Methods in Molecular Biology (2022) 2436: 183–192

DOI 10.1007/7651_2021_413

© Springer Science+Business Media, LLC 2021

Published online: 07 September 2021

Production of Extracellular Vesicles Using a CELLine

Adherent Bioreactor Flask

Anastasiia Artuyants, Vanessa Chang, Gabrielle Reshef, Cherie Blenkiron,

Lawrence W. Chamley, Euphemia Leung, and Colin L. Hisey

Abstract

The efficient production of extracellular vesicles (EVs) from adherent cells in vitro can be challenging when

using conventional culture flasks. Issues such as low cell density leading to low EV yield, and the inability to

completely remove bovine serum EVs without starvation contribute to this challenge. By comparison, the

two-chamber CELLine adherent bioreactor can produce significantly more EVs with improved time, space,

and resource efficiency. Furthermore, it is highly accessible and can continually produce EVs using long

term cultures without the need for passaging. Lastly, the 10 kDa semipermeable, cellulose acetate mem-

brane separating the cell and media chambers allows for the continual use of bovine serum in the media

chamber while preventing bovine EVs from contaminating the conditioned media.

Key words CELLine bioreactor, Exosomes, Extracellular vesicles, Microvesicles

1

Introduction

To date, most researchers producing EVs from cell cultures rely on

the use of multiple conventional culture flasks due to the

low-density monolayers of cells on the surface of the flasks. To

maintain multiple flasks, large volumes of culture media are

required, which must then be concentrated using ultrafiltration or

multiple ultracentrifuge spins for downstream characterization and

use of the EVs. In addition, this method requires the use of signifi-

cant amounts of single use polystyrene, user time, and incubator

space. Importantly, bovine or other animal serum is routinely used

in conventional cultures to support cell growth up to the point of

conditioned media collection, but the endogenous EVs and other

nanoparticles (e.g., protein/growth factor aggregates) within the

serum are nearly impossible to fully deplete, with researchers often

resorting to up to 72 h of serum starvation prior to media collec-

tion with confounding effects [1–3]. Lastly, these EV productions

are typically done as one-off experiments, causing the final EV

preparation to be seen as a precious material, ultimately discoura-

ging more creative and high-risk experimental designs.

183