Cell therapy is witnessing a notable shift toward cell-free treatments based on paracrine factors, in particular, towards small extracellular vesicles (sEV), that mimic the functional effect of the parental cells. While numerous sEV-based applications are currently in advanced preclinical stages, their promised translation depends on overcoming the manufacturing hurdles posed by the large-scale production of purified sEV. Unquestionably, the culture medium used with the parental cells plays a key role in the sEV’s secretion rate and content. An essential requisite is the use of a serum-, xeno-, and blood-free medium to meet the regulatory entity requirements of clinical-grade sEV’s production. Here, we evaluated OxiumTMEXO, a regulatory complying medium, with respect to production capacity and conservation of the EV’s characteristics and functionality and the parental cell’s phenotype and viability. A comparative study was established with standard DMEM and a commercially available culture medium developed specifically for sEV production. Under similar conditions, OxiumTMEXO displayed a three-fold increase of sEV secretion, with an enrichment of particles ranging between 51 and 200 nm. These results were obtained through direct quantification from the conditioned medium to avoid the isolation method’s interference and variability and were compared to the two culture media under evaluation. The higher yield obtained was consistent with several harvest time points (2, 4, and 6 days) and different cell sources, incluiding umbilical cord-, menstrual blood-derived mesenchymal stromal cells and fibroblasts. Additionally, the stem cell phenotype and viability of the parental cell remained unchanged. Furthermore, OxiumTMEXO-sEV showed a similar expression pattern of the vesicular markers CD63, CD9, and CD81, with respect to sEV derived from the other conditions. The in vitro internalization assays in different target cell types and the pharmacokinetic profile of intraperitoneally administered sEV in vivo indicated that the higher EV production rate did not affect the uptake kinetics or the systemic biodistribution in healthy mice. In conclusion, the OxiumTMEXO medium sustains an efficient and robust production of large quantities of sEV, conserving the classic functional properties of internalization into acceptor target cells and biodistribution in vivo, supplying the amount and quality of EVs for the development of cell-free therapies.
Bibliographical noteFunding Information:
The authors specially thank Dr. Jimena Cuenca and her group (Faculty of Medicine, Universidad de los Andes, Santiago, Chile) for their kind donation of the hPL and Daniel Meza for his collaboration in the preparation of OxiumTMEXO medium. The authors sincerely thank all the members of the laboratory for their important contributions to the scientific discussion of this work, especially Nicol?s Georges and Hugo Tobar. The authors acknowledge the technical expertise and assistance of Javiera Ponce and Camila S?nchez in the animal experiments. Funding. This work was funded in part by the Chilean National Agency for Research and Development (ANID), through FONDECYT Regular project #1190411 to FA-M, and by Consorcio Regenero, Chile.
© Copyright © 2021 Figueroa-Valdés, de la Fuente, Hidalgo, Vega-Letter, Tapia-Limonchi, Khoury and Alcayaga-Miranda.
- blood-free cell culture
- chemically defined
- culture medium
- mesenchymal stem (stromal) cells
- serum-free cell culture
- small extracellular vesicle
- xeno-free cell culture