Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins.

Extracellular vesicles (EVs) are released by all cells into biofluids such as plasma. The separation of EVs from highly abundant free proteins and similarly sized lipoproteins remains technically challenging. We developed a digital ELISA assay based on Single Molecule Array (Simoa) technology for ApoB-100, the protein component of several lipoproteins. Combining this ApoB-100 assay with previously developed Simoa assays for albumin and three tetraspanin proteins found on EVs (Ter-Ovanesyan, Norman et al., 2021), we were able to measure the separation of EVs from both lipoproteins and free proteins. We used these five assays to compare EV separation from lipoproteins using size exclusion chromatography with resins containing different pore sizes. We also developed improved methods for EV isolation based on combining several types of chromatography resins in the same column. We present a simple approach to quantitatively measure the main impurities of EV isolation in plasma and apply this approach to develop novel methods for enriching EVs from human plasma. These methods will enable applications where high-purity EVs are required to both understand EV biology and profile EVs for biomarker discovery.

Framework for rapid comparison of extracellular vesicle isolation methods.

Extracellular vesicles (EVs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. One of the main challenges in studying EVs is a lack of methods to quantify EVs that are sensitive enough and can differentiate EVs from similarly sized lipoproteins and protein aggregates. We demonstrate the use of ultrasensitive, single-molecule array (Simoa) assays for the quantification of EVs using three widely expressed transmembrane proteins: the tetraspanins CD9, CD63, and CD81. Using Simoa to measure these three EV markers, as well as albumin to measure protein contamination, we were able to compare the relative efficiency and purity of several commonly used EV isolation methods in plasma and cerebrospinal fluid (CSF): ultracentrifugation, precipitation, and size exclusion chromatography (SEC). We further used these assays, all on one platform, to improve SEC isolation from plasma and CSF. Our results highlight the utility of quantifying EV proteins using Simoa and provide a rapid framework for comparing and improving EV isolation methods from biofluids.

L1CAM is not associated with extracellular vesicles in human cerebrospinal fluid or plasma.

L1CAM is a transmembrane protein expressed on neurons that was presumed to be found on neuron-derived extracellular vesicles (NDEVs) in human biofluids. We developed a panel of single-molecule array assays to evaluate the use of L1CAM for NDEV isolation. We demonstrate that L1CAM is not associated with extracellular vesicles in human plasma or cerebrospinal fluid and therefore recommend against its use as a marker in NDEV isolation protocols.

Protection of tissue physicochemical properties using polyfunctional crosslinkers.

Understanding complex biological systems requires the system-wide characterization of both molecular and cellular features. Existing methods for spatial mapping of biomolecules in intact tissues suffer from information loss caused by degradation and tissue damage. We report a tissue transformation strategy named stabilization under harsh conditions via intramolecular epoxide linkages to prevent degradation (SHIELD), which uses a flexible polyepoxide to form controlled intra- and intermolecular cross-link with biomolecules. SHIELD preserves protein fluorescence and antigenicity, transcripts and tissue architecture under a wide range of harsh conditions. We applied SHIELD to interrogate system-level wiring, synaptic architecture, and molecular features of virally labeled neurons and their targets in mouse at single-cell resolution. We also demonstrated rapid three-dimensional phenotyping of core needle biopsies and human brain cells. SHIELD enables rapid, multiscale, integrated molecular phenotyping of both animal and clinical tissues.