Differentiating Human Induced Pluripotent Stem Cells into Neurons on Micro-Electrode Arrays

For the six-well MEAs, dilute the cells to obtain a cell suspension of 7.5 x 10⁵ cells per milliliter. Aspirate the diluted laminin from the six-well MEAs. For the six-well MEAs, plate the cells by adding 100 microliters of cell suspension to the active electrode area in each well.

Next, place the six-well MEAs in a humidified 37 degrees Celsius incubator. After two hours, carefully add 500 microliters of the prepared E8 medium to each well of the six-well MEAs. Subsequently, place the six-well MEAs overnight in a humidified 37 degrees Celsius incubator.

On day three, warm 0.05% Trypsin-EDTA to room temperature. Warm DPBS in DMEM/F-12 with 1% penicillin-streptomycin to 37 degrees Celsius. Then, aspirate the spent medium of the rat astrocyte culture. Wash the culture by adding 5 milliliters of DPBS, and swish it around gently.

Afterward, aspirate DPBS and add 5 milliliters of 0.05% Trypsin-EDTA. Swish the Trypsin-EDTA around gently. Then, incubate the cells in a humidified 37 degrees Celsius incubator for 5 to 10 minutes. Subsequently, check under the microscope to examine whether the cells are detached.

Detach the last cells by hitting the flask a few times. Then, add 5 milliliters of DMEM/F-12 to the flask. Triturate the cells gently inside the flask with a 10-milliliter pipette. Afterward, collect the cell suspension in a 15-milliliter tube. Spin the tube at 200 times g for eight minutes.

After that, aspirate the supernatant and resuspend the cells in 1 milliliter of DMEM/F-12. Determine the number of cells per milliliter using a hemocytometer.

Next, add 7.5 x 10⁴ astrocytes to each well of the six-well MEAs, and add 2 x 10⁴ astrocytes to each well of the 24-well plate. Incubate the cells overnight in a humidified 37 degrees Celsius incubator.