Multi-Module Cassette Tangential Flow Filtration Enables High-Throughput Isolation and Functional Analysis of Small...

Small extracellular vesicles (sEVs) are nanoscale, lipid bilayer-enclosed vesicles that carry diverse biomolecules and hold considerable potential in disease diagnosis, monitoring, and therapeutic interventions. Efficient and scalable isolation of sEVs is essential for both biomedical research and clinical translation; however, conventional techniques, such as ultracentrifugation (UC), are time-consuming, low-throughput, and insufficient for separating sEV subpopulations by size. In this study, we present a high-throughput isolation platform utilizing cassette-based tangential flow filtration (CTFF) with membrane encapsulation. This system enables rapid processing of 2 L of a conditioned medium in approximately 25 min, with a total workflow duration of ∼175 min─achieving a 3-fold reduction in processing time compared to UC. Furthermore, we developed a multimodule CTFF (MM-CTFF) system capable of fractionating sEVs into distinct size-based subpopulations. Functional assays revealed that mesenchymal stem cell-derived sEVs (MSC-sEVs) exhibit size-dependent biological activities, including differential cellular uptake, proliferation, and migration. These results offer valuable insights into the functional heterogeneity of MSC-sEVs and establish a foundation for the development of size-specific, sEV-based strategies in both biomedical research and translational medicine.