Biopolym. Cell. 2025; 41(3):200.
http://dx.doi.org/10.7124/bc.000B20
Molecular and Cell Biotechnologies
Comparison of EV isolation methods from MSCs
1, 2Khmelnytska Y. M., 1Pykhtieiev D. M., 1Yuriev B. V., 1Svoboda M. F., 1Gumeniuk M. I.
  1. Biotechnology Laboratory of the Bank of Umbilical Cord Blood, Tissues and Other Human Cells BU "Reocell"
    10, Amosova Str., Kyiv, Ukraine 03141
  2. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143

Abstract

Aim. To evaluate the impact of isolation methods and culture conditions on the purity, yield, and phenotype of extracellular vesicles (EVs) derived from human mesenchymal stromal cells (MSCs). Methods. EVs were isolated using tangential flow filtration (TFF) with membranes of different molecular weight cut-offs (100–300 kDa), combined with ultracentrifugation (UC), under serum-containing and serum-free conditions. Particle concentration and size distribution (40–1000 nm) were analyzed by flow cytometry, total protein content was measured fluorometrically, morphology was assessed by transmission electron microscopy, and expression of tetraspanins (CD9, CD63, CD81) was determined by immunophenotyping. Results. Serum-containing media yielded a higher number of EVs but resulted in substantial protein contamination. The purest isolates (0.09–0.2 mg/mL total protein) were obtained under serum-free conditions using TFF with <100 kDa membranes combined with ultracentrifugation. Under these conditions, CD63⁺ exosomes predominated (up to 98%). Transmission electron microscopy confirmed typical EV morphology with diameters of 30–200 nm. Conclusions. TFF combined with UC enables modulation of EV purity and yield. Serum-free culture significantly improves the biochemical purity and phenotypic specificity of EV isolates, making them more suitable for downstream applications. Optimization of filtration parameters remains critical for balancing vesicle concentration and quality.
Keywords: extracellular vesicles, mesenchymal stem cells, serum-free cultivation, tangential flow filtration, exosome characteristics, exosome purity
Full text: (PDF, in English)

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