Growth Media Conditions Influence the Secretion Route and Release Levels of Engineered Extracellular Vesicles

Abstract

Extracellular vesicles (EVs) are nanosized cell-derived vesicles produced by all cells, which provide a route of intercellular communication by transmitting biological cargo. While EVs offer promise as therapeutic agents, the molecular mechanisms of EV biogenesis are not yet fully elucidated, in part due to the concurrence of numerous interwoven pathways which give rise to heterogenous EV populations in vitro. The equilibrium between the EV-producing pathways is heavily influenced by factors in the extracellular environment, in such a way that can be taken advantage of to boost production of engineered EVs. In this study, a quantifiable EV-engineering approach is used to investigate how different cell media conditions alter EV production. The presence of serum, exogenous EVs, and other signaling factors in cell media alters EV production at the physical, molecular, and transcriptional levels. Further, it is demonstrated that the ceramide-dependent EV biogenesis route is the major pathway to production of engineered EVs during optimized EV-production. These findings suggest a novel understanding to the mechanisms underlying EV production in cell culture which can be applied to develop advanced EV production methods.

Unconventional Protein Secretion Dependent on Two Extracellular Vesicles: Exosomes and Ectosomes

“…In contrast the EVs, upon their release and during navigation, maintain their whole structure, composed by both membranes and cargos. Upon their fusion to specific target cells, their membranes are integrated into the plasma or endocytic membranes, while the cargos are discharged and diffuse in the cytoplasm ( Bost et al, 2021 ; Guan et al, 2021 ). Depending on their properties, the intracellular distribution of the received cargo proteins varies: a l fraction goes to the nucleus, others to the plasma membrane/intracellular membranes, or remain in the cytosol ( Somiya and Kuroda, 2021 ).…”

“…Additional mechanisms known to regulate the EV generation and function depend on the media of their culture. For example, addition of fetal bovine serum, widely used commercially, induces negative effects, whereas with various growth factors and also with glucose the induced effects are positive ( Bost et al, 2021 ; Guan et al, 2021 ).…”

“…Comparative analysis of the EVs derived from exosomes and ectosomes have revealed interesting aspects of heterogeneity. Proteins of important families, from their membranes (tetraspanins) and lumena (proteins associated to transport and fusion) are present in both types of EVs, however with differences in their levels and components ( Pizzirani et al, 2007 ; Van Niel et al, 2018 ; Bost et al, 2021 ; Guan et al, 2021 ; Mathieu et al, 2021 ). Differences have been reported also for weakly expressed proteins, some of which heterogeneous also among EVs from distinct subtypes of cellular vesicles ( Greening and Simpson, 2018 ; Gurunathan et al, 2021 ).…”

“…Differences have been reported also for weakly expressed proteins, some of which heterogeneous also among EVs from distinct subtypes of cellular vesicles ( Greening and Simpson, 2018 ; Gurunathan et al, 2021 ). Exosomes and ectosomes differ also in metabolites, a property relevant for navigation fluids, including blood plasma ( Bost et al, 2021 ; Gurunathan et al, 2021 ; Lim et al, 2021 ). In addition, recent evidence has demonstrated the ability of EVs to establish surface protein-protein interactions.…”

“…In many cases, however, binding is followed by insertion of the vesicle membrane in the plasma membrane, with enlargement of the cell surface area and cargo discharged into the cell cytoplasm ( Levy et al, 2021 ; An et al, 2021 ). Upon the introduction of new techniques to reveal the progressive changes of the EV protein distribution, the membrane and cargo events have been intensively investigated ( Lim et al, 2021 ; Bost et al, 2021 ). Where does the EV fusion occur in target cells?…”