The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles

Abstract

Background: Extracellular vesicles (EV), the collective term for vesicles released from cells, consist of vesicle species ranging in size from 30 nm to 5 µm in diameter. These vesicles are most commonly isolated by differential centrifugations, which pellets particles based on their differential movement through the liquid medium in which they are immersed. Multiple parameters, including the utilization of different rotor types, can influence the yield and purity of isolated vesicles; however, the understanding of how these factors affect is limited.

Materials and methods: Here, we compare the influence of multiple centrifugation parameters, including the use of swinging bucket and fixed angle rotors, as well as different centrifugation times, for the isolation of the smallest EVs, "exosomes." In particular, we determine the yields of exosomal RNA and protein, as well as the nature of the isolated vesicles and possible protein contamination with methods such as electron microscopy, western blot and flow cytometry.

Results: Our results show that application of a specific g-force or rotation speed by itself does not predict the ability of pelleting exosomes, and that prolonged centrifugation times can achieve greater yields of exosomal RNA and protein, whereas very long centrifugation times result in excessive protein concentrations in the exosome pellet.

Conclusion: In conclusion, rotor type, g-force and centrifugation times significantly influence exosome yield during centrifugation-based isolation procedures, and current commonly recommended isolation protocols may not be fully optimized for yield and purity of exosomes.

Keywords: exosomes; extracellular vesicles; isolation protocol; rotor; ultracentrifugation.

Fig. 1

Equations used for conversion of a centrifugal run between different rotors. Equation 1 is used to calculate the k-factor (clearance factor) for a rotor. If a rotor is run below its maximum velocity, equation 2 should be applied to calculate the rotor's k-factor. Equation 3 explains the sedimentation coefficient, which describes a particles movement through a liquid medium and is based on Stokes law, and is best applied to spherical particles. Shown in equation 4 is the relation between the k-factor, time of centrifugation, and sedimentation coefficient, which is expressed here in Svedberg units. Equation 5 shows how equation 4 can be used to convert a centrifugal run with one rotor so that an equivalent run can be applied with another rotor. However, this demands that S remains constant. These conversions can easily be performed with the help of online tools, such as the one provided at the Beckman Coulter web page (www.beckmancoulter.com, March 17th, 2014) RPM: revolutions per minute; R_max/R_min: maximum/minimum distance from the rotational axis; s; the sedimentation coefficient; m: the mass of the particle; η: the viscosity of the medium; r: the radius of the particle; T: centrifugation time (in hours); K: k-factor; S: Svedberg unit.

Fig. 2

Comparison between fixed angle (FA) rotor and swinging bucket (SW) rotor on exosome isolation. To equally deplete larger extracellular vesicles in all samples, one sample was centrifuged at 16,500×g in a FA rotor and filtered through 0.2 µm filters and then divided into three samples. These three samples were then centrifuged for either 70 minutes in a FA rotor (FA 70 min), 70 minutes in a SW rotor (SW 70 min) or for 114 minutes in a SW rotor (SW 114 min). The SW 114 minutes centrifugation is calculated to be equal to the FA 70 min centrifugation in terms of pelleting efficiency. (A) The exosomal yield presented as nanogram (ng) RNA/10⁶ cells for the three different centrifugation settings. (B) The exosomal yield presented as microgram (µg) protein/10⁶ cells for the three centrifugation settings. (C) The protein to RNA ration is calculated based on the measurements from A and B ((µg protein/10⁶ cells)/(μg RNA/10⁶ cells)). (D) Detection of the vesicular markers TSG101 and CD81 as well as the endoplasmatic reticulum marker calnexin with western blot in isolates from cells, FA 70 minutes and SW 114 minutes. (E) Detection of CD9, CD63 and CD81 by flow cytometry using CD63-coated beads with isolates from FA 70 minutes (blue) and SW 114 minutes (red). Repeated measures ANOVA followed by Tukey's post hoc test were used to determine significant differences. *p < 0.05, *p< 0.01, ***p < 0.001, ns; non-significant. Protein:RNA ratio data is presented as mean±SEM.

Fig. 3

Impact of centrifugation duration on exosomal RNA and protein yield. Conditioned media were centrifuged with a Type 70 Ti fixed angle (FA) rotor for 70 minutes, 155 minutes, 4 hours, 11 hours or 37 hours. The yield was determined by measuring RNA and proteins. (A) The exosomal yield presented as nanogram (ng) RNA/10⁶ cells for the different centrifugation durations. (B) The yield presented as microgram (µg) protein/10⁶ cells for the different centrifugation durations. The correlation between centrifugation duration and yield was also calculated giving a significant correlation coefficient of 0.8980 and 0.9036 for RNA and protein, respectively. (C) The RNA and protein yields from A and B were used to calculate a protein to RNA ratio ((µg protein/10⁶ cells)/(µg RNA/10⁶ cells)) to determine if increased centrifugation duration affected the purity of exosomes by also pelleting soluble proteins. (D) The increase of RNA (left Y-axis) and protein (right Y-axis) over time in minutes (X-axis). E and F) To determine the gain in yield made by prolonging the centrifugation time, a pelleting rate for different centrifugation time intervals for RNA (ng/minute) (E) and for protein (µg/minute) (F) was calculated based on 5 experiments where all samples were paired. G) Electron micrographs of samples from a 4 hours centrifugation performed on the supernatant from a regular 70 minutes centrifugation shows the presence of vesicles in the size range of 30–100 nm. The scale bar represents 200 nm. Data for C, E and F are presented as mean±SEM. One-way ANOVA followed by Tukey's post-hoc test were used to determine significant differences. *p < 0.05, ***p < 0.001, NS; non-significant.

Fig. 4

Characterization of vesicles isolated with longer centrifugation durations. (A) Size distribution of vesicles from 70 minutes, 155 minutes, 4 hours and 11 hours estimated by electron microscopy and presented as graphs with number of vesicles on y-axis and vesicle diameter in nm on x-axis. (B) Size distribution as percentage of vesicles larger than 100 nm (white), between 100–151 nm (grey) and between 20–50 nm (black), based on the same data as in A. (C) Western blot for isolates from 70 minutes, 155 minutes, 4 hours, 11 hours and 37 hours centrifugations. HMC-1 cell lysates were used as positive control and 25 µg was loaded per well with one extra set of 11 and 37 hours samples loaded with 100 µg protein (right hand side). (D) Flow cytometry on isolates from 70 minutes (blue), 155 minutes (red), 4 hours (green), 11 hours (purple) and 37 hours (black) probed for CD9, CD63 and CD81 markers as well as isotype control (grey).

Fig. 5

Alterations in the RNA profile during longer centrifugation duration. (A) RNase treatment of samples. Treated samples are presented as a percentage of RNA yield in comparison to yield of untreated control, which was kept on ice (red line marks 100%). (B) The RNA profile and yield of samples isolated with 70 minutes, 4 hours, 11 hours or a 37 hours centrifugation were determined with a Bioanalyzer. One representative experiment is shown per centrifugation time. (C) Profiles from B are shown in an overlay. Black arrows: 18 and 28 S ribosomal peaks; yellow dashed lines: 160–180 nt interval; blue dashed lines: 300–400 nt interval. (D) Ratios of the highest peak between 160 and 180 nt (yellow dashed lines in C) and the highest peak between 300 and 400 nt (blue dashed lines in C).