Production of extracellular vesicles from equine embryo-derived mesenchymal stromal cells

Abstract

Mesenchymal stromal cells (MSC) have recently been explored for their potential use as therapeutics in human and veterinary medicine applications, such as the treatment of endometrial inflammation and infertility. Allogeneic MSC-derived extracellular vesicles (EVs) may also provide therapeutic benefits with advantage of being an “off-the-shelf” solution, provided they can be produced in large enough quantities, without contamination from bovine EVs contained in fetal bovine serum that is a common component of cell culture media. Towards this aim, we demonstrated the successful isolation and characterisation of equine MSCs from pre-implantation embryos. We also demonstrate that many of these lines can be propagated long-term in culture while retaining their differentiation potential, and conducted a head-to-head comparison of two bioreactor systems for scalable EV production including in serum-free conditions. Based on our findings, the CELLineTM AD 1000 flasks enabled higher cell density cultures and significantly more EV production than the FiberCellTM system or conventional culture flasks. These findings will enable future isolation of equine MSCs and the scalable culture of their EVs for a wide range of applications in this rapidly growing field.

Investigating the consistency of extracellular vesicle production from breast cancer subtypes using CELLine adherent bioreactors

“…Due to the structure of the CELLine AD 1000 bioreactor, it is not possible to view the health or status of the cells besides inspecting for media colour (phenol red pH indicator, which is unreliable according to the manufacturer ( CELLine User Manual )), bubbles in the cell chamber, or analysis of the contents of the conditioned media once removed from the chamber. While glucose levels have been used in past studies as a means of monitoring (Jackson et al., 1996 ; Mittermaier, 2004 ; Tasma et al., 2022 ), media volume, shed cells and EVs can be easily monitored throughout the life of the bioreactor. Due to the natural turnover of cells on the growth surface and a limited live cell capacity ( CELLine User Manual ), we hypothesised that shed cells could act as a surrogate marker of the bioreactor's health.…”

“…In addition, in‐house depletion methods via ultracentrifugation and filtration are not capable of removing all of the exogenous EVs (Lehrich et al., 2021 ). The CELLine bioreactors avoid these issues by allowing FBS‐containing media to be used in the upper media chamber with contaminant bovine EVs excluded from entering the lower cell conditioned media by the 10‐kDa filter membrane (Tasma et al., 2022 ). However, we recommend seeding the cells in serum‐replete media to support adhesion to the growth surface prior to steady conversion of the media into serum depleted media to then commence EV collection.…”

“…While this bioreactor requires a pump system, it has been reported to produce significantly greater numbers of EVs per volume compared to conventional culture flasks. It has been primarily used for anti‐cancer or regenerative medicine therapeutic applications by producing EVs from mesenchymal stromal cells (Cao et al., 2020 ; Maji et al., 2017 ; Ouyang et al., 2019 ; Yan & Wu, 2020 ) or engineered HEK293 cells (Tasma et al., 2022 ; Watson et al., 2016 ; Watson et al., 2018 ; Yoo et al., 2018 ). Another interesting and related design is a 3D‐printed perfusable bioreactor that was used to produce EVs from endothelial cells and to determine whether ethanol conditioning could improve the bioactivity of the isolated EVs (Patel et al., 2019 ).…”

Light-induced Extracellular Vesicle Adsorption

“…CELLine AD 1000 bioreactors (Wheaton) were used for EV production following an established adaptation and EV collection and isolation protocol [54, 55, 68]. Briefly, bioreactors were adapted from complete media with 10 % fetal bovine serum (FBS) supplementation, then adapted to CDM-HD (Fiber-cell).…”

Manifold Learning Enables Interpretable Analysis of Raman Spectra from Extracellular Vesicle and Other Mixtures

“…These non-EV contaminant particles actually demonstrate the robust nature of label-free SERS combined with this analytical approach, as highly heterogeneous spectra can still be used for robust quantification. Additionally, a small amount of FBS was used in the upper bioreactor chamber throughout the MDA-MB-231 culture such that small molecules (¡10 kDa) could pass through the semi-permeable dialysis membrane and interact with the MDA-MB-231 cells and EVs while the FBS EVs were completely excluded from the cell chamber and the conditioned media [56]. Thus, these two EV samples are ideal, albeit simplified models that reflect unknown EV mixtures from conventional cell cultures.…”