Scholarship & Research
Permanent URI for this communityhttps://scholarworks.montana.edu/handle/1/1
Browse
9 results
Search Results
Item Single cell encapsulation, detection, and sorting of Pseudomonas syringae using drop-based microfluidics(Montana State University - Bozeman, College of Engineering, 2023) Lindsay, Travis Carson; Chairperson, Graduate Committee: Abigail Richards; Connie Chang (co-chair)Bacteria can survive antibiotic or bactericidal treatment through genetic mutations. Even within bacterial populations that are fully susceptible to treatment, a small proportion of cells can have enhanced survival capacity in a phenomenon called persistence. Traditional microbiology methods can fail to identify or isolate these persister cells present within the population. A novel method for high-throughput single cell analyses of microbial populations is that of drop-based microfluidics, in which individual cells can be isolated within picoliter-sized drops. In this work, fluorescent detection and dielectrophoresis-based sorting of drops was developed for isolating Pseudomonas syringae persister cells following antimicrobial treatment. We demonstrate: (1) the dielectrophoresis-based sorting of dye-filled 25 micron drops based upon two colors, (2) differences between laser-induced fluorescent detection of dyes compared to single bacterial cells, (3) single-cell isolation of P. syringae into 25 micron droplets with ~10% of droplets containing singlecells, and (4) the treatment, staining, and fluorescent characterization of P. syringae at 0.5x, 5x, and 50x the minimum inhibitory concentration of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), an antibiotic which resulted in 6.2%, 10.2%, and 88.6% cell death of the population, respectively. These results provide the groundwork for studying antibiotic-treated P. syringae and the isolation of surviving cells that will lend insight into the molecular basis of persistence for preventing recurrent infections and decreasing the likelihood of antibiotic resistance.Item Microgels for single-cell culturing of neurons and chondrocytes(Montana State University - Bozeman, College of Engineering, 2023) Fredrikson, Jacob Preston; Chairperson, Graduate Committee: Abigail Richards; This is a manuscript style paper that includes co-authored chapters.Tissue engineering is a multidisciplinary field that combines engineering and life sciences to restore, improve, or generate biological substitutes to replace damaged tissues or organs. This is often performed using hydrogels that serve as scaffolds for the growth and maintenance of target tissues. Hydrogels, crosslinked polymer networks composed primarily of water, are excellent tissue mimics with highly tunable mechanical and biochemical properties. Hydrogels can be fabricated at the microscale, termed microgels, using drop-based microfluidics, which enables the precise control of cell density within the microgels down to a single cell. Encapsulating cells in microgels allows for the manipulation of microgels after production for single cell analyses. In this dissertation, human articular cartilage (HAC) cells and neurons are cultured within and upon microgel particles that serve as microscale tissue models for the study of chondrocyte matrix production and Herpes Simplex Virus type -1 (HSV-1) infection studies. HAC is the load-bearing tissue that lines the interfaces of joints and is responsible for shock and wear resistance. Chondrocytes, the cells in HAC, are responsible for producing and maintaining HAC. The chondrocyte pericellular matrix (PCM) regulates the metabolism and mechanical strain of the cells, which is critical to cellular function and cartilage homeostasis. However, the PCM is challenging to produce in vitro. The first half of this work applies microgels for PCM formation in chondrocytes. Immunofluorescence and high-performance liquid chromatography-mass spectrometry data demonstrate that chondrocytes grown in alginate microgels form a collagen VI-rich PCM, significantly altering the cells' metabolic response to dynamic compression. Atomic force microscopy data demonstrates that when chondrocytes are grown in alginate microgels for ten days, the elastic modulus of the PCM increases an order of magnitude. HSV-1 is a human pathogen that invades the peripheral nervous system. Understanding the complexities of HSV-1 infection at the single-cell level could lead to better therapeutics and reduced disease outcomes. Drop-based microfluidics (DBM) has recently been adapted for studying single-cell viral infection but has not been applied to neurons and HSV-1. The second half of this work develops a method for growing individual neurons in microgels. These microgel-embedded neurons are isolated, encapsulated with precise inoculating doses of HSV-1 using DBM, and the kinetics of viral gene expression are tracked in individual neurons using a fluorescent-recombinant HSV-1 virus. The data demonstrate that microgels provide a solid scaffold for neuronal development that supports single-cell productive HSV-1 infection within droplets.Item Spatiotemporal mapping of oxygen in model porous media biofilms using 19 F magnetic resonance oximetry(Montana