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

Summary

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.

Abstract

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.

Introduction

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'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 activity^4,5,6. 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 circuitry^4,7,8. 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 communications^6,9. 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 activity^14,15. 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 in vitro pHGG models^{3,16,17,18,19}. Coupled with electrophysiological recordings using multielectrode arrays (MEA), microfluidic devices^20,21,22 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 device^23,24 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.

Protocol

For this protocol, the accreditation number related to the use of human materials is DC-2020-4203.

1. Microfluidic device fabrication, preparation and treatment

1. 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 µm high and 6 ± 1 µm wide) that defines asymmetric microgrooves under the patterning of main channels made in SU-8 2100 (200 µm high, 1 mm wide, and 13 mm long) (see Supplemental Figure S1).
2. 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.
3. 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 °C for 40 min.
4. 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.
5. 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.
6. Fill with a pipette the inlet of the microfluidic device before usage with 70% ethanol and empty it by pipetting aspiration.
7. Wash the microfluidic device by adding inlet reservoirs Dulbecco's Phosphate Buffered Saline (D-PBS) and empty it by pipetting aspiration three consecutive times.
8. Coat the device using 0.05 mg/mL Poly-D-Lysine and place it into an incubator (37 °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.
9. Fill the microfluidic chip with the cell culture medium and let it remain at room temperature until use for a maximum of 2 h.

2. Cell preparation and seeding in the microfluidic device

1. 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.
   1. Empty the inlet and outlet reservoirs of the microfluidic device by pipette aspiration before seeding, letting only the device's channels to be filled with medium.
   2. Seed the hiPS cells at Day 0 (D0) by putting 10 µL 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.
   3. After 15 min, fill both inlet and outlet reservoirs with 50 µL of D4 culture medium, whose entire composition is given in Table 1, and transfer the device into the incubator (37 °C, 5% CO₂).
   4. Maintain glutamatergic neuron differentiation for 23 days (Figure 1A(1), microscopy) under a controlled environment (37 °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.
   5. 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.
   6. Take microscopic pictures at D4, D21, and D23 using a standard phase-contrast microscope to assess cell viability and allow cell counting.
   7. 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.
   8. 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 °C.
   9. 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.
   10. Rinse the cells three times with PBS and counterstain with DAPI (4',6-diamino-2-phenylindole) for 10 min at room temperature.
   11. Acquire images with an inverted epifluorescence microscope fitted with a CMOS (Complementary metal-oxide-semiconductor) camera and analyze using appropriate image analysis software.
   12. Open the DAPI stained images and binarize with the thresholding routine in the software. To do this, click on Image > Adjust > Threshold, and set the appropriate parameters to distinguish the nucleus from the background. Then, click on Apply > Process > Watershed to split the aggregate nucleus.
   13. After that, use the Analyze Particles innate software'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 µm, bigger than 20 µm, or with a circularity smaller than 0.1 µm).
   14. Merge the contour images obtained from DAPI analysis to correspondent immunofluorescent pictures to validate the correct staining.
   15. 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.
2. Cell culture of commercialized pHGG line, UW479, and patient-derived cell line, BT35³
   1. 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.
   2. Maintain these pHGG cells under a controlled environment at 37 °C in normoxic conditions throughout the experiments.

3. Co-culture protocol

1. 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.
2. 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.
3. Maintain the co-cultures for 2 days under a controlled environment (37 °C, 5% CO₂) with glutamatergic neuron D11 and onward medium detailed in Table 1.
4. Count pHGG cells using the microscopic pictures analyzed with an image analysis software to assess their viability. Calculate the percentage of cells.
   ​NOTE: Use the following formula: 100 - ((number of cells at D23 / number of cells at D21) x 100).

4. Electrophysiological recording

1. 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 µm diameter electrodes spaced by 100 µm.
2. Perform a first electrophysiological recording on glutamatergic neurons at D21 before seeding of pHGG cells. Use the differentiated glutamatergic neurons as control and culture them alone in parallel to the co-culture. Do a second recording for both conditions as described in the next step numbered 3.
3. Perform a second electrophysiological recording at D23 (after 2 days of co-culture).

5. Electrophysiological data processing

1. Filter the raw data with a 2^nd order Band-pass Butterworth filter (with passband frequencies of 100 Hz and 2500 Hz).
2. 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's square value.
3. Apply a PTSD (Precision Timing Spike Detection²⁵) algorithm, considering a maximum duration of 2 ms for one action potential.
4. 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.
5. 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 (±SEM) and express it as a function on the day of recording.
6. 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.
7. Finally, generate bar graphs using the quantification software and perform comparisons using Kruskal Wallis tests.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
256MEA100/30iR-ITO-w/o	MCS	256MEA100/30iR-ITO-w/o
40 µm probe for Scepter counter	Dutscher	53750
60 µm probe for Scepter counter	Dutscher	51999
Accutase	Sigma	A6964
Ala -Gln (GlutaMAX)	Sigma	G8541
Axel Observer 7 Microscope	Zeiss	431007-9904-000
Cell culture flask with cap with filter membrane 70 mL Falcon®	Dutscher	353109
Class II Biological Safety Cabinet	Thermo Scientific	HERASafe type KS12
Colibri 7 LED	Zeiss	4230529710-000
Cortical Glutamatergic Neurons	BrainXell	BX-0300
DMEM/F-12 (1:1) GlutaMAX	Gibco	31331-028
DMEM/F12 Medium	Sigma	D8437
DPBS 1X	Dutscher	L0615-500
EasYFlaskTM cell culture flasks 75cm3	Nunc	156499
Foetal Bovine Serum (FBS)	Dutscher	500105
GDNF	Peprotech	450-10
Geltrex	Life Technologies	A1413201
Human BDNF	Peprotech	450-02
Incubator	Memmert	IC0150med
MCS InterFace Boarder	MCS	181205-MEA2100-11240
MEA2100	MCS	181205-MEA2100-11240
Micropipette P10	Sartorius	LH-729020
Micropipette P100	Sartorius	LH-729050
Micropipette P1000	Sartorius	LH-729070
Micropipette P200	Sartorius	LH-729060
Microtube Eppendorf 1,5 ml Safe-Lock	Dutscher	33290
MultiChannel Experimenter	MCS	-
N2 Supplement-A	StemCell	7152
Neurobasal Medium	Life Technologies	21103049
Neurocult SM1 neuronal supplement	StemCell	5711
Non filter tip 0.1 - 10 µl ClearLine® sterile in removable-lid rack	Dutscher	030570ACL
Non filter tip 1 - 200 µl ClearLine® sterile in removable-lid rack	Dutscher	032260CL
Non filter tip 50 - 1250 µl ClearLine® sterile in removable-lid rack	Dutscher	134760CL
Non-essential amino acids (NEAA) without L-glutamine	Dutscher	X0557-100
Pipeteur Pipet-Aid XP Gravity	Drummond	4000202/4038202
Pipette for cell culture 10 mL Falcon®	Dutscher	357551
Pipette for cell culture 5 mL Falcon®	Dutscher	357543
Plaque chauffante (CultureTemp)	Belart	370151000
Poly-D-Lysine	Sigma	P6407
Primovert microscope	Zeiss	415510-1100-000
Scepter (Handheld Automated Cell Counter)	Millipore	PHCC00000
TGF-β1	Peprotech	100-21C
Tube with conical bottom 15 mL (bulk) Falcon®	Dutscher	352096
Tube with conical bottom 50 mL (bulk) Falcon®	Dutscher	352070