Despite progress in technique, isolation of sEVs has been challenging. It would be wise to choose a strategy and develop isolation protocols depending on experiment purpose. Three concentration methods and four purification methods were evaluated in our study. A summarization of advantages, disadvantages and possible suggestions for isolation methods used in this study was shown in Table 1. For the concentration of sEVs, UC-based method showed high yield and purity of sEVs and was used to concentrate the sample before purification. For the purification of sEVs, IAC was effective and time-saving and yield purest sEVs among all methods evaluated, thus was recommended to be suitable for biomarker study13,14. DGUC method effectively produced the smallest and also purified sEVs with relatively high yield, thus may be suitable for EV therapeutic study as pure and large number of sEVs are required for therapeutic application 15,27−29. More importantly, sEVs obtained via DGUC method could avoid aggregation and precipitation 30 and were superior for cellular uptake 31.
In the presented study, the size, yield, morphology and protein were assessed for quality of sEVs. The size and yield of sEVs were crucial for therapeutic use, especially for drug delivery. It has been reported that smaller EVs could be uptaken by cells more efficiently31. The morphology showing the presence of EV was observed, sEVs obtained in this study was saucer-shaped under TEM images as reported32. CD63 was a marker for EVs and was used in IAC for purification9,33,34. Besides, in our fraction analysis, CD63 level was used to reflect EV-containing fractions, this method was also reported in previous studies 21,23,35. However, for SEC, CD63 may not fully represent the sEVs fractions as expected. Possibly, CD63 was not exclusive to the sEVs. Recent studies reported that microvesicles also expressed CD6336,37. A combined strategy of several sEVs marker proteins such as CD63, TSG101 and CD9 may reflect the sEVs-containing fractions more accurately.
Our study included the majority of current sEVs isolation methods, except commercial kits and microfluidic-based methods. Because of unstable quality of the extracted EVs, high price and unknown solutions38, commercial kits were not included in our study. The microfluidic technology was popular but not included in our study as the technique was mostly used for methodological studies, such as sEVs detection, instead of therapeutic application study, even if it was potentially available for isolation39,40.
In the presented study, an equal number of EVs particles was evaluated for each method in case of contaminant protein influencing the results. The level of target protein could be weak as detected if equal protein, with a high amount of contaminant protein, was loaded onto each well in the gel. This may explain why the level of EV marker protein was weak in study 41.
The UC-based technique remains the most common method for sEVs isolation42–44. However, our study results demonstrated that DGUC and IAC methods produced sEVs with better purity than UC. Besides, the interpretation of results should be cautious when commercial sEVs isolation kits based on the Co-P method was used38,45. The combination of several isolation methods may produce purer sEVs. Jeppesen et al. used ultracentrifugation-based technique, density gradient ultracentrifugation-based technique and immunoaffinity capture-based technique to purify sEVs 46. But more isolation steps may produce fewer sEVs, and combined isolation protocols were often hard to follow as the strategy could be complicated and fussy. The technique for evaluation of sEVs has been advancing. Tian et al. splendidly used nanoflow cytometry to evaluate the quality of sEVs38. But nanoflow cytometry-based analysis of sEVs needs further refinement of methods. For better comparisons between groups and future replication, we applied basic characterization of sEVs isolated by different methods23,35,47.
sEVs are a heterogenous group of vesicles 48. Single EV may carry distinct proteins, isolation method based on EV marker may lose EV subtypes of potential interest. For example, anti-CD63 conjugated-beads could be used to obtain CD63-enriched EVs, but effects of other vesicles may be neglected during subsequent experiments. This may explain why several metastasis and drug resistance-associated proteins were not highly expressed in purified sEVs obtained via IAC. However, future basic and clinical studies are likely to provide valuable information regarding their heterogeneity and advance our understanding of biological functions, thus reveal and harness their potentials for disease detection and therapy.