Isolation of exosomes from whole blood by integrating acoustics and microfluidics

Abstract

Exosomes are nanoscale extracellular vesicles that play an important role in many biological processes, including intercellular communications, antigen presentation, and the transport of proteins, RNA, and other molecules. Recently there has been significant interest in exosome-related fundamental research, seeking new exosome-based biomarkers for health monitoring and disease diagnoses. Here, we report a separation method based on acoustofluidics (i.e., the integration of acoustics and microfluidics) to isolate exosomes directly from whole blood in a label-free and contact-free manner. This acoustofluidic platform consists of two modules: a microscale cell-removal module that first removes larger blood components, followed by extracellular vesicle subgroup separation in the exosome-isolation module. In the cell-removal module, we demonstrate the isolation of 110-nm particles from a mixture of micro- and nanosized particles with a yield greater than 99%. In the exosome-isolation module, we isolate exosomes from an extracellular vesicle mixture with a purity of 98.4%. Integrating the two acoustofluidic modules onto a single chip, we isolated exosomes from whole blood with a blood cell removal rate of over 99.999%. With its ability to perform rapid, biocompatible, label-free, contact-free, and continuous-flow exosome isolation, the integrated acoustofluidic device offers a unique approach to investigate the role of exosomes in the onset and progression of human diseases with potential applications in health monitoring, medical diagnosis, targeted drug delivery, and personalized medicine.

Keywords: acoustic tweezers; blood-borne vesicles; exosomes; extracellular vesicles; surface acoustic waves.

Fig. 1.

Schematic illustration and mechanisms underlying integrated acoustofluidic device for isolating exosomes. (A) RBCs, WBCs, and PLTs are filtered by the cell-removal module, and then subgroups of EVs (ABs: apoptotic bodies; EXOs: exosomes; MVs: microvesicles) are separated by the exosome-isolation module. (B) An optical image of the integrated acoustofluidic device. Two modules are integrated on a single chip. (C) Size-based separation occurs in each module due to the lateral deflection induced by a taSSAW) field. The periodic distribution of pressure nodes and antinodes generates an acoustic radiation force to push large particles toward node planes.

Fig. 2.

Separation of synthetic microparticles and submicrometer particles using the acoustofluidic cell-removal module. Polystyrene particles with diameters of 5.84 µm (not labeled) and 970 nm (labeled with Dragon Green fluorescent dye) were processed through the acoustic field. The taSSAW field deflected microparticles to the waste outlets. The acoustic radiation force was not sufficiently large to move the submicrometer particles, which were therefore separated from microparticles at the outlet. White stripe in the two left panels indicates the centerline location of the CCD (charge-coupled device) image sensor. (Scale bar: 500 µm.)

Fig. S1.

Validation of the cell-removal module with polystyrene particles. (A) Polystyrene particles with diameters of 5 μm (not labeled) and 110 nm (labeled with Dragon Green fluorescent dye) are mixed and processed using the cell-removal module. (Scale bar: 500 μm.) (B) Particle size distribution of initial mixture and collected samples was measured by DLS. The initial mixture had two distinct size-distribution peaks; in contrast, the processed sample exhibited only one peak for both samples collected from the top and bottom outlets.

Fig. S2.

Isolation of EVs from whole blood using the cell-removal module. The images at outlet region when acoustic waves are (A) off and (B) on. Blood cells are pushed to bottom outlets when the acoustic field is on. White stripe in the figure indicates the centerline location of the CCD image sensor. (Scale bar: 500 μm.)

Fig. 3.

Characterization of the cell-removal module. (A) Separation of EVs from RBCs and other blood components. NTA was used to characterize the isolated EVs from the collection outlet. (B) RBCs and other blood components collected from waste outlet were characterized by DLS. The ordinate is the relative intensity of signals measured. (C) SEM image of isolated EVs sample loaded on a filter membrane. The EV sample contained vesicles of diameters from ∼50 to 300 nm. (D) Western blot with expression of RBC marker (GYPA), PLT marker (integrin β1), and EV markers (CD63). The proteins from blood, cell waste sample, and isolated EVs were extracted and prepared for electrophoresis.

