membrane may have ruptured and one should consider termi-

nating the bioreactor.

8. Once established, live and dead cells are shed into the cell

chamber culture media at a variable rate throughout the life

of the bioreactor culture. Do not misinterpret a large number

of shed cells as a “dead” reactor. Often, these shed cells can be

plated to demonstrate their viability or used as cell lysates for

comparative western blots. The rate at which cells are shed into

the conditioned media is highly dependent upon the cell type.

9. Testing for mycoplasma is recommended prior to plating as

well as an agar growth of cell media during the bioreactor

culture.

10. EVs can be isolated from conditioned media using several other

methods, depending on the application. For instance, a second

100,000
g spin can be performed by resuspending the pellet

from the first 100,000
g spin to further reduce contaminat-

ing proteins, or ultracentrifugation can be avoided altogether

by using ultrafiltration and/or a larger size exclusion column.

11. EVs should be resuspended in minimal volume and used fresh

when possible as all concentration processes have significant

inherent EV-loss. However, they can be stored at 4 ^C short

term, or 80 ^C for long term. Multiple freeze–thaw cycles are

not recommended.

12. We recommend using an automated fraction collector for SEC.

Fractions can be collected manually, but this will potentially

introduce more variability in volume between fractions. Den-

sity gradient ultracentrifugation is a suitable alternative to SEC

[15]. Other SEC columns are also suitable for use.

13. The use of biotinylated secondary antibodies and streptavidin-

conjugated horseradish peroxidase (HRP) can drastically

reduce the amount of required EV protein loading (1–5 μg/

lane). We recommend this approach for most EV workflows.

References

1. Lehrich BM et al (2018) Fetal bovine serum-

derived extracellular

vesicles persist within

vesicle-depleted culture media. Int J Mol Sci

19(11):3538

2. Shelke GVet al (2014) Importance of exosome

depletion protocols to eliminate functional and

RNA-containing

extracellular

vesicles

from

fetal bovine serum. J Extracell Vesicles 3

3. Lehrich BM, Liang Y, Fiandaca MS (2021)

Foetal bovine serum influence on in vitro extra-

cellular vesicle analyses. J Extracell Vesicles 10

(3):e12061

4. Yan IS, Shukla N, Borrelli DA, Patel T (2018)

Use of a hollow fiber bioreactor to collect evs

from

cells

in

culture.

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Walker

J

(ed) Extracellular RNA: methods in molecular

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1740.

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Protocols,

New York

5. Watson DC et al (2018) Scalable, cGMP-

compatible purification of extracellular vesicles

carrying

bioactive

human

heterodimeric

IL-15/lactadherin complexes. J Extracell Vesi-

cles 7(1):1442088

6. Watson DC et al (2016) Efficient production

and enhanced tumor delivery of engineered

Production of Extracellular Vesicles Using a CELLine Adherent Bioreactor Flask

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