# Origin and Composition of Exosomes as Crucial Factors in Designing Drug Delivery Systems
## 4 Exosomes Isolation Methods  
Currently, there are many available exosomes isolation methods (Table 1). The oldest and most commonly used exosome isolation method is ultracentrifugation stated by many as a gold standard. Other techniques are mostly based on size exclusion or antibody-mediated isolation of labeled exosomes. Every isolation method differs in terms of purity, amount, efficiency, and throughput. The most common disadvantages are inconsistency and damage or alteration in the structure of exosomes [6; 43]. Ultracetrifugation is based on the size (weight) of particles and their sedimentation under centrifugal force (100,000-110,000\(\times\)\(g\)). The previous removal of cellular debris and \begin{table}
\begin{tabular}{c c c c} \hline \hline  & **Method** & **Advantage** & **Disadvantage** \\ \hline \multirow{6}{*}{**Variant**} & Ultra- & \multirow{2}{*}{Large pools processing, high purity} & Long, low efficiency (5-25\%), poor repeatability, vesicle-damaging \\  & centrifugation & & \\ \cline{2-3}  & & & \\ \cline{2-3}  & & & \\ \cline{2-3}  & & & \\ \cline{2-3}  & & & \\ \cline{2-3}  & & & \\ \cline{2-3}  & & & \\ \cline{2-3}  & & & \\ \cline{2-3}  & & & \\ \cline{2-3}  & & & \\ \cline{2-3}  & & & \\ \cline{2-3}  & & & \\ \cline{2-3}  & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Exosome isolation methods. unwanted particles can be obtained by a few steps of slower-speed centrifugation named differential centrifugation. It can be used for large pool processing. This method can also be conducted in a sucrose gradient (1.13-1.19 g/mL) or cushions for higher enrichment, yield, and purity increase [44; 45]. Ultrafiltration methods include the utilization of filters with decreasing pore size; thus, larger particles will be excluded from the filtrate. It can be used in combination with centrifugation to accelerate exosomes purification by the initial removal of large particles such as cell debris. The pore clogging risk makes this method unfavorable for large pool processing [46; 47]. Size-exclusion chromatography is based on the size of gel pores and the size of exosomes. Particles larger than the gel pore size will be eluted in the first place; inversely, particles smaller than the gel pore size will be restrained. Isolation is commenced in a native environment and overall very high purity and yield are obtained. In combination with ultrafiltration, this method provides lesser protein contamination with a superior exosome recovery rate. The downside is the cost of the gel [48]. Polymer-based precipitation separation methods utilize super-hydrophilic polymers to enhance the precipitation of small-size particles such as exosomes. Polyethylene glycol is commonly used in concentrations varying between 8 and 15%. With the use of this method, the solution containing exosomes is overnight-incubated with polymer and further centrifuged at about 10,000\(\times\)\(g\). Commercial kits are also available [6]. Immunological separation methods utilize labeling of the exosomes with antibodies subjected to selected protein markers located in the exosomal membrane. These labeled exosomes can be further easily handled and purified, e.g., with microchips or magnetic beads [49; 50]. After magnetic separation, exosomes are purified and magnetic beads are dissolved and removed. With this simple and quick method, high-purity extracts can be obtained but exosomes lacking surface markers recognized by magnetic-labeled antibodies will not be isolated, which may lead to poor exosome pool representation [51]. This method is time-saving and straightforward with high-purity output but involves expensive reagents. In terms of affinity-isolation methods, there are also proposed lectins and synthetic Vn (venceremin) peptides. Lectins will bind glycosylated proteins presented at the surface of exosomes, leading to exosomes precipitation. Vn peptide isolation technology is based on its high affinity to HSP-containing extracellular particles.