An Ultrasensitive Flow Cytometry System for Multiparametric Analysis of Therapeutic Anti-Aging Extracellular Vesicles

PROJECT SUMMARY Extracellular vesicles (EVs) are small, lipid bound vesicles that are secreted by cells. Originally thought to serve little purpose, EVs have been gaining significant interest due to their promise as therapeutics for a wide range of indications including skin repair in wound healing and photoaging, and central nervous system (CNS) repair, including the treatment of neurogenerative diseases such as Alzheimer’s and Parkinson’s diseases. As EVs are found in a variety of physiological fluids including blood, urine, ascites and cerebral-spinal fluid, they also show great promise as minimally invasive disease biomarkers. However, due to their very small size, many ranging from 1 μm down to as small as 30 nm, they are exceedingly difficult to study. Characterization of EVs is critical to understanding function. This characterization includes not only EV size, but also the complement of molecular cargo that they carry. While some techniques such as nanoparticle tracking analysis (NTA) can accurately size particles, they have severe limitations in analyzing their molecular content. Other methods can determine molecular contents, but typically only as a bulk molecular analysis, preventing the cross-correlation of the expression of multiple markers in individual EV particles. Most of the current analysis methods are low throughput, laborious, and/or require significant technical expertise. However, one technique— nanoparticle flow cytometry (NP-FC)—has the capability to provide researchers with simultaneous investigation of size and multiparameter analysis of molecular cargo on a particle-by-particle basis. While most off the shelf flow cytometers are designed for the analysis of cells, many fail to perform adequately on EVs due to their small size. There is one built for purpose NP-FC on the market. While it has excellent size resolution, this comes at the expense of throughput (<200 events/sec), multiplexing capabilities (only two channels), fluorescence sensitivity, and ease of use. All of this leaves EV researchers wanting for a more comprehensive solution to better enable their critical studies correlating EV properties and reparative function. Kinetic River has developed a built for purpose NP-FC. Preliminary data shows size sensitivity (via light scattering) approaching that of the existing NP-FC. We propose to further develop this by improving three aspects of performance: i) scattering and fluorescence sensitivity; ii) optomechanical design to enable five lasers and up to six fluorescence detection channels; and iii) user experience, including throughput of up to 2,000 events/sec and a simple, automated size calibration method. In collaboration with EV researcher Dr. Fatah Kashanchi, we will evaluate the performance of the resulting instrument by addressing a key question in his lab regarding the reparative function of stem cell derived EVs. If successful, this Phase I will result in an EV analyzer with an unprecedented ability to fully identify and characterize EVs, paving the way for a Phase II where sorting capabilities can be incorporated, enabling isolation of EV subfractions for further downstream functional studies.