Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin.

Author(s): Veerman RE, Teeuwen L, Czarnewski P, Güclüler Akpinar G, Sandberg A, Cao X, Pernemalm M, Orre LM, Gabrielsson S, Eldh M

Publication: J Extracell Vesicles, 2021, Vol. 10, Page e12128

PubMed ID: 34322205 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the morphology, size distribution, concentration, and protein profile of particles isolated from the plasma of healthy donors using five EV isolation methods. K₂EDTA blood was collected from two healthy females and one healthy male 1-3 h after breakfast. Platelet-free plasma was obtained by dual centrifugation at 2500 x g for 15 min, aliquoted, and stored frozen at -80°C for <16 months. Plasma was thawed at room temperature, filtered through a 0.8 µm filter, and EVs were isolated using precipitation with ExoQuick Ultra, membrane-affinity with exoEasy Maxi Kit, size-exclusion with qEVoriginal 35 nm or 70 nm, and density gradient with OptiPrep. EV morphology and size were assessed by transmission electron microscopy. EV size was evaluated by nanoparticle tracking analysis. RNA was extracted from EVs using the miRNeasy Mini Kit and analyzed using the total RNA 6000 Pico Kit on a bioanalyzer. Levels of CD9, CD63, and CD81 on particles-bead complexes were analyzed by flow cytometry after binding to anti-CD9 and anti-CD63 beads. Proteins were separated using a Thermo Scientific Dionex nano LC-system coupled to a High Field QExactive-HF mass spectrometer and spectra were analyzed using the NextFlow proteomics analysis pipeline.

   Summary of Findings:

   Electron microscopy revealed three different particle sizes: <30 nm which were considered lipoproteins, 30-150 nm consisting of small EVs and lipoproteins, and >150 nm consisting of large EVs and lipoproteins. The most homogenous EV population was found when EVs were isolated using qEVoriginal 35 nm or 70 nm. The Optiprep isolation yielded the fewest particles. The size profile of the isolated EVs differed among the isolation methods with the majority of particles <30 nm when isolated using ExoEasy or ExoQuick, 30-150 nm when isolated using qEVoriginal 35 nm or 70 nm, and a similar amount of particles <30 nm and 30-150 nm when isolated using OptiPrep. Based on electron miscroscopy (EM), the proportion of EVs >150 nm was low using all methods but was higher using ExoEasy or qEVoriginal 35 nm or 70 nm than the other methods. Nanoparticle tracking analysis found more particles per mL when isolation was with ExoQuick than other methods (P<0.001). NTA identified particles of 30-350 nm using all methods but the peaks also differed between methods with the smallest mean particle size found using ExoQuick (119 nm) and the broadest distribution and mean size using qEVoriginal 35 nm (170 nm). The protein yield did not correspond to number of particles isolated. The highest protein yield was obtained when EVs were isolated using ExoEasy, regardless of input amount. For OptiPrep and qEVoriginal 70, the protein yields were much higher when 3 mL plasma was used rather the 250 µL, but the protein yield was only 3-fold higher for the ExoEasy method. The RNA yield from the EVs was significantly higher when EVs were isolated with ExoQuick than other methods (P<0.05). The relative signal of CD9, CD63, and CD81 differed among the isolation methods. CD9 signals were highest when EVs were isolated with qEVoriginal 35 followed by exoEasy OptiPrep. qEVoriginal 70 isolated EVs had low detection of all markers by flow cytometry but analysis of the flow-through found significant CD9 and CD63 signal indicating the loss of EVs using this method. CD9, CD81, and CD63 signals were also found in the low-density fraction from EVs isolated using OptiPrep. Mass spectrometry analysis of the EVs identified different numbers of proteins depending on isolation method with 250 µL qEVoriginal 70 and 3 mL ExoEasy isolated EVs clustering separately from the main cluster in principle component analysis. Comparison of isolated proteins and the Vesiclepedia top 100 list and vesicle-related GO list found the most overlap with either list when isolation of EVs was from 3 mL plasma using qEVoriginal 70 followed by ExoEasy. The fewest plasma proteins were isolated when EVs were obtained using ExoEasy. Hierarchical clustering clustered EVs isolated from 3 mL plasma using qEVoriginal 70 distinctly but showed enrichment of vesicle-related proteins, regardless of isolation method. Finally, different levels of proteins corresponding to the different cellular components were found in the EVs depending on the isolation method indicating the method can bias toward vesicles of a particular cellular compartment.  Isolation with exoEasy, ExoQuick, and qEVoriginal 35 or 70 enriched for plasma membrane. Levels of Endosomal Sorting Complexes Required for Transport (ESCRT) and multivesicular body (MVB) proteins were low, regardless of extraction method.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Morphology	Electron microscopy
   Morphology	Light scattering
   RNA	Fluorometry
   Protein	LC-MS or LC-MS/MS
   RNA	Automated electrophoresis/Bioanalyzer
   Protein	Flow cytometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Precipitation (ExoQuick Ultra) Membrane affinity (exoEasy Maxi Kit) Size exclusion (qEVoriginal 35nm and 70nm) Density gradient (OptiPrep)

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