State University - Bozeman, College of Engineering, 2019) Simkins, Jeffrey William; Chairperson, Graduate Committee: Philip S. Stewart and Joseph D. Seymour (co-chair); Philip S. Stewart and Joseph D. Seymour were co-authors of the article, 'Spatiotemporal mapping of oxygen in a microbially-impacted packed bed using 19 F nuclear magnetic resonance oximetry' in the journal 'The journal of magnetic resonance' which is contained within this dissertation.; Philip S. Stewart, Sarah L. Codd and Joseph D. Seymour were co-authors of the article, 'Non-invasive imaging of oxygen concentration in a complex in vitro biofilm infection model using 19 F MRI: persistence of an oxygen sink despite prolonged antibiotic therapy' submitted to the journal 'Magnetic resonance in medicine' which is contained within this dissertation.; Philip S. Stewart and Joseph D. Seymour were co-authors of the article, 'Microbial growth rates and local external mass transfer resistance in a porous bed biofilm system measured by 19 F magnetic resonance imaging of structure, oxygen concentration, and flow velocity' submitted to the journal 'Biotechnology and bioengineering' which is contained within this dissertation.Biofilms, microbial aggregates anchored to a surface using a sticky matrix of metabolic products called extracellular polymeric substances (EPS), are the dominant form of bacterial life and are widespread in nature, from glaciers to hot springs. The transition from the planktonic state to a biofilm is associated with striking changes to microbial phenotype which confer unique, biofilm-specific properties to resident cells that have important implications for medicine, industry, and environmental study. Many of these properties are caused in large part by oxygen transport limitation, which arises due to restriction of fluid flow in cell aggregates and consumption of oxygen for respiration. The balance of reactive and diffusive processes establishes strong spatial gradients in oxygen concentration which lead to profound spatial heterogeneity in bacterial species composition, growth yield, antimicrobial susceptibility, and reaction kinetics, among other traits. However, despite the importance of oxygen gradients in a host of highly-relevant biofilm phenomena, quantification of oxygen profiles in biofilms is difficult, both in the field and the lab, with the gold standard of measurement, the microelectrode, having significant limitations. 19 F Nuclear Magnetic Resonance (NMR) oximetry, a magnetic resonance-based technique for oxygen quantification that has been used to characterize oxygen usage in blood tissues and tumors, exploits the linear dependence of spin-lattice relaxation rate R 1 on local oxygen partial pressure for fluorine nuclei in perfluorocarbon (PFC) phases. In the current work, we apply 19 F NMR oximetry to a model packed bed biofilm system to generate novel insights into microbial oxygen usage and to introduce a complimentary oximetry tool for biofilm experimenters. We develop methodology for the introduction and fixation of a fluorinated oxygen sensor to facilitate long-term oxygen monitoring. We use 19 F oxygen distribution measurements in compliment to traditional NMR methods to correlate fluid flow with growth rate, generate spatial maps of oxygen utilization rate, identify differences in oxygen utilization behavior between different species, characterize infection persistence during antibiotic therapy, mathematically model macroscale oxygen sink development, and quantify local mass transfer phenomena.Item Gastrointestinal organoid structure and transport(Montana State University - Bozeman, College of Engineering, 2019) Sidar, Barkan; Chairperson, Graduate Committee: James Wilking; Thomas A. Sebrell was an author and Rachel Bruns, Royce A. Wilkinson, Blake Wiedenheft, Paul J. Taylor, Brian A. Perrino, Linda C. Samuelson, James N. Wilking and Diane Bimczok were co-authors of the article, 'Live imaging analysis of human gastric epithelial spheroids reveals spontaneous rupture, rotation, and fusion events' in the journal 'Cell and tissue research' which is contained within this dissertation.; Thomas A. Sebrell, Bengisu Kilic, David Brown, Mert Aytac, Brian A. Perrino, Linda C. Samuelson, Henry Fu, Diane Bimzcok, James N. Wilking were co-authors of the article, 'Rupturing of human gastric organoids' which is contained within this dissertation.; Brittany R. Jenkins, Sha Huang, Jason R. Spence, Seth T. Walk and James N. Wilking were co-authors of the article, 'Flow through human intestinal organoids with the gut organoid flow chip (GOFlowChip)' submitted to the journal 'Lab on a Chip' which is contained within this dissertation.; Dissertation contains two articles of which Barkan Sidar is not the main author.Organoids are three-dimensional (3D) self-assembled, mammalian tissue cultures derived from stem cells that differentiate to contain multiple cell types. These cells spatially organize within the 3D structure and are capable of recapitulating the structure and function of a particular organ. Organoids offer a variety of existing and potential applications in medicine and biotechnology, including drug formulation testing, regenerative