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1. Differentiation of&#160;rtTA/Ngn2-positive hiPSCs to Neurons on 6-well MEAs and Glass Coverslips
NOTE:&#160;In this protocol, the details are provided for differentiating&#160;reverse tetracycline-controlled transactivator (rtTA)/ Neurogenin-2 (Ngn2)-positive hiPSCs on&#160;6-well MEAs (devices composed of 6 independent wells with 9 recording and 1 reference embedded microelectrodes per well).
<ol>
	<li>Prepare the MEAs (day 0 and day 1)
	<ol>
		<li>The day before the start of the differentiation, sterilize the MEAs according to the manufacturer&#39;s recommendation.</li>
		<li>Dilute the adhesion protein Poly-L-Ornithine (PLO) in sterile ultrapure water to a final concentration 50 &#181;g/mL. Coat the active electrode area of 6-well MEAs by placing a 100 &#181;L drop of the diluted PLO in each well.</li>
		<li>Incubate the 6-well MEAs overnight (O/N) in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2. The next day, aspirate the diluted PLO. Wash the glass surfaces of the 6-well MEAs twice with sterile ultrapure water.</li>
		<li>Dilute laminin in cold Dulbecco&#39;s modified Eagle medium/nutrient mixture F-12 (DMEM/F12) to a final concentration of 20 &#181;g/mL. Immediately coat the active electrode area of the 6-well MEAs by placing a 100 &#181;L drop in each well.</li>
		<li>Incubate the 6-well MEAs in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2&#160;for at least 2 h.</li>
	</ol>
	</li>
	<li>Plate the hiPSCs (day 1) 
	NOTE:&#160;The volumes that are mentioned in steps 1.2.1 - 1.2.4 assume that the&#160;rtTA/Ngn2-positive hiPSCs are cultured in a 6-well plate and that the cells of one well are harvested. The volumes that are required for plating the cells on the 6-well MEAs depends on the number of 6-well MEAs that are used in the experiment; the numbers specified in steps 1.2.6 - 1.2.8 allow scaling to different experiment sizes.
	<ol>
		<li>Warm DMEM/F12, cell detachment solution (CDS) and E8 medium with 1% (v/v) penicillin/streptomycin to R/T. Add doxycycline to a final concentration of 4 &#181;g/mL and rho-associated protein kinase (ROCK) inhibitor to the E8 medium.</li>
		<li>Aspirate the spent medium of the&#160;rtTA/Ngn2-positive hiPSCs and add 1 mL CDS to the hiPSCs. Incubate 3 - 5 min in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2. Check under the microscope whether the cells are detaching from one another.</li>
		<li>Add 2 mL DMEM/F12 in the well, gently suspend the cells with a 1,000 &#181;L pipette and transfer the cells to a 15 mL tube. Add 7 mL DMEM/F12 to the cell suspension. Spin the cells at 200 x g for 5 min.</li>
		<li>Aspirate the supernatant and add 2 mL of the prepared E8 medium. Dissociate the hiPSCs by putting the tip of a 1,000 &#181;L pipette against the side of the 15 mL tube and resuspending the cells gently. Check under the microscope whether the cells are dissociated.</li>
		<li>Determine the number of cells (cells/mL) using a hemocytometer chamber. 
		NOTE:&#160;A 6-well plate well at 80 - 90% confluency will typically yield 3.0 - 4.0 x 106&#160;cells in total.</li>
		<li>Aspirate the diluted laminin. For the 6-well MEAs, dilute the cells to obtain a cell suspension of 7.5 x 105&#160;cells/mL. Plate the cells by adding a drop of 100 &#181;L of the cell suspension on the active electrode area in each well of the 6-well MEAs.</li>
		<li>Place the 6-well MEAs in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2&#160;for 2 h.</li>
		<li>After 2 h, carefully add 500 &#181;L of the prepared E8 medium to each well of the 6-well MEAs. Place the 6-well MEAs O/N in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2.</li>
	</ol>
	</li>
	<li>Change the medium (day 2)
	<ol>
		<li>The next day, prepare DMEM/F12 with 1% (v/v) N-2 supplement, 1% (v/v) non-essential amino acids and 1% (v/v) penicillin/streptomycin. Add human recombinant neurotrophin-3 (NT-3) to a final concentration of 10 ng/mL, human recombinant brain-derived neurotrophic factor (BDNF) to a final concentration of 10 ng/mL, and doxycycline to a final concentration of 4 &#181;g/mL. Warm the medium to 37 &#176;C.</li>
		<li>Add laminin to a final concentration of 0.2 &#181;g/mL to the medium. Filter the resulting medium. Aspirate the spent medium from the wells of the 6-well MEAs and the 24-well plate and replace it with the prepared medium. Incubate the 6-well MEAs O/N in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2.</li>
	</ol>
	</li>
	<li>Add rat astrocytes (day 3) 
	NOTE:&#160;The volumes that are mentioned in this protocol assume that the rat astrocytes are cultured in T75 culture flasks. It is critical that the rat astrocytes that are added to the cultures are of good quality. We use two criteria to check if the rat astrocytes are of good quality. First, the rat astrocyte culture should be able to grow confluent within ten days after the isolation from the rat embryonic brains. Second, after splitting the rat astrocyte culture, the rat astrocytes should be able to form a confluent, tessellated monolayer. If the rat astrocyte culture does not fulfill these two criteria, we advise not to use this culture for differentiation experiments.
	<ol>
		<li>Warm 0.05% trypsin- ethylenediaminetetraacetic acid (EDTA) to RT. Warm the Dulbecco&#39;s Phosphate-Buffered Saline (DPBS) and DMEM/F12 with 1% (v/v) penicillin/streptomycin to 37 &#176;C.</li>
