Dissociate iPSC colonies to single cells with Accutase for 0h 7m 0s at 37°C.

Re-suspend cells in day0 medium and plate 8.000 cells/well in 96-Wells Plate U-round-Bottom Low Attachment.

Carefully exchange the medium, without touching the EBs.

Dilute Matrigel in a 3:2 ratio with day 4 medium (used as an embedding mixture).

Wash EBs in day 4 medium. Transfer and mix 5-8 EBs into the embedding mixture and plate onto a 6-well ultra-low-attachment plate.

Incubate for 0h 30m 0s at 37°C and add day 7 medium.

Incubate at least 24h 0m 0s between d7 and d8.

Add fresh differentiation medium without disrupting the embedded EBs.

After Day 8 change medium twice a week.

At day 10 reduce CHIR to 3.0micromolar (µM) (1:1000).

Reduce CHIR to 0.7micromolar (µM). Remove CHIR from medium at day 15 and onwards.

Manually dissociate organoids from Matrigel, using two surgical needles.

Place on orbital shaker after dissociation (80rpm).