Selection of the optimal cell source for extracellular vesicles production in regenerative medicine or drug delivery

Extracellular vesicles (EVs) are vesicles secreted by cells from several biogenesis pathways. Extracellular vesicles are currently developed for therapies mainly in regenerative medicine to replace stem cell therapy1,2, and in the drug delivery field as nanocarriers3. They gather the multiple nomenclatures of exosomes, microvesicles, apoptotic bodies, and other natural “-osomes” products. They are ubiquitous, present in all biological fluids from all species (humans, animals, plants, microorganisms). They have attracted a lot of interest because they contain biomolecules, such as phospholipids, intraluminal and membrane-bound proteins, and different types of RNAs. One intrinsic characteristic of EVs is their heterogeneity. Cells produce different populations of extracellular vesicles, and each particle contains its proper combination of biomolecules.

To produce extracellular vesicles at a large scale, they must be collected from cell culture conditioned medium, or directly from a biological fluid such as milk or blood. The selection criteria for the EV cell source can be divided in two categories: i) the extracellular vesicle intrinsic properties, in line with their therapeutic activity, and ii) the cell line characteristics associated to their robustness for large production.

Each extracellular vesicle type is associated to endogenous biological properties, that will drive their pharmaceutical effect. It is related to their biomolecular content, and their physical properties. Two academic studies have compared several cell sources for extracellular vesicle bioactivity: Wiklander & al have followed the biodistribution of EVs in a mouse with 6 various cell lines from human, rat, or mouse. For each organ, the amplitude of EV proportion at 24h varied significantly, with between 45 and 75 % in the liver, or between 4 and 18 % in the Gastro-intestinal tract for instance5. Furthermore, Barile & al have compared extracellular vesicles from cardiomyocytes progenitor cells (CPC), bone-marrow derived mesenchymal stem cells, and dermal fibroblasts, for their cardiac regenerative properties. Only CPC-EVs showed a significant cardiac function improvement in two different pathological models6. These 2 examples show that cell source has a direct impact on extracellular vesicle properties.

Then, the cell source directly influences the ability to produce a large extracellular vesicle quantity, which is important in terms of collected doses. Different cells secrete very different quantities of EVs in the same culture conditions. In any cases, cells must be expanded widely, over multiple passages. It means that their availability, together with their stability during the scale-up is essential. Finally, using stably transfected cells can allow to engineer exosomes, and the ability to be transfected is variable among cell lines, HEK293 being the most famous.

Clinical trials using extracellular vesicles provide an overview of current EV sourcing. All except one phase II trials have used mesenchymal stem cells (MSC). This MSC proportion remains important at all stages but the diversity increases a lot with preliminary studies7.

Once the type of cell sourcing for extracellular vesicles has been selected, it remains to decide to work with primary cell line, which will be closer to the physiological properties but available only during a limited time, or with immortalized cells, with some safety risks and a potential derivation of the cell line. But immortalization allows a phenotypic and genotypic stability, increases the amplification potential, and batch consistency. At the same time, it will reduce costs associated to multiple master cell bank validations. Immortalization was not possible for cell therapy due to safety concerns, but with extracellular vesicles, it becomes straightforward.

1.         Nagelkerke A, Ojansivu M, van der Koog L, et al. Extracellular vesicles for tissue repair and regeneration: Evidence, challenges and opportunities. Advanced Drug Delivery Reviews. 2021;175:113775. doi:10.1016/j.addr.2021.04.013
2.         Lee JY, Kim HS. Extracellular Vesicles in Regenerative Medicine: Potentials and Challenges. Tissue Eng Regen Med. 2021;18(4):479-484. doi:10.1007/s13770-021-00365-w
3.         Meng W, He C, Hao Y, Wang L, Li L, Zhu G. Prospects and challenges of extracellular vesicle-based drug delivery system: considering cell source. Drug Delivery. 2020;27(1):585-598. doi:10.1080/10717544.2020.1748758
4.         Kim J, Song Y, Park CH, Choi C. Platform technologies and human cell lines for the production of therapeutic exosomes. EVCNA. Published online 2021. doi:10.20517/evcna.2020.01
5.         Wiklander OPB, Nordin JZ, O’Loughlin A, et al. Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting. Journal of Extracellular Vesicles. 2015;4(1):26316. doi:10.3402/jev.v4.26316
6.         Barile L, Cervio E, Lionetti V, et al. Cardioprotection by cardiac progenitor cell-secreted exosomes: role of pregnancy-associated plasma protein-A. Cardiovascular Research. 2018;114(7):992-1005. doi:10.1093/cvr/cvy055
7.         Silva AKA, Morille M, Piffoux M, et al. Development of extracellular vesicle-based medicinal products: A position paper of the group “Extracellular Vesicle translatiOn to clinicaL perspectiVEs – EVOLVE France.” Advanced Drug Delivery Reviews. 2021;179:114001. doi:10.1016/j.addr.2021.114001