This work was supported by grants from Satt Conectus program, Fondation de l&#39;Universit&#233; de Strasbourg, &#171;J&#39;ai demand&#233; la lune&#187;, &#171;Une roulade pour Charline&#187;, &#171;LifePink&#187;, &#171;Franck, Rayon de Soleil&#187; and &#171;Semeurs d&#39;Etoile&#187; associations. We thank the children and families affected by HGGs for their contributions to this research and their support.

For this protocol, the accreditation number related to the use of human materials is DC-2020-4203.
1. Microfluidic device fabrication, preparation and treatment
<ol>
	<li>Fabricate SU-8 molds using conventional photolithography techniques18. 
	NOTE: For this purpose, two photolithography masks have been designed to construct two layers of photoresist structures on silicon wafer substrate and a thin SU-8 2005 photoresist layer (3.2 &#181;m high and 6 &#177; 1 &#18...

<ol>
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AB, MG, JR, LM, ML, JV, DD are employed by NETRI, FL is Chief Technology Officer at NETRI, and TH is Chief Scientific Officer at NETRI. The other authors have nothing to disclose.

This work describes an accurate functional in vitro model to evaluate the interaction between human hiPS-derived cortical glutamatergic neurons and brain tumoral cells in microfluidic devices. One of the crucial steps in the present protocol was the hiPS differentiation in glutamatergic neurons, which was confirmed by the decrease of Nestin and Sox2 immunofluorescent staining and simultaneous appearance of mGluR2 and vGLUT1 staining. Nevertheless, few neural progenitors remained as only half of the glutamatergic...

Pediatric high-grade gliomas (pHGG) exhibit an extended genotypic and phenotypic diversity depending on patient age, tumor anatomical location and extension, and molecular drivers1. They are aggressive brain tumors that are poorly controlled with the currently available treatment options and are the leading cause of death related to brain cancers in children and adolescents2. So, more than 80% of patients are relapsing within 2 years after their diagnosis, and their median survival is 9-15 months, depending on brain locations and driver mutations. The absence of curative treatment is the primary urge for laboratory research and highlights the immediate need for new innovative therapeutic approaches. For this purpose, patient-derived cell lines (PDCL) were developed with the hope of providing the pHGG diversity3 in two-dimensional (2D) lines and/or three-dimensional (3D) neurospheres. Nevertheless, those patient-derived in vitro cell cultures do not mimic all brain variable situations. These models do not consider the macroscopic and microscopic neuro-anatomical environments typically described in pHGG.
Usually, pHGG in younger children is mainly developing in pontine and thalamic regions, whereas adolescent and young adult&#39;s HGG concentrate in the cortical areas, especially in frontotemporal lobes1. These location-specificities across pediatric ages seem to involve different environments leading to gliomagenesis and an intricating network between tumor cells and specific neuronal activity4,5,6. Although mechanisms are still not identified, pHGG mainly develops from neural precursor cells along the differentiation trajectory of astroglial and oligodendroglial lineages. While the role of these glial lineages has been for long restricted to simple structural support for neurons, it is now clearly established that they integrate entirely into neural circuits and exhibit complex bi-directional glial-neuronal interactions able to reorganize structural regions of the brain and remodel neuronal circuitry4,7,8. Moreover, increasing shreds of evidence indicate that the central nervous system (CNS) plays a critical role in brain cancer initiation and progression. Recent works focused on neuronal activity, which seems to drive growth and mitosis of glial malignancies through secreted growth factors and direct electrochemical synaptic communications6,9. Reciprocally, high-grade glioma cells seem to influence neuronal function with an increasing glutamatergic neuronal activity and modulate the operation of the circuits into which they are structurally and electrically integrated9. So, studies using patient-derived models and novel neuroscience tools controlling neuron action demonstrated a circuit-specific effect of neuronal activity on glioma location, growth, and progression. Most of these neuronal projections involved in gliomas are glutamatergic and communicate through glutamate secretions. Specific glutamatergic biomarkers such as mGluR2 or vGlut1/2 are commonly described6.
Interestingly, despite their molecular heterogeneity, pediatric and adult high-grade gliomas show a typical proliferative response to glutamatergic neuronal activity and other secreted factors such as neuroligin-3 or BDNF (brain-derived neurotrophic factor)4,6,10,11,12,13. In cortical regions, pediatric and adult HGGs can induce neuronal hyperexcitability through an increased glutamate secretion and inhibit GABA interneurons leading to gliomas associated with epileptic network activity14,15. On top of that, neural circuits can be remodeled by gliomas pushing specific neurological tasks, for instance, language, and can requisition additional organized neuronal activity9.
Based on this rationale, advancing the understanding of bidirectional communications between glioma cells and neurons must be fully elucidated and integrated with the early stages of in vitro pHGG approaches. Such innovative modeling is crucial in understanding and measuring the neuronal electrical activity impact during drug testing and anticipating pHGG response into brain circuitry. Recent developments in neuroscience tools, such as microfluidic devices and pHGG research works, are the bed to develop new modeling approaches and be able now to integrate brain microenvironment in&#160;in vitro pHGG models3,16,17,18,19. Coupled with electrophysiological recordings using multielectrode arrays (MEA), microfluidic devices20,21,22 offer the possibility to mimic physiological conditions while recording the electrical activity of the entire neural network and extract network connectivity parameters under several conditions. This device23,24 allows first the precise deposition of cells in a chamber directly on MEA. This technology enables the control of cell seeding density and homogeneity on MEA and the fine control of media exchange, which is a critical step for human neural progenitor differentiation directly into devices. Moreover, the present deposition chamber can be seeded with multiple cells at different time points.
So, this study aimed to develop a functional in vitro model co-culturing human Pluripotent Stem (hiPS)-derived cortical glutamatergic neurons and pHGG-derived cells into microfluidic devices and recording their electrical activity to evaluate electrical interactions between both cell populations. First, hiPS-derived cortical glutamatergic neurons were obtained and characterized in microfluidic devices at different stages of culture [day 4 (D4), as hiPS cells, and day 21 (D21) and day 23 (D23), as glutamatergic matured neurons]. For the second step of co-culture, two pHGG models were used: commercialized pediatric UW479 line and pHGG cells initiated from a patient tumor (BT35)3, bearing an H3.3 K27M driver mutation. Finally, we performed electrophysiological recordings of glutamatergic cells at D21 before pHGG cell seeding and D23 after 48 h of co-culture into the same microfluidic device. The interactions between glutamatergic neurons and pHGG cells were characterized by a significant increase in the recorded electrophysiological activity.

