Size exclusion chromatography (SEC) is widely used to isolate extracellular vesicles (EVs) from plasma; however, the EV-containing fractions obtained are often dilute, which can limit downstream analyses requiring low input volumes. This dilution becomes more pronounced as smaller input sample volumes are processed on an SEC column of fixed dimensions. Although the number of collected fractions can be adjusted to reduce the EV elution volume, the resulting EV concentration may remain suboptimal.
To address this challenge, we developed a streamlined workflow that integrates the Ascent automated fractionation instrument, Apex SEC columns, and direct fraction collection into centrifugal concentration filters. This approach enables immediate post-SEC concentration without additional manual transfer steps.
Instead of the standard tube holder used to collect fractions on the Ascent, we designed a custom holder compatible with Amicon® Ultra centrifugal filters (10 kDa MWCO; 2 mL capacity). In this workflow, the Ascent is first used to wash the columns, after which the sample is loaded, and the discard volume is directed to waste. The EV-enriched fraction is then collected directly into the Amicon filters, which are subsequently transferred to a centrifuge for concentration (Figure 1).
Figure 1. Isolating EVs directly into centrifugal concentration filters using the Ascent. A. Photograph of the Ascent instrument showing EV fractions from eight samples collected on Apex columns directly into Amicon Ultra 10 kDa centrifugal filters. B. Ascent run parameters used to collect a single pooled EV-enriched fraction from 0.5 mL sample input: column type = Apex 4B; number of columns = 8; wash volume = 17 mL; discard volume = 2 mL; number of fractions collected = 1; fraction volume = 1.5 mL. C. Following fractionation, the concentration filters are removed from the Ascent, sealed, and centrifuged for 45 min at 3,000 × g. The flow-through is discarded, and the filters are inverted and centrifuged for 2 min at 2,000 × g to recover the concentrated EVs.
To demonstrate this workflow, human plasma was fractionated on Apex 4B SEC columns using the Ascent instrument at four input volumes (0.125, 0.25, 0.5, and 1.0 mL). Each condition was performed in duplicate using independent columns (eight columns total). For each input volume, a single EV-enriched SEC fraction (1.5 mL) was collected directly into an Amicon Ultra centrifugal filter (10 kDa MWCO). A 50 µL aliquot of the pre-concentrated fraction was removed for analysis, and the remaining volume was concentrated by centrifugation for 45 min (Figure 2).
Figure 2. Concentration of EVs following SEC at increasing plasma input volumes. EV concentration was quantified using the Atlas EV ELISA before (green) and after (orange) centrifugal concentration.
Lower plasma input volumes resulted in proportionally smaller final concentrate volumes, with average final volumes of 75, 85, 110, and 180 µL for input volumes of 0.125, 0.25, 0.5, and 1.0 mL, respectively. Overall, this workflow enabled up to a 20-fold reduction in volume and up to a 12-fold increase in EV concentration after correcting for losses associated with the concentrator membranes.
In summary, coupling automated SEC fractionation with direct collection into centrifugal concentrators provides a simple and effective solution for overcoming dilution challenges commonly encountered in EV isolation workflows. This approach enables flexible handling of a wide range of input volumes while delivering EV fractions well-suited for downstream quantitative assays and method optimization. The workflow can be readily implemented on the Ascent platform using Apex SEC columns and commercially available centrifugal concentrators (e.g., Amicon filters), and compatibility with alternative filter formats can be accommodated.
For studies involving larger sample cohorts, this workflow can be further integrated into a fully automated, plate-based format using the Summit platform, enabling end-to-end automation of EV isolation and ultrafiltration-based concentration for up to 48 samples processed in parallel.
