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EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Cell preparation and seeding in the microfluidic device
<ol>
	<li>Culture, seeding, and immunofluorescent characterization of human hiPS-derived cortical glutamatergic neurons 
	NOTE: Conduct experiments with commercialized human induced pluripotent stem cells (hiPS)-derived cortical glutamatergic neurons (Figure 1A). Preserve human-derived materials and handle them with the approval and under the legislation guidelines.
	<ol>
		<li>Empty the inlet and outlet reservoirs of the microfluidic device by pipette aspiration before seeding, letting only the device&#39;s channels be filled with medium.</li>
		<li>Seed the hiPS cells at Day 0 (D0) by putting 10 &#181;L of a 6.5 x 106 hiPS cells/mL suspension (e.g., 900 cell/mm2 concentration) with the medium, whose composition is given in Table 1, and let the device be under the hood (at room temperature) for 15 min to allow cells to attach.</li>
		<li>After 15 min, fill both inlet and outlet reservoirs with 50 &#181;L of D4 culture medium, whose entire composition is given in Table 1, and transfer the device into the incubator (37 &#176;C, 5% CO2).</li>
		<li>Maintain glutamatergic neuron differentiation for 23 days (Figure 1A(1), microscopy) under a controlled environment (37 &#176;C, 5% CO2) and with a specific cell culture medium, whose composition is described in Table 1. Replace the medium regularly, as detailed in the next step 5 just below, and follow the steps in Figure 1B.</li>
		<li>Replace the medium every 3 to 4 days following Table 1 composition. Use seeding medium until D4, D4 medium until D7, D7 medium until D11, and D11 medium for the remaining culture time.</li>
		<li>Take microscopic pictures at D4, D21, and D23 using a standard phase-contrast microscope to assess cell viability and allow cell counting.</li>
		<li>For characterization, fix the differentiated glutamatergic neurons in 4% paraformaldehyde (PFA) for 30 min at room temperature after medium aspiration and follow the protocol for immunofluorescence staining.</li>
		<li>Wash cells three times with phosphate-buffered saline (PBS) and permeabilize them for 10 min with 0.1% Triton-X followed by 30 min with 3% bovine serum albumin (BSA). Add primary antibodies and incubate the device overnight at 4 &#176;C.</li>
		<li>Rinse the cells thrice with PBS and incubate further with the corresponding secondary antibodies for 2 h at room temperature.</li>
		<li>Rinse the cells thrice with PBS and counterstain with DAPI (4&#39;,6-diamino-2-phenylindole) for 10 min at room temperature.</li>
		<li>Acquire images with an inverted epifluorescence microscope fitted with a CMOS (Complementary metal-oxide-semiconductor) camera and analyze using appropriate image analysis software.</li>
		<li>Open the DAPI stained images and binarize them with the thresholding routine in the software. To do this, click Image &#62; Adjust &#62; Threshold and set the appropriate parameters to distinguish the nucleus from the background. Then, click on Apply &#62; Process &#62; Watershed to split the aggregate nucleus.</li>
		<li>Then, use the Analyze Particles innate software&#39;s function to interpret the binary images. To do this, click Analyze and then Analyze Particles and set the appropriate parameters after this: size and circularity filter (exclude from analysis objects smaller than 7 &#181;m, bigger than 20 &#181;m, or with a circularity smaller than 0.1 &#181;m).</li>
		<li>Merge the contour images obtained from DAPI analysis to correspondent immunofluorescent pictures to validate the correct staining.</li>
		<li>Finally, save the associated data (number of objects, mean area, % of coverage, etc.) for the different biomarkers and quantify the expression at D4 and/or D21 using appropriate quantification software.</li>
	</ol>
	</li>
	<li>Cell culture of commercialized pHGG line, UW479, and patient-derived cell line, BT353&#8203;
	<ol>
		<li>Culture both cell lines in Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) GlutaMAX supplemented with 10% Fetal Bovine Serum (FBS) in a cell culture flask. Wait for 80% confluence of each cell line, as presented in Figure 1C.</li>
		<li>Maintain these pHGG cells under a controlled environment at 37 &#176;C in normoxic conditions throughout the experiments.</li>
	</ol>
	</li>
</ol>
2. 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 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 D11 and onward medium detailed in Table 1.</li>
	<li>Count pHGG cells using the microscopic pictures analyzed with an image analysis software to assess their viability. Calculate the percentage of cells. 
	&#8203;NOTE: Use the formula: 100 - ((number of cells at D23 / number of cells at D21) x 100).</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>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>Cortical Glutamatergic Neurons</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 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>
		</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 filter tip 0.1 - 10 &#181;l ClearLine&#174; sterile in removable-lid rack</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</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;</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>
		<tr>
			<td>Scepter (Handheld Automated Cell Counter)</td>
			<td>Millipore</td>
			<td>PHCC00000</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF-&#946;1</td>
			<td>Peprotech</td>
			<td>100-21C</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube with conical bottom 15 mL (bulk) Falcon&#174;</td>
			<td>Dutscher</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube with conical bottom 50 mL (bulk) Falcon&#174;</td>
			<td>Dutscher</td>
			<td>352070</td>
			<td></td>
		</tr>
	</tbody>
</table>

