instance, 1 mL cell suspension in 10 mL CCM) in a centrifuge

tube. Gently pipette the mixture and then centrifuge at 300
g

for 5 min (see Note 6).

2. After centrifugation, carefully remove the supernatant and do

not disturb the cell pellet. Resuspend the cell pellet with

1–3 mL hiPSC-CCM carefully and distribute the cell suspen-

sion onto Geltrex-coated culture surface at 1–2
10⁵ cells/

cm². ROCK inhibitor Y27632 is added at 10 μM in the media.

The recovered hiPSCs are cultured in a standard incubator

(37 ^C, 5% CO2).

3. First medium change is performed 24-h later to remove ROCK

inhibitor. Cell attachment can be visualized under microscope.

hiPSC culture is maintained in CCM and medium is replaced

every 2–4 days. When cells are compacted and reach a high

density, medium change is performed daily.

4. For passaging hiPSCs, culture medium is removed, and the

cells are washed with sterile PBS. Then the cells are incubated

with Accutase solution at 37 ^C for 5 min. Gently pipet the

mixture to acquire single cell suspension. Transfer the mixture

to a 15 mL centrifuge tube, wash the culture surface once with

hiPSC-CCM, and transfer the medium to the centrifuge tube

(see Note 7).

5. Spin down the cells at 300
g for 5 min and remove the

supernatant. Resuspend the cell pellet with hiPSC-CCM and

determine the cell number by hemocytometer. Passaged

hiPSCs can be expanded on Geltrex-coated surface or prepared

for NPC differentiation in bioreactors.

3.2

Differentiation of

NPC Organoid from

hiPSCs in Spinner

Flasks

1. hiPSC suspension is collected and seeded in the 15 mL spinner

flask at 4–5
10⁵ cells/mL in NPC differentiation medium

containing 10 μM Y27632. The bioreactor is set up on a

programmable magnetic stirrer (Wheaton, #900701) and the

whole system is placed in a standard culture incubator (37 ^C,

5% CO2).

2. In initial aggregation phase (day 0), intermittent agitation is

used after cell seeding in the bioreactor. The stirrer is set to

80 rpm for 15 min and off for 15 min for a total of 10 cycles.

Then the agitation speed is set to 80 rpm for the rest of the

culture.

3. At day 1, stop the agitation and let the hiPSC aggregates to

settle down at the bottom; carefully remove the medium by

pipette and resuspend the aggregates with fresh NPC differen-

tiation medium containing 10 μM SB431542 and 100 nM

LDN193189 to induce neural lineage commitment. Restart

the agitation and culture.

Human Stem Cell-derived Extracellular Vesicles in Bioreactors

197