How to Culture Primary Rodent Neurons for MEA Analysis Using the Maestro MEA™ System

Electrophysiological analyses are widely used among researchers to investigate the electrical properties and activity of neurons and neural tissues. Micro-electrode array (MEA) systems are powerful electrophysiology tools that allow researchers to simultaneously record electrical activity from large populations of cells. They detect activity-associated voltage changes in the extracellular space around electrically active cells, such as neurons, that are grown on specialized cell culture plates containing a grid of electrodes in each well. By measuring activity from several electrodes simultaneously, researchers can obtain valuable insights into neural networks and cell function. Using MEAs to measure cellular activity is non-invasive and does not require cell labeling, allowing for long-term monitoring of electrophysiological maturation. This makes MEA systems ideal for both disease modeling and drug studies.

Since cells must cover the electrodes uniformly and consistently to produce high-fidelity data, culture substrates and seeding densities must be optimized for each cell type. Follow this step-by-step protocol to culture embryonic day 18 (E18) rat cortical neurons using BrainPhys™ Neuronal Medium on CytoView MEA™ plates for measurable, consistent, and stable neuronal activity using Maestro MEA™ systems.

Workflow Specifications

Obtaining Rodent Primary Neural Cells

This protocol outlines a method for preparing a single-cell suspension of primary neurons from the cortices of rat embryos at gestational day E18. Other brain regions isolated from rodent embryos of other gestational days may also be used.

Choosing an MEA Plate Format

This protocol is optimized for culturing cells on polyethyleneimine (PEI)-coated 24-, 48-, and 96-well CytoView MEA™ plates. Refer to Figure 1 and Table 1 below for a schematic of the bottom surfaces of the wells for each of these plate formats, as well as plate specifications.

We recommended seeding at least 3 - 8 replicate wells per condition to calculate sample-to-sample variability alongside well-to-well variability.

Figure 1. CytoView MEA™ Plate Electrode Layouts

Schematic diagrams of the bottom surfaces of different CytoView MEA™ Plate formats depicting recording electrodes (blue), grounds (orange), and on-plate spotting guides (gray), and where present, a large dedicated stimulation electrode (blue; 24-well format).

Table 1. CytoView MEA™ Plate Specifications and Compatibility

CytoView MEA™ Plate Format
Growth Area Per Well (cm2)
Recording Area Per Well (mm x mm)
# Electrodes Per Well
Maestro System Compatibility
24-well
0.23
1.1 x 1.1
16
Pro, Edge
48-well
0.22
1.1 x 1.1
16
Pro
96-well
0.32
0.8 x 0.8
8
Pro

Materials and Reagents


Protocol

Part I. Reagent Preparation

Borate Buffer, pH 8.4

  1. Add 100 mL of distilled water to a glass beaker and heat to 50°C.
  2. Stir at 300 rpm and add the following to the beaker of water:
    • 310 mg of boric acid
    • 475 mg of sodium tetraborate
  3. Continue to stir at 300 rpm at 50°C for 30 minutes.
  4. Adjust pH to pH 8.4 with 12 N HCl.
    Note: Adjust pH while the chemicals are dissolving. pH adjustment can accelerate dissolution.
  5. Stir until all chemicals are dissolved and the solution becomes clear.
  6. Measure pH and adjust to pH 8.4 with HCl or NaOH accordingly.
  7. Remove the beaker from the hot plate and allow the solution to equilibrate to room temperature (15 - 25°C).
  8. Filter sterilize and store at room temperature until use.

5% Polyethyleneimine (PEI) Intermediate Stock Solution in Borate Buffer

Note: The following instructions are to prepare a 5% PEI intermediate stock solution through dilution of a commercially provided 50% PEI stock solution in borate buffer. The 5% intermediate stock solution will be further diluted to a working concentration of 0.1% immediately before use. Due to the viscosity of the 50% PEI solution, which can make pipetting difficult, it is recommended to weigh the material to ensure accuracy during preparation.
  1. Pour approximately 2 mL of 50% (w/v) PEI stock solution into a tared 50 mL tube.
  2. Centrifuge at 1000 rpm for 5 minutes.
  3. Re-weigh the tube to determine the weight (in grams) of 50% PEI solution added to the tared tube.
  4. Add borate buffer (pH 8.4) to dilute the 50% PEI solution to a final concentration of 5% (w/v). Vortex to mix.
    • e.g. 2 g of 50% (w/v) PEI solution is diluted with 18 mL of borate buffer to reach a final volume of 20 mL, resulting in a 5% PEI solution.
  5. Store the 5% PEI solution at 2 - 8°C for up to 6 months.

