4. Medium sample can be collected every day for the analysis of

glucose and lactate concentration by YSI2950 biochemical

analyzer. Half medium change is performed every 2 days with

fresh

NPC

differentiation

medium

containing

10

μM

SB431542 and 100 nM LDN193189.

5. On day 8, NPC aggregates or spheres can be collected for the

evaluation of neural progenitor markers. Both SB431542 and

LDN193189 are removed by medium change and fresh NPC

differentiation medium is added to the bioreactor for continu-

ous agitation culture (see Note 8).

6. For EV collection, conditioned medium collection can be per-

formed by defined time interval, for example, every 2 days.

Cultures can be maintained in this manner for a very long

time (25–70 days) (Fig. 1a). Collected media are preserved

under 4 ^C for further processing.

DAY 25

DAY 27

Mean

EV size (nm)

Mode

0

50

100

150

200

250

hiPSC-NPC EV size

A

B

C

Fig. 1 (a) hiPSC-NPC organoid growth in spinner flask at day 25 and day 27 under optical light microscope.

Scale bar: 400 μm. (b) Transmission electron microscopy (TEM) images of EVs from hiPSC-NPC bioreactor

culture. Scale bar: 200 nm. (c) The size of hiPSC-NPC secreted EVs determined by nanoparticle tracking

analysis (NTA)

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Xuegang Yuan et al.