assessing glutamatergic neurons and glioma cells interactions in a co-culture

Assessing Glutamatergic Neurons and Glioma Cells Interactions in a Co-Culture

Seed the trypsinized UW479 and BT35 cells on top of the mature adherent glutamatergic neurons in each dedicated microfluidic device. Maintain the co-cultures for two days under a controlled environment at 37 degrees Celsius and 5% carbon dioxide with glutamatergic neuron D11 and onward medium.
Count pediatric high-grade glioma cells using the microscopic pictures analyzed with the image analysis software to assess their viability, and calculate the percentage of cells. Place the MFD in the recording device, and use a commercially available software to perform the electrophysiological recording of glutamatergic neurons on the 21st day.
Before seeding pediatric high-grade glioma cells, perform a second electrophysiological recording of the differentiated glutamatergic neurons cultured as control, parallel to the co-culture.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.&#160;
1. Co-culture protocol
<ol>
	<li>Adjust the seeding day and concentration for UW479 and BT35 cell lines to reach 80% of confluent cells when co-culture should start, corresponding to 21 days of culture for glutamatergic neurons.</li>
	<li>Trypsinize UW479 and BT35 cells and seed them on the top of glutamatergic neurons in each dedicated microfluidic device (with a target density of 900 cell/mm2 for each cell type) before seeding the pediatric high-grade gliomas (pHGG) cells into the microfluidic device containing matured glutamatergic neurons.</li>
	<li>Maintain the co-cultures for 2 days under a controlled environment (37 &#176;C, 5% CO2) with glutamatergic neuron Day11 and onward medium detailed in Table 1.</li>
	<li>Count pHGG cells using the microscopic pictures analyzed with image analysis software to assess their viability. Calculate the percentage of cells. 
	&#8203;NOTE: Use the following formula: 100 - ((number of cells at D23 / number of cells at D21) x 100).</li>
</ol>
2. Electrophysiological recording
<ol>
	<li>Perform the electrophysiological recording with a commercial system and commercially available software. 
	NOTE: The experiments were carried out with a multielectrode array (MEA) that consisted of 30 &#181;m diameter electrodes spaced by 100 &#181;m.</li>
	<li>Perform a first electrophysiological recording on glutamatergic neurons at D21 before seeding of pHGG cells. Use the differentiated glutamatergic neurons as control and culture them alone in parallel to the co-culture. Do a second recording for both conditions as described in the next step numbered 3.</li>
	<li>Perform a second electrophysiological recording at D23 (after 2 days of co-culture).</li>
</ol>
Table 1: Culture media composition for hiPS-derived glutamatergic neurons.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Components</td>
			<td>Seeding medium</td>
			<td>D4 medium</td>
			<td>D7 medium</td>
			<td>D11 and onward medium</td>
		</tr>
		<tr>
			<td>DMEM/F-12 Medium</td>
			<td>0.5x</td>
			<td>0.25x</td>
			<td>0.125x</td>
			<td>&#216;</td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>0.5x</td>
			<td>0.25x</td>
			<td>0.125x</td>
			<td>&#216;</td>
		</tr>
		<tr>
			<td>BrainPhys Medium</td>
			<td>&#216;</td>
			<td>0.5x</td>
			<td>0.75x</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>SM1 Supplement</td>
			<td>1x</td>
			<td>1x</td>
			<td>1x</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>N2 Supplement-A</td>
			<td>1x</td>
			<td>1x</td>
			<td>1x</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Ala-Gln (GlutaMax)</td>
			<td>0.5 mM</td>
			<td>0.5 mM</td>
			<td>0.5 mM</td>
			<td>0.5 mM</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>10 ng/mL</td>
			<td>10 ng/mL</td>
			<td>10 ng/mL</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>10 ng/mL</td>
			<td>10 ng/mL</td>
			<td>10 ng/mL</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td>TGF-&#946;1</td>
			<td>1 ng/mL</td>
			<td>1 ng/mL</td>
			<td>1 ng/mL</td>
			<td>1 ng/mL</td>
		</tr>
		<tr>
			<td>Geltrex</td>
			<td>30 &#181;g/mL</td>
			<td>&#216;</td>
			<td>&#216;</td>
			<td>&#216;</td>
		</tr>
		<tr>
			<td>Seeding Supplement</td>
			<td>1x</td>
			<td>&#216;</td>
			<td>&#216;</td>
			<td>&#216;</td>
		</tr>
		<tr>
			<td>Day 4 Supplement</td>
			<td>&#216;</td>
			<td>1x</td>
			<td>&#216;</td>
			<td>&#216;</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>256MEA100/30iR-ITO-w/o</td>
			<td>MCS</td>
			<td>256MEA100/30iR-ITO-w/o</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m probe for Scepter counter</td>
			<td>Dutscher</td>
			<td>53750</td>
			<td></td>
		</tr>
		<tr>
			<td>60 &#181;m probe for Scepter counter</td>
			<td>Dutscher</td>
			<td>51999</td>
			<td></td>
