Limitations and Leads for the Future Exosomes are potentially future avenues in therapeutics and drug delivery systems. we highlight current perspectives that primarily focus on their effect on various diseases and their potential as a drug delivery vehicle. S2 cells, depletion of the Q-SNARE syntaxin 1A (Syx1A) decreased the release of EV enriched v exosomes . Wei et al. reported that pyruvate kinase type M2 (PKM2) phosphorylates SNAP-23, thus enabling exosome release . Although most studies around the molecular mechanism of exosome release are on cancer, few (almost none) have reported on mesenchymal stem cell exosomes [21,22]. Rab GTPases, the largest family of small GTPases, regulate many actions of membrane trafficking, including vesicle budding, transport of vesicles along actin and tubulin, and membrane fusion , are also involved in exosome secretion. Several studies exhibited that Rab family proteins (Rab2b, Rab5a, Rab27a, Rab27b, Rab35, and Rab11) are involved in this process . Additionally, it has also been shown by Yu et al. that this tumor suppressor protein p53 may also influence exosome secretion through regulating transcription genes such as TSAP6 and CHMP4C . Apart from that, various stimuli and changes like cell membrane pH and the concentration of K+ may also trigger the secretion of exosomes [26,27]. 2.3. Isolation of Exosomes: The First Step towards Pharmaceuticalization MSC-derived exosomes are being considered a novel tool for cell-free therapeutics [28,29,30,31]; however, the cardinal step in evaluating the extent of their competence is usually to successfully isolate and purify exosomes and obtain a good yield. Although a great deal of experimentation has been performed, there is still no uniformity in isolation methods; but, by far, the technique considered best is usually ultracentrifugation due to the superlative quality of exosomes isolated within it and the ubiquity of its D609 use [32,33]. Basic ultracentrifugation as an exosome isolation technique was introduced by Johnstone et al.  to infer that vesicle shedding was an intermediate process during maturation to erythrocytes. There have been several advancements to this process, such as modulation in the number of cycles of centrifugation  and optimization in protocols of differential ultracentrifugation [36,37], density gradient ultracentrifugation [32,38,39,40], Vegfc etc. Certain isolation kits have also been devised to be considered a time-saving alternative showing reasonable results [41,42,43]. The possibility of combining the beneficial effects of ultracentrifugation and precipitation-based kits was explored by Ryu et al. . They inferred that combining the potential of both techniques was expedient for the isolation of small EVs, provided a good output, and held no lags about their constitution, hence utilizable for catering to massive sample-based critical clinical evaluations. Common protocols used for exosome isolation are shown in Figure 2. Open in a separate D609 window Figure 2 Isolation of Exosomes: exosomes are commonly isolated from the conditioned media. Some common preprocessing steps are required for both the techniques, including collecting conditioned media from MSCs, performing a centrifugation round at 2000 for 30 min to remove debris. Furthermore, the conditioned media D609 can be subject to any of the two techniques including, Ultracentrifugation (1) or Kit-based methods (2) for isolation of exosomes. These exosomes can be further used for characterization, aliquoting, and storage for future experiments. Despite abounding attempts to find a robust technique for uniform, use globally, many shortcomings exist that need to be addressed, such as long duration, complicated protocols, need for special equipment, lack of cost-effectiveness, limited utility, D609 the requirement of large volumes of sample, lack of specificity, truncated yield, low rate of recovery, dubious purity, and risk of mechanical damage. These techniques, in their current form, are not suitable for standardization. All the techniques have their advantages and drawbacks; however, a technique that could satisfactorily channel the benefits of all pre-existing technologies collectively while facilitating exosome isolation for downstream processing at a translational level to visualize the use of exosomes for future applications like drug formulation and delivery D609 of therapeutics, is yet to be devised. 2.4. Characterization and Visualization.