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 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&#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>
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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...

Watch this Scientific Journal Video about Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions at JoVE.com

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 ...

co-culture-glutamatergic-neurons-pediatric-high-grade-glioma-cells

Research

JoVE Journal

Cancer Research

3.4K Views.  UMR CNRS 7021 - Laboratory of Bioimaging and Pathologies. 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.

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 ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

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 ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using three-dimensional (3D) cell-based invasion and migration assays. In this study, we have optimized these 3D assays for live-cell immunocytochemistry. An Antibody Labeling Reagent was used to detect in real-time the expression of the adhesion molecule CD44, on the plasma membrane of migrating and invading cells of a DMG H3K27M primary patient-derived cell line. CD44 is associated with cancer stem cell phenotype and tumor cell migration and invasion and is involved in the direct interactions with the central nervous system (CNS) extracellular matrix. Neurospheres (NS) from the DMG H3K27M cell line were embedded into the basal membrane matrix (BMM) or placed onto a thin coating layer of BMM, in the presence of an anti-CD44 antibody in conjunction with the antibody labeling reagent (ALR). The live-3D-cell immunocytochemistry image analysis was performed on a live-cell analysis instrument to quantitatively measure the overall CD44 expression, specifically on the migrating and invading cells. The method also allows visualizing in real-time the intermittent expression of CD44 on the plasma membrane of migrating and invading cells. Moreover, the assay also provided new insights into the potential role of CD44 in the mesenchymal to amoeboid transition in DMG H3K27M cells.

This study presents a protocol of live-3D-cell immunocytochemistry applied to a pediatric diffuse midline glioma cell line, useful to study in real-time the expression of proteins on the plasma membrane during dynamic processes like 3D cell invasion and migration.

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the discovery of novel therapeutics and predictive biomarkers for ovarian cancer. These models recapitulate clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Additionally, they have been shown to match the primary tumor morphologically, cytologically, immunohistochemically, and genetically. Thus, organoids facilitate research on tumor cells and the tumor microenvironment and are superior to cell lines. The present protocol describes distinct methods to generate patient-derived ovarian cancer organoids from patient tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The patient samples are separated into cellular suspensions by both mechanical and enzymatic digestion. The cells are then plated utilizing a basement membrane extract (BME) and are supported with optimized growth media containing supplements specific to the culturing of high-grade serous ovarian cancer (HGSOC). After forming initial organoids, the PDOs can sustain long-term culture, including passaging for expansion for subsequent experiments.

Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics.

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...

Quantifying Telomere Length Using Non-Radioactive Chemiluminescence Detection

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.

Author Spotlight: Optimization of Performance Parameters of the TAGGG Telomere Length Assay  

Here, we describe in detail the protocol for quantifying telomere length using non-radioactive chemiluminescent detection, with a focus on the optimization of various performance parameters of the TAGGG telomere length assay kit, such as buffer quantities and probe concentrations.

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...

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Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4). As retroviral transduction requires dividing cells for transgene integration, OP9-DL4 provides a suitable in vitro environment for cultivating hematopoietic progenitor cells. This is particularly advantageous when studying the effects of the expression of a specific gene during normal T cell development and leukemogenesis, as it allows researchers to circumvent the time-consuming process of generating transgenic mice. To achieve successful outcomes, a series of coordinated steps involving the simultaneous manipulation of different types of cells must be carefully performed. Although these are very well-established procedures, the lack of a common source in the literature often means a series of optimizations are required, which can be time-consuming. This protocol has been shown to be efficient in transducing primary thymocytes followed by differentiation on OP9-DL4 cells. Detailed here is a protocol that can serve as a quick and optimized guide for the co-culture of retrovirally transduced thymocytes on OP9-DL4 stromal cells.

This protocol describes the isolation of double-negative thymocytes from the mouse thymus followed by retroviral transduction and co-culture on the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4) for further functional analysis.

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line ...