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	<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&#160;</td>
			<td >Dutscher</td>
			<td >53750</td>
			<td ></td>
		</tr>
		<tr >
			<td >60 &#181;m probe for Scepter counter&#160;</td>
			<td >Dutscher</td>
			<td >51999</td>
			<td ></td>
		</tr>
		<tr >
			<td >Accutase</td>
			<td >Sigma</td>
			<td >A6964</td>
			<td ></td>
		</tr>
		<tr >
			<td >Ala -Gln (GlutaMAX)</td>
			<td >Sigma</td>
			<td >G8541</td>
			<td ></td>
		</tr>
		<tr >
			<td >Axel Observer 7 Microscope</td>
			<td >Zeiss</td>
			<td >431007-9904-000</td>
			<td ></td>
		</tr>
		<tr >
			<td >Cell culture flask with cap with filter membrane 70 mL Falcon&#174;</td>
			<td >Dutscher</td>
			<td >353109</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 >Colibri 7 LED</td>
			<td >Zeiss</td>
			<td >4230529710-000</td>
			<td ></td>
		</tr>
		<tr >
			<td >
			<div>Cortical Glutamatergic Neurons</div>
			</td>
			<td >BrainXell</td>
			<td >BX-0300</td>
			<td ></td>
		</tr>
		<tr >
			<td >DMEM/F-12 (1:1) GlutaMAX</td>
			<td >Gibco</td>
			<td >31331-028</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&#160;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 >MCS InterFace Boarder</td>
			<td >MCS</td>
			<td >181205-MEA2100-11240</td>
			<td ></td>
		</tr>
		<tr >
			<td >MEA2100</td>
			<td >MCS</td>
			<td >181205-MEA2100-11240</td>
			<td ></td>
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		<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 filter tip 0.1 - 10 &#181;l ClearLine&#174; sterile in removable-lid rack&#160;</td>
			<td >Dutscher</td>
			<td >030570ACL</td>
			<td ></td>
		</tr>
		<tr >
			<td >Non filter tip 1 - 200 &#181;l ClearLine&#174; sterile in removable-lid rack&#160;</td>
			<td >Dutscher</td>
			<td >032260CL</td>
			<td ></td>
		</tr>
		<tr >
			<td >Non filter tip 50 - 1250 &#181;l ClearLine&#174; sterile in removable-lid rack</td>
			<td >Dutscher</td>
			<td >134760CL</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;&#160;</td>
			<td >Dutscher</td>
			<td >357551</td>
			<td ></td>
		</tr>
		<tr >
			<td >Pipette for cell culture 5 mL Falcon&#174;&#160;</td>
			<td >Dutscher</td>
			<td >357543</td>
			<td ></td>
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		<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>
		<tr >
			<td >Scepter (Handheld Automated Cell Counter)</td>
			<td >Millipore</td>
			<td >PHCC00000</td>
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co-culture of glutamatergic neurons and pediatric high-grade glioma cells into microfluidic devices to assess electrical interactions

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to overcome the resistance to current treatments and find a new way of cure, modeling the disease as close as possible in an in vitro setting to test new drugs and therapeutic procedures is highly demanding. Studying their fundamental pathobiological processes, including glutamatergic neuron hyperexcitability, will be a real advance in understanding interactions between the environmental brain and pHGG cells. Therefore, to recreate neurons/pHGG cell interactions, this work shows the development of a functional in vitro model co-culturing human-induced Pluripotent Stem (hiPS)-derived cortical glutamatergic neurons pHGG cells into compartmentalized microfluidic devices and a process to record their electrophysiological modifications. The first step was to differentiate and characterize human glutamatergic neurons. Secondly, the cells were cultured in microfluidic devices with pHGG derived cell lines. Brain microenvironment and neuronal activity were then included in this model to analyze the electrical impact of pHGG cells on these micro-environmental neurons. Electrophysiological recordings are coupled using multielectrode arrays (MEA) to these microfluidic devices to mimic physiological conditions and to record the electrical activity of the entire neural network. A significant increase in neuron excitability was underlined in the presence of tumor cells.