1 M Glucose Stock Solution

Notes:
  • The following instructions are for preparing 10 mL of 1 M D-(+)-glucose stock solution from D-(+)-glucose powder. If preparing other volumes, adjust accordingly. Alternatively, a sterile 10% w/v D-(+)-glucose solution can be used by diluting to a 1 M solution with distilled water.
  • Use sterile technique to prepare 1 M glucose stock solution.
  1. Dispense 7 mL distilled water into a 50 mL tube.
  2. Weigh out 1.8 g of D-(+)-glucose powder and add to the tube containing 7 mL water.
  3. Mix to dissolve.
  4. Check the volume of the solution and add distilled water to bring the total volume up to 10 mL.
  5. Filter sterilize the glucose solution and store at 4°C.

Complete Plating Medium

Note: BrainPhys™ Primary Neuron Kit is used for culturing primary tissue-derived neurons in this protocol.

Refer to the Product Information Sheet for BrainPhys™ Neuronal Medium (page 3) for detailed instructions on preparing the complete plating medium.

Glucose-Supplemented Complete Maturation Medium

Notes:
  • BrainPhys™ contains glucose at a physiological level of 2.5 mM. However, in applications with high cellular metabolic demands, this glucose level may deplete rapidly. To ensure cell survival and support neuronal activity, supplementing the cultures with additional glucose is recommended. For MEA cultures of primary tissue-derived neurons, add 12.5 mM glucose to achieve a final concentration of 15 mM. This process is described below.
  • Use sterile techniques to prepare Glucose-Supplemented Complete Maturation Medium (BrainPhys™ Neuronal Medium + NeuroCult™ SM1 Neuronal Supplement + glucose). The following example is for preparing 10 mL of complete medium. If preparing other volumes, adjust accordingly.
  1. Thaw one bottle of NeuroCult™ SM1 at room temperature (15 - 25°C) for 1 hour.
    Note: If not used immediately, aliquot and store at -20°C. Do not exceed the shelf life of the supplement. After thawing aliquots, use immediately. Do not re-freeze.
  2. Add 0.2 mL of NeuroCult™ SM1 to 9.8 mL of BrainPhys™ Neuronal Medium.
    Note: For other volumes, note that you require a 1 in 50 dilution.
  3. Add 0.125 mL of 1 M glucose stock solution and mix thoroughly.
    Note: If not used immediately, store Glucose-Supplemented Complete Maturation Medium at 2 - 8°C for up to 1 month. Warm medium to 37°C before use. Protect from light.

Part II. Coating MEA Plates

Note: Use sterile technique for this part of the protocol.
  1. Prepare a suitable volume of 0.1% PEI solution in borate buffer (pH 8.4). Filter sterilize and use immediately.
    • e.g. To prepare 10 mL of 0.1% PEI solution, add 200 μL of 5% PEI solution to 9.8 mL of borate buffer (pH 8.4).
  2. Pipette a 10 µL droplet of 0.1% PEI solution over the electrode array at the center of each well.
    Note: 10 µL of PEI solution is sufficient to cover the electrode array, for any of the 24-, 48- or 96-well MEA plates.
  3. Incubate the MEA plate for at least 2 hours at room temperature.
  4. Gently tilt the PEI-coated cultureware onto one side. Aspirate the excess solution, ensuring that the coating is not scratched.
  5. Wash the PEI-coated cultureware 3 times (refer to Table 2 for recommended volumes) by pipetting sterile distilled water toward the edge of each well to avoid scratching the PEI substrate. To remove each wash volume, tilt the plate and aspirate carefully from the bottom edge of the well.
  6. Replace the plate lid and allow the plate to air dry overnight at room temperature in a biosafety cabinet.
    Note: This step may be shortened to ~ 2 hr at room temperature if desired, provided the plate completely dries out during this time.

Table 2. Recommended Volumes for CytoView MEA™ Plates

Plate Type
Recording Area Per Well (mm x mm)
Coating & Seeding Volume (μL)
Wash Volume (μL)
Final Culture Volume (μL)
24-well
1.1 x 1.1
10
300 - 500
500
48-well
1.1 x 1.1
10
200 - 300
300
96-well
0.8 x 0.8
10
100 - 200
200

Part III. Preparing a Single-Cell Suspension of Primary Rodent Neurons

Note: Use sterile technique for this part of the protocol.
  1. Prepare papain solution immediately before use by dissolving a vial containing ≥ 100 units of papain in 5 mL of NeuroCult™ Neuronal Plating Medium. Incubate at 37°C for 10 minutes, swirling the vial every 5 minutes to mix.
  2. Add 2 mL of papain solution to a vial of E18 rat cortex.
  3. Incubate at 37°C for 10 minutes. Mix the suspension every 5 minutes by swirling or inverting the vial.
  4. Remove papain and replace with 1 mL of Glucose Supplemented Complete Plating Medium.
  5. Using a 1 mL micropipette, triturate the tissues by pipetting up and down several times to obtain a single-cell suspension. Avoid bubbles when triturating.
  6. Transfer cell suspension to a 15 mL tube. Rinse the vial 2 times with 1 mL of Glucose Supplemented Complete Plating Medium to collect any remaining cells, and transfer to the same 15 mL tube.
  7. Centrifuge cells at 2000 rpm for 3 minutes.
  8. Discard supernatant and resuspend cells in 1 mL of Glucose Supplemented Complete Plating Medium.
  9. Filter cells through a 40 µm cell strainer over a 50 mL tube.
  10. Perform a viable cell count using trypan blue and a hemocytometer.

