3.6 Coculturing with Astrocytes

In order to increase the maturation of neurons for electrophysiological measurements, coculturing with astrocytes is highly recommended [4, 15]. We adapted the protocol from Kaech and Banker [16] to our cell culture.

Rat primary cortical astrocytes are cultured in astrocyte medium at 37°C and 5% according to the manufacturer’s instructions.

For passaging, aspirate the culture medium and store it in a Falcon tube as a washing solution ( see Note 26 ). Rinse the cells once with 1x.

Add prewarmed Accutase and incubate the cells at 37°C until all of them are detached (usually 0h 5m 0s are sufficient).

Stepwise add the cell culture medium stored in step 3 to flush cells and collect all cells to a prerinsed 15 ml Falcon tube.

Centrifuge at 400x g. Aspirate the supernatant and resuspend the pellet in prewarmed astrocyte growth medium.

Count the cells using Trypan Blue and seed the appropriate amount in uncoated tissue-culture treated dishes at a seeding density of approximately 5000 cells per cm². Change the growth medium every 3–4 days.

For the coculture with neurons, prepare astrocytes to be ~80% confluent at day 4 of neuronal differentiation. One day before the reseeding of neurons, wash the astrocytes three times with 1x and add BrainPhys™ medium with minimal supplements.

Thoroughly clean the coverslips in a big glass petri dish. First, rinse the coverslips in ddH₂O for 2h 0m 0s and then shake in 50mL 2h 0m 0s.

Rinse three times with ddH₂O by shaking for 0h 2m 0s, and rinse another two times with ddH₂O by shaking for 2h 0m 0s. Shake three times in 100% for 0h 2m 0s and one time 0h 2m 0s.

Sterilize the coverslips at 225°C for 6h 0m 0s–16h 0m 0s (can be done overnight) ( see Note 27 ) [16].

Autoclave ~100mL in a 500 ml bottle. Melt in a boiling water bath (1 l beaker with 400mL on a heat plate with 350°C). Use a 2 ml aspiration pipette with a 200 μl pipette tip attached and soak it in melted paraffin. Shake off extra drops and place small drops on coverslips to create paraffin feet as spacer ( see Note 28 ). Coat the coverslips with PLL and laminin ( see protocol 3.5) on the side with the paraffin dots.

Differentiate the iPSCs on Matrigel-coated cell culture dishes using 0.5μg/mL for 4 days ( see protocol 3.5). Add 5micromolar (µM) to the culture for 1 day to remove occasionally undifferentiated cells.

At day 5, reseed predifferentiated neurons on PLL and laminin coated coverslips with paraffin feet. Collect the old medium from the culture well and wash the cells very carefully with prewarmed 1x.

Dissociate the cells by adding Accutase and place in the incubator for approximately 0h 5m 0s until the neuronal network detaches. Add the cell culture medium stored in step 3 and transfer the cell solution to a 15 ml Falcon tube.

Rinse the well 1–2 times with 1% to collect all cells and centrifuge at 400x g.

Aspirate the supernatant and resuspend the cell pellet in 200µL slowly and carefully (this step is crucial for the survival of single cells).

Add 800µL to a total volume of 1mL.

Centrifuge at 20x g and collect 800µL supernatant in a fresh tube (this is the single cell suspension).

Repeat the dissociation of the pellet for a maximum of five times until no pellet is visible any more.

Centrifuge the single cell suspension at 400x g and resuspend the pellet in 0.5mL. Count the cells if necessary and seed on coated coverslips on the side with the paraffin feet ( see Note 29 ).

After 2h 0m 0s, place the coverslips with the differentiated iPSCs upside down into culture wells containing 80% confluent rat astrocytes. Every 7 days, exchange 50% of the BrainPhys™ medium and compensate the volume loss due to evaporation with ddH₂O ( see Note 30 ).