(176g) 3D Dynamic Culture Enhances Extracellular Vesicle Production with Altered Biogenesis and Cargo Contents in Human Mesenchymal Stem Cells

none;text-autospace:none">3D
Dynamic Culture Enhances Extracellular Vesicle Production with Altered
Biogenesis and Cargo Contents in Human Mesenchymal Stem Cells

none;text-autospace:none">

none;text-autospace:none"> font-family:" times new roman>Xuegang Yuan^{1, 2}, Dingani Nkosi³, Qin Fu¹, Yuan Liu¹,
Richard Jeske¹, Li Sun³, David Meckes³ and
Teng Ma^1,2

none;text-autospace:none"> ^{10.0pt;font-family:" times new roman>}

none;text-autospace:none"> ^{10.0pt;font-family:" times new roman>1}Chemical
and Biomedical Engineering; ²The National High Magnetic Field
Laboratory; ³Department of Biomedical Sciences, College of Medicine

none;text-autospace:none"> font-family:" times new roman>The Florida State University, Tallahassee,
Florida, USA

none;text-autospace:none">

font-family:" times new roman>Key words: Human mesenchymal stem cell; 3D aggregation; extracellular
vesicles; exosome; immunomodulation; manufacturing normal">

none;text-autospace:none"> " times new roman>

none;text-autospace:none">Abstract:

Human mesenchymal stem cells (hMSCs) have demonstrated significant therapeutic potential and
are major candidate in cell therapy in a wide range of diseases including stroke,
multiple sclerosis, cardiovascular, graft versus host disease.  The clinical efficacy of hMSCs is mainly attributed to the paracrine effects thorough
hMSC secretome.  Among various components of secretome, extracellular vehicles (EVs) are cell-derived
vesicles responsible for cell-cell communication and regulation of cellular
events.  hMSCs
derived EVs, including microvesicles (MVs) and
exosomes, contain various cargos such as DNA, proteins, miRNA and organelles, exerting
paracrine effect and cell-cell communication to facilitate immunomodulatory
activity [1]. With several clinical trials ongoing, hMSC
derived EVs possess the potential as cell-free therapeutic strategy in
regenerative medicine, with feasibility of biomanufacturing. However, scale up
of hMSC derived EVs has become a major challenge for
clinical research. Studies have shown low efficiency of EVs secretion under
conventional monolayer culture. Unstable EV properties with regards to the cargo
and functional difference are also reported and may be due to the heterogeneity
and senescence during culture expansion. In current study, we employ dynamic 3D
aggregation culture of hMSCs to improve the
bioprocessing strategy of EVs production. hMSCs were
cultured as 3D aggregates under rocking motion to facilitate aggregation [2].
EVs production was significantly improved under 3D dynamic culture of hMSCs. Moreover, 3D hMSC-EVs also
exhibited smaller size compared to 2D hMSC-EV from nanoparticle
tracking analysis (NTA), with higher exosome enrichment. Our results also
demonstrated enhanced biogenesis of EVs in 3D hMSC
aggregates. Proteomic and transcriptomic analysis were also conducted to
demonstrate the difference of cargos between 3D dynamic culture and monolayer
culture. For functional evaluation, 3D hMSC aggregate
derived EVs has similar effect of immunomodulation compared to EVs derived from
hMSCs under monolayer culture. Moreover, 3D hMSC aggregate derived EVs exhibited enhanced
anti-senescence when cultured with hMSCs with
replicative senescence. Together, our study provides a promising bioprocess for
hMSCs derived EVs production with enhanced
therapeutic potentials compared to conventional monolayer culture.

Reference:

inter-ideograph"> font-family:" times new roman mso-font-kerning:12.0pt>1. Rider MA., Hurwitz S., Meckes
D. ExtraPEG: A Polyethylene Glycol-Based Method for
Enrichment of Extracellular Vesicles. Sci
Rep. 2016; 6: 23978.

inter-ideograph"> font-family:" times new roman mso-font-kerning:12.0pt>2. Tsai AC., Liu Y., Yuan X., Chella
R., Ma T. Aggregation Kinetics of Human Mesenchymal Stem Cells Under Wave
Motion. Biotechnol J. 2017;
12(5).