Comparative analysis of exosome isolation methods using culture supernatant for optimum yield, purity and downstream applications

Abstract

Exosomes have received significant attention for their role in pathobiological processes and are being explored as a tool for disease diagnosis and management. Consequently, various isolation methods based on different principles have been developed for exosome isolation. Here we compared the efficacy of four kits from Invitrogen, 101Bio, Wako and iZON along with conventional ultracentrifugation-based method for exosome yield, purity and quality. Cell culture supernatant was used as an abundant source of exosomes, and exosome quantity, size-distribution, zeta-potential, marker-expression and RNA/protein quality were determined. The Invitrogen kit gave the highest yield but the preparation showed broader size-distribution likely due to microvesicle co-precipitation and had the least dispersion stability. Other preparations showed <150 nm size range and good stability. Preparation from iZON column; however, had a broader size-distribution in the lower size range suggestive of some impurities of non-vesicular aggregates. RNA quality from all preparations was comparable; however, proteins from Invitrogen method-based exosomal preparation showed polyethylene glycol (PEG) contamination in mass spectrometry. Chemical impurities from the precipitant could also be the cause of toxicity of Invitrogen method-based exosomal preparation in biological growth measurement assay. Together, these findings should serve as a guide to choose and further optimize exosome isolation methods for their desired downstream applications.

Figure 1

Comparison of various methodologies for exosome isolation. (a) Schematic representation of exosomes isolation methods. MiaPaCa cells (3 × 10⁶) were cultured in regular media, after 24 h, media was replaced with 5% exosome depleted FBS. After 48 h condition media was collected, centrifuged at 300 × g for 10 min to remove cells and cell-debris. Thereafter, exosomes were isolated using ultracentrifugation and four commercial kits following the manufacturer’s instructions. (b) Levels of proteins were estimated in intact exosomes by DC protein assay. Highest exosome yield was detected for Invitrogen Kit, while MagCapture yielded the least amount of exosomes. Data presented as mean ± s.d.; n = 3, p-values **<0.0093, and ****<0.0001.

Figure 2

Size distribution and Polydispersity index (PDI) analysis of using dynamic light scattering. To determine the size distribution, freshly isolated exosomes were subjected to dynamic light scattering measurements using DelsaMax Pro (Backman Coulter Inc.). Exosomes isolated using Ultracentrifugation, 101Bio (PureExo), MagCapture and iZON gel-filtration, were under the size range (<150 nm), while the exosomes isolated from Invitrogen precipitation method showed broader size distribution with a shift towards the bigger size (182 ± 13.93). Data presented as mean ± s.d.; n = 3.

Figure 3

Measurement of dispersion stability. Zeta potential was estimated by Dynamic Light Scattering equipped with Phase Analysis Light Scatter (PALS). Values were calculated from measured velocities using Smoluchowski equation. Data presented as mean ± s.d.; n = 3, p-value **<0.012, ***<0.0002, and ****<0.0001.

Figure 4

Measurement of exosome purity. A total of 25 µg protein from different exosomal preparations was resolved on 12% SDS-PAGE gel and immunoblotted using antibodies against specific marker proteins (CD9 for exosomes) and (ARF6 for microvesicles). Chemo-luminescence signal was detected under ChemiDoc gel imager (Bio-Rad) and photographed. Representative images are shown.

Figure 5

RNA quantification and quantitative real-time PCR. (a) RNA was isolated from different exosomal preps using total exosome RNA isolation kit and quantified using nanodrop-1000. Differential RNA yield reflected different exosome quantity obtained from various procedures. (b) cDNA was prepared using 40 ng of RNA using specific RT primer for microRNA while Random primer were used for GAPDH and β-actin and expression of U6, miR-21, β-actin and GAPDH was examined by qRT-PCR. Ct values are plotted for each transcripts and data presented as mean ± s.d.; n = 3, p-values *≤0.0246, and ****<0.0001.

Figure 6

Mass spectrometry analysis of the exosomal proteins. To evaluate the compatibility of exosomes preps for proteomics study, the isolated exosome samples were dried using speed-vac and suspended into 20 µl of 8 M urea to solubilize membrane proteins followed by sonication. Subsequently, samples were incubated at −80 °C for 30 min and diluted four times by adding 60 µl of 50 mM Ammonium biocarbonate (ABC) and 10 mM Tris(2-carboxyethyl)phosphine (TCEP). Overnight digestion was done at 37 °C with sequencing grade trypsin. Samples were subjected to 1 h MS analysis on Thermo QExactive Plus mass spectrometer.

Figure 7

Biological effect of isolated exosomes on cells. To assess the quality of exosomal preps for functional analyses, we used isolated exosomes (10 µg/ml) for the treatment of MiaPaCa cells (5 × 10³) grown in 96-well plate. After 72 h, the cell proliferation assay was performed using WST-1 reagent along with control without exosome treatment. As expected we observed some growth promoting effect on MiaPaCa cells of all exosomal preps except the one isolated using Invitrogen method, which exhibited some toxicity. Data presented as mean ± s.d.; n = 3, p-value *≤0.0117, and **≤0.0065.