Busatto, Sara

Extracellular vesicles (EVs) play important roles in (patho)physiological processes by mediating cell communication. Although EVs contain glycans and glycosaminoglycans (GAGs), these biomolecules have been overlooked due to technical challenges in comprehensive glycome analysis coupled with EV isolation. Conventional mass spectrometry (MS)-based methods are restricted to the assessment of N-linked glycans. Therefore, methods to comprehensively analyze all glyco-polymer classes on EVs are urgently needed. In this study, tangential flow filtration-based EV isolation was coupled with glycan node analysis (GNA) as an innovative and robust approach to characterize most major glyco-polymer features of EVs. GNA is a molecularly bottom-up gas chromatography-MS technique that provides unique information that is unobtainable with conventional methods. The results indicate that GNA can identify EV-associated glyco-polymers that would remain undetected with conventional MS methods. Specifically, predictions based on GNA identified a GAG (hyaluronan) with varying abundance on EVs from two different melanoma cell lines. Enzyme-linked immunosorbent assays and enzymatic stripping protocols confirmed the differential abundance of EV-associated hyaluronan. These results lay the framework to explore GNA as a tool to assess major glycan classes on EVs, unveiling the EV glycocode and its biological functions.

AbstractWith an exponential increase in extracellular vesicle (EV) studies in the past decade, focus has been placed on standardization of experimental design to ensure inter‐study comparisons and validity of conclusions. In the case of in vitro assays, the composition of cell culture media is important to consider for EV studies. In particular, levels of lipoproteins, which are critical components of the interstitial fluid, should be taken into consideration. Results from this study reveal that lipoprotein levels in cell culture medium impact the effects that EVs have on recipient cells. Additionally, evidence of EV binding and fusion to lipoprotein‐like structures in plasma is provided. However, it is unclear whether the impact of lipoproteins in cell culture is due to direct interactions with EVs, indirect effects, or a combination of both mechanisms. Taken together, cell culture studies performed in the absence of physiological levels of lipoproteins are unlikely to reflect interactions that occur between EVs and recipient cells in an in vivo environment.

Extracellular vesicles (EVs) mediate intercellular transport of biomolecular cargo in the body, making them promising delivery vehicles for bioactive compounds. Genetic engineering of producer cells has enabled encapsulation of therapeutic proteins in EVs. However, genetic engineering approaches can be expensive, time-consuming, and incompatible with certain EV sources, such as human plasma and bovine milk. The goal of this study was to develop a quick, versatile, and simple method for loading proteins in EVs post-isolation. Proteins, including CRISPR associated protein 9 (Cas9), were bound to cationic lipids that were further complexed with MDA-MB-231 cell-derived EVs through passive incubation. Size-exclusion chromatography was used to remove components that were not complexed with EVs. The ability of EVs to mediate intracellular delivery of proteins was compared to conventional methods, such as electroporation and commercial protein transfection reagents. The results indicate that EVs retain native features following protein-loading and obtain similar levels of intracellular protein delivery as conventional methods, but display less toxicity. This method opens up opportunities for rapid exploration of EVs for protein delivery.

Blood plasma is a readily accessible source of extracellular vesicles (EVs), i.e., cell-secreted nanosized carriers that contain various biomolecules, including glycans. Previous studies have demonstrated that glycans play a major role in physiological and pathological processes, and certain plasma glycans have been associated with disease conditions. However, glycome studies have been limited by a lack of analytical techniques with the throughput capacity necessary to study hundreds of clinical samples. This study is the first to characterize the EV plasma glycome based on all major glycan classes. The results based on glycan node analysis revealed, as expected, that plasma-derived EVs have distinct glycan features from donor-matched whole plasma. Specifically, glycan nodes corresponding to those observed in chondroitin sulfate, dermatan sulfate, type I keratan sulfate, and type II keratan sulfate were enriched on EVs. The identification of specific differences in glycan features in plasma vs. plasma-derived EVs is relevant for understanding the physiological role of EVs and as a reference for future diagnostic studies. Additionally, the results indicate that EV glycan nodes do not substantially differ among a small set of healthy donors. These results lay the framework for the further evaluation of all EV glycan classes as diagnostic markers, therapeutic targets, and biologically active components in health and disease.

Abstract Extracellular vesicles secreted from adipose‐derived mesenchymal stem cells (ADSCs) have therapeutic effects in inflammatory diseases. However, production of extracellular vesicles (EVs) from ADSCs is costly, inefficient, and time consuming. The anti‐inflammatory properties of adipose tissue‐derived EVs and other biogenic nanoparticles have not been explored. In this study, biogenic nanoparticles are obtained directly from lipoaspirate, an easily accessible and abundant source of biological material. Compared to ADSC‐EVs, lipoaspirate nanoparticles (Lipo‐NPs) take less time to process (hours compared to months) and cost less to produce (clinical‐grade cell culture facilities are not required). The physicochemical characteristics and anti‐inflammatory properties of Lipo‐NPs are evaluated and compared to those of patient‐matched ADSC‐EVs. Moreover, guanabenz loading in Lipo‐NPs is evaluated for enhanced anti‐inflammatory effects. Apolipoprotein E and glycerolipids are enriched in Lipo‐NPs compared to ADSC‐EVs. Additionally, the uptake of Lipo‐NPs in hepatocytes and macrophages is higher. Lipo‐NPs and ADSC‐EVs have comparable protective and anti‐inflammatory effects. Specifically, Lipo‐NPs reduce toll‐like receptor 4‐induced secretion of inflammatory cytokines in macrophages. Guanabenz‐loaded Lipo‐NPs further suppress inflammatory pathways, suggesting that this combination therapy can have promising applications for inflammatory diseases.

Concentration of extracellular vesicles (EVs) from biological fluids in a scalable and reproducible manner represents a major challenge. This study reports the use of tangential flow filtration (TFF) for the highly efficient isolation of EVs from large volumes of samples. When compared to ultracentrifugation (UC), which is the most widely used method to concentrate EVs, TFF is a more efficient, scalable, and gentler method. Comparative assessment of TFF and UC of conditioned cell culture media revealed that the former concentrates EVs of comparable physicochemical characteristics, but with higher yield, less single macromolecules and aggregates (<15 nm in size), and improved batch-to-batch consistency in half the processing time (1 h). The TFF protocol was then successfully implemented on fluids derived from patient lipoaspirate. EVs from adipose tissue are of high clinical relevance, as they are expected to mirror the regenerative properties of the parent cells.

ABSTRACT The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell‐released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV‐associated functional activities. Finally, a checklist is provided with summaries of key points.