Physicochemical Modulation Strategies for Mass Production of Extracellular Vesicle

Abstract

Background: Extracellular vesicles (EVs) have attracted expanded attention as vehicles for the diagnosis and therapy of diseases and regenerative medicine due to their biocompatibility, efficient cellular uptake ability, and capacity to transport biologically active molecules. However, the low secretion yield of EVs and the challenges of large-scale production remain the main barriers to their extensive clinical use.

Methods and results: This review explores recent strategies to enhance EV production in cell culture systems, focusing on chemical stimulation, mechanical stimulation, and structural stimulation. First, we review chemical stimulation strategies for modulating culture conditions using chemical stimulation, including nutrient composition, pH, temperature, oxygen levels, intracellular cholesterol, and oxidative stress. Second, we examine mechanical stimulation strategies, including shear stress, irradiation, and ultrasound. Third, we explore structural stimulation strategies, such as three-dimensional (3D) culture systems involving spheroid-based culture, as well as the use of bioreactors and scaffolds. In addition, cell-derived nanovesicles containing cell membrane and cellular component, which can be more easily mass-produced compared to EVs, are proposed as an alternative to EVs.

Conclusion: Future research should focus on developing cost-effective and scalable EV production methods while improving purification techniques to ensure a high yield without compromising functional integrity. Moreover, integrating optimized stimulation strategies-such as refining 3D culture systems, bioreactor designs, and mechanical stimulation methods-could further enhance EV secretion. Addressing these challenges is essential for advancing EV-based applications in both research and clinical practice.

Keywords: 3D culture system; Chemical modulation; EV mimetic nanovesicles; Extracellular vesicle; Mass production; Mechanical modulation.

Fig. 1

Strategies for the large-scale production of extracellular vesicles

Fig. 2

Chemical modulation strategies for increasing EV production. A Nutrients starvation. B pH regulation C Temperature change. D Hypoxia. E Cholesterol regulation. A adapted from Wang et al., CC BY-NC 4.0 [26], B adapted from Nakase et al., CC BY 4.0 [27], C adapted from Otsuka et al., CC BY 4.0 [28] D adapted from Wang et al., CC BY-NC-ND 4.0 [29] E reproduced from [30] with permission from Elsevier

Fig. 3

Mechanical modulation strategies for EV mass production. A Shear stress induces EV production through calcium influx. B Irradiation exposure increases EV secretion. C LIUS stimulation enhances EV yield in A2780 cells. A Reproduced from [74] with permission from The Royal Society of Chemistry. B reproduced from [75] with permission from American Association for Cancer Research. C reproduced from [76] with permission from Elsevier