Part IV. Plating and Culturing Primary Rodent Neurons on MEA Plates

Note: Use sterile technique for this part of the protocol.
  1. Dilute cell suspension to 5 x 106 live cells/mL in Glucose Supplemented Complete Plating Medium.
  2. Place a 10 µL droplet of cell suspension (containing 5 x 104 cells) over the electrode array at the center of each well.
    Note: 10 µL of cell suspension is sufficient to cover the electrode array, for any of the 24-, 48-, or 96-well MEA plates.
  3. Add sterile distilled water to reservoirs around the edge of the plate to prevent evaporation.
  4. Incubate the MEA plate with seeded cells at 37°C and 5% CO2 for 1 hour.
  5. Gently add half of the final culture volume of warm (37°C) Glucose Supplemented Complete Plating Medium to each well (refer to Table 2 for recommended volumes). Expel the medium slowly down the wall of the well to avoid cell attachment.
  6. Immediately repeat step 5.
  7. Incubate MEA plate at 37°C, 5% CO2 for 5 days.
  8. After 5 days of culture, perform a half-medium change with warm (37°C) Glucose Supplemented Complete Maturation Medium.
  9. Repeat half-medium changes every 2 days for the remainder of the culture period with warm (37°C) Glucose Supplemented Complete Maturation Medium.
    Notes:
    • Ensure that the MEA culture is incubated in a humidified environment to avoid medium evaporation. Evaporation may increase the osmolality of the culture medium and can negatively affect cell survival and neuronal activity. Placing the MEA plate into a larger covered tray containing additional water dishes can further help to maintain humidity and avoid evaporation of the culture medium.
    • To avoid weekend feedings, add twice the required medium volume on Friday. Remove the same volume of spent medium on Monday and resume half medium changes as described above (e.g. for a 96-well plate, remove 100 µL per well and add 200 µL of fresh medium on Friday. On Monday, remove 200 µL per well and replace with 100 µL of fresh medium).

Part V. MEA Data Acquisition and Analysis

Notes:
  • Neurons are sensitive cell types. Medium changes and fluctuations in environmental temperature can introduce stress to the neurons that may negatively impact cell survival, overall culture health, and measured activity.
  • It is advisable to standardize the length of time between medium changes and subsequent recordings, e.g. if a recording is planned for the afternoon, perform a medium change approximately 4 - 6 hours before the recording, keeping this time frame as consistent as possible between different recording days.
  1. Transfer the plate from the incubator to a Maestro MEA™ system pre-equilibrated to 37°C and 5% CO2.
  2. Place the MEA plate into the instrument and allow an additional 5 - 10 minutes before starting the acquisition to let the system and culture equilibrate.
  3. Refer to the AxIS Navigator™ Software Manual for the Maestro MEA™ systems for detailed guidance on data acquisition.
  4. Return the plate to the incubator to maintain the culture until the next recording day.
  5. Analyze recorded data by using either commercially available software (e.g. Neural Module for Maestro Pro™ or Maestro Edge™) or customized algorithms (e.g. MATLAB).

Part VI. Example Results

Early drug development often involves screening compounds for neurotoxic effects to assess their impact on nervous system activity. This poster shows how primary rodent neurons cultured in physiological conditions in a high-throughput format can be used in an in vitro MEA assay to assess the neurotoxic effects of pharmacological compounds.

Figure 2 below illustrates the evaluation of neurotoxic effects, using picrotoxin and GABA to assess neuronal stimulation and inhibition, respectively. For details on the experimental design, please refer to the poster.

Figure 2. Effects of Pharmacological Compounds on Neuronal Spike Activity

In the study from the poster, both caffeine and ibuprofen were used as negative control compounds, which showed no significant effects on neuronal activity and firing patterns. In contrast, spike and burst activity were predictably stimulated and inhibited when neurons were treated with picrotoxin (seizurogenic compound) and GABA (inhibitory neurotransmitter), respectively.

Note: Compounds were tested at concentrations from 0.03 to 300 µM, with vehicle controls (Ibuprofen and Caffeine). This range is recommended; however, optimizing these conditions to fit your specific experimental methods and cell lines is essential.
  • Document #PR00096
  • Version 1.0.0
  • December 2024


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