Investigation of on-line electrokinetic enrichment strategies for capillary electrophoresis of extracellular vesicles.

This work explores strategies for electrokinetic preconcentration of extracellular vesicles (EVs) that are potential source of biomarkers for different diseases. The first approach that led to successful preconcentration of EVs is based on large volume sample stacking (LVSS), allowing an enrichment factor of 7 for CE of EVs with long-end injection (using a capillary with an effective length of 50 cm). Attempts were also made to perform multiple cycles of LVSS, field amplified sample stacking (FASS) and field amplified sample injection (FASI), to improve EVs preconcentration performance. The focus was then put on development of capillary isotachophoresis under high ionic strengths (IS) for electrokinetic enrichment of slow migrating EVs having heterogeneous mobilities. This approach relies on the use of extremely high concentrations of the terminating electrolyte (TE) to slow down the mobility of TE co-ions, rendering them slower than those of EVs. The limit of detection for intact EVs using the developed ITP-UV method reached 8.3 × 108 EVs/mL, allowing an enrichment of 25 folds and a linear calibration up to 4 × 1010 EVs/mL. The ITP-UV and ITP-LIF approaches were applied to provide the electrokinetic signature of EVs of bovine milk and human plasma as well as to visualize more specifically intravesicular fluorescently labelled EVs. The investigation of these strategies shredded light into the challenges still encountered with electrokinetic preconcentration and separation of heterogeneous EVs sub-populations which are discussed herein based on our results and other attempts reported in the literature.

Fast, simple and calibration-free size characterization and quality control of extracellular vesicles using capillary Taylor dispersion analysis.

This study reports the development of a Taylor Dispersion Analysis (TDA) method for the size characterization of Extracellular Vesicles (EVs), which are highly heterogeneous nanoscale cell-derived vesicles (30-1000 nm). Here, we showed that TDA, conducted in uncoated fused silica capillaries (50 µm i.d.) using a conventional Capillary Electrophoresis instrument, is able to provide absolute sizing (requiring no calibration) of bovine milk-derived EVs in a small sample volume (∼ 7 nL) and over their entire size range, even the smallest ones (< 70 nm) not accessible via other techniques that provide nanoparticle sizing in suspension. TDA size measurements were repeatable (RSD < 10%) and the average EV sizes were found in the range of 120-210 nm, in very good agreement with those measured with Nanoparticle Tracking Analysis, commonly used for EV characterization. TDA allowed quantitative estimation of EVs for concentrations ≥ 2 × 1011 EVs/mL. Furthermore, TDA was able to detect minor changes in EV size (i.e. by ∼25 nm upon interaction with specific anti-CD9 antibodies of ∼150 kDa), and to highlight the impact of extraction methods (i.e. milk pretreatment: freezing, acid precipitation or centrifugation; the type of size-exclusion chromatography column) and of fluorescent labeling (i.e. intravesicular or surface labeling) on the isolated EV population size. In parallel to EV sizing, TDA allowed to detect molecular contaminants (average sizes ∼1-13 nm) present within the sample, rendering this method a valuable tool to assess the quality and quantity of EV isolates.

Development of a microfluidic droplet platform with an antibody-free magnetic-bead-based strategy for high through-put and efficient EVs isolation.

In this study, we present a novel microfluidic droplet-based strategy for high performance isolation of extracellular vesicles (EVs). For EVs capture and release, a magnetic bead-based approach without having recourse to any antibody was optimized in batch and then adapted to the microfluidic droplet system. This antibody-free capture approach relies on the presence of a water-excluding polymer, polyethylene glycol (PEG), to precipitate EVs on the surface of negatively charged magnetic beads. We significantly improved the reproducibility of EV recovery and avoided positive false bias by including a washing step and optimizing the protocol. Well-characterized EV standards derived from pre-purified bovine milk were used for EVs isolation performance evaluation. An EVs recovery of up to 25% estimated with nanoparticle tracking analysis (NTA) was achieved for this batchwise PEG-based approach. The confirmation of isolated EVs identity was also made with our recently developed method using capillary electrophoresis (CE) coupled with laser-induced fluorescent (LIF) detection. In parallel, a purpose-made droplet platform working with magnetic tweezers was developed for translation of this PEG-based method into a droplet microfluidic protocol to further improve the performance in terms of EVs capture efficiency and high throughput. The droplet-based protocol offers a significant improvement of recovery rate (up to 50%) while reducing sample and reagent volumes (by more than 10 folds) and operation time (by 3 folds) compared to the batch-wise mode.

