A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents

Abstract

Exosomes play a role in cell-to-cell signaling and serve as possible biomarkers. Isolating exosomes with reliable quality and substantial concentration is a major challenge. Our purpose is to compare the exosomes extracted by three different exosome isolation kits (miRCURY, ExoQuick, and Invitrogen Total Exosome Isolation Reagent) and differential ultracentrifugation (UC) using six different volumes of a non-cancerous human serum (5 ml, 1 ml, 500 μl, 250 μl, 100 μl, and 50 μl) and three different volumes (1 ml, 500 μl and 100 μl) of six individual commercial serum samples collected from human donors. The smaller starting volumes (100 μl and 50 μl) are used to mimic conditions of limited availability of heterogeneous biological samples. The isolated exosomes were characterized based upon size, quantity, zeta potential, CD63 and CD9 protein expression, and exosomal RNA (exRNA) quality and quantity using several complementary methods: nanoparticle tracking analysis (NTA) with ZetaView, western blot, transmission electron microscopy (TEM), the Agilent Bioanalyzer system, and droplet digital PCR (ddPCR). Our NTA results showed that all isolation techniques produced exosomes within the expected size range (40–150 nm). The three kits, though, produced a significantly higher yield (80–300 fold) of exosomes as compared to UC for all serum volumes, except 5 mL. We also found that exosomes isolated by the different techniques and serum volumes had similar zeta potentials to previous studies. Western blot analysis and TEM immunogold labelling confirmed the expression of two common exosomal protein markers, CD63 and CD9, in samples isolated by all techniques. All exosome isolations yielded high quality exRNA, containing mostly small RNA with a peak between 25 and 200 nucleotides in size. ddPCR results indicated that exosomes isolated from similar serum volumes but different isolation techniques rendered similar concentrations of two selected exRNA: hsa-miR-16 and hsa-miR-451. In summary, the three commercial exosome isolation kits are viable alternatives to UC, even when limited amounts of biological samples are available.

Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next‐generation sequencing

“…In line with this, we report precipitation-based isolation to yield samples with lower purity and significant protein contamination, but excellent potential for transcriptomics-driven biomarker discovery. We agree with previous publications stating that enriching serum EVs by precipitation might be a viable strategy for biomarker discovery studies [21,23]. Alvarez et al presented similar findings for profiling RNA biomarkers in urinary EVs [9].…”

“…Additionally, modifications of EVs during isolation, including coating with precipitation polymers or serum proteins, might mask antigens and impede detection of marker proteins as observed elsewhere [83,84]. Even though increasing the input for protein analysis helped other investigators to detect markers in crude EV samples [23], it was not sufficient for samples from precipitation and sedimentation in our study. Additional purification by density gradient centrifugation, however, established the presence of EV markers syntenin and CD63 in fractions with a density of 1.18 g/ml (Figure 9).…”

“…Rekker et al, Andreu et al and Crossland et al relied on RT-qPCR to profile vesicular miRNAs, comparing isolation based on UC, precipitation and filtration [12,21,22]. Helwa et al isolated EVs from different starting volumes by precipitation and UC and quantified associated miRNAs by droplet digital PCR [23]. Analysing EV miRNAs using PCR-based assays is an important and well-established approach supported by excellent protocols and methods [24–26].…”

Extracellular vesicles from bone marrow‐derived mesenchymal stromal cells support ex vivo survival of human antibody secreting cells

“…The supernatant was aspirated using a sterilized glass Pasteur pipette, with the resulting pellet, which we refer to as EV fractions (Supplemental Figure 1), resuspended in 500–600 µL sterile PBS and stored at −80°C until use. Our method, which did not use a sucrose gradient, was adapted from other similar publications [30,54]. …”

Extracellular vesicles as a platform for membrane‐associated therapeutic protein delivery

“…This protocol has been considered a gold standard because it yields UC-separated small EVs of high purity. However, the run time of the technique exceeds 6 h, the physicochemical functions of the vesicles can be damaged by the high relative centrifugal field (RCF), and the production yield is low [170]. Thus, it is currently impractical to use UC-isolated vesicles in therapeutic studies.…”