Recording Electrophysiological Activity of the Neuronal Network in a Neuron-Astrocyte Co-culture

Abstract

Source: Taga, A., et al. Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons. J. Vis. Exp. (2021).

This video demonstrates establishing a neuron-astrocyte co-culture for recording neuronal network activity. Upon establishing the co-culture of motor neurons and astrocytes on a multi-electrode array (MEA) plate, the synchronous rhythmic electrical activity of the neuronal network was recorded extracellularly using the MEA.

Protocol

1. Co-culturing MN and spinal cord astrocytes in multi-electrode array plates

1. Differentiate and plate the motor neurons and astrocytes when they are ready to be used on the same day and aged to day in vitro (DIV) 60 for the motor neurons and DIV 90 for the astrocytes.
2. Coat the MEA plates on the day before or on the day of plating as follows if working with 24-well plastic plates (Table of Materials).
3. Dilute polyornithine (PLO) in water or phosphate-buffered saline (PBS) to 100 µg/mL.
   1. Add 15-20 µL to each well (dependent on pipette comfort level), forming a droplet on the center of the well covering the area of the electrodes and surrounding area but not the entirety of the well.
   2. Take care not to damage electrodes with the pipette tip. Be consistent with volume from well to well to ensure coverage of the same surface area in each well.
   3. Incubate PLO at 37 °C for a minimum of 1 h (preferably 2 h).
      NOTE: The small volumes will dry up if there is not sufficient humidity in the plates. Add water to the compartments surrounding wells to ensure sufficient humidity throughout the course of coating and recordings.
4. Aspirate as much PLO as possible using a plastic micropipette tip. Take care not to touch the electrodes. Wash with 250 µL of water three times. If using a vacuum aspirator for washes, do not allow the tip near the electrode array. After the third wash, remove as much water as possible, using a pipette tip as necessary. Let the surface dry under the cell culture hood with the lid removed.
5. Once plate surfaces are dry, add laminin diluted to 10 µg/mL in PBS. Use 15-20 µL to cover each electrode array. Add water to the humidity compartments, replace the lid, and return the plate to 37 °C incubation for a minimum of 2 h up to overnight.
6. On the day of plating, rinse the motor neuron (MN) and astrocyte cultures once with PBS and add trypsin 0.05% at 37 °C to lift cells (5 min). Collect into a 15 mL conical tube containing trypsin inhibitor and wash plates with medium or base to ensure that all the cells are collected. Centrifuge at 300 x g for 5 min and resuspend with a 1000 µL pipette to generate 1 mL of a single cell suspension. When re-suspending MN and astrocytes, switch to the co-culture medium (Table 1), with the addition of 20 µM Rho-associated coiled-coil forming protein serine/threonine kinase inhibitor (ROCK-I).
7. Count the MN and astrocytes in parallel using a hemocytometer. While counting and making calculations, cap the cell suspensions and place them in a Styrofoam rack at 4 °C.
8. Calculate the volume required to resuspend the cultures to a concentration of 5 x 10⁴ cells per 5 µL for motor neurons and 2.5 x 10⁴ cells per 5 µL for astrocytes. Centrifuge the 1 mL cell suspensions at 300 x g for 5 min and resuspend in the calculated volume.
9. Calculate the number of desired wells to be seeded and multiply it by 5 µL of each cell suspension. Combine the required volume of neuron and astrocyte suspensions at a 1:1 ratio and mix by pipetting until thoroughly combined (usually twice) but avoid being too aggressive.
10. Remove the laminin from each well of the MEA plate using a pipette tip. Transfer 10 µL of the final combined cell suspension to each well, forming a small droplet covering the electrode array to provide a cell density of 5 x 10⁴ MN and 2.5 x 10⁴ astrocytes per well.
    NOTE: High cell density in the combined suspension requires frequent resuspension in between wells during the seeding step to ensure accurate and consistent cell counts.
11. Return the plates to the incubator for 20-30 min. Take care not to disturb the cell droplet and allow the cells to form initial attachments on the plates.
12. After 20-30 min, add warm co-culture media + ROCK-I to each well by pipetting 250 µL down on the wall of each well, followed by an additional 250 µL down the wall of the same well on the opposite side. Return the plate to the incubator.
13. Examine plates the day after seeding (Co-culture Day 1). If there is significant cell debris or dead cells, exchange the medium with a fresh co-culture medium containing ROCK-I. Otherwise, change the medium on the second day after seeding (Co-Culture Day 2) to the co-culture medium without ROCK-I. Perform half-medium exchanges (aspirate 50% and add 60% of the final volume to account for evaporation) twice a week.
14. If using MEA systems with single well 30 mm glass plates (Table of Materials), the plate preparation may take 2 or more days. Follow the steps below to perform this.
15. Lift the cells and cell debris from the previous MEA cultures with 0.05% Trypsin for 5-15 min.
16. Aspirate trypsin and rinse three times with water.
17. Sterilize by adding 70% ethanol and incubate in the hood for 10 min. Aspirate the ethanol, and then rinse with water three times.
18. Apply 1% anionic detergent with protease enzyme in water. Cover the plates with a lid and wrap with thermoplastic film and aluminum foil, and then leave them on a rocker at room temperature overnight.
19. Aspirate the detergent, and then rinse three times with water.
20. If planning to store the plates, add a sufficient volume of water. Cover the plates with a lid and wrap them with thermoplastic film and aluminum foil and leave them in the refrigerator until next use.
21. If planning to coat, let the surface dry under the cell culture hood.
22. If extra sterilization is needed (e.g., previous infection or non-recent use), at this point only, expose MEA plates to ultraviolet (UV) light under the hood for 30-60 min.
    NOTE: Avoiding frequent UV light will prevent damage to the electrodes. The use of UV light when plates have serum on them will result in nonfunctional electrodes, which is why it should only be used on cleaned plates.
23. When the plates are completely dry, proceed with plasma cleaning for 1 min to charge the glass surface of MEA plates and enhance coating effectiveness. Plasma-clean at least once every 4-5 cycles of MEA plate cleaning-plating cycles.
    1. As an alternative, when plasma cleaning is not feasible or not warranted, add a sufficient volume of fetal bovine serum (FBS)-containing medium to cover the MEA plates' surface and return them to the incubator overnight.
24. Use PLO (polyornithine)/Laminin coating. Add PLO solution (100 µg in PBS) immediately after plasma cleaning or aspirating and rinsing out the FBS-containing medium.
25. Plate the cells as above at the following densities: 1 x 10⁵ human pluripotent stem cell-derived astrocytes (hiPSC-A) / plate and 5 x 10⁵ human pluripotent stem cell-derived motor neurons (hiPSC-MN)/plate.

