Rapid inertial solution exchange for enrichment and flow cytometric detection of microvesicles

Despite the significant interest and widespread implications of exosomes, their clinical utility has been limited and biological roles obscured due to the difficulty in their isolation. The current gold standard for isolation is a label-free method that involves repeated lengthy ultracentrifugation steps, totaling several hours of sample processing. While ultracentrifugation is label-free ensuring no molecular bias in isolation, tetraspanin expression on exosomes has been well-characterized (such as CD63 and CD81). Additionally, epithelial cell adhesion molecule (EpCAM) expression has been found on the surface of some exosomes (specifically tumorderived exosomes). This has enabled novel affinity-based methods exploiting the levels of surface markers on exosomes that can expedite their isolation and potentially provide pure samples. These methods involve either immunomagnetic capture or affinity extraction on the surface of microchannels. Immunomagnetic methods require magnetic-activated cell sorting (MACS) separators and also an ultracentrifugation step to account for dilutions that occur during operation. Similarly, the microchannel method also requires an ultracentrifugation step prior to operation for some samples and operates at low flow-rates, limiting the volume of fluid it can process. Both of these techniques additionally do not integrate quantification or detection inline with isolation.

Subsequent to isolation, several methods of quantification and characterization can be employed. These include analysis of protein content, RNA content, surface marker expression, and total count. Accordingly, there is a need to standardize techniques in exosome isolation, handling, detection, and quanitification.

Here, researchers from UCLA present a tool for affinity isolation of exosomes that operates at high-throughput (orders of magnitude greater volumes than previously reported microfluidic isolation methods) and requires just one benchtop centrifugation step prior to operation, which they utilized to isolate exosomes from various biofluids of interest (e.g., cell culture supernatants and blood). The typical flow rate achieved in our device is greater than five-fold higher than previous methods. Highthroughput isolation will be crucial for fully dissecting the roles of exosomes in various biological contexts. Finally, they integrated an inline fluorescence detection system for immediate detection of exosomes.