Comprehensive isolation of extracellular vesicles and nanoparticles

Extracellular vesicles (EVs) and nonvesicular (NV) extracellular nanoparticles (NVEPs) play pivotal roles in both physiological and pathological conditions1,2,3,4. However, a major challenge in the field of EVs and NVEPs is their heterogeneity and the methods used to isolate and purify distinct populations3,5,6,7,8,9,10,11,12. Furthermore, the field has largely focused on studies related to EVs, while studies on extracellular amembranous NVEPs, including the recently discovered exomeres5,10 and supermeres13,14,15, are limited. However, it is becoming increasingly clear that different classes of EVs may contain specific cargo and, equally important, that NVEPs, such as exomeres and supermeres, contain many of the biomolecules, including proteins, RNA and DNA, that have previously been ascribed to exosomes3,9,15,16. EVs range in size from small EVs (sEVs; <200 nm), including exosomes generated from endosomal compartments, to large EVs (lEVs; >200 nm), including microvesicles and large oncosomes, which are shed from the plasma membrane3,6,7,9,11. NVEPs include a wide range of size of particles, including lipoproteins, exomeres and supermeres5,15,17,18,19. Fluid-phase atomic force microscopy (AFM) has revealed that supermeres have distinct morphological features in comparison to both sEVs and exomeres15. To understand the roles of EVs and NVEPs in basic cell biology, as well as to realize their full clinical potential, robust and reliable methods are needed to separate distinct populations of these particles. Diverse EV isolation methods have been extensively described, including differential ultracentrifugation, size-exclusion chromatography, ultrafiltration, immunocapture and microfluidics9,20,21,22,23,24. Ultracentrifugation is the gold standard for isolation of EVs and NVEPs from cells, tissues and plasma9,10,15, and high-resolution density-gradient purification has been shown to further separate NV material from purified vesicles9,15,18,25,26,27,28. Centrifugation-based isolation schemes have the advantage of being relatively high yield, with ultracentrifuges being widely available to basic and clinical research laboratoriess. In the protocol herein, we provide a detailed description of how to reproducibly obtain highly purified lEVs, sEVs, exomeres and supermeres from human cell lines and human plasma9,10,15. In addition, we describe how to improve the purification of exomeres and supermeres from plasma by the addition of an albumin-depletion step. Methods for isolation of lipoproteins from plasma are not described here; however, methods for their isolation have been described and reviewed elsewhere17,19,27.

Development and overview of the protocol

Major challenges in the field involve the heterogeneity of EVs and NVEPs and the various methods used to isolate and purify distinct populations5,6,7,8,9,10,11. It is increasingly clear that traditionally isolated ‘exosome’ or ‘EV’ samples contain a heterogeneous mixture of EVs and NV components9. Progress in the EV field has been hampered by the lack of methods to separate the various secreted vesicles from NV components. Furthermore, the focus has been on EVs and lipoproteins6,9,17,18,19,26,27,29,30,31,32,33,34, while studies of the recently discovered exomeres and supermeres are very limited5,10,15,35. Isolation of EVs and NVEPs from plasma and other body fluids is challenging. Most EV studies have been focused on isolation of EVs from cell-conditioned media, while reports for EV and lipoprotein isolation from plasma and other body fluids are more limited, but a number of protocols using combinations of different techniques are available18,19,20,26,27,30,33,34,36,37,38,39.

Our comprehensive protocol is based on methods introduced in three articles by our group9,10,15. These protocols entail a series of sequential centrifugation, concentration, filtration and high-resolution density-gradient centrifugation steps to sequentially isolate lEVs, sEVs, exomeres and supermeres from cell-conditioned medium (Figs. 1 and 2) and human plasma (Fig. 3). Some of the major steps and modifications are listed below:

1

Removal of cells and cell debris from cell culture medium and human plasma by a series of centrifugation steps. We also describe an albumin-depletion step (Fig. 4) that increases the purity of exomeres and supermeres from human plasma.

2

Isolation of lEVs by a combination of ultracentrifugation and high-resolution 12–36% (wt/vol) iodixanol density-gradient fractionation (Figs. 13). Bottom loading of the high-resolution gradient is important to our method because it removes contaminating NV fractions from EV samples.

3

A filtration step to ensure that any remaining lEVs are removed so that only sEVs, exomeres and supermeres remain for subsequent steps.

4

A dual-purpose concentration step. A 100,000-molecular-weight cutoff concentrator is used to concentrate the sample from a large to a small volume and to facilitate the removal of free proteins. This step is omitted from most published EV protocols but is crucial to maximize the yield of exomere and supermere fractions15 and to remove free proteins that would otherwise contaminate exomere and supermere samples.

5

Isolation of sEV samples by a combination of high-speed ultracentrifugation and high-resolution 12–36% (wt/vol) iodixanol density-gradient fractionation9,15 (Figs.13). The bottom-loaded high-resolution gradient has been demonstrated to remove contaminating NV fractions from sEV samples, including contaminating vault structures and nucleosomes9. Bottom loading is necessary because the centrifugation time (15 h) and speed (120,000g) is insufficient for NV components to reach their buoyant densities if samples are top loaded.

6

Isolation of exomeres by 167,000g ultracentrifugation10,28. This simple step is an alternative to asymmetric flow field-flow fractionation (AF4)5,35, which is more costly and results in a lower yield (Figs. 1 and 3).