co-culturing glutamatergic neurons and pediatric high-grade glioma cells

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 showcases a technique for co-culturing glutamatergic neurons and pediatric high-grade glioma (pHGG) cells within a microfluidic device. Human induced pluripotent stem cells are introduced into the microfluidic system and incubated to promote their differentiation into mature cortical glutamatergic neurons. Following this, the pHGG cells are introduced onto the mature neurons, establishing a co-culture and enabling interactions between neurons and pHGG cells.

<img alt="Figure 1" src="/files/ftp_upload/22351/22351_Figure1.jpg" /> 
Figure 1: Cell characterization and co-culture protocol of glutamatergic neurons and high-grade pediatric glioma (pHGG) lines. (A) Prerequisite microscopic and immunofluorescent characterization of glutamatergic neurons. (1) Microscopic pictures at day 21 (D21) and day 23 (D23) of glutamatergic neurons. (2) Nestin labeling in green and DAPI in bl...

Co-culturing Glutamatergic Neurons and Pediatric High-Grade Glioma Cells

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Cell preparation and seeding in ...

Begin with a polymer-coated microfluidic device comprising reservoir wells connected by channels and filled with culture medium.
Remove the medium from the reservoirs and seed human induced pluripotent stem cells.
The cells enter the channels and adhere to the coated surface.
Now, fill the reservoirs with a differentiation medium.
Growth factors in the medium induce stem cell differentiation into neuronal progenitor cells.
Next, add a medium containing specific nutrients and neurotrophic factors that induce neuronal progenitor maturation into glutamatergic neurons.
These neurons release the excitatory neurotransmitter glutamate from their presynaptic terminals, which bind to postsynaptic neuron receptors, generating excitatory signals.
Now, take pediatric high-grade glioma cells, cancer cells derived from aggressive brain tumors.
Add the cells to the reservoirs and incubate. The glioma cells enter the channels and adhere.
Gradually, these cells form functional synapses with the glutamatergic neurons, facilitating neuronal-glioma cell crosstalk and leading to neuronal hyperactivity.

co-culturing-glutamatergic-neurons-pediatric-high-grade-glioma

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Co-culture and Interaction Studies

50 Views. Source: Fuchs, Q., et al. Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions. J. Vis. Exp. (2021). This video showcases a technique for co-culturing glutamatergic neurons and pediatric high-grade glioma (pHGG) cells within a microfluidic device. Human induced pluripotent stem cells are introduced into the microfluidic system and incubated to promote their differentiation into mature cortical glutamatergic neurons...

Video: Co-culturing Glutamatergic Neurons and Pediatric High-Grade Glioma Cells - Concept

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.

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

JoVE Journal

Cancer Research

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

Immunology and Infection

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Co-culturing Glutamatergic Neurons and Pediatric High-Grade Glioma Cells

Transcript

Co-culturing Glutamatergic Neurons and Pediatric High-Grade Glioma Cells

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

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons