Human induced pluripotent stem cell (hiPSC)-derived neurons enable the study of neuronal cell biology and electrophysiology in both healthy and pathological contexts. While studying mono-cultures of excitatory (glutamatergic) or inhibitory (GABAergic) neurons can be helpful, the functions of both these types of neurons are intimately connected in the human brain. To understand the interconnected relationships of these neuronal cells in healthy conditions and during neurological diseases it is necessary to culture them together in vitro, along with supporting glial cells such as astrocytes.

A multielectrode array (MEA) is a tool used in neuroscience to record electrical activity from multiple points in a cell culture simultaneously. It consists of a grid of small electrodes that can detect electrical signals from individual cells or groups of cells. MEAs are particularly beneficial in cell research as they allow for high-throughput, non-invasive monitoring of neural activity, enabling detailed studies on neural development, disease modelling, and drug screening in a controlled environment. Electrophysiological assays are especially useful for studying functional phenotypes associated with neurodegenerative disorders, such the reduced electrical activity seen in the ioGlutamatergic Neurons Huntington’s disease. Co-culture cell models can be used tangentially with MEA to study the electrical activity in a heterogeneous neuronal network, to further shed light on the pathogenesis of diseases such as autism, epilepsy, frontotemporal dementia, and Alzheimer’s disease.

ioGlutamatergic Neurons and ioGABAergic Neurons are human iPSC-derived excitatory and inhibitory neurons, precision reprogrammed using opti-ox™ technology. The following protocol describes the establishment of a tri-culture system with ioGlutamatergic Neurons, ioGABAergic Neurons and supporting hiPSC-derived astrocytes with a specific emphasis on preparing the tri-culture for MEA analysis with the Axion Maestro Pro system. 

See the functional MEA data on tri-cultures on the product page >

• ioGlutamatergic Neurons (io1001)

• ioGABAergic Neurons (io1003)

• hiPSC-derived astrocytes*

• MEA plates (Axion, 48-well: M768-tMEA-48W)

• Geltrex (Reduced GF) (ThermoFisher, A1413202)

• Poly-D-lysine hydrobromide (Sigma, P6407)

• Borate buffer (20x) (ThermoFisher, 28341)

• Sterile water (Sigma, W3500)

• DMEM/F-12 (ThermoFisher, 11330032)

• Neurobasal (ThermoFisher, 21103049)

• B27 (ThermoFisher, 17504044)

• Glutamax (ThermoFisher, 35050061)

• 2-Mercaptoethanol (ThermoFisher, 31350010)

• NT3 (R&D, 267-N3-005)

• BDNF (R&D, 248-BDB-005)

• DAPT (Biotechne, 2634)

• Doxycycline (Sigma, D9891)

• Bovine Serum Albumin (Sigma, A7906)

• Brain Phys medium (Stem Cell Technologies, 5790)

• Standard tissue culture wares (pipettes, tips)

• 50 mL centrifuge tube, conical (Greiner Bio-one, 227261)

• Axion Maestro Pro MEA platform

• Biological safety cabinet

• Normoxic cell culture incubator (37°C, 5% CO₂)

• Bench top centrifuge

• 37°C water bath

• Automatic cell counter

* Please note that the astrocyte thawing procedure may require optimisation and that the characteristics of astrocytes may vary depending on the supplier used.

This protocol is split into five steps:
Step 1: Primary plate coating (PDL)
Step 2: Secondary plate coating (Geltrex)
Step 3: Cell thawing and seeding
Step 4: Culture maintenance
Step 5: Acute compound dosing and measuring electrical activity

1. Primary plate coating (PDL)

Tip: See the tutorial video for how to prepare tissue culture vessels with PDL-Geltrex™ for advice on coating the plates.

1.1. Resuspend a 5 mg vial of PDL in 50 mL of the 1x borate buffer, for a working concentration of 100 µg/mL.

Tip: PDL coating solution can be kept at -20°C for long-term storage.

1.2. Coat the surface area of your culture vessel with the PDL coating solution. We recommend 70 µL for each well of the 48-well CytoView plate.

1.3. Incubate the coated plates overnight at 37 °C, or for at least 3 hours at 37 °C.

1.4. Aspirate PDL solution and wash 3 times with sterile water. For each wash, use the same volume used for coating.

1.5. Aspirate the water and allow coated surfaces to dry completely in a biological safety cabinet, this typically requires 60 minutes.

2. Secondary plate coating (Geltrex)

2.1. Thaw an aliquot of Geltrex on ice for 30 minutes (refer to ioGlutamatergic Neurons user manual section 6.5 for details on Geltrex aliquot preparation).

2.2. In the meantime, add 4 mL of sterile water to the surrounding areas outside of the wells to reduce media evaporation during incubation.

2.3. Dilute the Geltrex 1:100 in chilled DMEM/F-12 (e.g. 100 μL in 10 mL).

2.4. Coat the surface area of your PDL-coated culture vessel with the Geltrex:DMEM/F-12 coating solution. We recommend 70 µL for each well of a 48-well CytoView plate.

2.5. Incubate the coated plates at 37 °C for a minimum of 60 minutes.

2.6. Following the incubation, carefully aspirate off the excess Geltrex, then immediately plate the cells.

3. Cell thawing and seeding

3.1. For each vial of cryopreserved cells (all cell types), thaw the vial according to the following procedure:

3.1.1. Place the vial in a 37°C water bath with gentle agitation for ~2 minutes. Keep the cap out of water to minimize the risk of contamination.

3.1.2. Transfer the cells into a 50 mL tube containing 4 mL pre-warmed (37°C) b:GN medium.

3.2. Count each cell type, including a viability marker such as Trypan Blue or AOPI, using an automated cell counter.

3.3. Calculate the volume required to seed each well with the number of cells outlined in Table 1 and multiply this by the number of wells in the experiment. Include an extra 50,000 cells to account for pipetting error.

Table 1: Suggested tri-culture cell seeding densities for an Axion Maestro Pro Cytoview 48-well plate.

3.4. Pool the adequate volumes of each cell type into a new 50 mL tube, and centrifuge at 200 x g for 3 minutes at room temperature.

3.5. Aspirate the supernatant and resuspend the cells in the required volume of comp:GN+D media. We recommend a seeding volume of 50 µL for each well of a 48-well CytoView plate.

3.6. Add 50 μL of pooled cell mix to each well of the previously coated 48-well CytoView plate.

3.7. Incubate seeded MEA plate in a cell culture incubator at 37°C, 5% CO₂, and 95% humidity.

3.8. 24 hours post-plating, add 250 μL of fresh, pre-warmed comp:GN+D media to each well.

3.9. 48 hours post-plating, perform a 100% media change with 300 µL of fresh, pre-warmed comp:GN+D+DAPT.

3.10. 96 hours post-plating, perform a 100% media change with 300 µL of fresh, pre-warmed comp:GN.

3.11. From day 6 onwards, a 50% media change is performed by removing 150 µL of media from each well and replacing with 150 µL of Brain Phys complete media.

4. Culture maintenance

4.1. Perform 50% media changes with Brain Phys complete media every 48 to 72 hours .

4.2. Carry out 5-minute recordings after 7 days of culture, the day after feeding. For example: Feed Monday/Wednesday/Friday and record Tuesday/Thursday.

Tip: recording can be carried out a minimum of 4 hours post-feeding where this is required.

4.3. Cells may be imaged regularly using a brightfield microscope.

5. Acute compound dosing and measuring electrical activity

5.1. 24 hours prior to compound application, perform a 100% media change to ensure the volume in the well is correct.

Tip: If required, cells may be imaged pre-application on a brightfield microscope.

5.2. Create intermediate compound plate.

5.2.1. Make up 10X stocks of each compound to be used for treatment, at an appropriate DMSO concentration (10x final assay concentration of vehicle), in Brain Phys complete media. Please use the lowest working DMSO concentration possible.

5.3. Record electrophysiological activity for each well, for a pre-application baseline. Label this timepoint “-5 min”.

5.4. Calculate 10% of the volume of the media in each well of the MEA assay plate. Apply this 10% volume from the intermediate compound plate to the MEA assay plate using multi-channel automatic pipette. This will result in 1x concentrations for the compounds in the MEA assay plates.

5.5. Place the plate in the recording chamber that has been allowed to equilibrate to the correct CO₂ and temperature. Begin recording as soon as possible. If possible, compound dosing should occur on the machine itself. Label this timepoint “0 min”.

5.6. Take recordings in accordance with experimental plan (for example, 5 minutes every 5.1 minutes for 12 total recordings). Label these timepoints according to your recording times.

5.7. If required, cells may be imaged on a brightfield microscope following the compound dosing.

See MEA data on tri-cultures after addition of GABAA receptor modulatory compounds >

Table 2: Preparation of basal co-culture medium.

Table 3 – Media recipes

This protocol was optimised using the wild type ioGlutamatergic Neurons and ioGABAergic Neurons and can also be used for the related ioCells listed in here.

Table 4 – ioCells that may be used in this protocol.

If you have any questions or need assistance, please reach out to technical@bit.bio and we will do our best to support you.

Published April 2024, version 1