Browsing by Author "Phelps, Jolene"

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    Bioprocessing of Mesenchymal Stem Cells and Their Derivatives: Toward Cell-Free Therapeutics
    (2018-09-12) Phelps, Jolene; Sanati-Nezhad, Amir; Ungrin, Mark; Duncan, Neil A.; Sen, Arindom
    Mesenchymal stem cells (MSCs) have attracted tremendous research interest due to their ability to repair tissues and reduce inflammation when implanted into a damaged or diseased site. These therapeutic effects have been largely attributed to the collection of biomolecules they secrete (i.e., their secretome). Recent studies have provided evidence that similar effects may be produced by utilizing only the secretome fraction containing extracellular vesicles (EVs). EVs are cell-derived, membrane-bound vesicles that contain various biomolecules. Due to their small size and relative mobility, they provide a stable mechanism to deliver biomolecules (i.e., biological signals) throughout an organism. The use of the MSC secretome, or its components, has advantages over the implantation of the MSCs themselves: (i) signals can be bioengineered and scaled to specific dosages, and (ii) the nonliving nature of the secretome enables it to be efficiently stored and transported. However, since the composition and therapeutic benefit of the secretome can be influenced by cell source, culture conditions, isolation methods, and storage conditions, there is a need for standardization of bioprocessing parameters. This review focuses on key parameters within the MSC culture environment that affect the nature and functionality of the secretome. This information is pertinent to the development of bioprocesses aimed at scaling up the production of secretome-derived products for their use as therapeutics.
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    Physiological oxygen conditions enhance the angiogenic properties of extracellular vesicles from human mesenchymal stem cells
    (2023-08-23) Phelps, Jolene; Hart, David A.; Mitha, Alim P.; Duncan, Neil A.; Sen, Arindom
    Abstract Background Following an ischemic injury to the brain, the induction of angiogenesis is critical to neurological recovery. The angiogenic benefits of mesenchymal stem cells (MSCs) have been attributed at least in part to the actions of extracellular vesicles (EVs) that they secrete. EVs are membrane-bound vesicles that contain various angiogenic biomolecules capable of eliciting therapeutic responses and are of relevance in cerebral applications due to their ability to cross the blood–brain barrier (BBB). Though MSCs are commonly cultured under oxygen levels present in injected air, when MSCs are cultured under physiologically relevant oxygen conditions (2–9% O2), they have been found to secrete higher amounts of survival and angiogenic factors. There is a need to determine the effects of MSC-EVs in models of cerebral angiogenesis and whether those from MSCs cultured under physiological oxygen provide greater functional effects. Methods Human adipose-derived MSCs were grown in clinically relevant serum-free medium and exposed to either headspace oxygen concentrations of 18.4% O2 (normoxic) or 3% O2 (physioxic). EVs were isolated from MSC cultures by differential ultracentrifugation and characterized by their size, concentration of EV specific markers, and their angiogenic protein content. Their functional angiogenic effects were evaluated in vitro by their induction of cerebral microvascular endothelial cell (CMEC) proliferation, tube formation, and angiogenic and tight junction gene expressions. Results Compared to normoxic conditions, culturing MSCs under physioxic conditions increased their expression of angiogenic genes SDF1 and VEGF, and subsequently elevated VEGF-A content in the EV fraction. MSC-EVs demonstrated an ability to induce CMEC angiogenesis by promoting tube formation, with the EV fraction from physioxic cultures having the greatest effect. The physioxic EV fraction further upregulated the expression of CMEC angiogenic genes FGF2, HIF1, VEGF and TGFB1, as well as genes (OCLN and TJP1) involved in BBB maintenance. Conclusions EVs from physioxic MSC cultures hold promise in the generation of a cell-free therapy to induce angiogenesis. Their positive angiogenic effect on cerebral microvascular endothelial cells demonstrates that they may have utility in treating ischemic cerebral conditions, where the induction of angiogenesis is critical to improving recovery and neurological function.
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    The Impact of Culture Environment on the Composition and Function of Mesenchymal Stem Cell Derived Extracellular Vesicles
    (2022-05-30) Phelps, Jolene; Sen, Arindom; Duncan, Neil A.; Ungrin, Mark; Sanati-Nezhad, Amir
    Mesenchymal stem cells (MSCs) have attracted research interest due to their ability to induce tissue repair and reduce inflammation. More recently, their therapeutic benefits have been attributed to small membrane bound vesicles called extracellular vesicles (EVs) that they release. The implantation of MSC-EVs offers significant advantages over the implantation of viable MSCs. Their nonliving nature eliminates many of the safety concerns related to cell therapies and enables them to be stored and transported more efficiently. There is a significant need to develop clinically applicable bioprocesses that can produce large numbers of therapeutically relevant MSC-EVs. This involves the translation of small-scale studies to large scale processes, which is limited by considerations such as the medium and platform in which the cells are cultured. Due to the novelty of the EV field, current research in this area is very limited, and thus the goal of this thesis was to evaluate the efficacy of producing MSC-EVs in a clinically relevant medium, and to gain insight into the impact of culture parameters needed for the scale-up of MSC-EVs. This thesis demonstrated, for the first time, that functional MSC-EVs could be produced using a clinically relevant serum-free culture medium for the applications of articular cartilage repair, cerebral ischemia, and intracranial aneurysm repair. Further, it was found that functional MSC-EVs could be produced in scalable stirred suspension bioreactors. These studies were among the first to evaluate the use of such a platform for EV production and were the first to present a scalable model for EV production specifically for the applications of articular cartilage repair and cerebral ischemia. In addition, the culture of MSCs at physiological oxygen conditions was evaluated and found to enhance their angiogenic potential, without damaging physiological function, in the application of cerebral ischemia. Finally, important considerations relevant to the upstream production of MSC-EVs were studied, including the confluence of cultured MSCs and the medium in which the EVs were isolated. This work enabled a proposed workflow for producing EVs in serum-free medium and will contribute highly to the development of clinically applicable bioprocesses aimed at producing MSC-EVs for applications in regenerative medicine.