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

Area of Science:

• Extracellular Vesicles Research
• Biotechnology
• Biochemistry

Background:

• Exosome isolation methods vary, necessitating direct comparisons of common techniques.
• Centrifugal molecular weight cutoff (MWCO) filters are widely used but require standardized evaluation.

Purpose of the Study:

• To quantitatively compare 10, 50, and 100 kDa Amicon MWCO filters for exosome isolation.
• To assess the impact of MWCO selection on exosome yield, size, homogeneity, and purity.

Main Methods:

• Exosomes were isolated from A549 conditioned medium using 10, 50, and 100 kDa Amicon filters.
• Multimodal characterization included Z-average size, polydispersity index (PDI), FluoroCet quantification, and Western blotting for canonical markers (CD63, TSG101).

Main Results:

• The 100 kDa filter yielded significantly larger (90.83 nm) and more homogeneous exosomes (PDI = 0.455) compared to 10 and 50 kDa filters.
• Exosome yield was highest with the 100 kDa filter (∼1.3 × 10^9 exosomes/µg protein), significantly outperforming the 10 and 50 kDa filters.
• Western blotting confirmed superior enrichment of CD63 and TSG101 markers in 100 kDa isolates.

Conclusions:

• MWCO selection critically influences exosome yield, purity, and quality.
• 100 kDa ultrafiltration represents a pragmatic and cost-effective approach for isolating exosomes from cell culture media.