Before studying electrical interactions between glutamatergic neurons and glioma cells, hiPS-derived cortical glutamatergic neurons were characterized to validate the feasibility of culturing them in microfluidic devices (Figure 1A). Their characterization was assessed using Nestin, Sox2, mGlurR2 (metabotropic Glutamate Receptors 2), and vGLUT1 immunostaining, represented in Figure 1A(2-7). As Nestin is an intermediate f...

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Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions

Recent works uncover the neuronal impact on high-grade pediatric glioma (pHGG) cells and their reciprocal interactions. The present work shows the development of an in vitro model co-culturing pHGG cells and glutamatergic neurons and recorded their electrophysiological interactions to mimic those interactivities.

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to ...

The interactions between the malignant glioma cells and their surrounding neurons are gaining interest. Here, we show the development of an in vitro model co-culturing derived cortical glutamatergic neurons and tumor cells. The microfludic devices used for co-cultures coupled to multielectrode arrays allow the study of different cell types and perform electrical activity recordings at different time points of the cultures.This method is particularly helpful for functional and mechanistic studies and for analyzing the effects of pharmacological agents that block pediatric high-grade glioma cell migration and interaction with neurons. To begin culturing the commercialized human IPS-derived cortical glutamatergic neurons, empty the inlet and outlet reservoirs of the microfluidic device by pipette aspiration, letting only the device channels be filled with medium. Seed the human IPS cells by adding 10 microliters of 6.5 million human IPS cells per milliliter suspension in the medium and let the device be under the hood for 15 minutes to allow cells to attach.After 15 minutes, fill both inlet and outlet reservoirs with 50 microliters of day four culture medium, then transfer the device into the incubator to maintain the cells at 37 degrees Celsius and 5%carbon dioxide for 23 days. Replace the medium every three to four days as described in the text. To count the cells and assess viability using a standard phase contrast microscope, take microscopic pictures at 4th day, 21st day and 23rd day.Culture both UW479 and BT35 cell lines in DMEM and F-12 GlutaMAX supplemented with 10%fetal bovine serum in a cell culture flask. Maintain cell culture under a controlled environment at 37 degrees Celsius in normoxic conditions throughout the experiment. Observe each cell line to reach 80%confluency.On day 21 post-culture of glutamatergic neurons, start co-culture by trypsinizing UW479 and BT35 cells. 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.Perform another electrophysiological recording on the 23rd day after two days of the co-culture. Human IPS-derived cortical glutamatergic neurons were characterized and assessed using nestin, SOX2, metabotropic glutamate receptors II, and VGLUT1 immunostaining. Nestin is an intermediate filament protein and it was expressed in undifferentiated CNS cells.SOX2 was also expressed in undifferentiated cells of the neural epithelium in CNS. Nestin and SOX2-positive cells percentage decreased from the fourth day to the 21st day, confirming the differentiated state of glutamatergic neurons. The microscopic images revealed a progressive distribution of glutamatergic neurons across microfluidic devices.When BT35 and UW479 were added to the culture, it was observed that the floating cells were present after glioma cell seeding on the 21st day, which progressively disappeared until the 23rd day and became adherent in the device. The percentages of viable cells for BT35 and UW479 were comparable and implied that patient-derived cell lines could be used appropriately. Spike detection and raster plot on the 23rd day of culture showed increased electrical activity after adding pediatric high-grade glioma cells.To monitor the synchronicity of the neural network activity, the instantaneous firing rate in BT35 or glutamatergic neurons was recorded. The difference in electrical activity between the individually cultured and co-cultured glutamatergic neurons was significant on the 23rd day. Further methodological development may include the functional assessment of the network with burst analysis and special temporal cross-correlations of the network.Now, researchers have a promising tool to explore interactions and pharmacological targeting of pediatric high-grade glioma tumor lines and high PSC neurons. Several tumor lines and several neuron types might be used.

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3.3K ビュー.  UMR CNRS 7021 - Laboratory of Bioimaging and Pathologies. 悪性グリオーマ細胞とその周囲のニューロンとの相互作用が関心を集めています。ここでは、インビトロモデルの共同培養由来皮質グルタミン酸ニューロンと腫瘍細胞の開発を示す。多電極アレイに結合された共培養に使用されるマイクロフルディックデバイスは、異なる細胞タイプの研究を可能にし、培養の異なる時点で電気活動記録を行う。この方法は、機能?...