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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Nanyang Technological University

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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.remotexs.ntu.edu.sg/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.

21 ビュー. 共培養におけるグルタミン酸作動性ニューロンと神経膠腫細胞の相互作用の評価

ビデオ: 共培養におけるグルタミン酸作動性ニューロンと神経膠腫細胞の相互作用の評価 - 実験

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

腫瘍細胞の挙動および薬物応答の長期的研究のためのゼブラフィッシュ小児脳腫瘍の免疫適格宿主への移植

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

JoVE Journal

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Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

パターン化ニューロン上の神経膠芽腫幹細胞の共培養による移動と細胞相互作用の研究

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

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.

マルチ電極アレイ上のらせん神経節ニューロン外植片培養と電気生理学

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

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

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