particles secreted by cells, have been shown to carry specific cellular

characteristics from the original cells and thus could represent

lineage commitment or exhibit potential therapeutic effects

[5]. EVs generated from undifferentiated hiPSCs carry specific

cargos indicating the difference from the original cells being repro-

grammed [6]. Potentially, EVs from hiPSCs exert homeostatic

regulation on stressed human umbilical vein endothelial cells

(HUVECs) to maintain cell viability and reduce senescence

[7]. Moreover, EVs from hiPSC-derived neural progenitor cells

(hiPSC-NPCs) not only carry lineage-specific information but

also exhibit pro-neurogenesis and circuit assembly potentials

[8, 9]. For hMSCs, there is an increasing body of evidence indicat-

ing the secreted EVs capture the therapeutic potentials of hMSCs

and

remain

biologically

active

after

transplantation

[10–

12]. Human umbilical cord MSC-secreted EVs have been shown

to exert liver protection under culture stress and ameliorate auto-

immune symptoms via immunomodulation in rodent uveoretinitis

models [13, 14]. Clinical trials have approached to study steroid

refractory graft-versus host disease and grade III-IV chronic kidney

disease patients with hMSC-EV administrations [15, 16].

Two-dimension (2D) conventional culture of human stem cells

is sufficient and robust in general laboratory scale. However, indus-

trial or clinical applications require a large amount of cells as well as

stem cell derivatives beyond what the 2D culture can provide. For

instance, 2–8
10⁶ cells/kg patient weight would be needed for

graft-versus-host diseases in stem cell based therapy [17]. To fulfill

the clinical requirement, three-dimensional (3D) suspension bior-

eactors are designed for biomanufacturing of human stem cells and

their derivatives. Microcarriers have been applied for anchorage-

dependent cells such as hMSCs. Studies have reported up to

43-fold increase of hMSCs in a 50 L stir-tank bioreactor

[18]. Moreover, advanced bioreactors, such as wave bioreactor

and PBS vertical wheel (PBS-VW) bioreactors have been designed

to reduce shear stress to maintain hMSC homeostasis while still

provide rapid expansion [19]. With the success of cell expansion in

bioreactors, stem cell derivatives such as EVs should also be able to

be scaled up for biomanufacturing purpose, though not many cases

have been published.

In this protocol chapter, detailed procedures and handling

notes are described for EV isolation from hiPSC-NPCs and

human umbilical cord-derived MSCs (hUC-MSCs) grown in sus-

pension bioreactors. For hiPSC-NPC differentiation and expan-

sion, a 50 mL spinner flask is selected. For hUC-MSC expansion,

Cytodex-1 microcarrier and a 100 mL PBS-VW bioreactor is

selected. EV-free medium preparation and the downstream EV

isolation is modified and described based on our previous studies

[20–22].

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