TABLE 1.

Different sources used to isolate EVs, methods of isolation, and effectiveness of each project.

Source of EVs	Application/purpose	Method of EVs isolation	Quantity of EVs used/obtained*	Effectiveness/results	References
Human plasma	Develop a single-step protocol to isolate EVs from plasma aiming at less contamination by proteins and high-density lipoproteins (HDL)	Size-exclusion chromatography (SEC) with Sepharose CL-2B column	1.5 ml of platelet-free plasma was collected in 26 fractions of 0.5 ml. Fractions 9–12 presented the highest concentrations of particles (46%) and 4 μg EV protein (Western blot)	With this method, there is no risk of protein complex formation, being fast and inexpensive, with the recovery of vesicles with Sepharose CL-2B SEC being 43%, compared to 2%–80% by ultracentrifugation. Display good vesicle recovery with almost complete removal of contaminants	Böing et al. (2014)
Visceral adipose tissue	Evaluate how a high-fat diet influences colitis through exosomes derived from visceral adipose tissue. This work also isolated exosomes from the liver, skeletal muscle, and subcutaneous adipose tissue	Ultracentrifugation and ExoQuick Kit	Tissues with less than 1 cm3 cultured for 24 h in a serum-free medium. Recovery not specified	A high-fat diet changed miRNA-exosome profile switching from an anti-inflammatory to a pro-inflammatory phenotype, increasing intestinal inflammation via M1 macrophage	Wei M et al. (2020)
Human and rat plasma	Compare the efficiency of SEC and ultracentrifugation to isolate exosomes with minimum content of albumin as contaminant	size exclusion chromatography (SEC) versus ultracentrifugation (UC)	UC: 7 ml twofold diluted plasma SEC: 0.5 ml of filtered platelet-free plasma was diluted and loaded with 10 ml of bed volume (results presented by Western blot)	SEC is more suitable than UC for the exos isolation from blood without significant albumin contamination, but the efficiency needs to be improved	Baranyai et al. (2015)
Conditioned medium (CM) from mouse neuroblastoma and myoblast cells (N2a and C2C12 lines)	Evaluated a novel liquid chromatography technique for EV purification: using core bead chromatography	Bind-elute (BE) size-exclusion chromatography (SEC)	20 ml of filtered conditioned medium supernatant recovery about 80% with values equal to (N2a) 1.17 × 1010 P/ml and (C2C12) 1.32 × 1010 P/ml	BE-SEC columns can purify EVs in a reliable and scalable fashion with yields ranging from 70% to 80%, purity comparable to UC, and it is a fast procedure (85–150 min)	Corso et al. (2017)
CM of breast cancer MCF7 and MDA-MB-231 line	Use ultrafiltration/diafiltration for the purification of exosomes from the CM cell lysate with 3 kDa or 100 kDa Amicon® Pro device Purification + TEI	Ultrafiltration-based method	—	Higher protein concentration and higher relative absorbance units for lipids in the 3-kDa or 10-kDa membrane indicated that this device was a better choice for exosome sample preparation	Gutierrez et al. (2013)
Porcine milk	Evaluated whether porcine milk-derived exosomes could attenuate PLS-induced intestinal epithelial inflammation by downregulating inflammatory and apoptosis pathways via miRNAs	Ultracentrifugation	∼50 ml fresh porcine milk	Porcine milk-derived exosomes appear to protect the intestine from LPS-induced injury by decreasing inflammation and apoptosis through the inhibition of the NF-ĸβ and p53 pathways	Xie et al. (2019)
			Exosome recovery was not specified
Urine EVs	Propose a nanowire-based methodology for collecting urine EV-encapsulated miRNAs that unveils massive numbers of urinary miRNAs of different sequences that potentially serve as biomarkers for cancer not only for urologic but also for non-urologic malignancies	Nanowires anchored into a microfluidic substrate	1 ml urine	The device could achieve higher efficiency for in situ extraction of urine EV-encapsulated miRNAs compared to the most popular conventional method of ultra- centrifugation. Could extract a much larger variety of species of miRNAs despite the smaller volume and could find cancer-related miRNAs from urine samples of just 1 ml for not only urologic malignancies but also non-urologic ones	Yasui et al. (2017)
			Collection efficiency (small RNA yield)
			0.194 ± 0.028 ng/ml
			Extraction species of urinary miRNAs being identified
			749, 822, 1,111 (n = 3)
Human blood serum	Evaluation of six different kit isolation methods	ExoQuick, ExoEasy, Exospin, ME kit, ExoQuick Plus, ExoFlow	The serum volume follows the manufacturer’s instructions but varies from 250 μL to 4,000 μl. The exosome concentration, also, varies: 44 × 1013 P/L; 18 × 1013 P/L; 64 × 1013 P/L; 0,13 × 1013 P/L; 59 × 1013 P/L; 10 × 1013 P/L (values ordered as in column of Methods)	Regarding particle counts, Exo-spin, exoEasy, and ExoQuick isolated similar values, on the contrary, exoEasy and ExoQuick Plus presented the highest purity levels. An important observation raised was that depending on the KIT chosen, the molecules and their respective concentration could vary	Macías et al. (2019)
Human-breast milk	Analyze the potential protective effect of human-breast milk exosomes from oxidative stress in intestinal epithelial cells	Ultracentrifugation	Initial volume not specified. Recovery of 3–9 × 108 particles/ml	Breast milk exosomes seem to protect against cell toxicity induced by H2O2 with their own cargo. Also, human milk exosomes could be used in neonates with intestine injury	Martin et al. (2018)
M2-macrophage	Investigate the roles of M2-macrophage-derived exosomes in vascular smooth muscle cell (VSMC) differentiation, including the mechanisms after stent implantation	Ultracentrifugation	Not specified	M2-macrophage-derived exosomes promote re-endothelization and VSMC differentiation via the MAPK (AP-1) pathway	Yan et al. (2020)
Wistar rats’ blood plasma	Investigate the effectiveness of UC and SEC methods of exosome isolation	Ultracentrifugation (UC) and Size Exclusion Chromatography (SEC) with Sepharose CL-4B, Sepharose 2B, and Sephacryl S-400	Ultracentrifugation	SEC is more suitable than UC, especially due their low albumin contamination. However, SEC efficiency should be improved	Baranyai et al. (2015)
			7 ml of plasma
			Recovery not specified
			Size exclusion chromatography (SEC)
			0.5 ml of filtered plasma
			Recovery not specified
Human blood plasma and cell culture medium	Characterize and compare extracellular vesicles isolated from plasma and cell culture medium using SEC, polyethylene glycol (PEG), and protein organic solvent precipitation (PROSPR)	SEC PEG PROSPR	Size exclusion chromatography (SEC)	Comparing all three methods, the SEC protocol was the most efficient and most capable of removing most protein contamination and maintaining the vesicular structure and conformation of extracellular vesicles	Gámez-Valero et al. (2016)
			2 ml of filtered plasma
			Recovery not specified
			Polyethylene glycol (PEG)
			2 ml of plasma in 50% PEG6000 (final concentration: 10%)
			Recovery not specified
			Protein organic solvent precipitation (PROSPR)
			2 ml of plasma
			Recovery not specified