3.5
EV Isolation from
the Collected Media
1. Collected media can be stored at 4 C for 1 week before
processing, otherwise they should be stored at 50 C. Col-
lected media, both from hiPSC-NPC organoid culture and
hMSC bioreactor culture, undergo modified differential cen-
trifugation at 4 C: 500 g for 5 min, 2000 g for 10 min,
and 10,000 g for 30 min. Supernatants are collected from
each step (see Note 11).
2. For hiPSC-NPC organoid conditioned media, 10% EV-free
FBS and 16% PEG6000 solution are added to the medium to
a final concentration of 8% PEG6000. For hMSC conditioned
media, only 16% PEG6000 solution is added to the media at a
final concentration of 8% PEG6000. All samples are well mixed
and stored at 4 C for 12–24 h.
3. Next, medium samples are centrifuged at 3200 g for 60 min
at 4 C to collect PEG-enriched EV pellets. The supernatants
are discarded, and the pellets are resuspended in sterile, EV-free
PBS for washing out PEG 6000, then ultracentrifuge is per-
formed at 120,000 g for 70 min at 4 C.
4. After ultracentrifuge, supernatants are discarded and the EV
pellets are resuspended in sterile, EV-free PBS (usually at
100 μL, but the volume can vary). The morphology of the
isolated EVs can be examined by transmission electron micros-
copy (Figs. 1b and 2b). EV size distribution and concentrations
can be determined by Nanoparticle Tracking Analysis (NTA)
using a Malvern NanoSight LM10 instrument (Figs. 1c and 2c)
(see Note 12).
5. Stem cell-secreted EVs are aliquoted and stored at 80 C for
further downstream analysis, culture experiments, or animal
transplantation.
4
Notes
1. For long time storage, cryopreserved hiPSCs should be stored
in liquid nitrogen.
2. Proper Geltrex coating is critical for hiPSC recovery and expan-
sion. Geltrex coating solution should be well mixed in cold
medium before applied to culture surface.
3. mTeSR™1 medium should be properly prepared and stored
following the manufacturer’s instruction. The hiPSCs are very
sensitive to the quality of the medium. Degradation in both
basal medium and 5 supplement would result in cell detach-
ment and spontaneous differentiation.
4. Differentiation protocols can be modified to reflect the pur-
pose of neural lineage commitment. Small molecules treatment
can be modified to generate neural cells with specific brain
Human Stem Cell-derived Extracellular Vesicles in Bioreactors
201