Bioreactors originally designed for monoclonal antibody pro-

duction have shown immense promise as a solution to many of the

aforementioned issues when appropriately repurposed for EV pro-

duction. These include hollow-fiber flow systems like the FiberCell

bioreactor [4–6], and two-chambered static systems like the CEL-

Line AD 1000 bioreactor flask [7–14]. Both approaches eliminate

the risk of endogenous serum EV contamination by separating cell

and media chambers using membranes with molecular cutoff sizes

much smaller than EVs, while also eliminating the need for cell

passaging. However, the CELLine AD 1000 system (Fig. 1) differs

in that it is a static system which requires no powered pumps and

takes up only slightly more space than a T-175 flask. Furthermore,

the 15 mL cell chamber and 1 L media chamber provide a highly

concentrated conditioned media product with minimal mainte-

nance. In combination, these benefits will enable researchers to

freely explore many other applications of EVs by producing much

larger amounts of EVs from long-term cultures, with significantly

less space and time requirements. In this protocol, we demonstrate

a simple workflow to inoculate, maintain, and monitor the CEL-

Line AD 1000 bioreactor flask for EV production, as well as the

basic steps for isolation and characterization of the resulting EVs as

defined by the MISEV guidelines [15]. Lastly, we present a method

for imaging the 3D growth of cells on the bioreactor surface once

EV production has ended.

Fig. 1 CELLine AD 1000 Bioreactor flask schematic illustration. (Figure created with BioRender.com)

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