Fig. 4.

Separation of exosomes from microvesicles using the exosome-isolation module. (A) Size distribution of original mixture (MIX), isolated EXO, and MV samples. The data were obtained from at least three NTA assays. The black line and the red area represent the fitting curve and the error bar, respectively. The y axis is the concentration of particles. The peak positions are marked. The green dashed line is located at 140 nm, which is set as the cutoff size. (B) Quantitative characterization of exosome/microvesicle separation, showing the concentrations of vesicle subgroups (cutoff size at 140 nm) in the mixture and processed samples. The concentration is expressed as the number of particles per microliter. (C) TEM image of isolated exosome samples.

Fig. S3.

Separation of 340- and 110-nm particles using the exosome isolation module. White stripe in the two left panels indicates the centerline location of the CCD image sensor. (Scale bar: 500 μm.)

Fig. 5.

Isolation of exosomes from whole blood using the integrated device using acoustofluidics. In our experiments, inlet A is for whole blood; inlets B, C, and E are for sheath flows. Outlet D is cell waste. Outlets F and G are for isolated exosomes and vesicle waste, respectively. Images were taken under the microscope at the corresponding areas of the device. Blood components were directed to each corresponding outlet when the acoustic wave was on. White stripe in the four grayscale panels indicates the centerline location of the CCD image sensor. (Scale bar: 500 μm.)

Fig. S4.

Isolation of EV subgroups from whole blood using the integrated acoustofluidic device. The inlets and outlets are (A) whole-blood inlet; (B, C, and E) sheath flow inlets; (D) cell waste outlet; (F) exosome outlet; (G) vesicle waste outlet. The blood cells are deflected to outlet D; EV subgroups other than exosomes are pushed to G when the acoustic field is on. Purified exosomes are collected from outlet F.

Fig. 6.

Characterization of exosome isolation from whole blood using the integrated acoustofluidic chip. (A) Removal of blood cells and PLTs. In the original sample (undiluted whole blood), RBCs occupied approximately half of the volume. The isolated exosome sample and vesicle waste sample contain a minimal amount of blood cells. (B) EVs in blood plasma showed a dispersed size distribution that ranged between 30 nm and 1 µm. The size distribution of collected exosome sample exhibited a major peak at <100 nm. (C) Western blot of exosome markers, showing a prominent expression in the isolated exosome and blood samples, while the other samples (vesicle waste and cell waste) exhibited low expression level of exosomal proteins. (D and E) The expression (expressed as relative fold difference) of individual mRNAs (D) and miRNAs (E) in human blood and isolated exosomes. The data represent three independent experiments. *P < 0.05 (ANOVA) (F) TEM images of isolated exosomes. The exosomes (red arrows) have a characteristic round shape and a cup-like structure.

Fig. S5.

Size distribution of isolated human plasma exosomes using the Opti-Prep-based gradient ultracentrifugation technique (16). The mean size of the exosomes was 104.15 ± 7.60 (n = 3), which was slightly larger than that of exosomes isolated from human blood using the acoustofluidic device.

Fig. S6.

Relative levels of mRNA and miRNA transcripts in human blood versus isolated exosomes using OptiPrep gradient ultracentrifugation. Fold changes of individual mRNAs (A) and miRNAs (B) in human blood and isolated exosomes were quantified by qPCR. Results were derived from three different blood samples and analyzed using a one-way ANOVA post hoc test. The asterisk indicates statistical significance with adjusted P value < 0.05.

Fig. S7.

Simulation results showing that by using our acoustic methods: (A) exosome can be first isolated from blood cells and other EVs based on size difference; (B) isolated exosomes can then be purified based on the difference in acoustic contrast factor by isolating exosomes from nonexosomal particles and proteins that have negative acoustic contrast factor, such as LDLs, IDLs, VLDL, and chylomicrons; (C) exosomes can be further purified based on the size difference by isolating exosomes from particles smaller than exosomes such as HDLs.