Exosomes can be isolated from the conditioned media of cultured cells and almost any biological fluid. Current methods of exosome isolation and purification involve ultracentrifugation, precipitation, immunoaffinity and size-based isolation techniques. Ultracentrifugation is currently considered to be the most widespread method by which to obtain exosomes, as it is low cost, and yet it can still isolate particles from large volumes of biological fluids. However, the centrifugation processes may pellet non-exosome contaminants of similar densities [
26,
27]. Therefore, additional purification steps may be applied, such as a density gradient or ultrafiltration, to further separate out the specific exosome populations. Precipitation is another method of isolating and purifying exosomes. In this method, water-excluding polymers such as polyethylene glycol (PEG) are used to bind water molecules and force less soluble particles such as exosomes out of the aqueous solution [
28,
29]. This is a relatively simple process and does not require the use of specialized equipment. However, the use of foreign polymers can introduce impurities into the sample that is difficult to remove, and may adversely impact downstream applications [
30,
31]. A third method, immunoaffinity, relies on the interactions between exosome membrane proteins and their respective antibodies [
29] This is a more precise method of capturing specifically exosomes, but is limited in that it cannot be used for large sample volumes. Therefore, it is more commonly used in conjugation with the conventional ultracentrifugation methods where the concentrated pellet is subsequently purified using immunoaffinity. Size exclusion methods include techniques such as size exclusion chromatography and ultrafiltration [
26,
29]. In both, samples are passed through membranes with predefined size or molecular weight limits, and exosomes are separated from other particles based on these physical characteristics. Size exclusion chromatography and ultrafiltration are relatively simple procedures for exosome isolation. However, size exclusion chromatography requires long columns for ideal size separation, which may be impractical for large scale manufacturing. In ultrafiltration, the use of force that is required to pass the samples through the membrane can potentially damage or deform exosomes. Additionally, exosomes can be lost or otherwise remain adhered to the membrane following this procedure.
As a result of the imprecise and heterogenous nature of exosome isolation, quality control becomes of the utmost importance when developing and establishing clinical-grade exosome treatments. Current good manufacturing practices (cGMPs) can be adopted to standardize exosome isolation procedures by keeping constant the cell sources, as exosomal cargo content can be altered by cell state [
26]. Therefore, environmental conditions, such as cell passage number, seeding density or culture conditions, must be kept uniform in order avoid inconsistencies in the quality of the final exosome yield. Purity can be quantitatively assessed by measuring levels of contaminants (e.g., endotoxin, sterility and mycoplasma) through standard clinical tests [
32]. Following isolation, current assessments of exosome identity involve an extensive profiling of characteristic surface proteins on both the exosomes and parental cells. Western blotting or flow cytometry can be used to assess the presence of exosome surface proteins (e.g., CD9, CD63, CD81) on the isolated particles, while parental cells are characterized for viability and cell surface marker profiles [
33]. Microscopy techniques, such as electron microscopy (EM) and atomic force microscopy (AFM), may be useful for additional morphology and size distribution characterization [
32]. Current methods of exosome quantitation mainly rely on the quantification of EV numbers or of EV protein weight [
34]. Exosome particle numbers have been commonly calculated through the NTA or other similar concentration measuring instruments and technologies. Exosome total protein content has also been used as a parameter for quantification [
32,
34]. These quantitative measurements are often used when calculating dosages. Most approaches currently involve administering EVs that are cell equivalent in number (compared to a control treatment group consisting of cells only), as calculated from concentration measurements [
31]. Doses can also be administered based on total protein content, though this may be a less than accurate method due to the variation in protein content between individual exosomes [
31,
35]. Exact clinical doses are dependent on the disease at hand as well as the potency of the exosomes. To assess potency, functional assays can be conducted as a downstream validation process. Otherwise, proteomic or transcriptomic studies can be conducted to directly assess exosomal contents as a way to standardize the bioactive cargo between different batches [
32].