A. James Clark School of Engineering

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The collections in this community comprise faculty research works, as well as graduate theses and dissertations.

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    Assessment of Mechanical Cues to Enhance the Clinical Translation of Extracellular Vesicles
    (2022) Kronstadt, Stephanie Marie; Jay, Steven M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Mesenchymal stem cells (MSCs) are a common source for cell-based therapies due to their innate regenerative properties. However, these cells often die shortly after injection and, if they do survive, run the risk of forming tumors. Cell-secreted nanoparticles known as extracellular vesicles (EVs) have been identified as having therapeutic effects similar to those of their parental cells without the safety risks. Specifically, MSC EVs have emerged as a promising therapeutic modality in a multitude of applications, including autoimmune and cardiovascular diseases, cancer, and wound healing. Despite this promise, low levels of naturally occurring EV cargo may necessitate repeated doses to achieve clinical benefit, countering the advantages of EVs over MSCs. The current techniques to combat low EV potency (e.g., loading external molecules or using chemicals) are not agreeable to large-scale manufacturing techniques and would substantially increase the regulatory burden associated with EV translation. Fortunately, mechanical cues within the microenvironment have potential to overcome these translational barriers as they can alter EV therapeutic effects but are also cost-effective and can be precisely manipulated in a reproducible manner. The goal of this project is to understand how these cues impact MSC EV secretion and physiological effects. We showed that flow-derived shear stress applied to MSCs seeded within a 3D-printed scaffold (i.e., the bioreactor) can significantly upregulate EV production (EVs/cell) while maintaining the in vitro pro-angiogenic effects of MSC EVs. Interestingly, we demonstrated that MSC EVs generated using the bioreactor system significantly improved wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture-generated MSC EVs. Furthermore, for the first time, we showed that mechanical confinement of MSCs within micropillars could augment MSC EV production and bioactivity. Lastly, we demonstrated that soft substrates composed of various polydimethylsiloxane (PDMS) formulations could increase MSC EV production and activity as well. Through the work performed here, we have laid the groundwork to elucidate the relationship between cell mechanobiology and EV activity that will ultimately enable an adaptable and scalable EV therapeutic platform.
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    Dynamic Coculture of a Prevascularized Engineered Bone Construct
    (2016) Nguyen, Bao-Ngoc Bich; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The generation of functional, vascularized tissues is a key challenge for the field of tissue engineering. Before clinical implantations of tissue engineered bone constructs can succeed, in vitro fabrication needs to address limitations in large-scale tissue development, including controlled osteogenesis and an inadequate vasculature network to prevent necrosis of large constructs. The tubular perfusion system (TPS) bioreactor is an effective culturing method to augment osteogenic differentiation and maintain viability of human mesenchymal stem cell (hMSC)-seeded scaffolds while they are developed in vitro. To further enhance this process, we developed a novel osteogenic growth factors delivery system for dynamically cultured hMSCs using microparticles encapsulated in three-dimensional alginate scaffolds. In light of this increased differentiation, we characterized the endogenous cytokine distribution throughout the TPS bioreactor. An advantageous effect in the ‘outlet’ portion of the uniaxial growth chamber was discovered due to the system’s downstream circulation and the unique modular aspect of the scaffolds. This unique trait allowed us to carefully tune the differentiation behavior of specific cell populations. We applied the knowledge gained from the growth profile of the TPS bioreactor to culture a high-volume bone composite in a 3D-printed femur mold. This resulted in a tissue engineered bone construct with a volume of 200cm3, a 20-fold increase over previously reported sizes. We demonstrated high viability of the cultured cells throughout the culture period as well as early signs of osteogenic differentiation. Taking one step closer toward a viable implant and minimize tissue necrosis after implantation, we designed a composite construct by coculturing endothelial cells (ECs) and differentiating hMSCs, encouraging prevascularization and anastomosis of the graft with the host vasculature. We discovered the necessity of cell to cell proximity between the two cell types as well as preference for the natural cell binding capabilities of hydrogels like collagen. Notably, the results suggested increased osteogenic and angiogenic potential of the encapsulated cells when dynamically cultured in the TPS bioreactor, suggesting a synergistic effect between coculture and applied shear stress. This work highlights the feasibility of fabricating a high-volume, prevascularized tissue engineered bone construct for the regeneration of a critical size defect.
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    Protein Production Development with Recombinant Vaccinia Virus
    (2004-04-29) Bleckwenn, Nicole Aleece; Bentley, William E; Chemical Engineering
    The vaccinia virus expression system was developed into a scaleable recombinant protein production process in perfused mammalian cell culture. Growth of anchorage dependent HeLa cells on microcarriers and the suspension adapted HeLa S3 cell line were studied in bioreactor cultures utilizing the ATF System or hollow fiber filter, respectively, for perfusion. Recombinant vaccinia virus expressing enhanced green fluorescent protein (EGFP) as a model protein was used to study the effects of several process parameters on expression. These included multiplicity of infection (MOI), volume during infection, serum concentration during infection, inducer concentration, timing of inducer addition relative to infection, and dissolved oxygen and temperature during the protein production phase. Increases in protein yield were made as each of these parameters was studied. The microcarrier based system reached 20 mg/l EGFP while the suspension based system achieved 27 mg/l under the conditions found through experiment. A second virus containing the gene for gp120, an HIV envelope coat protein with complex post-translational modifications, was produced in microcarrier based bioreactor culture with HeLa cells. The protein produced was purified and analyzed for post-translational modifications which found that half of the molecular weight was contributed through N-linked glycans. The reactor culture produced 10.5 mg/l gp120 at 96 hours post infection with an ID(50) of 3.1 µg/ml. A survey of expression, using both EGFP and gp120 expressing viruses, was conducted on several mammalian cell lines which may be more appropriate for commercial manufacturing processes. Results varied, depending on the protein produced, with HeLa cells producing the most EGFP and BS-C-1 the most gp120. 293 cells performed fairly well in both cases and their use in other manufacturing processes and ability to grow in serum-free suspension culture lead to a recommendation that they be considered for further process development. These studies have provided insight into the vaccinia virus expression system as a potentially large-scale production method for complex human proteins. Further optimization of the process could continue to increase the yields and potentially bring this viral process into the arena of available technologies for production.