RESUMEN
We report spatial separation of extracellular vesicle (EVs) populations based on particle size by using an approach that exploits Marangoni flow and the coffee-ring effect in microdroplets. Sequential transfer of a drying droplet progressively increases the mean size of EVs in the sample by repeated subsampling of a droplet during coffee-ring formation. This method allows size-based sorting, separation, and eventual retrieval of EVs for RNA and protein analysis. To demonstrate the biomedical relevance of this method, EVs from prostate cancer patients were analyzed; results revealed that the expression of cancer-associated genes and proteins was higher in small EVs than in large EVs. This ability to sort EVs using a combination of coffee ring with Marangoni flow and sequential droplet-transfer allows analysis of subpopulations of EVs, and will facilitate further studies of EVs.
Asunto(s)
Vesículas Extracelulares/química , Neoplasias de la Próstata/química , Vesículas Extracelulares/genética , Humanos , Células MCF-7 , Masculino , Nanopartículas/química , Tamaño de la Partícula , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/genética , Propiedades de Superficie , Células Tumorales CultivadasRESUMEN
Extracellular vesicles are categorized in subsets according to their biogenesis processes. To facilitate the investigation of subsets, an effective method is needed for isolating subpopulations. The efficacy of existing density and size-based isolation methods is limited, and as a result, the correlation of properties within separated subpopulations is modest. Here, we introduced size separation with â¼48 nm resolution that exploits Marangoni flow and the coffee-ring effect in microdroplets in which extracellular vesicles are spatially deposited at different location according to size of extracellular vesicle. Interestingly, the analysis of tetraspanin proteins of the extracellular vesicles facilitated by this method reveals that the size of extracellular vesicles is correlated with expression of tetraspanin proteins (CD9, CD63, CD81) that are associated with the size of extracellular vesicles. The findings show that CD9 and CD81 are uniformly expressed regardless of size, CD63 is highly expressed only in larger extracellular vesicles. This evidence indicates that extracellular vesicles can be classified based on size and expression of CD63.
RESUMEN
We propose a microfluidic system that generates nanovesicles (NVs) by slicing living cell membrane with microfabricated 500 nm-thick silicon nitride (SixNy) blades. Living cells were sliced by the blades while flowing through microchannels lined with the blades. Plasma membrane fragments sliced from the cells self-assembled into spherical NVs of ~100-300 nm in diameter. During self-assembly, the plasma membrane fragments enveloped exogenous materials (here, polystyrene latex beads) from the buffer solution. About 30% of beads were encapsulated in NVs, and the generated NVs delivered the encapsulated beads across the plasma membrane of recipient cells, but bare beads could not penetrate the plasma membrane of recipient cells. This result implicates that the NVs generated using the method in this study can encapsulate and deliver exogenous materials to recipient cells, whereas exosomes secreted by cells can deliver only endogenous cellular materials.