What do you recommend for using iCell DopaNeurons on Axion Maestro MEAs?

We have an Application Protocol for culturing iCell® DopaNeurons on the 48-well multielectrode arrays (MEAs) from Axion BioSystems and subsequent recording on their Maestro MEA System. This Application Protocol describes how to thaw, plate, and culture iCell DopaNeurons on the Maestro MEA System and provides basic instructions for data acquisition and analysis.

General Protocol Recommendations:

  • Assay ECM: Precoat each well the night before cell addition with polyethyleneimine (PEI) then add laminin as a "pre-dot" prior to cell addition as well as to cell suspension during thaw. Note that both the culture and assay media will also contain laminin.
  • Assay density: Plate at 60,000 - 70,000 cells/well
  • Assay media (you can sterilize maintenance or assay medium through 0.22um filter if you prefer, but it is not necessary):
    • Complete DopaNeurons Maintenance Medium
Component Amount (ml)
iCell DopaNeurons Maintenance Medium 100
iCell DopaNeurons Medium Supplement 2
iCell Nervous System Supplement 1
    • Complete DopaNeurons Maintenance Medium containing 10 μg/ml Laminin
Component Amount
Complete DopaNeurons Maintenance Medium 25 ml
Laminin (11 µg/ml final) 250 µl
    • Complete BrainPhys Medium, filtered through 0.22 µM filter 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
  • Assay timing: Record MEA activity and begin compound application at Day 18+.  Optimal synchronous neuronal activity is observed from day 18 post-plating.
droplet-mea-illustration4

Figure 1: illustration of the optimal method for pipetting into Maestro MEA plates.

Important Notes:

  • It is critical to let the plate dry overnight for optimal cell attachment. To avoid damaging the MEA plate, do not turn on the UV light in the biological safety cabinet.
  • Thaw stock laminin solution at room temperature or at 4°C overnight. Do not thaw the stock laminin solution in a 37°C water bath. Do not vortex the stock laminin solution.
  • Due to the length of incubation (≥18 days) we recommend adding Penicillin-streptomycin to the Complete Maintenance Medium at 1X final concentration.
  • Dot one row at a time and then invert cell suspension tube to mix cells. Do not allow droplets to dry.
  • When adding Complete DopaNeurons Maintenance Medium containing 10 µg/ml laminin, tilt the pipette to a 75-degree angle and dispense the fluid slowly down the side of the well of the plate to avoid cell detachment.
  • After complete media removal, slowly add 150 µl to the side of each well, one row at a time. Repeat for a final volume of 300 µl/well.
  • Dotting angle.  Carefully add 10 µl cell suspension to each well with the MEA plate tilted at 30°. See Figure 1.

References:

  1. Application Protocol: Measuring Synchronous Neuronal Activity on the Maestro Multielectrode Array
  2. Webinar Presentation: Measuring Pharmaco Influences on Human iPSC-derived Neuronal Networks with a Novel MEA Analysis Tool