absenteeism. The major challenge of suppression is that this type of intensive intervention packageor something equivalently effective at reducing transmission -will need to be maintained until a vaccine becomes available (potentially 18 months or more) -given that we predict that transmission will quickly rebound if interventions are relaxed. We show that intermittent social distancingtriggered by trends in disease surveillance -may allow interventions to be relaxed temporarily in relative short time windows, but measures will need to be reintroduced if or when case numbers rebound. Last, while experience in China and now South Korea show that suppression is possible in the short term, it remains to be seen whether it is possible long-term, and whether the social and economic costs of the interventions adopted thus far can be reduced.

It is known that culture media (CM) promotes cellular growth, adhesion, and protects explanted primary brain cells from in vitro stresses. The fetal bovine serum (FBS) supplement used in most CM, however, contains significant quantities of extracellular vesicles (EVs) that confound quantitative and qualitative analyses from the EVs produced by the cultured cells. We quantitatively tested the ability of common FBS EV-depletion protocols to remove exogenous EVs from FBS-supplemented CM and evaluated the influence such methods have on primary astrocyte culture growth and viability. We assessed two methodologies utilized for FBS EV removal prior to adding to CM: (1) an 18-h ultracentrifugation (UC); and (2) a commercial EV-depleted FBS (Exo-FBS™). Our analysis demonstrated that Exo-FBS™ CM provided the largest depletion (75%) of total FBS EVs, while still providing 6.92 × 109 ± 1.39 × 108 EVs/mL. In addition, both UC and Exo-FBS™ CM resulted in poor primary astrocyte cell growth and viability in culture. The two common FBS EV-depletion methods investigated, therefore, not only contaminate in vitro primary cell-derived EV analyses, but also provide a suboptimal environment for primary astrocyte cell growth and viability. It appears likely that future CM optimization, using a serum-free alternative, might be required to advance analyses of cell-specific EVs isolated in vitro.

Foetal bovine serum influence on in vitro extracellular vesicle analyses  INTRODUCTION Extracellular vesicles (EVs) are nanosized lipid bilayer vesicles most notably from either endosomal (i.e., exosomes) or plasma membrane origins (i.e., microvesicles/ectosomes) and released from nearly all mammalian cells (Colombo et al., 2014). An interest in EV research has increased over the past decade, in part due to their participation in complex intercellular communication (Roy et al., 2018). Though EVs are abundant in blood and other biofluids, the investigation of in vitro-derived EVs provides a critical tool for understanding various mechanisms associated with their biogenesis, molecular composition, packaging of specific payloads, and cellular trafficking. Once released, EVs traffic to target cells where they may be taken up to release their payloads via specific mechanisms, and thereby exert their physiological influence (Colombo et al., 2014; Kowal et al., 2014). Although engineered micelles and liposomes have previously been utilized as lipid nanocarriers (Fiandaca & S., 2013; Fiandaca et al., 2011) for many therapeutic applications, EVs have garnered recent interest as drug delivery vehicles (Elsharkasy et al., 2020). Currently, there exist vastly heterogeneous cell culture conditions for EV production and isolation (Consortium, 2017). Therefore, there is a current need to define more standard cell culture conditions for investigating EVs that may accelerate the translation of therapeutic clinical-grade EVs (Lener et al., 2015; Lötvall et al., 2014; Théry et al., 2018). Herein, we present a mini-review on recent investigations reporting the influence of foetal bovine serum (FBS)-supplemented media formulations on cultured cell physiology, EV production/release, and its contaminating presence of vesicular and non-vesicular particles. Additionally, we describe potential solutions and provide recommendations to aid in vitro EV investigators.  CELL CULTURE CONDITIONS FOR EV INVESTIGATIONS: SERUM USAGE AND CONCERNS An international survey observed 83% of International Society for Extracellular Vesicles (ISEV) respondents utilize conditioned cell culture media as their starting material (Gardiner et al., 2016). FBS is a common additive in cell culture and 52% of ISEV respondents utilize serum-containing media for downstream EV analyses, with 59% and 57% of those respondents performing in vitro and in vivo functional studies, respectively (Gardiner et al., 2016). Serum usage, in part due to its ill-defined composition, provides a variety of contaminating particles (e.g., EVs, lipoproteins, and protein complexes, which differ in their physical properties, yet also have similar size, density, and/or RNA components) that confound these investigative results.  FBS SUPPLEMENTATION AND GENERAL CONCERNS The growth factors and other constituents within FBS appear to provide a nourishing ecosystem for many cultured cells (Bettger & Mckeehan, 1986). Despite this nourishing milieu, the presence of FBS in culture has raised spec...

Extracellular vesicles (EVs) are released by many cell types and distributed within various biofluids. EVs have a lipid membrane-confined structure that allows for carrying unique molecular information originating from their parent cells. The species and quantity of EV cargo molecules, including nucleic acids, proteins, lipids, and metabolites, may vary largely owing to their parent cell types and the pathophysiologic status. Such heterogeneity in EV populations provides immense challenges to researchers, yet allows for the possibility to prognosticate the pathogenesis of a particular tissue from unique molecular signatures of dispersing EVs within biofluids. However, the inherent nature of EV's small size requires advanced methods for EV purification and evaluation from the complex biofluid. Recently, the interdisciplinary significance of EV research has attracted growing interests, and the EV analytical platforms for their diagnostic prospect have markedly progressed. This review summarizes the recent advances in these EV detection techniques and methods with the intention of translating an EV-based liquid biopsy into clinical practice. This article aims to present an overview of current EV assessment techniques, with a focus on their progress and limitations, as well as an outlook on the clinical translation of an EV-based liquid biopsy that may augment current paradigms for the diagnosis, prognosis, and monitoring the response to therapy in a variety of disease settings.