Comparative evaluation of ultrafiltration membranes for exosome isolation from A549 cell-conditioned medium

Although numerous exosome isolation protocols are available, a practical need remains for a direct, quantitative comparison of commonly used centrifugal molecular weight cutoff (MWCO) filters under identical and standardized conditions. In this study we isolated exosomes with 10, 50 and 100 kDa Amicon filters using A549 conditioned medium and performed multimodal characterization. The Z-average sizes of the exosomes were 49.24 nm (10 kDa), 61.64 nm (50 kDa) and 90.83 nm (100 kDa). Statistical analysis showed that the size differences between 10 kDa and 100 kDa (<i>p</i> &lt; 0.0001) and between 50 and 100 kDa (<i>p</i> = 0.0026) were highly significant. The 100 kDa isolates were most homogeneous (PDI = 0.455; 100 kDa vs. 10 kDa <i>p</i> = 0.0046). FluoroCet quantification yielded ∼ 1.3 × 10⁹, 7.0 × 10⁸ and 4.5 × 10⁸ exosomes/µg protein for 100, 50 and 10 kDa filters, respectively, with 100 kDa significantly higher than 10 kDa (<i>p</i> = 0.0079) and 50 kDa (<i>p</i> = 0.0279). Western blotting showed highest enrichment of canonical markers CD63 and TSG101 in 100 kDa isolates. Overall, this study highlights the impact of MWCO selection on exosome yield, purity, and quality and concludes that 100 kDa ultrafiltration is a pragmatic, low-cost alternative for cell-culture exosome preparations.