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 &#181;m wide) that defines asymmetric microgrooves under the patterning of main channels made in SU-8 2100 (200 &#181;m high, 1 mm wide, and 13 mm long) (see Supplemental Figure S1).</li>
	<li>Activate the wafer by using a plasma cleaner (5.00e-1 torr, high radio frequency (RF) level) for 1 min and silanize it using 1 mL of (trichloro(1H,1H,2H,2H-perfluorooctyl)silane in an aluminum folder into a desiccator for 30 min.</li>
	<li>Prepare a 10:1 ratio of Polydimethylsiloxane (PDMS), mix the prepolymer with catalyst, pass it in a vacuum desiccator to remove trapped bubbles, and cast it slowly onto the mold before being cured in an oven at 80 &#176;C for 40 min.</li>
	<li>Cut it after that using a razor at the desired size before peeling off the mold. Punch out the inlet and the outlet zones to obtain 3 mm wide holes, clean the PDMS, and protect it using adhesive tape. 
	NOTE: The thickness of the final PDMS device is approximately 5 mm.</li>
	<li>Treat the device using a plasma cleaner (5.00e-1 torr, High RF level during 1 min), assemble it with a polystyrene Petri dish, and expose the vacuum under UV light for 30 min.</li>
	<li>Fill with a pipette the inlet of the microfluidic device before usage with 70% ethanol and empty it by pipetting aspiration.</li>
	<li>Wash the microfluidic device by adding inlet reservoirs Dulbecco&#39;s Phosphate Buffered Saline (D-PBS) and empty it by pipetting aspiration three consecutive times.</li>
	<li>Coat the device using 0.05 mg/mL Poly-D-Lysine and place it into an incubator (37 &#176;C, 5% CO2). After 24 h, rinse the device by adding into the inlet reservoir the neurobasal medium and empty it by pipetting aspiration three consecutive times.</li>
	<li>Fill the microfluidic chip with the cell culture medium and let it remain at room temperature until use for a maximum of 2 h.</li>
</ol>
2. Cell preparation and seeding in the microfluidic device
<ol>
	<li>Culture, seeding, and immunofluorescent characterization of human hiPS-derived cortical glutamatergic neurons 
	NOTE: Carry out experiments with commercialized hiPS-derived cortical glutamatergic neurons (Figure 1A). Preserve human-derived materials and handle them with the approval and under the guidelines of legislation.
	<ol>
		<li>Empty the inlet and outlet reservoirs of the microfluidic device by pipette aspiration before seeding, letting only the device&#39;s channels to 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 as 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 time of the culture.</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 three times with PBS and incubate further with the corresponding secondary antibodies for 2 h at room temperature. 
		NOTE: Immunofluorescent antibodies used in the study are listed in Table 2.</li>
		<li>Rinse the cells three times 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 with the thresholding routine in the software. To do this, click on 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>After that, use the Analyze Particles innate software&#39;s function to interpret the binary images. To do this, click on Analyze and then on 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
	<ol>
		<li>Culture both cell lines in 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>
3. 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&#160;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>
4. Electrophysiological recording
<ol>
	<li>Perform the electrophysiological recording with a commercial system and commercially available software. 
	NOTE: The experiments were carried out with a 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&#160;described in the next step numbered 3.</li>
	<li>Perform a second electrophysiological recording at D23 (after 2 days of co-culture).</li>
</ol>
5. Electrophysiological data processing
<ol>
	<li>Filter the raw data with a 2nd order Band-pass Butterworth filter (with passband frequencies of 100 Hz and 2500 Hz).</li>
	<li>For spike detection, compute the root mean square of the signal over the 10 min recording for each electrode and set an amplitude threshold corresponding to eight times this root mean&#39;s square value.</li>
	<li>Apply a PTSD (Precision Timing Spike Detection25) algorithm, considering a maximum duration of 2 ms for one action potential.</li>
	<li>Consider as an active electrode, each electrode detecting at least 5 spikes/min. Continue the assay if at least 10% of the recording electrodes are active.</li>
	<li>Divide the number of detected spikes by the time-period of recording to calculate the mean firing rate of each active electrode. Calculate an average mean firing rate for each independent experiment. Ultimately, calculate an average by condition (&#177;SEM) and express it as a function on the day of recording.</li>
	<li>Compute a raster plot for each experiment representing the events detected for each active electrode as a function of time. Then, calculate the temporal array-wide firing rates (or instantaneous firing rates) of all the active electrodes, allowing to monitor synchronicity of the neural network activity.</li>
	<li>Finally, generate bar graphs using the quantification software and perform comparisons using Kruskal Wallis tests.</li>
</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.

