Comparative analysis of extracellular vesicle isolation methods from human AML bone marrow cells and AML cell lines

Abstract

Cellular crosstalk between hematopoietic stem/progenitor cells and the bone marrow (BM) niche is vital for the development and maintenance of myeloid malignancies. These compartments can communicate via bidirectional transfer of extracellular vesicles (EVs). EV trafficking in acute myeloid leukemia (AML) plays a crucial role in shaping the BM microenvironment into a leukemia-permissive niche. Although several EV isolation methods have been developed, it remains a major challenge to define the most accurate and reliable procedure. Here, we tested the efficacy and functional assay compatibility of four different EV isolation methods in leukemia-derived EVs: (1) membrane affinity-based: exoEasy Kit alone and (2) in combination with Amicon filtration; (3) precipitation: ExoQuick-TC; and (4) ultracentrifugation (UC). Western blot analysis of EV fractions showed the highest enrichment of EV marker expression (e.g., CD63, HSP70, and TSG101) by precipitation with removal of overabundant soluble proteins [e.g., bovine serum albumin (BSA)], which were not discarded using UC. Besides the presence of damaged EVs after UC, intact EVs were successfully isolated with all methods as evidenced by highly maintained spherical- and cup-shaped vesicles in transmission electron microscopy. Nanoparticle tracking analysis of EV particle size and concentration revealed significant differences in EV isolation efficacy, with exoEasy Kit providing the highest EV yield recovery. Of note, functional assays with exoEasy Kit-isolated EVs showed significant toxicity towards treated target cells [e.g., mesenchymal stromal cells (MSCs)], which was abrogated when combining exoEasy Kit with Amicon filtration. Additionally, MSC treated with green fluorescent protein (GFP)-tagged exoEasy Kit-isolated EVs did not show any EV uptake, while EV isolation by precipitation demonstrated efficient EV internalization. Taken together, the choice of EV isolation procedure significantly impacts the yield and potential functionality of leukemia-derived EVs. The cheapest method (UC) resulted in contaminated and destructed EV fractions, while the isolation method with the highest EV yield (exoEasy Kit) appeared to be incompatible with functional assays. We identified two methods (precipitation-based ExoQuick-TC and membrane affinity-based exoEasy Kit combined with Amicon filtration) yielding pure and intact EVs, also suitable for application in functional assays. This study highlights the importance of selecting the right EV isolation method depending on the desired experimental design.

Rigorous Comparison of Extracellular Vesicle Processing to Enhance Downstream Analysis for Glioblastoma Characterization

“…Higher salt concentrations within the elution buffer may compound these modifications and may be mediated by buffer exchange, as described by Lang and colleagues. [30] These findings support the use of size exclusion chromatography isolation as a non-destructive modality for EV pulldown that may benefit applications such as immunobased assays.…”

Enhancing Extracellular Vesicle Analysis by Integration of Large-Volume Sample Stacking in Capillary Electrophoresis with Asymmetrical Flow Field-Flow Fractionation

“…UC is a well-established method but is time-consuming and may result in coprecipitation of protein aggregates and lipoproteins . The exoEasy Maxi Kit takes only 30 min to isolate EVs, providing higher yields, but the resultant EVs exhibit a larger median diameter and broader size range than those obtained by SEC …”

Bifurcated Asymmetric Field Flow Fractionation of Nanoparticles in PDMS-Free Microfluidic Devices for Applications in Label-Free Extracellular Vesicle Separation

“…To ensure no EV adsorption and release take place, all microfluidic channels were used only once. While variation between replicates may seem huge, this is an expected result in comparison to standard EV isolation method efficacy, where EV recovery can vary up to 90% [ 33 , 34 , 35 , 36 ]. OSTE-COC devices showed lower variation than PDMS, while in flowthrough from PDMS devices particles, were constantly higher than in the reference sample, suggesting significant particle leakage from PDMS that cannot be detected in blank measurements due to the total amount being out of the NTA working range.…”