Place a 5 μl droplet of PDL solution (4.5 µg/cm2 ) to each well in the MEA plate. Tip: Recommended to add 6-8 mL of sterile water to the on-plate reservoirs to increase humidity.
Incubate the PDL-coated MEA plate at 37°C, 5% CO2 for 1 hour.
Rinse PDL from the culture surface with 200 µl of sterile DI water 4 times, then allow the MEA plate to air dry overnight.
Culturing Rat Cortical Neurons
Prepare Neurobasal Plus Complete Media according to the Gibco Primary Rat Cortex and Hippocampus Neurons User Guide.
Thaw primary rat cortical neurons according to the Gibco Primary Rat Cortex and Hippocampus Neurons User Guide.
Remove a sample of the cell suspension and count the neurons using a hemocytometer to determine both the viability and total number of viable cells. Transfer the cell suspension to a 15 ml conical tube. Tip: Ensure the neurons are evenly suspended before removing an aliquot to count.
Centrifuge the cell suspension at 100 x g for 5 minutes. Tip: Neurons are sensitive to centrifugation, so care should be taken to monitor speed and duration during this step. The cell provider does not recommend centrifugation and is not responsible for cell death induced by centrifugation.
Aspirate the supernatant, being careful not to disturb the cell pellet.
Dilute the cell suspension in Neurobasal Plus Complete Medium to 16,000,000 neurons/ml.
Plating Gibco Rat Cortical Neurons onto the MEA
Place an 5 µl droplet of the rat cortical neuron suspension over the recording electrode area of each well of the MEA. See Figure 1 on page 2 for appropriate drop placement.
Incubate the MEA plate with the seeded neurons in a cell culture incubator at 37°C, 5% CO2 for 1 hour.
Gently add 1/2 of the final volume of the medium to each well of the MEA. Adding the medium too quickly will dislodge the adhered neurons. Recommended final well volumes for each plate type are: 6- and 12-well = 1000 µl, 24-well = 500 µl, 48-well = 300 µl, 96 well = 200 µl. Tip: Using a pipettor, add medium first in a semi-circle along the outer edge of the well. Progressively add medium to either side of the well so it fills evenly towards the center. The goal is to prevent a rush of medium in either direction that might dislodge the neurons.
Repeat step 12 a second time to reach the final recommended volume of medium.
Incubate in a cell culture incubator at 37°C, 5% CO2 .
For optimal cell health, be sure to exchange 50% of the medium every 2-3 days. Though neural spikes may be detectable within 4 days, optimal neural network structure is typically achieved after 28 days in culture.
Drop Placement
Figure 1: Drop Placement Diagram - The layouts above represent the bottom surfaces of wells in (A) a 48-well MEA, (B) a 6- or 12-well MEA, (C) a 24-well MEA or 48 well E-Stim+ MEA, (D) a 48-well AccuSpot MEA, (E) a 96-well MEA, and (F) a 48-well CytoView MEA. The number of electrodes per well is different across the plate formats, however the drop placement is the same, with the drop (red circle) centered on the recording electrodes and staying within the ground electrodes. On plate types with the addition of the stim-paddle in the lower right corner of the array, it is important to make sure the droplet covers this feature. The droplet may need to be manipulated after placement of the pre-treatment to ensure stim-paddle coverage.
Visualization of Typical Neuron Seeding Results
Figure 2: Gibco Rat Cortical Neuron Morphology Gibco Rat Cortical Neurons at day 2 in vitro in a 48-well CytoView MEA, 160K cells, 10x magnification. Notice that neuron morphology is easily recognizable.
