A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option?
Abstract
:1. Introduction
2. Methods of Exosome Isolation
2.1. Differential Ultracentrifugation (dUC)
2.2. Ultrafiltration (UF)
2.3. Poly-Ethylene Glycol (PEG)-Based Precipitation
2.4. Immunoaffinity Capture
2.5. Microfluidics
2.6. Size-exclusion Chromatography (SEC)
3. Ideal Method for Exosome Isolation: SEC
4. Application of SEC for Isolation of Exosomes from Adipose Tissue
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
EV | Extracellular vesicles |
sEVs | Small EVs |
m/lEVs | Medium/large EVs |
dUC | Differential ultracentrifugation |
SEC | Size-exclusion chromatography |
UF | Ultrafiltration |
PEG | Poly-ethylene glycol |
MVB | Multivesicular bodies |
ESCRT | endosomal sorting complexes required for transport |
NSF | N-ethylmaleimide sensitive factor |
SNARE | soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor |
ISEV | International Society for Extracellular Vesicles |
MISEV | Minimal Information for Studies of Extracellular Vesicles |
PBS | phosphate-buffered solution |
EPCAM | epithelial cellular adhesion molecule |
Go/PDA | graphene oxide/polydopamine |
iPSC | induced pluripotent stem cell |
SVF | stromal vascular fraction |
DMC | dual-mode chromatography |
LPPs | lipoproteins particles |
HDLs | high-density lipoproteins |
EXO | exosomes |
MV | microvesicles |
AB | apoptotic bodies |
AFSCs | amniotic fluid stem cells |
MSCs | mesenchymal stromal cells |
SVF | stromal vascular fraction |
ATGL | adipose triglyceride lipase |
PPARγ | peroxisome proliferator-activated receptor gamma |
FABP4 | fatty acid binding protein 4 |
CAV1 | caveolin 1 |
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dUC | UF | PEG | IA | MF | SEC | |
---|---|---|---|---|---|---|
Mechanism of separation | Size, density | Size and molecular weight; through a filter membrane | Surface charge, solubility | Specific binding of antibodies to exosome markers | Immuno- affinity, density, and size | Size, shape, and molecular weight; large particles are eluted first |
Specificity 1 | ++ | + | + | +++ | +++ | ++ |
Recovery 1 | ++ | +++ | +++ | ++ | + | +++ |
Purity 1 | +++ | + | + | +++ | +++ | +++ |
Sample volume 1 | ++ | ++ | + | ++ | + | + |
Time 1 | +++ | +++ | ++ | +++ | ++ | + |
Cost 1 | + | ++ | + | +++ | +++ | + |
Specialized equipment 2 | ++ | + | + | + | ++ | + |
Complexity 1 | ++ | + | + | ++ | +++ | + |
Efficiency 1 | ++ | ++ | ++ | ++ | +++ | +++ |
Functionality of EVs 2 | ++ | ++ | ++ | + | + | +++ |
Scalability 1 | ++ | ++ | +++ | + | + | +++ |
Sample Type | Type of Column | Sample Volume (mL) | Fractions Used | Size of Isolated Evs | Type of Cargo | References |
---|---|---|---|---|---|---|
Plasma | Sepharose CL-2B, qEV original | 1–2 | 4–6, 8–10, 4–7, 10–12, 7–10 | 20–200 nm | Proteins, miRNAs | [20,21,22,23,24,25] |
Serum | qEV original, Sepharose CL-2B | 0.5–1 | 7–9, 8–10 | 50–200 nm | miRNAs, proteins | [26,27,28] |
Milk | qEV original, Sephacryl S-500 | 0.5 | 7–10 | <200 nm | RNAs | [29,30] |
Urine | qEV, Sepharose CL-4B/2B | 0.5–3 | 8–11, 9–10, 7–10, 7–19 | 40–200 nm | miRNAs, proteins, RNAs | [31,32,33,34] |
Saliva | miniPURE-EVs, qEV | 1 | 7–11, 8–10 | 50–200 nm | miRNAs, proteins | [35,36] |
CSF | Exo-spin™ mini-column, qEV single | 0.1–3 | 5–6, 3–4 | 30–150 nm | Protieins | [37,38] |
Synovial fluid | Sephacryl S-500 HR | – | 2–4 | <200 nm | Proteins | [39] |
Tears | qEV | 1 | 8–10 | <200 nm | Proteins | [35] |
Seminal fluid | Exo-spin™ column | 1 | 5–9 | <200 nm | – | [40] |
Nasal lavage | qEV original | 0.5 | 7–9 | <200 nm | miRNAs | [41] |
Stromal vascular fraction; adipose tissue | qEV70s single, Illustra Sephacryl S-1000 | 0.15–0.7 | 8–11, 8–16 | 50–700 nm, <250 nm | miRNAs, neutral lipids | [42,43] |
Conditioned media | qEV original, Sepharose CL-2B, Sepharose CL-4B | 0.5–1.5 | 3–7, 7–9, 7–10, 6–12 | 30–200 nm | mRNAs, proteins, miRNAs | [41,44,45,46,47,48,49] |
Acceleration (g) | The acceleration of the centrifuge, also known as the g force, refers to the speed and determines the separation efficiency. |
Rotor (k) | The k-factor represents the relative pelleting efficiency of a rotor at maximum speed. The lower the k factor, the better the pelleting efficiency of the rotor, and the shorter the centrifugation time. The pelleting time (T) is determined by the equation T = k/s, where T is the time in hours required for centrifugation, s is the sedimentation coefficient in Svedberg units, and k is the k-factor. Sedimentation coefficients depend on the size and shape of the vesicle being isolated, and the viscosity of the sample media. The smaller the s, the longer it takes to pellet the particle. There are two types of rotors that are commonly used for exosome isolation: swinging bucket (SW) and fixed-angle (FA) rotors, principally differing in sedimentation efficiency. A SW rotor stands out horizontally during centrifugation, and thus has a larger sedimentation path than FA rotors. While this lowers the pelleting efficiency of SW rotors (higher k value) resulting in lower yield, SW rotors have better resolution, i.e., they can separate vesicles with small differences in size more effectively than FA rotors. |
Viscosity | Reducing viscosity of the sample increases the efficiency of isolation, as the higher the viscosity, the more difficult it would be for the exosomes to travel through the sample and pellet. |
Time | The amount of time a biological fluid is centrifuged is determined by the viscosity, rotor g value, and desired purity of the exosomal fraction. The duration can be extended to yield greater quantities of exosome-based contents such as protein and RNA, though this is limited by the possibility of condensing the pellet to such an extreme that they aggregate, making them hard to resuspend and it may thus interfere with the functional integrity of the final product. Longer time of centrifugation also co-precipitates non-exosomal proteins and reduces purity of the end product. |
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Sidhom, K.; Obi, P.O.; Saleem, A. A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option? Int. J. Mol. Sci. 2020, 21, 6466. https://doi.org/10.3390/ijms21186466
Sidhom K, Obi PO, Saleem A. A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option? International Journal of Molecular Sciences. 2020; 21(18):6466. https://doi.org/10.3390/ijms21186466
Chicago/Turabian StyleSidhom, Karim, Patience O. Obi, and Ayesha Saleem. 2020. "A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option?" International Journal of Molecular Sciences 21, no. 18: 6466. https://doi.org/10.3390/ijms21186466