Bomb, Kartik2023-10-092023-10-092023https://udspace.udel.edu/handle/19716/33410The immune system, consisting of innate and adaptive immunity, acts as the body's defense mechanism to protect against disease-causing pathogens. Innate immune cells, such as macrophages and dendritic cells, act as an initial line of defense in the human body to protect against pathogens non-specifically. Adaptive immune cells, such as B and T-cells, are a part of acquired immunity that specifically targets the disease-causing agent. In a healthy state, innate and adaptive immune cells work together to protect the body against pathogens. However, in a diseased state, innate and adaptive immune cells execute a complex cascade of events that can reinforce or exacerbate disease progression. Engineered systems are needed to understand these complex processes and direct them for improved treatments. Synthetic culture platforms, such as hydrogels, present opportunities to dissect this complexity and mimic key aspects of tissue microenvironments in both i) fundamental studies of immune cell responses to specific extracellular stimuli (e.g., microenvironment changes during disease progression) and ii) applied studies that utilize these extracellular cues to direct immune cell phenotype and enable their engineering and expansion. In this thesis, I designed and applied hydrogel-based platforms with tunable biophysical and biochemical properties to investigate and modulate responses of both innate and adaptive immune cells with the goal of developing new therapeutic strategies and cell manufacturing approaches. ☐ Focusing first on innate immunity, I studied the role of two individual components that are critical in developing the hydrogel-based in vitro culture platform: cells and biomaterials. For innate immune cells, I studied how tissue of origin can impact cell response, particularly for macrophages. By culturing cells from different origins, including lungs, peritoneal cavity, and monocyte-derived, I documented aspects of both phenotype and function that are heavily influenced by macrophage tissue origin. Next, I studied how to tune the properties of our hydrogel platform based on the application of interest, with a focus on probing macrophage responses to microenvironment cues. To develop a 2D hydrogel culture platform with tunable degradation or stability for macrophage culture, I investigated how the chirality of the amino acids plays a role in the degradability and cytocompatibility of both peptides and peptide-linked hydrogels. By systematically substituting D-amino acids for L-amino acids within a commonly used linker peptide sequence (VPMS↓MRGG), I successfully created a library of peptide sequences with increased resistance to enzymatic degradation. However, this trend was accompanied by increased cytotoxicity for immune cells. This work established strategies that could be easily employed to design and evaluate other enzymatically responsive linker peptides with tunable degradation properties and the important interplay between peptide D-AA content, degradability, and cytotoxicity. ☐ Based on the insights gained from these studies, I created a culture platform with tunable biophysical and biochemical properties inspired by the pulmonary microenvironment to study the role of macrophages in the initiation and progression of fibrosis. I utilized a 2-factorial design of experiment (DOE) approach to study how macrophages respond to pro-fibrotic stimuli: increased matrix stiffness and IL-13, a profibrotic soluble factor linked with disease severity. This approach allowed us to study the individual and combinatorial effects of profibrotic stimuli on macrophage phenotype and function. I found that macrophage morphology, phenotype, and particle uptake were influenced by substrate stiffness and IL13 independently. In addition, both stiffness and IL13 worked synergistically to further influence macrophage phenotype and diminish efferocytosis. These results demonstrate how bioinspired hydrogel platforms can be effectively utilized for investigating immune cell responses in diseased states. ☐ Focusing next on adaptive immunity, I established a bioinspired hydrogel platform that can be integrated within a flow-based device for T cell activation and engineering for the production of cell therapies. I first collaboratively created a soft hydrogel platform with bioactive antibodies (anti-CD3 and anti-CD28) to stimulate T cells and found that cells interacting with these platforms had increased proliferation, similar activation, and a more desirable memory phenotype with lower exhaustion compared to cells activated on stiff plastic. I then showed how these soft hydrogels can be incorporated with existing membrane-based flow devices to increase the transduction efficiency of the cells. Overall, I showed how a soft bioinspired material system can effectively tune immune cell responses for cell therapy applications. ☐ In conclusion, my dissertation delves into the use of bioinspired hydrogel platforms to understand and manipulate both innate and adaptive immune cells. I demonstrated new opportunities for tailoring the hydrogels to the application of interest, studying complex immune responses, and manipulating the microenvironment for improved cell therapy manufacturing.BiomaterialsCell therapiesFibrosisHydrogelsImmune cellsThesis1409204403https://doi.org/10.58088/xj7x-hd682023-09-20en