7

Isolation of supermeres by 367,000g high-speed ultracentrifugation15. This method of purification was used in our recent article15 that described the discovery of supermeres and is the only published method for their isolation (Figs. 1 and 3).

8

Albumin-depletion using a commercially available kit. When isolating sEVs, exomeres and supermeres from plasma, the biggest challenge for downstream analysis is albumin contamination. The albumin-depletion step greatly improves the purity of exomere and supermere samples, thereby aiding downstream analysis and characterization.

Fig. 1: Overview of steps for isolation of EVs and NVEPs from cell-conditioned medium.figure 1

a, Schematic for isolation of large EV pellets (lEV-Ps), small EV pellets (sEV-Ps), exomeres and supermeres. Serum-free conditioned medium is centrifuged (500g and 2,000g) to remove dead cells, cellular debris and apoptotic bodies. The lEV-P is obtained after centrifugation of the supernatant at 10,000g centrifugation for 40 min. The leftover supernatant is first concentrated and then subjected to ultracentrifugation at 167,000g for 4 h to obtain the sEV-P (washed one time in PBS by ultracentrifugation at 167,000g for 4 h). The supernatant from the previous step is centrifuged at 167,000g for 16 h to isolate the exomeres (washed one time in PBS by ultracentrifugation at 167,000g for 16 h). The supernatant from the previous step is centrifuged at 367,000g for 16 h to isolate supermeres. b, Representative photographs of the most important steps during the concentrator procedure from a.

Fig. 2: Overview of high-resolution density-gradient fractionation of EVs.figure 2

a, Schematic of the generation of lEVs or sEVs and NV fractions by high-resolution iodixanol density-gradient fractionation (12–36%, wt/vol). Crude pellets of lEV-Ps or sEV-Ps were resuspended in ice-cold PBS and mixed with ice-cold iodixanol (OptiPrep)/PBS for a final 36% (wt/vol) iodixanol solution. The suspension was added to the bottom of a centrifugation tube, and solutions of descending concentrations of iodixanol (30%, 24%, 18% and 12%) in PBS were carefully layered on top, yielding the complete gradient. The bottom-loaded 12–36% (wt/vol) gradient was subjected to ultracentrifugation at 120,000g for 15 h. Twelve individual fractions of 1 ml were collected from the top of the gradient. The first six fractions are pooled in a tube, and the last five fractions are pooled in a second tube. The tubes are filled with PBS and mixed. After ultracentrifugation at 120,000g for 4 h, the two pellets represent purified EVs and NVs, respectively9. b, Representative photographs of the most important steps during the high-resolution gradient fractionation procedure from a. Panel a adapted with permission from ref. 9, Elsevier.

Fig. 3: Schematic of the isolation procedure for lEVs, sEVs, exomeres and supermeres from human plasma described in Box 1.figure 3

a, Schematic for isolation of different fractions from plasma. Plasma is generated by centrifugation of the blood at 2,500g for 15 min twice at room temperature. The resulting plasma samples are immediately diluted ∼1:10–20 in ice-cold PBS-HEPES (PBS-H) and centrifuged at 10,000g for 40 min to pellet lEV-Ps. The supernatant is filtered through a 0.22-μm pore polyethersulfone (PES) filter, and the resulting supernatants are subjected to sequential ultracentrifugation at 167,000g for 4 h and 16 h and then at 367,000g for 16 h to isolate sEV-Ps, exomeres and supermeres, respectively. b, Schematic of the generation of purified plasma lEVs, sEVs and NV fractions by high-resolution iodixanol density-gradient fractionation (12–36%, wt/vol). Crude pellets of lEV-Ps or sEV-Ps are processed as described in Fig. 2. The research conducted as part of this protocol complies with all the relevant ethical regulations. The use of the human samples was approved by the Vanderbilt University Medical Center Institutional Review Board (IRB; IRB nos. 161529 and 151721). RBC, red blood cell; WBC, white blood cell. Figure adapted with permission from ref. 9, Elsevier.

Fig. 4: Flowchart of albumin depletion steps from human plasma-derived sEV-Ps, exomeres and supermeres described in Box 1.figure 4

Albumin spin columns are equilibrated in albumin-binding buffer by centrifugation at 1,500g for 1 min at room temperature (RT). The samples are then applied to the albumin-binding columns and incubated for 1 h at RT with rotation. The columns are centrifuged, and the albumin-depleted samples are collected in the flowthrough. Albumin is eluted by centrifugation by using albumin-elution buffer. This protocol was modified from the albumin depletion kit procedure (Abcam). The research conducted as part of this protocol complies with all the relevant ethical regulations. The use of the human samples was approved by the Vanderbilt University Medical Center IRB (IRB nos. 161529 and 151721).

The protocol we previously described15 allows reasonable quantities of supermeres to be isolated from cell-conditioned medium and plasma. We detail the necessary steps, including crucial filtration and concentration steps, to maximize yield (Figs. 1 and 3 and Steps 1–38).

A further development of the EV purification schema is the use of a high-resolution density gradient9,15 to increase the resolving power of separating both lEVs and sEVs from NV contaminants such as nucleosomes, vault structures and nanoparticles (Fig. 2 and Steps 39–51). For purification of EVs from human plasma, the gradient allows separation of EVs and HDL particles

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