Coat a 6 well plate coat with gelatin 0.1% or 1:100

Collect 10-20 spheres and place in a 6 well plate (day 40+ spheres)

Wash in PBS twice

Aspirate the PBS

Add 1mL of for 0h 5m 0s

Triturate using glass a pipette (2-3 up and down)

Transfer the cells and tissue (even the chunks, the trituration is more effective if you have chunks of tissue) to the coated 6 well plate

Aspirate the extra TrypLe

Add 3mL of astrocyte media (http://www.sciencellonline.com/astrocyte-medium.html)

Half media change media every 3 days or until it turns orange.

Keep changing media every 3d until you observe good amount of cell attached to the plate (may vary according to the patterning)

Passage the astrocytes to a gelatin or geltrex coated 15 cm plate in Astrocyte media once they reach 80% confluency

For the experimental protocols after passage cell will be incubated in maturation media (TBD)