Impact of cell culture parameters on production and vascularization bioactivity of mesenchymal stem cell‐derived extracellular vesicles

Abstract

Mesenchymal stem cell (MSC)‐derived extracellular vesicles (EVs) have emerged as potential therapeutic agents for numerous applications. EVs offer potential advantages over cell‐based therapies with regard to safety, stability and clearance profiles, however production and potency limitations must be addressed to enable eventual translation of EV‐based approaches. Thus, we sought to examine the role of specific cell culture parameters on MSC EV production and bioactivity toward informing rational design parameters for scalable EV biomanufacturing. We report significantly reduced MSC EV vascularization bioactivity, as measured by an endothelial cell gap closure assay, with increasing passage in culture by trypsinization, especially beyond passage 4. We further show that increased frequency of EV collection yielded higher numbers of EVs from the same initial number of MSCs over a 24 hr period. Finally, we demonstrate that decreased cell seeding density in culture flasks resulted in increased production of EVs per cell in MSCs and other cell types. Overall, these studies highlight the need for careful consideration of the parameters of cell passage number and cell seeding density in the production of therapeutic EVs at laboratory scale and for rational design of large‐scale EV biomanufacturing schemes.

Physical confinement alters cytoskeletal contributions towards human mesenchymal stem cell migration

“…Previous reports have shown that increasing hMSC passage number decreases the invasiveness and bioactivity of hMSCs (Patel et al, ; Spagnol et al, ), so we investigated hMSC speed as a function of passage and of confinement. hMSCs at passage 3, 4, or 5 (P3, P4, or P5) were seeded in the microfluidic device, and the speed of hMSCs migrating within microchannels was subsequently tracked.…”

“…Previous reports have indicated that hMSC behavior can be passage dependent, as increased passage leads to loss of hMSC proliferative capacity and multipotency (Banfi et al, ), and also reduced exosome production and invasiveness (Patel et al, ; Spagnol et al, ). Therefore, we hypothesized that passage number would influence hMSC migration, possibly as a function of confinement.…”

Therapeutic Advances of Stem Cell-Derived Extracellular Vesicles in Regenerative Medicine

“…Low-density inoculation (1.5 cells/cm 2 ) may result in faster stem cell proliferation [84]. Low-density cultures may activate EV-mediated paracrine signaling of stem cells and promote EV secretion [85].…”

“…Stem cells alter phenotypes and functions as the in vitro expansion time increases. Early MSCs have the potential for osteogenic and adipogenic differentiation, and the differentiation potential tilts toward adipogenesis as the in vitro culture is extended [85]. MSCs secrete less EVs after aging [85].…”

Editorial: The launch phase of Bioengineering & Translational Medicine

“…In terms of scientific topics, BioTM has focused on subjects where Engineering, Chemical Engineering in particular, has made a strong fundamental and translational impact on the field. These topics include drug delivery, Nucleic acid delivery, Regenerative Medicine, Biotechnology, Nanomedicine, Biomedical Devices, and immunotherapy . We look forward to continue building our strengths in these fields and expand the list to include additional key areas.…”