Culture Condition of Bone Marrow Stromal Cells Affects Quantity and Quality of the Extracellular Vesicles

Abstract

Extracellular vesicles (EVs) released by bone marrow stromal cells (BMSCs) have been shown to act as a transporter of bioactive molecules such as RNAs and proteins in the therapeutic actions of BMSCs in various diseases. Although EV therapy holds great promise to be a safer cell-free therapy overcoming issues related to cell therapy, manufacturing processes that offer scalable and reproducible EV production have not been established. Robust and scalable BMSC manufacturing methods have been shown to enhance EV production; however, the effects on EV quality remain less studied. Here, using human BMSCs isolated from nine healthy donors, we examined the effects of high-performance culture media that can rapidly expand BMSCs on EV production and quality in comparison with the conventional culture medium. We found significantly increased EV production from BMSCs cultured in the high-performance media without altering their multipotency and immunophenotypes. RNA sequencing revealed that RNA contents in EVs from high-performance media were significantly reduced with altered profiles of microRNA enriched in those related to cellular growth and proliferation in the pathway analysis. Given that pre-clinical studies at the laboratory scale often use the conventional medium, these findings could account for the discrepancy in outcomes between pre-clinical and clinical studies. Therefore, this study highlights the importance of selecting proper culture conditions for scalable and reproducible EV manufacturing.

MSC-EV therapy for bone/cartilage diseases

“…Similarly, donor variability has been identified in MSC-EVs from different donors. Proteomic and transcriptomic analyses of MSC-EVs from different donors demonstrated differences in their contents ( McBride et al, 2021 ; Ragni et al, 2019 ; Scheiber et al, 2022 ). These differences in cargo lead to the functional alteration of MSC-EVs, raising the challenge of reproducibility of EV therapies using EVs from different donors ( Chance et al, 2019 ; Kordelas et al, 2014 ; Kordelas et al, 2019 ).…”

“…Since there is no raw material derived from animals or humans, such media will be more consistent between lots, which potentially makes EV quality more reproducible. Xeno-free media not only helps the rapid expansion of MSCs but also yields significantly greater amounts of EVs compared to conventional FBS-containing media ( Scheiber et al, 2022 ). However, miRNA profiles in EVs from such media are altered ( Scheiber et al, 2022 ).…”

“…Xeno-free media not only helps the rapid expansion of MSCs but also yields significantly greater amounts of EVs compared to conventional FBS-containing media ( Scheiber et al, 2022 ). However, miRNA profiles in EVs from such media are altered ( Scheiber et al, 2022 ).…”

“…EVs have been demonstrated to contain a complex array of bioactive molecules that includes proteins ( Tkach and Théry, 2016 ), lipids ( Skotland et al, 2017 ), and nucleic acids such as messenger RNA (mRNA) ( Valadi et al, 2007 ), micro RNA (miRNA) ( Nazari-Shafti et al, 2020 ; Scheiber et al, 2022 ), long noncoding RNA (lncRNA) ( Wen et al, 2022 ), transfer RNA (tRNA) ( Baglio et al, 2015 ), genomic DNA( Kahlert et al, 2014 ; Thakur et al, 2014 ), and mitochondrial DNA (mtDNA) ( Guescini et al, 2010 ). EVs mediate intercellular communication by transferring such cargo to each other ( Tkach and Théry, 2016 ; Valadi et al, 2007 ).…”

“…In addition to the quality of EVs, the quantity must be considered for clinical usage involving multiple injections throughout the treatment ( Adlerz, 2020 ; Colao et al, 2018 ; Phinney and Pittenger, 2017 ). However, scalable manufacturing of EVs also affects the quality of EVs ( Haraszti et al, 2018 ; Patel et al, 2017 ; Scheiber et al, 2022 ). Therefore, in this review, to overcome these challenges and achieve effective and reliable EV therapy, we highlight the key elements that can affect EV quality followed by a summary of the pre-clinical and clinical EV studies surrounding skeletal diseases.…”