		<li>Aspirate the spent medium of the rat astrocyte culture. Wash the culture by adding 5 mL DPBS and swish it around gently.</li>
		<li>Aspirate the DPBS and add 5 mL 0.05% trypsin-EDTA. Swish the trypsin-EDTA around gently. Incubate in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2&#160;for 5 - 10 min.</li>
		<li>Check under the microscope whether the cells are detached. Detach the last cells by hitting the flask a few times.</li>
		<li>Add 5 mL of DMEM/F12 to the flask. Triturate the cells gently inside the flask with a 10 mL pipette. Collect the cell suspension in a 15 mL tube. Spin the tube at 200 x g for 8 min.</li>
		<li>Aspirate the supernatant and resuspend the cells in 1 mL of DMEM/F12. Determine the number of cells (cells/mL) using a hemocytometer chamber.</li>
		<li>Add 7.5 x 104&#160;astrocytes per well of the 6-well MEAs. Incubate the MEAs O/N in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2.</li>
	</ol>
	</li>
	<li>Change the medium (day 4)
	<ol>
		<li>Prepare neurobasal medium with 2% (v/v) B-27 supplement, 1% (v/v) L-alanyl-L-glutamine and 1% (v/v) penicillin/streptomycin. Add NT-3 to a final concentration of 10 ng/mL, BDNF to a final concentration of 10 ng/mL, and doxycycline to a final concentration of 4 &#181;g/mL. In addition, add cytosine &#946;-D-arabinofuranoside to a concentration of 2 &#181;M. 
		NOTE:&#160;Cytosine &#946;-D-arabinofuranoside is added to the medium to inhibit astrocyte proliferation and to kill the remaining hiPSCs that are not differentiating into neurons.</li>
		<li>Filter the medium and warm to 37 &#176;C. Aspirate the spent medium from the wells of the 6-well MEAs and replace it with the prepared medium. Maintain the 6-well MEAs in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2.</li>
	</ol>
	</li>
	<li>Refresh the medium (day 6 - 28) 
	NOTE:&#160;Starting from day 6, refresh half of the medium every two days. From day 10 onwards, the medium is supplemented with fetal bovine serum (FBS) to support the astrocyte viability.
	<ol>
		<li>Prepare neurobasal medium with 2% (v/v) B-27 supplement, 1% (v/v) L-alanyl-L-glutamine and 1% (v/v) penicillin/streptomycin. Add NT-3 to a final concentration of 10 ng/mL, BDNF to a final concentration of 10 ng/mL, and doxycycline to a final concentration of 4 &#181;g/mL. From day 10 onwards, also supplement the medium with 2.5% (v/v) FBS. Filter the resulting medium and warm to 37 &#176;C.</li>
		<li>Remove half of the spent medium from the wells of the 6-well MEAs using a 1000 &#181;L pipette and replace it with the prepared medium. Maintain the 6-well MEAs in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2.</li>
	</ol>
	</li>
</ol>
2. Establish the Neurophysiological Profile of hiPSC-derived Neurons
NOTE:&#160;Two to three weeks after the induction of differentiation, the hiPSC-derived neurons can be used for different downstream analyses. In this section, examples of some downstream analyses are given that can be performed to establish the neurophysiological profile of the hiPSC-derived neurons.
<ol>
	<li>Characterize the neuronal network activity using MEAs
	<ol>
		<li>Record 20 min of electrophysiological activity of hiPSC-derived neurons cultured on MEAs. During the recording, maintain the temperature at 37&#160;&#176;C, and prevent evaporation and pH changes of the medium by inflating a constant, slow flow of humidified gas (5% CO2, 20% O2, 75% N2) onto the MEA.</li>
		<li>After 1200X amplification (MEA 1060, MCS), sample the signal at 10 kHz using the MCS data acquisition card. Analyze the data (spike and burst detection) using a custom software package.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>B-27 supplement</td>
			<td>Gibco</td>
			<td>0080085SA</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05 % Trypsin-EDTA&#160;</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>High-glucose DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F2442-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320-074</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell detachment solution</td>
			<td>Sigma-Aldrich</td>
			<td>A6964</td>
			<td></td>
		</tr>
		<tr>
			<td>E8 medium</td>
			<td>Gibco</td>
			<td>A1517001</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK inhibitor</td>
			<td>Gibco</td>
			<td>A2644501&#160;</td>
			<td>Alternatively, ROCK inhibitors like thiazovivin can be used.</td>
		</tr>
		<tr>
			<td>6-well MEAs</td>
			<td>Multi Channel Systems</td>
			<td>60-6wellMEA200/30iR-Ti-tcr</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-ornithine</td>
			<td>Sigma-Aldrich</td>
			<td>P3655-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Sigma-Aldrich</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>NT-3, human recombinant</td>
			<td>Promokine</td>
			<td>C66425</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF, human recombinant</td>
			<td>Promokine</td>
			<td>C66212</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>L-alanyl-L-glutamine</td>
			<td>Gibco</td>
			<td>35050-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytosine &#946;-D-arabinofuranoside&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C1768-100MG</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring the electrophysiological activity of neuronal networks using micro-electrode arrays

