A Chemically Defined, Xeno- and Blood-Free Culture Medium Sustains Increased Production of Small Extracellular Vesicles From Mesenchymal Stem Cells

Abstract

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.

Understanding molecular characteristics of extracellular vesicles derived from different types of mesenchymal stem cells for therapeutic translation

“…Culturing MSCs under GMP-compliant-XF/SFM not only enables production of EVs with conserved classical functional properties but can also enhance EV quantities. 76 In fact, some studies suggest that XF/SFM may even enhance MSC-EVs’ cardiomyogenic and angiogenic potential. 77 A comprehensive comparison of the current FBS-free alternatives was done by Oikonomopoulos et al Their results led to the following conclusions: (1) both human platelet lysate (HPL) and XF/SFM increased the proliferation of MSCs; (2) HPL diminished the immunosuppressive properties for MSCs; (3) BM-MSCs and AT-MSCs in FBS and serum/xeno-free media showed potent immunosuppressive properties when primed with interferon (IFN)-γ; (4) both HPL and SF/XFM primed with IFN-γ increased levels of indoleamine 2,3-dioxygenase 1 (IDO-1) compared to FBS.…”

Current Strategies for Promoting the Large-scale Production of Exosomes

“…Studies have shown MSC-EXO cultured in a serum-free medium to exhibit better effects in terms of promoting wound healing and angiogenesis [ 77 ]. Another study reported better results in terms of sEVs yield using a new serum-free medium (Oxium TM EXO) [ 78 ].…”

Cell culture‐derived extracellular vesicles: Considerations for reporting cell culturing parameters

“…Considering the complex nature and important functions of serum in cell culture media, any manipulation including replacing the medium with a serum‐free medium, or heat treatment to inactivate the complement system, may alter the physiology of the cultured cells and derivative EVs (Alcolea et al., 2016 ; Angelini et al., 2016 ; Aswad et al., 2016 ; Bhat et al., 2021 ; Cavallari et al., 2017 ; Clabaut et al., 2015 ; Eitan et al., 2015 ; Frigerio et al., 2021 ; Lehrich et al., 2021 , 2018 ; Li et al., 2015 ; Liao et al., 2017 ; Mehta et al., 2010 ; Obrochta et al., 2015 ; Pham et al., 2021 ; Urzì et al., 2023 ). Some reports showed that serum‐free or chemically defined medium enhance EV production (Biadglegne et al., 2021 ; Bost et al., 2022 ; Faruqu et al., 2021 ; Figuero, 2021 ).…”