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

Abstract

Extracellular vesicles (EVs) are nanosized lipid bilayer-bound vesicles that are naturally secreted from most cell types as a communication mechanism to deliver proteins, lipids, and genetic material. Despite the therapeutic potential of EVs, there is limited information on EV uptake kinetics and specificity. Here, we optimized an imaging flow cytometry (IFC)-based platform to quantitatively assess dose, time, and recipient cell specificity effects on human embryonic kidney cell (HEK293T) EV internalization in a high-throughput manner. We found that HEK293T EV uptake is an active process that is dose and time dependent. Further, the selectivity of EV uptake was quantified in vitro, and we found that HEK293T EVs were internalized at higher quantities by cells of the same origin. Lastly, neural stem cells internalized significantly more HEK293T EVs relative to mature neurons, suggesting that stem cells or progenitors, which are more metabolically active than terminally differentiated cells, may have higher rates of active EV internalization. The characterization of EV uptake, notably specificity, dose and time dependence, and kinetic assays will help inform and develop targeted and efficient EV-based therapeutics.

Extracellular Vesicle Transportation and Uptake by Recipient Cells: A Critical Process to Regulate Human Diseases

“…Despite advances in our understanding of the molecular mechanisms underlying EV internalization, the long-standing question of EV uptake specificity remains to be comprehensively addressed in the field. While studies have indicated the prevalence of EV uptake into any cell type tested [73,80], results from other studies demonstrated that EV uptake is a highly specific process in which the recipient cells and EVs would require the 'right' type of surface receptors and ligands for the coordinated protein-protein interactions [43,50,[80][81][82][83][84]. For example, the fusion of anti-epidermal growth factor receptor (EGFR) nanobodies to glycosylphosphatidylinositol (GPI) anchor signal peptides on the EV surface was sufficient to alter their cell targeting behavior and promote efficient binding to tumor cells that are dependent on EGFR density [81].…”

“…In addition, CD63-positive EVs were specifically bound to neuronal and glial cells, whereas CD63-negative EVs targeted only to the dendritic cells of neurons [82]. Recent evidence also indicated that the transfer of secreted exosomes is selective to the cell type of origin i.e., exosomes are preferentially taken up by cell types where they were originally secreted from [83,84]. Furthermore, EV uptake can also be affected by factors such as metabolic status of the recipient cells, and attributes including the types and characteristics of secreted EVs [47,84].…”

“…Recent evidence also indicated that the transfer of secreted exosomes is selective to the cell type of origin i.e., exosomes are preferentially taken up by cell types where they were originally secreted from [83,84]. Furthermore, EV uptake can also be affected by factors such as metabolic status of the recipient cells, and attributes including the types and characteristics of secreted EVs [47,84]. For example, it was observed that neural stem cells tend to exhibit a significantly higher capacity of internalizing EVs in comparison to mature neurons, implying that metabolically active cells may display higher rates of active EV uptake than terminally differentiated cells [84].…”

“…Furthermore, EV uptake can also be affected by factors such as metabolic status of the recipient cells, and attributes including the types and characteristics of secreted EVs [47,84]. For example, it was observed that neural stem cells tend to exhibit a significantly higher capacity of internalizing EVs in comparison to mature neurons, implying that metabolically active cells may display higher rates of active EV uptake than terminally differentiated cells [84]. Alternatively, the modifications of EV surface glycosylation patterns, leading to either changes in glycosylation states or vesicular charges, were found to affect the subsequent uptake of the EVs.…”

Mesenchymal stem cell‐glioblastoma interactions mediated via kinin receptors unveiled by cytometry

“…Image flow cytometry has been used for the detection of exosome and vesicle transference. For this study, Jurgielewicz et al transfected HEK293T cells with a plasmid coding for the CD63‐eGFP fusion protein [113]. CD63, an exosomal marker present in exosome membranes, fused with eGFP can be easily tracked by flow cytometry.…”