Source: Frega, M. et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54900">Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays.</a> J. Vis. Exp. (2017).
The video demonstrates how to use a microelectrode array (MEA) to record the electrophysiological activity of neurons derived from human induced pluripotent stem cells (hiPSCs). Electrophysiological activity from a multiwell MEA containing a neuron-astrocyte co-culture is recorded to detect action potentials from multiple neurons in the network, confirming the successful differentiation of hiPSCs into neurons.

Measuring the Electrophysiological Activity of Neuronal Networks Using Micro-Electrode Arrays

1. Differentiation of&#160;rtTA/Ngn2-positive hiPSCs to Neurons on 6-well MEAs and Glass Coverslips
NOTE:&#160;In this protocol, the details are provided ...

Begin with a neuron-astrocyte co-culture in a multi-well micro-electrode array or MEA.
Each well contains a network of neurons derived from human induced pluripotent stem cells, or hiPSCs. The rat astrocytes provide support for maintaining the network.
The base of the wells contains a grid of tiny electrodes.
Place the MEA in a recording device to capture the spontaneous electrophysiological activity of the neuronal network.
Flow a humidified gas mixture into the culture to prevent media evaporation and stabilize the pH, ensuring cell viability.
Neurons transmit signals via electrical impulses, known as action potentials, that travel along their axons.
Upon reaching the synapse, the impulse triggers the release of neurotransmitters, which propagate the signal to the next neuron.
The electrodes within each well simultaneously record action potentials generated by multiple synaptically connected neurons.
The spontaneous electrophysiological activity of the neuronal network confirms the differentiation of hiPSCs into neurons.

measuring-electrophysiological-activity-neuronal-networks-using-micro

Universidad de Cartagena UDEC

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JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

86 Views. Source: Frega, M. et al. Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays. J. Vis. Exp. (2017). The video demonstrates how to use a microelectrode array (MEA) to record the electrophysiological activity of neurons derived from human induced pluripotent stem cells (hiPSCs). Electrophysiological activity from a multiwell MEA containing a neuron-astrocyte co-culture is recorded to detect action potentials from multiple neuron...

Video: Measuring the Electrophysiological Activity of Neuronal Networks Using Micro-Electrode Arrays - Concept

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

JoVE Journal

Developmental Biology

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.

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 ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Measuring the Electrophysiological Activity of Neuronal Networks Using Micro-Electrode Arrays

Transcript

Measuring the Electrophysiological Activity of Neuronal Networks Using Micro-Electrode Arrays

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

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

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation