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

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.