Methods in Molecular Biology (2022) 2436: 193–204

DOI 10.1007/7651_2021_416

© Springer Science+Business Media, LLC 2021

Published online: 07 September 2021

Extracellular Vesicle Collection from Human Stem Cells

Grown in Suspension Bioreactors

Xuegang Yuan, Xingchi Chen, Changchun Zeng, David G. Meckes Jr,

and Yan Li

Abstract

Extracellular vesicles (EVs) are particles with 100–1000 nm sizes which are secreted by cells for intercellular

communication. Meanwhile, studies have found that EVs secreted by human stem cells carry similar

characteristics (microRNAs, proteins, metabolites, etc.) from their cell counterpart. Thus, EVs derived

from stem cells, especially human induced pluripotent stem cells (hiPSCs) and human mesenchymal

stromal/stem cells (hMSCs) are promising candidates for cell-free therapy. However, conventional planar

culture is insufficient to produce a large amount of cells or EVs to satisfy clinical requirements. In this

chapter, we described feasible approaches to harvest EVs secreted by lineage-specific hiPSCs and undiffer-

entiated hMSCs in suspension bioreactors. Differentiation of hiPSCs to cortical organoids can be per-

formed in suspension bioreactors and the corresponding EVs can be isolated and purified. This scale-up

protocol can be applied to a majority of stem cell types with EV collection thus provides useful information

for both experimental and biomanufacturing purposes.

Key words Biomanufacturing, Differential centrifugation, Extracellular vesicles, Human stem cells,

Suspension bioreactors

1

Introduction

In past decades, human stem cells including pluripotent stem cells

and adult multipotent stem cells have drawn significant attentions

pre-clinically and clinically [1]. Human induced pluripotent stem

cells (hiPSCs) exhibit robust differentiation potential for modeling

of disease pathology, drug discovery, and act as potential cell

sources for therapeutic applications [2]. On the other hand,

human mesenchymal stromal/stem cells (hMSCs) have been

widely acknowledged for their regenerative potentials mediated

by their secretome and paracrine effects. Studies have demonstrated

promising strategies by using both types of stem cells to understand

disease progression and therapeutic mechanisms, as well as in pre-

clinical/clinical

trials

following

biomanufacturing

regulations

[3, 4]. Recently, extracellular vesicles (EVs) or exosomes, the nano-

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