What recommendations do you have for using iCell GlutaNeurons on the Axion Maestro MEA?

We have optimized an Application Protocol for culturing iCell® GlutaNeurons on the Axion 48-well MEA and subsequent recording on the Maestro system. This Application Protocol describes how to thaw, plate, and culture iCell GlutaNeurons on the Maestro MEA system and provides basic instructions for data acquisition and analysis.

General Protocol Recommendations:

  • Assay ECM: Pre-coat each well the night before cell addition with polyethylenimine (PEI, large molecular weight) and include laminin at 1 µg/ml final concentration in Complete BrainPhys Medium; also include laminin at 11 µg/ml final concentration in the dotting medium.
    The 50% PEI solution (w/v) (Sigma-Aldrich) is very viscous. We recommend that you pour ~1 ml 50% PEI into a 15 ml conical tube and let it sit (?at room temperature?) for several hours to allow the solution to settle in the bottom of the tube, and then ascertain the actual volume (why not centrifuge?). Use the settled solution and volume measurement to prepare an intermediate 10% PEI solution by adding 4 volumes of 20X Borate Buffer (Thermo Fisher Scientific) to the tube. Prepare the final 0.05% PEI solution by adding 500 μl 10% PEI Solution to 99.5 ml 1X Borate Buffer.
  • Assay density: Plate at 120,000 cells/well in 10 µl plating volume (12,000 cells/µl)
  • Assay medium: Complete BrainPhys Medium (100 ml), filtered through 0.22 µM filter and equilibrated to room temperature before use:
Component Amount (ml)
BrainPhys Neuronal Medium 95
N2 Supplement 1
iCell DopaNeurons Medium Supplement 2
iCell Nervous System Supplement 1
Laminin (1 µg/ml final concentration) 0.1
100X Pen/Strep 1

On days 0 and 1 post-plating, perform a 50% medium exchange, and then perform a 50% medium exchange every other day.

  • Dotting medium: Complete BrainPhys Medium with Laminin (1 ml)
Component Amount (µl)
Complete BrainPhys Medium 990
Laminin (11 µg/ml final) 10
  • Assay timing: Acquire data as needed. You can begin detection on day 4 of neural activity and on day 18 of synchronous firing from a macro network. Optimal performance from synchronous network activity can be seen on days 20 to 24.

Thawing and Dotting:

  1. Prepare Complete BrainPhys Medium and Complete BrainPhys Medium with Laminin.
  2. Thaw iCell GlutaNeurons in a 50 ml centrifuge tube and dilute the cell suspension to a final volume of 10 ml in Complete BrainPhys Medium.
  3. Remove a sample of the cell suspension and count the iCell GlutaNeurons with a hemocytometer to verify the viability and total number of cells listed in the Certificate of Testing.
  4. Centrifuge the tube at 400 x g for 5 minutes.
  5. Carefully aspirate supernatant, taking care to not disturb the cell pellet, and leave ~50 µl in the tube. Measure the amount remaining with a pipette.
  6. Add Complete BrainPhys Medium with Laminin (11 μg/ml) to reach a concentration of 12 million cells/ml and mix by gentle pipetting.
  7. Transfer cell suspension to a 1.5 ml centrifuge tube, gently invert tube to mix cells, and immediately add 10 µl/well directly onto recording electrode area with the MEA plate tilted at 30°, dotting one row at a time and then inverting cell suspension tube to mix cells. Note that after incubation and washing with DPBS without Ca2+ and Mg2+, it is critical to let the plate dry overnight for optimal cell attachment. Also, to avoid damaging the MEA plate, do not turn on the UV light in the biological safety cabinet.
  8. Add ~2 ml sterile water to the area surrounding the wells.
  9. Cover the 48-well MEA plate with a sterile, hydrated Microclime lid and incubate in a cell culture incubator at 37°C, 5% CO2, 95% humidity for 60 minutes but no longer than 75 minutes, assuring that you do not allow droplets to dry.
  10. At ~75°C, gently add 150 µl/well of Complete BrainPhys Medium slowly down the side of the well of the plate to avoid cell detachment.
  11. Return plate to flat position and add an additional 150 µl/well of Complete BrainPhys Medium.
  12. Cover plate and incubate at 37°C, 5% CO2, 95% humidity.

Preparing the 48-well MEA PLATE:

  1. Add 150 µl of the ~0.05% PEI solution to each well directly onto the center electrodes and incubate at 37°C for 1 hour. Note that currently, we do not include a laminin pre-dotting step.

Aspirate PEI solution and quickly rinse 3X with 300 μl D-PBS (without Ca2+ or Mg2+) and once with 300 μl sterile water. Aspirate all wells and air-dry the MEA plate in a biological safety cabinet overnight with the lid off. Make sure plate is completely dry.

References:

  1. Poster Presentation: Excitatory Cortical Neurons (iCell GlutaNeurons) Derived from Human iPS Cells Create Functional Macro Networks in vitro
  2. Poster Presentation: Development of Assays for Neurotoxicity Using Human iPSC-derived Neurons
  3. Poster Presentation: Investigating Neural Networks Using Human iPSC-derived Neurons on MEA