2. Multi-electrode array recording

1. Extract the raw voltage data at a 12.5 kHz sampling frequency, using a Butterworth filter with a 200 Hz high pass and 3 kHz low pass filter. Set spike recognition as instantaneous time points of voltages ≥6 standard deviations from baseline. Identify bursts as an activity with >5 spikes in 100 ms. Define network activity when over 35% of total active electrodes fire within 100 ms with a minimum of 50 spikes per network burst.
2. Start recording as soon as possible after Co-Culture (CC) Day 1.
   NOTE: Electrical activity will be rarely noted before CC Day 3.
3. Plate parallel cultures at similar densities and replicates in the appropriate culture plates or coverslips for biochemical assays, immunocytochemistry (ICC), or quantitative PCR (qPCR).
4. Perform recordings with the temperature set to 37 °C and CO₂ at 5%. Transfer plates to the machine and allow them to equilibrate for at least 5 min prior to recording.
5. Record baseline activity either every other day or weekly, over 1-15 min depending on the experimental design. Do not record for at least 1 h after a medium exchange, and ideally wait for 24 h after every media exchange.
6. To prevent contamination, change the medium (i.e., half medium exchange as in step 1.14) after any recording in which the lid of the sterile plate has to be opened outside of the sterile hood (i.e., the application of drug compounds). Perform complete medium exchanges and washes to wash out drugs if plates are going to continue to be used beyond drug treatment.
7. For analysis purposes, use at least three technical and biological replicates for each condition. Express electrophysiological data as means of n ≥ 3 replicates.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 cm sterile culture plates	Falcon	353003
25 cm2 sterile culture flasks	Falcon	353136
500 mL 0.2 µm CA Filter System	Corning	430769
5 mL pipette	Falcon	357543
6 well sterile culture plates	Falcon	3046
Axion CytoView MEA 24 plates (M384-tMEA-24W)	Axion	OPT-24
Axion Edge MEA platform	Axion	Maestro Edge
Basement Membrane matrix - Matrigel	Corning	354277	Details in the protocol
Benchtop microscope (sterile under cell culture hood)	Zeiss	415510-1100-000	Primo Vert
Ciliary neurotrophic factor (CNTF)	Peprotech	450-13	Dissolve 100 µg in 1mL of sterile PBS, and then add 9 mL of sterile 0.1% BSA-PBS to 10 µg/mL. Aliquot and freeze at -80 ºC. Dilute at 1: 1000 for use. (working concentration 10 ng/mL).
CO2 tanks and regulator for Axion Edge	AirGas/Harris	9296NC
DMEM/F12	Thermofisher	113300	Working concentration 1x
Essential 8 Medium + Essential 8 Supplement	Thermofisher	A1517001	Combine 10 mL of Essential 8 (10x)  supplement with 500 mL of Essential 8 Growth Medium (1x)
Fetal Bovine Serum (FBS)	Thermofisher	16140071	Working concentration 1x
Hemocytometer	Election Microscopy Sciences	63510-20
Humidity controlled Cell culture incubator	ThermoFisher	370	set to 37 ºC, 5 % CO2
Laminin	Thermofisher	23017-015	Stock solution 1 mg/mL, working concentration 10 µg/mL (coating), 1 µg/mL (cell media)
L-Glutamine	Thermofisher	25030	Dissolve 2 mg of LDN into 500 µL of Chloroform to get 10 mM stock. Aliquot this and freeze at -80 ºC. For using, dilute the stock 1 to 10 into DMSO [to 1 mM] first, then dilute 1:5000 of 1 mM into the desired media to get 0.2 µM working solution
MEA glass plates	MultiChannel Systems	60MEA200/30iR-Ti-gr	Working concentration 100x
Multichannel Pipet P200	Gilson	PJ22224
Neurobasal	Thermofisher	21103049
Non-Essential Amino Acids (NEAA)	Thermofisher	11140050	Working concentration 1x
Pencillin/Streptomycin	Thermofisher	15140122	Working concentration 100x
Polyornithine (PLO)	Sigma-Aldrich	P3655	Working concentration 100x
Potassium chloride (KCl)	NA	NA	Dissolve 100 mg in 1 mL of ddW to get 100 mg/mL stock solution. Aliquot the stock in 100 µL tubes. (working concentration 100 µg/mL)
Purmorphamine (PMN)	Millipore-Sigma	540223	Working concentration100 mM
ROCK-Inhibitor	Peprotech	1293823	Centrifuge briefly before reconstitution. Dissolve 100 µg in 1 mL of PBS to 100 µg/mL, and then add 9 mL of sterile 0.1% BSA-PBS to 10 µg/mL. Aliquot and freeze at -80 ºC. Dilute at 1: 1000 for use. (working concentration 10 ng/mL).
Sterile cell culture hoods	Baker Company	SG-600	Dissolve 5 mg in 1480 µL of dH2O to get 10 mM stock, aliquot and freeze at -80 ºC. Dilute at 1: 500 for use. (working concentration 20 µM).
Supplement B - B27 Supplement	Thermofisher	21985023
Table top cell culture centrifuge	ThermoFisher	75004261	Working concentration 50x
Thermoplastic film - Parafilm	PARAFILM	P7793	Sorvall Legend X1R
Trypsin-EDTA (0.05%)	Thermofisher	2530054	Working concentration 1x
Waterbath	ThermoFisher	2332	Isotemp