medicine, and microbiome research. Despite their value, knowledge of how organoid structure impacts dynamics, mechanics, and transport is lacking. This is particularly true for gastrointestinal organoids, which are composed of a monolayer-thick epithelial sheet wrapped into a closed sphere. The primary goals of this dissertation are to understand the impact of gastrointestinal organoid structure on organoid function, develop a millifluidic chip platform to improve their viability and reliability as a model system and to explore their uses as model co-culture systems. To achieve this, we use a combination of time-lapse microscopy, image analysis, modeling, and fluidics fabrication techniques to develop an understanding of organoid growth and development in addition to expanding current uses as model systems. Our observations revealed that human gastric organoid growth was associated with cyclic rupture of the epithelial shell, rotational movement around their axes within the Matrigel matrix and luminal fusion by adjacent organoids. Furthermore, the rupture events are an indirect result of osmotic swelling carried out by the diffusion of water due to osmolyte concentration regulation by the epithelial shell. To overcome the advection limitation due to the topologically closed spherical structure of the organoids, we developed a millifluidic device called the Gut Organoid Flow Chip (GOFlowChip). This represents the first demonstration of established liquid flow through the luminal space of a gastrointestinal organoid. Given that organoids show great potential as model systems, established co-culture system consisting of dendritic cells (DC) with infected human gastric organoids shows the gastric epithelium actively recruits DCs for immunosurveillance with increased recruitment upon active Helicobacter pylori infection. Finally, investigation on CD103 attachment protein in gastric DCs revealed that CD103 engages in DC-epithelial cell interactions upon contact with epithelial E-cadherin but is not a significant driver of DC adhesion to gastrointestinal epithelia.Item Complexation of lipids with cyclodextrin carriers for fully defined supplementation of cell culture(Montana State University - Bozeman, College of Letters & Science, 2019) Corbin, Elizabeth Dale; Chairperson, Graduate Committee: Edward Dratz and Renee Reijo Pera (co-chair)Induced Pluripotent Stem Cells (iPSCs) hold great promise for revolutionizing medicine and research. Scientists are currently able to reprogram adult cells of almost any type to a genetically 'open' state, pluripotency, wherein they lose the characteristics of their original cell type, and revert to a state that can reproduce indefinitely, and can be differentiated to many different cell types. IPSCs are currently grown in 'chemically defined' media that contains no animal derived components. This media eliminates animal and human sera because these tend to be quite variable and confound the reprogramming process, but the chemically defined media in use has almost no lipid content. The central goal of this project was to develop methods for chemically defined addition of lipids to cell culture media. The methods developed promise to be an advance in stem cell and general cell culture methodology, providing more reproducible experimental results, and supporting cells in culture with optimized lipid contents. In order to facilitate the addition of lipids to cell culture media without animal serum or serum albumin, complexation of individual lipids with a methyl beta-cyclodextrin starch was accomplished without addition of other molecules or oxidation of delicate lipids at a 1:1 stoichiometry. The lipid/MBCD complexes are soluble in aqueous media, and can be added individually or as a mixture to cell cultures. Application of complexed lipids to stem cells and fibroblasts revealed significant differences in lipid responses. Supplementation of human fibroblasts with a mixture of complexed lipids and other elements caused a 60% increase in proliferation over a 10 day period. Supplementation of stem cells with complexed lipids significantly increased proliferation, without reduction of pluripotency. Differences in lipid responses were also found between iPSC and embryonic stem cells, that may help elucidate differences between genetic or metabolic states which may point the way for more effective reprogramming of adult cells to pluripotency.Item The use of plant tissue culture in agricultural education in the northwestern states(Montana State University - Bozeman, 1993) Lombardi, Bill L.; Chairperson, Graduate Committee: C. Van ShelhamerItem Transformation of Gaeumannomyces graminis and the fate of transforming DNA(Montana State University - Bozeman, College of Agriculture, 1991) Pilgeram, Alice LaRayne; Co-chairs, Graduate Committee: Don E. Mathre and Joan HensonItem Biology of the eremophilanes produced by Drechslera gigantea(Montana State University - Bozeman, College of Agriculture, 1989) Bunkers, Gregory JamesItem Grasshopper hemagglutinin : immunochemical localization in hemocytes and confirmation of non-opsonic properties(Montana State University - Bozeman, College of Letters & Science, 1987) Bradley, Roger Steven