Browsing by Author "Bomb, Kartik"
Now showing 1 - 3 of 3
Results Per Page
Sort Options
Item Designing hydrogel-based tunable platforms to investigate and modulate immune cell responses(University of Delaware, 2023) Bomb, KartikThe 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.Item Macrophage variance: investigating how macrophage origin influences responses to soluble and physical cues with immortalized vs. primary cells in 2D and 3D culture(Frontiers in Biomaterials Science, 2024-05-22) Graf, Jodi; Bomb, Kartik; Trautmann-Rodriguez, Michael; Jarai, Bader M.; Gill, Nicole; Kloxin, April M.; Fromen, Catherine A.Macrophages are phagocytic innate immune cells capable of phenotypical switching in response to the local microenvironment. Studies often use either primary macrophages or immortalized cell lines for hypothesis testing, therapeutic assessment, and biomaterial evaluation without carefully considering the potential effects of cell source and tissue of origin, which strongly influence macrophage response. Surprisingly, limited information is available about how, under similar stimuli, immortalized cell lines and primary cells respond in both phenotypical and functional changes. To address this need, in this work, we cultured immortalized macrophage cell lines derived from different origins (i.e., blood, lung, peritoneal) to understand and compare macrophage phenotypical responses, including polarization and plasticity, morphological changes, and phagocytic functionalities, as well as compared primary macrophages extracted from peritoneal and bone marrow to their immortalized cell line counterparts. We found significant differences in baseline expression of different markers (e.g., CD86, MHCII, CD206, and EGR2) amongst different cell lines, which further influence both polarization and repolarization of the cells, in addition to their phagocytic functionality. Additionally, we observed that, while RAW 264.7 cells behave similarly to the primary bone marrow-derived macrophages, there are noticeable phenotypical and functional differences in cell line (IC-21) and primary peritoneal macrophages, highlighting tissue-specific differences in macrophage response amongst cell lines and primary cells. Moving to three-dimensional (3D) culture in well-defined biomaterials, blood-derived primary and cell line macrophages were encapsulated within hydrogel-based synthetic extracellular matrices and their polarization profiles and cell morphologies were compared. Macrophages exhibited less pronounced polarization during 3D culture in these compliant, soft materials compared to two-dimensional (2D) culture on rigid, tissue culture plastic plates. Overall, our findings highlight origin-specific differences in macrophage response, and therefore, careful considerations must be made to identify the appropriate cell source for the application of interest.Item Nanoparticle pre-treatment for enhancing the survival and activation of pulmonary macrophage transplant(Drug Delivery and Translational Research, 2023-03-14) Jarai, Bader M.; Bomb, Kartik; Fromen, Catherine A.Despite recent clinical successes of chimeric antigen receptor T cell therapies in treating liquid cancers, many lingering challenges stand in the way of therapeutic translation to broader types of malignancies. Macrophages have been proposed as alternatives to T cells given macrophages’ advantages in promoting tumor infiltration, acquiring diverse antigens, and possessing the ability to continuously stimulate adaptive responses. However, the poor survival of macrophages upon transplantation in addition to transient anti-tumor phenotypical states have been major obstacles standing in the way of macrophage-based cell therapies. Given recent discoveries of nanoparticle strategies in improving macrophage survival and promoting phenotype retention, we herein report the ability to extend the survival and phenotype of macrophage transplants in murine lungs via pre-treatment with nanoparticles of varying degradation rates. Macrophages pre-treated with 100 µg/ml dose of poly(ethylene glycol) diacrylate nanoparticle formulations improve pulmonary macrophage transplant survival over untreated cells beyond 7 days, where degradable nanoparticle formulations result in over a 50% increase in retention of transplanted cell counts relative to untreated cells. Furthermore, pre-treated macrophages more efficiently retain an imposed pro-inflammatory-like polarization state following transplantation out to 7 days compared to macrophages pre-treated with a classical pro-inflammatory stimulus, interferon-gamma, where CD86 costimulatory molecule expression is greater than 150% higher in pre-treated macrophage transplants compared to untreated counterparts. These findings provide an avenue for a major improvement in the lifespan and efficacy of macrophage-based cell therapies and have broader implications to other phagocyte-based cellular therapeutics and administration routes. Graphical abstract available at: https://doi.org/10.1007/s13346-023-01319-6