Refer to the publish protocols to obtain the cell materials required.

http://www.protocols.io/view/midbrain-organoid-differentiation-in-spinner-flask-rm7vzbnr4vx1/v1

http://www.protocols.io/view/astrocyte-extraction-from-brain-organoids-261ge364wl47/v2

From a 90% confluent plate of astrocytes.

Wash with PBS.

Add 1 mL of TrypLE™ Select Enzyme (1X), no phenol red Thermo Fisher Catalog #12563011.

Transfer the cells to 15 ml falcon tubes and add 5 ml of media.

300rcf,25°C

Resuspend the cell pellet in 1ml of

Count the cells using

Equipment

Value	Label
Countess II	NAME
Life Technologies	BRAND
AMQAX1000	SKU
http://www.thermofisher.com/us/en/home.html	LINK

Individually add day 40 midbrain organoids to a Corning 96 well V bottom shape plate.

Add 50K astrocyte to the organoids.

Add 100 ul of organoid media.

After a week transfer the organoids to a 96 well plate coated with 1/100 .