Two-step magnetic bead-based (2MBB) techniques for immunocapture of extracellular vesicles and quantification of microRNAs for cardiovascular diseases: A pilot study.

Shu-Chu Shiesh,Chihchen Chen,Wei-Chun Chin,Gwo-Bin Lee,Shi Chen

doi:10.1371/journal.pone.0229610

Shu-Chu Shiesh, Chihchen Chen + Show 3 more

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https://doi.org/10.1371/journal.pone.0229610

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Abstract

Extracellular vesicles (EVs) have attracted increasing attention because of their potential roles in various biological processes and medical applications. However, isolation of EVs is technically challenging mainly due to their small and heterogeneous size and contaminants that are often co-isolated. We have thus designed a two-step magnetic bead-based (2MBB) method for isolation a subset of EVs as well as their microRNAs from samples of a limited amount. The process involves utilizing magnetic beads coated with capture molecules that recognize EV surface markers, such as CD63. Captured EVs could be eluted from beads or lyzed directly for subsequent analysis. In this study, we used a second set of magnetic beads coated with complementary oligonucleotides to isolate EV-associated microRNAs (EV-miRNAs). The efficiencies of 2MBB processes were assessed by reverse transcription-polymerase chain reaction (RT-PCR) with spiked-in exogenous cel-miR-238 molecules. Experimental results demonstrated the high efficiency in EV enrichment (74 ± 7%, n = 4) and miRNA extraction (91 ± 4%, n = 4). Transmission electron micrographs (TEM) and nanoparticle tracking analysis (NTA) show that captured EVs enriched by 2MBB method could be released and achieved a higher purity than the differential ultracentrifugation (DUC) method (p < 0.001, n = 3). As a pilot study, EV-miR126-3p and total circulating cell-free miR126-3p (cf-miR126-3p) in eight clinical plasma samples were measured and compared with the level of protein markers. Compared to cf-miR126-3p, a significant increase in correlations between EV-miR126-3p and cardiac troponin I (cTnI) and N-terminal propeptide of B-type natriuretic peptide (NT-proBNP) was detected. Furthermore, EV-miR126-3p levels in plasma samples from healthy volunteers (n = 18) and high-risk cardiovascular disease (CVD) patients (n = 10) were significantly different (p = 0.006), suggesting EV-miR126 may be a potential biomarker for cardiovascular diseases. 2MBB technique is easy, versatile, and provides an efficient means for enriching EVs and EV-associated nucleic acid molecules.

Highlights

Many cell types release membrane-enclosed particles, known as extracellular vesicles (EVs), into the extracellular space as a means to transmit signaling and genetic information [1,2,3]
The efficiency of enrichment and elution of plasma EVs using anti-CD63 magnetic beads was assessed by comparing the nanoparticle levels using nanoparticle tracking analysis (NTA)
Our results revealed that plateletpoor plasma (PPP) from the healthy donor contained 15.2 ± 1.42 × 1010 particles/mL and following mixing with anti-CD63 magnetic beads the number decreased to 1.4 ± 0.06 × 1010 particles/mL in the supernatant (Fig 2A, n = 3)

Summary

Introduction

Many cell types release membrane-enclosed particles, known as extracellular vesicles (EVs), into the extracellular space as a means to transmit signaling and genetic information [1,2,3]. Based on their biogenesis pathways, EVs are typically categorized into three main subgroups: exosomes, microvesicles, and apoptotic bodies [4]. It remains technically challenging to isolate EVs into a homogeneous subgroup and we use either EV as the generic term or terms for EV subtypes reflecting their physical characteristics, such as small EVs, or biochemical composition, such as CD63-positive EVs, following the ISEV guidelines (MISEV2018) [5]. There is a need for the improvement of methodologies that can consistently generate pure and intact EVs to provide reproducibility within and among laboratories [15]

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