3.5
EV Isolation
and Purification
(See Note 10)
1. Ultracentrifuge the supernatant from the 2000 g spin at
10,000 g for 30 min at 4 C (or optionally at 20,000 g).
Transfer the supernatant to a new ultracentrifuge tube and
resuspend the 10,000 g pellet in PBS or other suitable buffer
if interested in large EVs and store (Fig. 1).
2. Ultracentrifuge the 10,000 g supernatant at 100,000 g for
70 min at 4 C.
3. Discard the supernatant. Resuspend the resulting crude small
EV pellet (in 500 μL buffer for SEC) and store (see Note 11).
4. Purify the crude small EV suspension with SEC using a 35 nm
qEV Original or other appropriate column (see Note 12).
5. See Size Exclusion Chromatography: A Simple and Reliable
Method for Exosome Purification for extensive details [16].
3.6
Characterizing
EVs
EVs should be characterized according to the MISEV2018 guide-
lines [15] prior to downstream molecular profiling or functional
use in order to enhance rigor and reproducibility in EV studies.
1. Quantify protein using BCA (high sensitivity standards are
recommended) or other appropriate assay.
Fig. 3 Example HDFa (human dermal fibroblast) bioreactor data showing monitoring of shed cells, EV
characterization based on MISEV guidelines [15], and SEM bioreactor growth surface. (a) The number of
shed cells and their viability over the bioreactor’s 5 month lifetime, (b) SEC purification profile showing protein
and particles amounts in each collected fraction, (c) NTA size distribution of pooled HDFa small EVs, (d) TEM
image of purified HDFa small EVs (scale bar ¼ 100 nm), (e) Western blot showing CD81, CD9, GRP94, and
GAPDH for HDFa small EVs (sEV) and cell lysates, and (f) SEM images of a growth surface of the HDFa
bioreactor with and without cells (scale bars ¼ 100 μm)
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