Combining established techniques enables large-scale production of potentially therapeutic extracellular vesicles enriched with specific miRNAs.
Extracellular vesicles (EVs) are currently being intensively studied for their therapeutic potential following promising clinical results and recent regulatory approvals of cell-based therapies. However, for the excitement surrounding EVs to ultimately yield useful therapies, critical challenges remain to be overcome. Specifically, although microRNA (miRNA) is often cited as a critical component of EV therapeutic activity, specific miRNA amounts in native EVs can be quite low (far less than one miRNA per one EV on average in many cases), raising concern about the potency of EV-based therapies. Further, scalable biomanufacturing of therapeutic EVs is nontrivial and could present a barrier to translation.
To address these issues, Yoo et al. used a combination of established, commercially available technologies to define a method for producing large quantities of EVs enriched with specific miRNAs. First, they used lentiviral vectors to generate stable HEK293 cell lines capable of producing EVs with more than 2000-fold enrichment of specific miRNAs. Then, a hollow fiber bioreactor was employed for continuous production of EVs from the same stable cell lines for up to 30 days, with additional gains in miRNA levels observed compared with EVs harvested from cells grown in conventional cell culture flasks. Last, tangential flow filtration was used to concentrate miRNA-enriched EVs by ~200-fold without precipitate formation. To validate the potential therapeutic utility of EVs produced through this scheme, miR-133a-3p–enriched EVs were injected intraperitoneally in mice. The result was an increase in the level of circulating miR-133a-3p after four hours. The broad applicability of the techniques used in this process suggests that it could be used to increase blood levels of any desired miRNA via EV association.
Further optimization of this method will be necessary to enable production of EVs from different primary cell types, and this production scheme still contains potential manufacturing bottlenecks, such as lentiviral transfection. The ultimate therapeutic potential of miRNA delivery via EVs produced by the process still remains to be established. However, the general approach described is widely applicable to platform production of miRNA-enriched EVs. More importantly, all the technologies employed are commercially available and should be within reach for a majority of academic labs and small companies to access or acquire. Thus, this process could serve as an important template for advancing research and overcoming the lack of method standardization in development of EV therapeutics, taking the entire field closer to clinical translation.
K. Yoo, N. Li, V. Makani, R. Singh, A. Atala, & B. Lu. Large-scale preparation of extracellular vesicles enriched with specific microRNA. Tissue Eng. Part C: Methods (2018) 24: 637-644. doi: 10.1089/ten.TEC.2018.0249 PMID: 30306827.