<table><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 &amp;micro;m probe for Scepter counter&amp;nbsp;</td><td>Dutscher</td><td>53750</td><td></td></tr><tr><td>60 &amp;micro;m probe for Scepter counter&amp;nbsp;</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&amp;reg;</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&amp;nbsp;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 &amp;micro;l ClearLine&amp;reg; sterile in removable-lid rack&amp;nbsp;</td><td>Dutscher</td><td>030570ACL</td><td></td></tr><tr><td>Non filter tip 1 - 200 &amp;micro;l ClearLine&amp;reg; sterile in removable-lid rack&amp;nbsp;</td><td>Dutscher</td><td>032260CL</td><td></td></tr><tr><td>Non filter tip 50 - 1250 &amp;micro;l ClearLine&amp;reg; 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&amp;reg;&amp;nbsp;</td><td>Dutscher</td><td>357551</td><td></td></tr><tr><td>Pipette for cell culture 5 mL Falcon&amp;reg;&amp;nbsp;</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-&amp;beta;1</td><td>Peprotech</td><td>100-21C</td><td></td></tr><tr><td>Tube with conical bottom 15 mL (bulk) Falcon&amp;reg;&amp;nbsp;</td><td>Dutscher</td><td>352096</td><td></td></tr><tr><td>Tube with conical bottom 50 mL (bulk) Falcon&amp;reg;&amp;nbsp;</td><td>Dutscher</td><td>352070</td><td></td></tr></tbody></table>

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 ...

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Cancer Research

3.4K Views.  UMR CNRS 7021 - Laboratory of Bioimaging and Pathologies. De interacties tussen de kwaadaardige glioomcellen en hun omringende neuronen winnen aan belangstelling. Hier tonen we de ontwikkeling van een in vitro model dat afgeleide corticale glutamaterge neuronen en tumorcellen co-cultiveert. De microfludische apparaten die worden gebruikt voor coculturen gekoppeld aan multi-elektrode arrays maken de studie van verschillende celtypen mogelijk en voeren elektrische activiteitsregistraties uit op verschillende tijdstippen van de culturen.Deze met...

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

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.

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

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

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 ...

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Neuroscience

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Biology

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Direct-Contact Co-culture of Astrocytes and Glioblastoma Cells Patterned using Polyelectrolyte Multilayer Templates

Glioblastoma Multiforme (GBM) is the most abundant and fatal malignant brain cancer. There are more than 13,000 cases projected in the United States in 2020 and 2021. GBM tumors most often arise from astrocytes and are characterized by their invasive nature, often recruiting healthy tissues into tumor tissue. Understanding communication between astrocytes and glioblastoma cells is vital for the molecular understanding of tumor progression. This protocol demonstrates a novel patterned co-culture method to investigate contact-mediated effects of astrocytes on GBM employing layer-by-layer assembly and micro-capillary-force driven patterning. Advantages include a protein-free cell culture environment and precise control of cellular interaction dictated by the pattern dimensions. This technique provides a versatile, economical, reproducible protocol for mimicking cellular interaction between glioma and astrocytes in glioma tumors. This model can further be used to tease apart changes in GBM molecular biology due to physical contact with astrocytes or with non-contact mediated soluble cofactor communication.

This protocol describes a patterned direct contact glioma-astrocyte co-culture utilizing micro-contact printing on polyelectrolyte multilayers (PEMs) to pattern U87 or A172 GBM cells and primary astrocytes.

Glioblastoma Multiforme (GBM) is the most abundant and fatal malignant brain cancer. There are more than 13,000 cases projected in the United States in ...

Bioengineering

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.

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

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Co-culture and Interaction Studies

Co-Culturing Glioblastoma Cells on Patterned Neurons: A Technique to Monitor the Migration of Glioblastoma Cells on Rat Hippocampal Neurons In Vitro

Source: Guyon, J. et al. <a href="https://www.jove.com/video/62213" target="_blank">Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions.</a> J. Vis. Exp. (2021)
This video describes a co-culture assay of glioblastoma cells on micropatterned neurons, which mimic the myelinated neuronal tracts invasion by the glioblastoma stem-like cells as they migrate on neurons in the peritumoral spaces. This assay can help analyze the effects of pharmacological drugs that inhibit glioblastoma cells&#39; migration on neurons in a glioma.

Source: Guyon, J. et al. Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions. J. Vis. Exp. 
...

Cancers of the Nervous System

In vitro studies

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

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

	...

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

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