2. Measure particle size and a concentration with nanoparticle

tracking

analysis

(NTA),

tunable

resistive

pulse

sensing

(TRPS), or other counting and sizing system.

(a)

Settings will be sample-dependent, but setting consistency

is important to be able to compare between different EV

preparations.

(b)

If doing SEC for the first time, validate which fractions

contain EVs (Fig. 3b) and pool EV-rich fractions for

downstream experimentation (Fig. 3c). If necessary,

EV-rich fractions can be concentrated using ultracentrifu-

gation or 10–100 kDa cutoff filters.

3. Visualization of EV morphology using TEM (Fig. 3D).

(a)

See Detection and Characterization of Extracellular Vesi-

cles by Transmission and Cryo-Transmission Electron

Microscopy for extensive details [17].

4. Western Blotting for EV and contaminant markers (see Note

13).

(a)

To prepare EV lysates, mix EV samples with Protease

Inhibitor and radioimmunoprecipitation assay buffer

(RIPA buffer). Some antibodies require reduction of pro-

tein using DTT or 2-mercaptoethanol.

(b)

At least two proteins from a category of EV-enriched

proteins (CD9, CD63, CD81, Alix, TSG101, etc.) and

one from the category of EV-depleted proteins (calnexin,

GRP94, etc.) are recommended to be analyzed to dem-

onstrate the EV nature and the degree of purity of an EV

preparation [15] (Fig. 3e).

(c)

See Extracellular Vesicle Isolation and Analysis by Western

Blotting for extensive details [18].

3.7

Imaging the

Bioreactor Growth

Surface

1. Before terminating the bioreactor culture, wash 3
with

prewarmed PBS.

2. Add 15 mL 4% glutaraldehyde or Karnovsky’s fixative to the

cell chamber using a 25 mL serological pipette and 30 mL to

the media chamber and leave at 4 ^C overnight.

3. Aspirate fixative from cell chamber and pour out from media

chamber, then cut the bioreactor open from the bottom using a

Dremel™circular blade or similar tool using the outline of the

growth surface as a guide. Carefully cut out regions of the

growth surface of approximately 25 mm² using a scalpel or

scissors.

4. Transfer the regions of interest to the wells of a 6 well culture

plate and gradually dehydrate from 100% PBS to 100% ethanol

Production of Extracellular Vesicles Using a CELLine Adherent Bioreactor Flask

189