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

Figures & data

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.

Table I Rotor settings

Isolation setting	Rotor	RCF (average)	RPM	Time (minutes)	k-factor
FA 70 minutes	Type 70 Ti (Fixed angle)	118,000	40,000	70	133.7
SW 70 minutes	SW 32 Ti (Swinging bucket)	118,000	31,000	70	217.6
SW 114 minutes	SW 32 Ti (Swinging bucket)	118,000	31,000	114	217.6

The three different centrifugation settings used for the comparison of rotors. The FA 70 minutes setting is used as a reference to which the SW 70 minutes setting (which is not compensated for the change in rotor) and the SW 114 minutes setting (which is compensated by prolonged centrifugation duration) were compared to.

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.

Table II Rotor types and their properties

Rotor	gavg	RPM	Migration path length	k-factor	Equivalent centrifugation duration (minutes)
Type 70 Ti	118,000	40,045	52.4	133.4	70
Type 45 Ti	118,000	38,837	67.9	178.3	93
SW 41 Ti	118,000	30,913	85.7	217.5	114
SW 32 Ti	118,000	30,998	85.7	217.6	114
TLA-100.3	118,000	52,724	20.8	51.3	27

Some common rotors and their characteristics, adjusted to a centrifugal force of 118,000×g at the average distance from the rotational axis. Seventy minutes with the Type 70 Ti rotor is set as a reference and the equivalent centrifugation time (calculated with the equations in Fig. 1) for the other rotors are presented in the last column to illustrate the differences.

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).