		</tr>
		<tr>
			<td>Ala -Gln (GlutaMAX)</td>
			<td>Sigma</td>
			<td>G8541</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II Biological Safety Cabinet</td>
			<td>Thermo Scientific</td>
			<td>HERASafe type KS12</td>
			<td></td>
		</tr>
		<tr>
			<td>Cortical Glutamatergic Neurons</td>
			<td>BrainXell</td>
			<td>BX-0300</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 Medium</td>
			<td>Sigma</td>
			<td>D8437</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS 1X</td>
			<td>Dutscher</td>
			<td>L0615-500</td>
			<td></td>
		</tr>
		<tr>
			<td>EasYFlaskTM cell culture flasks 75cm3</td>
			<td>Nunc</td>
			<td>156499</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum (FBS)</td>
			<td>Dutscher</td>
			<td>500105</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Peprotech</td>
			<td>450-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex</td>
			<td>Life Technologies</td>
			<td>A1413201</td>
			<td></td>
		</tr>
		<tr>
			<td>Human BDNF</td>
			<td>Peprotech</td>
			<td>450-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Memmert</td>
			<td>IC0150med</td>
			<td></td>
		</tr>
		<tr>
			<td>MEA2100</td>
			<td>MCS</td>
			<td>181205-MEA2100-11240</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette P10</td>
			<td>Sartorius</td>
			<td>LH-729020</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette P100</td>
			<td>Sartorius</td>
			<td>LH-729050</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette P1000</td>
			<td>Sartorius</td>
			<td>LH-729070</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette P200</td>
			<td>Sartorius</td>
			<td>LH-729060</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtube Eppendorf 1,5 ml Safe-Lock</td>
			<td>Dutscher</td>
			<td>33290</td>
			<td></td>
		</tr>
		<tr>
			<td>MultiChannel Experimenter</td>
			<td>MCS</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Supplement-A</td>
			<td>StemCell</td>
			<td>7152</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Life Technologies</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurocult SM1 neuronal supplement</td>
			<td>StemCell</td>
			<td>5711</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids (NEAA) without L-glutamine</td>
			<td>Dutscher</td>
			<td>X0557-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipeteur Pipet-Aid XP Gravity</td>
			<td>Drummond</td>
			<td>4000202/4038202</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette for cell culture 10 mL Falcon&#174;</td>
			<td>Dutscher</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette for cell culture 5 mL Falcon&#174;</td>
			<td>Dutscher</td>
			<td>357543</td>
			<td></td>
		</tr>
		<tr>
			<td>Plaque chauffante (CultureTemp)</td>
			<td>Belart</td>
			<td>370151000</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Primovert microscope</td>
			<td>Zeiss</td>
			<td>415510-1100-000</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Fuchs, Q. et. al., <a href="https://app.jove.com/v/62748">Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions.</a> J. Vis. Exp. (2021)
This video demonstrates the assessment of neuronal electrical activity in a co-culture of hiPSC-derived cortical glutamatergic neurons and glioma cells using a microfluidic device with multielectrode arrays.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.&#160;
1. Co-culture protocol

	...

Begin with a laminin-coated, compartmentalized microfluidic device positioned over a patterned multielectrode array or MEA.
The device carries cultured iPSC-derived cortical glutamatergic neurons adhered to microelectrodes of an MEA.
These neurons communicate via electrical signals. Record these signals as baseline neuronal activity.
Next, seed the device with glutamate -rich pediatric high-grade glioma cells, a type of brain tumor cell, and incubate.
Over time, glioma cells enter the channel and attach, establishing the co-culture. Later, glioma cells release glutamate, an excitatory neurotransmitter.
The released glutamate interacts with neuronal receptors, leading to an influx of calcium and sodium ions that causes neuronal membrane depolarization.
This induces neuronal excitability and enhances neuronal activity.
Re-record the electrical signals, and a significant increase after co-culture indicates successful neuron and glioma cell interaction.

18 vues. Évaluation des interactions entre les neurones glutamatergiques et les cellules de gliome dans une co-culture

Vidéo: Évaluation des interactions entre les neurones glutamatergiques et les cellules de gliome dans une co-culture - Expérience

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Assessing Glutamatergic Neurons and Glioma Cells Interactions in a Co-Culture

Transcription

Assessing Glutamatergic Neurons and Glioma Cells Interactions in a Co-Culture