(Life Technologies, #12000-063), 2.2 g sodium bicarbonate

(Sigma-Aldrich, #S5761), 10 mL penicillin–streptomycin (Life

Technologies, #15070-063), and 100 mL fetal bovine serum

(FBS, Atlanta Biologicals, Inc. #S11110) in 1 L deionized

water; then filtered by 0.2 μm pore size filter in sterile bottle

and stored under 4 ^C for future use. The 150 mm diameter

tissue culture petri dishes are obtained from VWR Interna-

tional (#25382-442). 0.25% trypsin/EDTA solution is from

ThermoFisher Scientific (#25200056).

2.3

Bioreactor

Preparation

1. A lab scale of 50 mL glass spinner flask bioreactor (Wheaton,

#356875) is used for hiPSC-NPC differentiation, as well as

medium collection for EV isolation. Prior to culture, the glass

vessel is coated with 1 mL sigmacote (Sigma-Aldrich, #SL2)

and then dried overnight at room temperature. The spinner

flask is autoclaved for future use.

2. For PBS-VW bioreactor, PBS mini system is purchased from

PBS Biotech™, Inc. (Camarillo, CA). This system includes

magnetic agitation base and a sterile single-use vessel in

100 mL. The system is assembled and placed in standard cell

culture 37 ^C incubator with 5% CO2. Cytodex 1 microcarriers

(GE Healthcare Life Sciences, #17-0448-01) are prepared by

hydrating the microcarriers in phosphate buffered saline (PBS)

for overnight and washed twice with PBS before autoclave.

Then the microcarriers are washed again with PBS and ready

to use.

2.4

Materials for EV

Isolation from Human

Stem Cells Grown in

Bioreactor Cultures

1. EV-free FBS is prepared by ultracentrifuge. FBS is spun at 4 ^C,

100,000
g for 20 h. The supernatant is carefully collected as

EV-free FBS for EV collection.

2. All centrifuges are pre-cooled to 4 ^C during EV isolation and

purification. Polyethylene glycol 6000 (PEG 6000, VWR

International, Radnor, PA., #80503) solution is prepared by

mixing 160 g PEG6000 with 1 M sodium chloride (NaCl,

VWR International, #470302) in 1 L milli-Q water, then fil-

tered with 0.2 μm pore size filter, resulting in 16% PEG6000

solution. EV-free PBS is prepared by double-filter of sterile

PBS for future use (see Note 5).

3

Methods

3.1

Culture and

Expansion of hiPSCs in

Planar Culture

1. Frozen iPSK3 cells are recovered by immediately thawing in a

37 ^C water bath for 30 s until a small piece of ice remains.

Spray the cryopreserve vial with 70% ethanol and open the vial

in biological safety cabinet. Transfer the cell suspension care-

fully into at least ten times of volume of hiPSC-CCM (for

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