Neuronal co-culture

Coat chambers with 200µL per well with 0.1 Poly-L-Ornithine (PLO) in PBS.

Incubate plates overnight at 37°C.

Wash the chambers thrice with PBS.

Coat chambers with 200µL per well 10 Laminin plus 2 fibronectin, both diluted in PBS.

Incubate plates overnight at 37°C . Do not store coated plates. Proceed with preparation of plates for seeding cells.

Prepare enough amount of NB/B27 medium.

1. For 500mL

• NB/B27 medium (see Method section of paper), add:

A	B
Neurobasal medium	484 mL
B27 supplement without vitamin A	10 mL
GlutaMAX	5 mL
Penicillin-Streptomycin	1 mL

Storage: NB/B27 medium can be stored for 5 days at or for up to one month at .

• Warm NB/B27 medium at 37°C.
• Make NB/B27 complete medium by adding:

A	B
BDNF	20 ng/ml
Ascorbic acid	0.2 mM
GDNF	20 ng/ml
db-cAMP	0.5 mM
TGFβ3	1 ng/ml
DAPT	10 uM
Y-27632	10 uM

Discard coating reagents and add 200µL per well of NB/B27 complete medium.

Keep the plate at 37°C for 0h 15m 0s before seeding cells.

Replate cultured iPSC-derived dopaminergic neurons (day 30, see Method section of paper) on one side of the two-chamber microfluidic compartmentalization device (OMEGA4, eNuvio) at a cell concentration of 3×10⁵. Only the axons of DA neurons can migrate through the microfluidic channels connected to the adjacent chamber.

Feed neurons with fresh NB/B27 media every 3 days. Add 10 Laminin to NB/B27 media every 10 days before feeding the neurons.

After an additional 25 days in the co-culture device, thaw frozen iPSC-derived medium spiny neurons (MSN) from BrainXell and plate on the other half of the device (where only the axons of DA neurons are present) at a cell concentration of 3x10⁵ cells.

Fix the DA-MSN co-cultures till 7-10 days later for immunofluorescence (see Method section of paper).