Efficient extracellular vesicle isolation by combining cell media modifications, ultrafiltration, and size-exclusion chromatography

Abstract

Extracellular vesicles (EVs) are a heterogeneous population of biological particles released by cells. They represent an attractive source of potential biomarkers for early detection of diseases such as cancer. However, it is critical that sufficient amounts of EVs can be isolated and purified in a robust and reproducible manner. Several isolation methods that seem to produce distinct populations of vesicles exist, making data comparability difficult. While some methods induce cellular stress that may affect both the quantity and function of the EVs produced, others involve expensive reagents or equipment unavailable for many laboratories. Thus, there is a need for a standardized, feasible and cost-effective method for isolation of EVs from cell culture supernatants. Here we present the most common obstacles in the production and isolation of small EVs, and we suggest a combination of relatively simple strategies to avoid these. Three distinct cell lines were used (human oral squamous cell carcinoma (PE/CA-PJ49/E10)), pancreatic adenocarcinoma (BxPC3), and a human melanoma brain metastasis (H3). The addition of 1% exosome-depleted FBS to Advanced culture media enabled for reduced presence of contaminating bovine EVs while still ensuring an acceptable cell proliferation and low cellular stress. Cells were gradually adapted to these new media. Furthermore, using the Integra CELLine AD1000 culture flask we increased the number of cells and thereby EVs in 3D-culture. A combination of ultrafiltration with different molecular weight cut-offs and size-exclusion chromatography was further used for the isolation of a heterogeneous population of small EVs with low protein contamination. The EVs were characterized by nanoparticle tracking analysis, immunoaffinity capture, flow cytometry, Western blot and transmission electron microscopy. We successfully isolated a significant amount of small EVs compatible with exosomes from three distinct cell lines in order to demonstrate reproducibility with cell lines of different origin. The EVs were characterized as CD9 positive with a size between 60–140 nm. We conclude that this new combination of methods is a robust and improved strategy for the isolation of EVs, and in particular small EVs compatible with exosomes, from cell culture media without the use of specialized equipment such as an ultracentrifuge.

Metabolic signature of extracellular vesicles depends on the cell culture conditions

“…When the concentrations of the C‐EVs and BR‐EVs were determined by NTA, both the PC‐3 and VCaP cell line‐derived BR‐20K and BR‐110K samples showed >100‐fold increase in yield (measured as a total of one isolation round), when compared to the respective C‐EVs (Figure 2(a)). The improved yield is in line with the results from two previous studies, where bioreactors were shown to improve the EV yield 10–12‐fold (presented per volume) compared to conventional cell culture seemingly without major effects to the EV populations and properties [16,26]. The main methodological differences of the studies are (i) the cell lines and media composition in the BR cell compartment (containing FBS), (ii) the EV isolation method, and (iii) the analytical methods, and these factors may well explain the differences between studies.…”

“…cell type, cell confluence and cellular stress in cell culture conditions on EV preparations, has been previously reviewed [53]. One factor for the observed differences in the EV metabolomes in our study may be confluence, as during C‐EV production, confluence was maintained below 80%, while in bioreactors, culture is continued without passaging, and the platform has been shown to double the cell numbers and enable spheroid‐like development [16], which may affect the metabolic activity of the cells [54,55]. Furthermore, increasing harvesting intervals with concomitant exchange of fresh medium may stabilize culture conditions and increase yields of more conventional cell culture ‐like EVs minimizing apoptotic or stress conditions.…”

“…omics analyses) requires large amounts of EVs, for which conventional cell culture systems are too inefficient, requiring active maintenance and processing of large volumes of cell conditioned media. Bioreactors, such as two‐chamber or hollow‐fibre bioreactors, in which cells can be grown in high densities in a 3D‐like platform, present an attractive alternative for producing more EVs [15,16]. However, it is currently unknown how well the properties of the bioreactor‐derived EVs translate to the current knowledge of EVs, mostly derived from EVs from cells grown in conventional cell cultures.…”

Engineering of Exosomes to Target Cancer Metastasis

“…Ultrafiltration, including syringe-driven filtration, may be the most straightforward method for exosome isolation. 31 Exosomes can be separated from other components in the sample using membrane filters with defined pore size or molecular weight (MW) limits. Due to the size heterogeneity of the components in exosome-containing samples, sequential filtration is needed to remove other components that are significantly larger or smaller than exosomes.…”

Towards establishing Extracellular Vesicle-associated RNAs as biomarkers for HER2+ breast cancer

“…Here, we present a simple solution for obtaining high quantities of cancer-associated EVs by culturing the HER2-positive breast cancer cell line BT-474 in a CELLine AD 1000 two-chamber bioreactor flask. The CELLine bioreactor system mimics physiological growth conditions by allowing 3D cell growth on a fibre-mimetic surface, resulting in increases in cell number as well as EV production [6].…”