PhD Scientific Days 2025

Poster Session II. - G: Pharmaceutical Sciences and Health Technologies

Translation of Combined Density Gradient Ultracentrifugation and Size Exclusion Chromatography-based EV Isolation from Rat to Human Plasma for Metabolomic and Proteomic Analyses

Name of the presenter

Kapui Dóra

Institute/workplace of the presenter

Semmelweis University, Department of Pharmacology and Pharmacotherapy

Authors

Dóra Kapui^1,2, Szabolcs Hambalkó^1,2, Tamás Visnovitz^3,4, Péter Ferdinandy^1,2,5, Csenger Kovácsházi^1,2, Zoltán Giricz^1,2,5

1: Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest
2: Center for Pharmacology and Drug Research & Development, Semmelweis University, Budapest
3: Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest
4: ELTE Eötvös Loránd University, Department of Plant Physiology and Molecular Plant Biology, Budapest
5: Pharmahungary Group, Szeged

Text of the abstract

Introduction: Isolation of high purity extracellular vesicle (EV) samples is needed to implement EV-based biomarker analyses for diagnostics and prognostics. An established method based on iodixanol density gradient ultracentrifugation (DGUC) followed by CaptoCore700 bind-elute size exclusion chromatography (BE-SEC) was successfully applied to identify EV-based metabolic alterations in hypercholesteremia in rats.
Aims: Our aim was to apply this technology for human blood plasma samples.
Methods: EVs were isolated from human platelet-free plasma by iodixanol DGUC, qEV and Exo-spin SEC columns, or iodixanol DGUC followed by Sepharose CL-2B, CL-4B or 4 Fast Flow (4FF) SEC columns. EV isolates were analyzed by nanoparticle tracking analysis, bicinchoninic acid protein assay and Western blot (WB).
Results: By isolating EVs from human plasma with DGUC, EV markers could not be detected by WB. After isolating EVs with qEV or Exo-spin column, EV markers could be detected by WB, however, significant lipoprotein contaminants were present. DGUC followed by Sepharose CL-2B SEC resulted in isolates positive for CD81 with ApoB below the detection limit. By comparing Sepharose CL-2B, CL-4B and 4FF SEC columns for EV isolation, 4FF column with 70 mL volume and 26 mm diameter resulted in the strongest CD81 and TSG101 signals, low relative ApoA and ApoB signals and good process time.
Conclusions: Isolating EVs with DGUC followed by Sepharose 4FF SEC column, a methodology similar to earlier used technology, resulted in EVs with ApoB contamination below detection limit. This method could be used to test the diagnostic potential of circulating EV metabolites in cardiovascular diseases. To test the effect of the difference in Sepharose 4FF versus CaptoCore700 SEC, further studies are needed.
Funding: The Ministry for Innovation and Technology of Hungary from the source of National Research, Development and Innovation Fund supported CK (EKÖP-2024-226 New National Excellence Program), ZG(K139105) and the project (2020-1.1.5-GYORSÍTÓSÁV-2021-00011). The project was further supported by the European Union (RRF-2.3.1-21-2022-00003). KD was supported by SE 250+ Excellence PhD Scholarship.