Table 1.

Stimulation methods for increasing EV secretion.

Methods	Cell type	Treatment	Endosomal markers	Altered endogenous cargo	Increase in release	Scalability in GMP environment	Therapeutic use	Refs
Physical Signals
Mechanical loading	HDMECs, DPSCs, hSkMCs, PDL	0.5 ml/min flow induction for 48 h, 25% 1 Hz cyclic stretch for 48 h	CD9, CD63, CD81	RhoG, ITGAV, CAPZA2, CKAP5, CDH13, ARPC2/4, MYH11, TUBB ↑	11 to 150 fold	+++	drug delivery platform, neuronal regeneration, muscle regeneration	(Guo et al., 2021)
Geometry	MSCs, DPSCs	3D aggregates, 3D constructs support by polymeric scaffold	CD9, CD63, CD81	miR-210 ↑, miR-134, miR-137, miR-184↓	3 to 10 fold	+++	pro-angiogenic properties	(Cha et al., 2018; Guo et al., 2021)
Acoustic stimulation	U87-MG human glioblastoma cells and A549 adenocarcinomic human alveolar basal epithelial cells	7 repeated cycles of 10 min SRBWs stimulation (4 W, 10 MHz) followed by 30 min of cell incubation	CD63, Alix	syntenin-1	8 to 10 fold	+	drug delivery platform	(Ambattu et al., 2020)
Electric stimulation	murine melanoma and murine fibroblast	0.34 mA/cm2 electrical field for 60 min	CD9, HSP70, and CD81	−	ca. 1.7 fold	+	beneficial effects of transcranial direct current stimulation (tDCS) in the brain	(Fukuta et al., 2020)
Molecular Interference
Glycolysis & oxidative phosphorylation inhibition	cancer cell lines: UMSCC47, PCI-13 and MEL526,	10 μM IAA/DNP for over 48 h	TSG101	−	3 to 16-fold	++	−	(Ludwig et al., 2020)
Endolysosomal trafficking	SKOV-3 cells, cardiac progenitor cells	NDRG1 knocked down	Alix, TSG101, CD9, CD81	−	up to 2 fold	++	Myocardial ischemia and ischemia/reperfusion injury.	(Ortega et al., 2019)
Adiponectin	MSCs	20 mg/ml HMW-APN for 48 h	Syntenin, MFG-E8, Alix, CD63, TSG101	↑ miRNAs: let-7 family, miR-21, −100, −148a, −10, −26, and − 199, and others	ca. 3 fold	++	Pressure-overload heart failure	(Nakamura et al., 2020)
Small molecule modulators	MSCs	100 μM NE/MeDA for 48 h	CD9, CD63, Hrs, TSG101, Stam1, Alix	↑ proteins: COL15A1, COL11A1, LOXL2, AGRN, NID2, HSPG2, COM	ca. 3 fold	++	Ischemic or inflammatory diseases	(Wang et al., 2020)
Environmental Factors
Hypoxia	Breast cancer cell lines	1% for 48 h or 0.1% O2 for 24 h	CD63, TSG101, CD9	miR-210 ↑	up to 2 fold	+++	−	(Hamish W. King et al., 2012)
	Pancreatic cancer cell lines	1% O2 for 48-72 h	CD63	circZNF91↑, miR-23b-3p ↓	up to 2 fold	+++	−	(Zeng et al., 2021)
	MSCs	1% O2 for 48 h	CD63, TSG101, CD9, CD81	miR-126 ↑	ca. 1.5 fold	+++	bone fracture healing	(Liu et al., 2020)
Acidity	metastatic prostate carcinoma LNCaP, metastatic melanoma Me30966, SaOS2 osteosarcoma, SKBR3 metastatic breast adenocarcinoma, and HCT116 colorectal carcinoma	pH 6.5 for 5 days	CD9, CD63, CD81, TSG101, Alix	−	sKBR3–6 fold, LNCaP - 9 fold, SaOS2–14 fold, HTC116–52 fold, Me30966–102 fold	+++	−	(Logozzi et al., 2018)
	metastatic melanoma cells	pH 6 for 4 days	Lamp-2, CD81, and Rab 5B	caveolin-1 ↑, membrane rigidity ↑, sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content ↑, exosome fusion ↑	up to 4 fold	+++	delivery system for paracrine diffusion of tumor malignancy	(Parolini et al., 2009)
	HEK 293	pH 4 for 3 days	CD9, CD63, HSP70	−	up to 6 fold	+++	−	(Ban et al., 2015)
Starvation or hyperglycemia	immortalized H9C2 cardiomyocytes	glucose starved for 48 h	CD9, CD63, CD81	22 different miRs ↑	ca. 3 fold	+++	pro-angiogenic properties	(Garcia et al., 2015)
	First-trimester trophoblast cells	1% O2, hyperglycemia (25 mM glucose) for 48 h	CD63	−	ca. 3 fold	+++	induction if cytokine release	(Rice et al., 2015)
External Inducers
Liposomes	cancer cell lines:C26, B16BL6, MKN45, DLD-1	0.5-2 mM PEGylated/NL/CL1 for 48 h	TSG101, CD63 or CD81	−	up to 3-fold	++	tumor metastasis	(Emam et al., 2018)
Nanoparticles	MSCs	5-20 μg/ml PLGA-PEI PCS NPs (+) for 24 h	CD63, CD9, and CD81	mmu-miR-2137, mmu-miR-3473b, mmu-miR-3473e, mmu-miR-3960, mmu-miR-5126, mmu-miR-5126, mmu-miR-455-3p ↑	5 to 20-fold	++	tissue regeneration or antioxidant efficacy	(Park et al., 2020)