Mesenchymal progenitor cells (MPCs) have shown promise initiating articular cartilage repair, with benefits largely attributed to the trophic factors they secrete. These factors can be found in the conditioned medium (CM) collected from cell cultures, and it is believed that extracellular vesicles (EVs) within this CM are at least partially responsible for MPC therapeutic efficacy. This study aimed to examine the functionality of the EV fraction of CM compared to whole CM obtained from human adipose-derived MPCs in an in vivo murine cartilage defect model. Mice treated with whole CM or the EV fraction demonstrated an enhanced cartilage repair score and type II collagen deposition at the injury site compared to saline controls. We then developed a scalable bioprocess using stirred suspension bioreactors (SSBs) to generate clinically relevant quantities of MPC-EVs. Whereas static monolayer culture systems are simple to use and readily accessible, SSBs offer increased scalability and a more homogenous environment due to constant mixing. This study evaluated the biochemical and functional properties of MPCs and their EV fractions generated in static culture versus SSBs. Functionality was assessed using in vitro MPC chondrogenesis as an outcome measure. SSBs supported increased MPC expression of cartilage-specific genes, and EV fractions derived from both static and SSB culture systems upregulated type II collagen production by MPCs. These results suggest that SSBs are an effective platform for the generation of MPC-derived EVs with the potential to induce cartilage repair.

Small extracellular vesicles (EVs) have emerged as a focal point of EV research due to their significant role in a wide range of physiological and pathological processes within living systems. However, uncertainties about the nature of these vesicles have added considerable complexity to the already difficult task of developing EV‐based diagnostics and therapeutics. Whereas small EVs have been shown to be negatively charged, their surface charge has not yet been properly quantified. This gap in knowledge has made it challenging to fully understand the nature of these particles and the way they interact with one another, and with other biological structures like cells. Most published studies have evaluated EV charge by focusing on zeta potential calculated using classical theoretical approaches. However, these approaches tend to underestimate zeta potential at the nanoscale. Moreover, zeta potential alone cannot provide a complete picture of the electrical properties of small EVs since it ignores the effect of ions that bind tightly to the surface of these particles. The absence of validated methods to accurately estimate the actual surface charge (electrical valence) and determine the zeta potential of EVs is a significant knowledge gap, as it limits the development of effective label‐free methods for EV isolation and detection. In this study, for the first time, we show how the electrical charge of small EVs can be more accurately determined by accounting for the impact of tightly bound ions. This was accomplished by measuring the electrophoretic mobility of EVs, and then analytically correlating the measured values to their charge in the form of zeta potential and electrical valence. In contrast to the currently used theoretical expressions, the employed analytical method in this study enabled a more accurate estimation of EV surface charge, which will facilitate the development of EV‐based diagnostic and therapeutic applications.

Interrupted blood flow in the brain due to ischemic injuries such as ischemic stroke or traumatic brain injury results in irreversible brain damage, leading to cognitive impairment associated with inflammation, disruption of the blood–brain barrier (BBB), and cell death. Since the BBB only allows entry to a small class of drugs, many drugs used to treat ischemia in other tissues have failed in brain-related disorders. The administration of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) has shown promise in improving the functional recovery of the brain following cerebral ischemia by inducing blood vessel formation. To facilitate such a treatment approach, it is necessary to develop bioprocesses that can produce therapeutically relevant MSC-EVs in a reproducible and scalable manner. This study evaluated the feasibility of using stirred suspension bioreactors (SSBs) to scale-up the serum-free production of pro-angiogenic MSC-EVs under clinically relevant physioxic conditions. It was found that MSCs grown in SSBs generated EVs that stimulated angiogenesis in cerebral microvascular endothelial cells, supporting the use of SSBs to produce MSC-EVs for application in cerebral ischemia. These properties were impaired at higher cell confluency, outlining the importance of considering the time of harvest when developing bioprocesses to manufacture EV populations.

The development of novel treatments that restore brain function and improve patient outcomes for Alzheimer's disease (AD) is necessary, given the complications and lack of improvement in recently approved amyloid beta (Aβ)‐targeting drugs. Cell‐derived extracellular vesicles (EVs) have been found to improve cognitive function through reduced inflammation, oxidative stress, and apoptosis, restoring neuronal and blood‐brain barrier function, and inhibiting Aβ and phosphorylated tau build‐up in the brain. Given the recent emergence of EVs into clinical trials, it is essential to provide the field with an update on proposed mechanisms of action, gaps in knowledge for further study, and recommendations for producing EVs with high therapeutic efficacy to ensure success in subsequent clinical trials. This systematic review summarizes original research to date that reports effects of mammalian cell‐derived EVs for the treatment of AD. Evidence of therapeutic benefits and reported mechanisms of action are discussed. Further, methods for engineering EVs to increase their therapeutic efficacy and produce high‐quality EVs relevant to the AD field are outlined. The quality of evidence is discussed in terms of reporting guidelines from the Minimal Information for Studies of Extracellular Vesicles (MISEV). The review further discusses current preclinical AD models and provides direction to improve the quality of AD models for testing novel therapeutics.

The immunomodulatory and regenerative effects of mesenchymal stem cell (MSC) extracellular vesicles (EVs) have spurred the development of strategies to manufacture these EVs as therapeutics. Clinical‐grade EV manufacturing requires defined serum‐free media (SFM) to reduce heterogeneity and improve batch reproducibility, yet standard protocols for collecting EVs from SFM are lacking. A primary concern is the co‐isolation of SFM‐derived proteins with EVs during separation, which can interfere with the characterization of the collected EVs, and their subsequent application. In this study, we evaluated how removing proteins from SFM affects EV yield and purity, and examined how collection timing and cell confluence influence EV yield and proteomic profile. A defined SFM (PPRF‐msc6) was compared to (i) an ultracentrifuged medium (PPRF‐msc6 after overnight ultracentrifugation), and (ii) a starvation medium (PPRF‐msc6 without albumin and fetuin). MSC growth and viability were reduced in starvation medium, but were not adversely impacted when using ultracentrifuged medium. Compared to unmodified PPRF‐msc6, ultracentrifuged medium improved EV purity but lowered EV yield. MSC confluence impacted the proteomic profile of EV fractions, demonstrating the importance of determining when EVs are collected during the culture period. Defining protocols for MSC‐EV collection contributes to standardization within the rapidly growing EV field, and informs the development of clinically relevant bioprocesses.

1. Growing practical and ethical concerns regarding the use of animals for disease modeling and drug testing are motivating improvements to the current drug development process. Drug development ta...