Abstract

Characterization of Cell-Derived Vesicles: Are They Really Different from Small EVs?

Ilaria Passalacqua, Scientist II, Process Development, LONZA

Cell-derived vesicles (CDVs) are one of the most promising non-viral drug delivery platforms. High amounts of these vesicles are produced through mechanical extrusion of cells and used to carry payloads with therapeutic benefit. CDVs share molecular signatures found in the producer cells but their cargo may differ from other classes of extracellular vesicles (EVs) due to their peculiar genesis. In this study, we ought to identify biophysical and molecular characteristics unique to CDVs or shared with small-EVs. Lonza’s HEK293 cells were used to produce CDVs using MDimune’s proprietary extrusion technology and to purify small-EVs from conditioned media. Both EV types were purified using the same downstream process and characterized by mass spectrometry and nano-flow cytometry (nFCM) to identify CDV-specific markers and evaluate membrane integrity. CDV-enriched proteins were found by proteomic analysis and confirmed by nFCM. Specifically, LAMP-1 and CD63 were identified as CDV-enriched markers and further confirmed by western blot analysis and flow cytometry (FACS). Notably, a partial overlap was observed between CD63 and LAMP-1 positive EV subpopulations , suggesting distinct molecular properties and, perhaps, functional roles. Further work is warranted to confirm these observations and to better understand the potential of CDVs a non-viral vector modality for cell-and-gene therapies.