics and it can also be adapted for use with other conductive inks besides Ti3C2 MXene. This protocol enables simple and scalable fabrication of microelectrodes from solution-based conductive inks, and specifically allows harnessing the unique properties of hydrophilic Ti3C2 MXene to overcome many of the barriers that have long hindered the widespread adoption of carbon-based nanomaterials for high-fidelity neural microelectrodes.

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

We describe here a method for fabricating Ti3C2 MXene microelectrode arrays and utilizing them for in vivo neural recording.

In Vivo Nöral Kayıt için Ti3C2 MXen Mikroelektrot Dizilerinin İmalatı

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural ...

Biyomühendislik

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Kayıt ve modülasyon Epileptiform aktivite elektrot dizilerin birleştiğinde kemirgen beyin dilimleri içinde

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Building Three-Dimensional Neuronal Networks Coupled to Micro-Electrode Arrays

TRANSKRIPT

Building Three-Dimensional Neuronal Networks Coupled to Micro-Electrode Arrays