3. Sampling of the bioreactor can be performed every day by

taking 1 mL of mixture from PBS-VW bioreactor under agita-

tion. Cells on microcarriers can be visualized by nucleus stain-

ing (Fig. 2a) and culture medium supernatants can be used for

glucose/lactate measurement.

4. Medium collection is performed every 2 days with 50% fresh

medium change. The agitation is stopped to let cells/micro-

carriers to settle down. 50 mL media are collected from the

PBS-VW bioreactor. Then 50 mL of pre-warmed EV-free

hMSC-CCM is added to the bioreactor and agitation is started

again. Collected medium is ready for EV isolation.

Day 1

Day 7

Mean

EV size (nm)

Mode

0

100

200

300

400

hMSC EV size

A

B

C

Fig. 2 (a) hUC-MSCs growth in PBS-VW bioreactors at day 1 and day 7, stained by Hoechst 33342 for cell

nuclei and visualized under fluorescent microscope. Scale bar: 200 μm. (b) Transmission electron microscopy

(TEM) images of EVs from hUC-MSC bioreactor culture. Scale bar: 100 nm. (c) The size of hUC-MSC secreted

EVs determined by nanoparticle tracking analysis (NTA)

200

Xuegang Yuan et al.