Biological membranes in EV biogenesis, stability, uptake, and cargo transfer: an ISEV position paper arising from the ISEV membranes and EVs workshop

Abstract

Paracrine and endocrine roles have increasingly been ascribed to extracellular vesicles (EVs) generated by multicellular organisms. Central to the biogenesis, content, and function of EVs are their delimiting lipid bilayer membranes. To evaluate research progress on membranes and EVs, the International Society for Extracellular Vesicles (ISEV) conducted a workshop in March 2018 in Baltimore, Maryland, USA, bringing together key opinion leaders and hands-on researchers who were selected on the basis of submitted applications. The workshop was accompanied by two scientific surveys and covered four broad topics: EV biogenesis and release; EV uptake and fusion; technologies and strategies used to study EV membranes; and EV transfer and functional assays. In this ISEV position paper, we synthesize the results of the workshop and the related surveys to outline important outstanding questions about EV membranes and describe areas of consensus. The workshop discussions and survey responses reveal that while much progress has been made in the field, there are still several concepts that divide opinion. Good consensus exists in some areas, including particular aspects of EV biogenesis, uptake and downstream signalling. Areas with little to no consensus include EV storage and stability, as well as whether and how EVs fuse with target cells. Further research is needed in these key areas, as a better understanding of membrane biology will contribute substantially towards advancing the field of extracellular vesicles.

Kinetics and Specificity of HEK293T Extracellular Vesicle Uptake using Imaging Flow Cytometry

“…There are challenges when analyzing EV uptake. For example, as we and others observed, results can differ if the EV dose and exposure time are altered (20). We addressed HEK293T EV dose and concentration as a kinetic variable.…”

“…A group of international experts on EVs emphasized a need to effectively determine the minimal effective dose of EVs for uptake assays and here we have developed a system that can be effectively adopted by the field (20,22,24). There are challenges when analyzing EV uptake.…”

“…As the ISEV position paper suggests, the choice of an EV label may affect uptake, necessitating less disruptive techniques such as the GFP tagging methods used in our study. Specifically, 72% of researchers participating in a survey claim that lipid dye experiments are unreliable unless proper controls are used (20). EV dyes do not reliably correlate with small EV content and may even increase vesicle size.…”

“…A potential reason for conflicting EV uptake results is the lack of standardization in measurement platforms, including analyses of dose and time effects. Recently, an International Society for Extracellular Vesicles (ISEV) group of experts, released a position paper emphasizing the need for analysis of dose and time, amongst other confounding factors on EV uptake (20). The group stated that 'one dose does not fit all,' and that dose may affect EV uptake or selectivity.…”

“…Traditional flow cytometers have been designed to measure biological particles in the cellular range, cannot differentiate EV swarm or coincidence, and have increased noise due to triggering (28)(29)(30)(31). As mentioned by the ISEV group, there is growing awareness of the physical limitations of traditional flow cytometry and highlight the demand for specialized flow cytometry with detection limits in the 100 nm range (20,28). Imaging flow cytometry (IFC) combines the high throughput quantitative nature of flow cytometry along with fluorescence imaging technology which can resolve inherently small fluorescent particles, down to 100 nm in diameter (31).…”