Electrokinetic characterization of extracellular vesicles with capillary electrophoresis: A new tool for their identification and quantification.

This work reports on the development of the first capillary electrophoresis methodology for the elucidation of extracellular vesicles' (EVs) electrokinetic distributions. The approach is based on capillary electrophoresis coupled with laser-induced fluorescent (LIF) detection for the identification and quantification of EVs after their isolation. Sensitive detection of these nanometric entities was possible thanks to an 'inorganic-species-free' background electrolyte. This electrolyte was made up of weakly charged molecules at very high concentrations to stabilize EVs, and an intra-membrane labelling approach was used to prevent EV morphology modification. The limit of detection for EVs achieved using the developed CE-LIF method reached 8 × 109 EV/mL, whereas the calibration curve was acquired from 1.22 × 1010 to 1.20 × 1011 EV/mL. The CE-LIF approach was applied to provide the electrokinetic distributions of various EVs of animal and human origins, and visualize different EV subpopulations from our recently developed high-yield EV isolation method.

Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection.

BACKGROUND: Laser-capture microdissection (LCM) that enables the isolation of specific cell populations from complex tissues under morphological control is increasingly used for subsequent gene expression studies in cell biology by methods such as real-time quantitative PCR (qPCR), microarrays and most recently by RNA-sequencing. Challenges are i) to select precisely and efficiently cells of interest and ii) to maintain RNA integrity. The mammary gland which is a complex and heterogeneous tissue, consists of multiple cell types, changing in relative proportion during its development and thus hampering gene expression profiling comparison on whole tissue between physiological stages. During lactation, mammary epithelial cells (MEC) are predominant. However several other cell types, including myoepithelial (MMC) and immune cells are present, making it difficult to precisely determine the specificity of gene expression to the cell type of origin. In this work, an optimized reliable procedure for producing RNA from alveolar epithelial cells isolated from frozen histological sections of lactating goat, sheep and cow mammary glands using an infrared-laser based Arcturus Veritas LCM (Applied Biosystems®) system has been developed. The following steps of the microdissection workflow: cryosectioning, staining, dehydration and harvesting of microdissected cells have been carefully considered and designed to ensure cell capture efficiency without compromising RNA integrity. RESULTS: The best results were obtained when staining 8 µm-thick sections with Cresyl violet® (Ambion, Applied Biosystems®) and capturing microdissected cells during less than 2 hours before RNA extraction. In addition, particular attention was paid to animal preparation before biopsies or slaughtering (milking) and freezing of tissue blocks which were embedded in a cryoprotective compound before being immersed in isopentane. The amount of RNA thus obtained from ca.150 to 250 acini (300,000 to 600,000 µm2) ranges between 5 to 10 ng. RNA integrity number (RIN) was ca. 8.0 and selectivity of this LCM protocol was demonstrated through qPCR analyses for several alveolar cell specific genes, including LALBA (α-lactalbumin) and CSN1S2 (αs2-casein), as well as Krt14 (cytokeratin 14), CD3e and CD68 which are specific markers of MMC, lymphocytes and macrophages, respectively. CONCLUSIONS: RNAs isolated from MEC in this manner were of very good quality for subsequent linear amplification, thus making it possible to establish a referential gene expression profile of the healthy MEC, a useful platform for tumor biomarker discovery.