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

Samenvatting

Source: Fuchs, Q. et. al., Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions. J. Vis. Exp. (2021)

This video 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.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 

1. 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 pediatric high-grade gliomas (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 Day11 and onward medium detailed in Table 1.
4. Count pHGG cells using the microscopic pictures analyzed with 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).

2. Electrophysiological recording

1. Perform the electrophysiological recording with a commercial system and commercially available software.
   NOTE: The experiments were carried out with a multielectrode array (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).

Table 1: Culture media composition for hiPS-derived glutamatergic neurons.

Components	Seeding medium	D4 medium	D7 medium	D11 and onward medium
DMEM/F-12 Medium	0.5x	0.25x	0.125x	Ø
Neurobasal Medium	0.5x	0.25x	0.125x	Ø
BrainPhys Medium	Ø	0.5x	0.75x	1x
SM1 Supplement	1x	1x	1x	1x
N2 Supplement-A	1x	1x	1x	1x
Ala-Gln (GlutaMax)	0.5 mM	0.5 mM	0.5 mM	0.5 mM
BDNF	10 ng/mL	10 ng/mL	10 ng/mL	10 ng/mL
GDNF	10 ng/mL	10 ng/mL	10 ng/mL	10 ng/mL
TGF-β1	1 ng/mL	1 ng/mL	1 ng/mL	1 ng/mL
Geltrex	30 µg/mL	Ø	Ø	Ø
Seeding Supplement	1x	Ø	Ø	Ø
Day 4 Supplement	Ø	1x	Ø	Ø

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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
Ala -Gln (GlutaMAX)	Sigma	G8541
Class II Biological Safety Cabinet	Thermo Scientific	HERASafe type KS12
Cortical Glutamatergic Neurons	BrainXell	BX-0300
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
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-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