Theses and Dissertations at Montana State University (MSU)
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Item Bacterial cultivation in microscale drops and capsules to resolve single-cell growth physiology(Montana State University - Bozeman, College of Engineering, 2023) Pratt, Shawna Leigh; Chairperson, Graduate Committee: Ross Carlson; This is a manuscript style paper that includes co-authored chapters.Single-cell heterogeneity contributes to the complex population dynamics of infectious microbial communities. Improving our understanding of single-cell physiology and heterogeneity may aid in mitigating microbial infections; however, assaying large populations of single cells can be challenging. Despite recent developments in single-cell assaying, tracking the physiology of large numbers of individual cells and their lineages over time is difficult to achieve using current technologies. Here, I apply drop-based microfluidics to develop microscale tools for improving high-throughput single-cell microbial growth assays. Drop-based microfluidics is a technology that generates and manipulates microscale drops. In this work, I create water-in-oil drops and hydrogel-shelled microcapsules using drop-based microfluidics to study the growth of P. aeruginosa bacteria, a key pathogen implicated in chronic lung infections and wounds. The growth of single bacterial cells inside drop microcompartments is observed via time-lapse confocal microscopy. Bacteria were cultured in water-in-oil drops and prepared for long-term storage in a novel microfluidic device environment, which we call a DropSOAC (Drop Stabilization on a Chip) chamber. The DropSOAC method prevents drop destabilization by saturating microfluidic devices with equilibrated water and oil, maintaining phase equilibrium in the drop emulsion. Using DropSOAC, the single-cell growth of starved P. aeruginosa wildtype and hibernation promotion factor mutants were characterized, revealing significant growth heterogeneity in the mutant strain. Finally, we present a method for generating hydrogel-shelled microcapsules that enables the culturing of single cells in microscale environments where nutrients and waste can diffuse in and out of the microculture environment. A 3-D microfluidic device and capsule generation protocol are designed, resulting in an optimized approach for capsule production using phase-separating polymer systems and rapid hydrogel crosslinking. The growth of hundreds of individual P. aeruginosa cells is observed over time with the hydrogel- shelled microcapsules. Due to the permeability of the microcapsules, antibiotics can be introduced at various times during growth to investigate single and biofilm P. aeruginosa physiology. Overall, this work introduces novel approaches for high-throughput, single-cell microbial growth characterization that enables a deeper understanding of the role of heterogeneity in bacterial populations.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 Colloids, diagnostics, and 3D-printed hydrogels(Montana State University - Bozeman, College of Engineering, 2021) LeFevre, Thomas Brian; Chairperson, Graduate Committee: James Wilking; This is a manuscript style paper that includes co-authored chapters.Colloidal suspensions are dispersions of microscopic particles in liquid. Their properties have broad impacts in industry, medicine, and biology. In Chapter 2, we focus on measuring the interactions between colloidal particles suspended in water and a glass surface. We measure these interactions using a custom-built fluorescence centrifuge force microscope (F-CFM). This is the first CFM built with fluorescence capability, the first CFM used to measure colloidal interaction forces, and the first CFM capable of operating at speeds above 2000 RPM - and up to 5000 RPM - in a centrifuge. The F-CFM enables colloidal scale objects to be discriminated by fluorescence, which opens potential applications for biological samples that fluoresce under different phenotypic states. In Chapter 3, we focus on designing a point-of-care (POC) saliva collection, metering, and mixing system for detecting viral pathogens. The device was designed for the specific purpose of testing for the presence of SARS-CoV-2 in saliva using molecular amplification methods but could be applied to any pathogen whose constituents can be detected in saliva. The design to prioritizes ease of use, low cost, and scalability in order to facilitate massively widespread testing, which was absent during the first years since the emergence of SARS-CoV-2, In Chapter 4, we describe a method of formulating and printing hydrogel resins with high resolution channels using light-based 3D printing. In Chapter 5, we describe a leak-resistant, pressurized connector platform for connecting modular hydrogels that can be used to create complex assemblies of hydrogel components. In Chapter 6, we describe a microscope sample temperature control platform that fits into standard upright microscope stages in order to heat and cool samples in a controlled manner under the microscope in order to observe temperature dependent reactions like polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP). In Chapter 7, we describe a LAMP formulation that can be used to detect the presence of SARS-CoV-2 RNA in saliva despite the inhibitory components present in saliva and demonstrate its comparable accuracy to the gold standard of pathogenic testing: nasopharyngeal PCR testing.Item Improving transport in hydrogels for 3D bioprinting applications(Montana State University - Bozeman, College of Engineering, 2021) Abbasi, Reha; Chairperson, Graduate Committee: James Wilking; Aaron D. Benjamin was an author and Madison Owens, Robert J. Olsen, Danica J. Walsh, Thomas B. LeFevre and James N. Wilking were co-authors of the article, 'Light-based 3D printing of hydrogels with high-resolution channels' in the journal 'Biomedical physics & engineering express' which is contained within this dissertation.; Thomas B. LeFevre was an author and Aaron D. Benjamin, Isaak J. Thornton, and James N. Wilking were co-authors of the article, 'Coupling fluid flow to hydrogel fluidic devices with reversible "pop-it" connections' in the journal 'Lab on a chip' which is contained within this dissertation.; Zahra Mahdieh was an author and Galip Yiyen, Robert A. Walker and James N. Wilking were co-authors of the article, 'Light-based 3D bioprinting of hydrogels containing colloidal calcium peroxide' submitted to the journal 'Bioprinting' which is contained within this dissertation.Hydrogels are soft, water-based gels with widespread applications in medicine, tissue engineering, and biotechnology. Many of these applications require structuring hydrogels in three-dimensional space. Light-based 3D printers offer exquisite spatial control; however, technologies for light-based 3D-printing of hydrogels remain limited. This is mainly caused by poor material transportation through the hydrogel. For example, limited transport of oxygen and other nutrients through 3D printed tissue constructs containing living cells leads to low cell viability. Here, we describe three experimental research studies focused on improving material transport in 3D-printed hydrogels. In the first part of this thesis, we describe a generalizable method for light-based 3D printing of hydrogels containing open, well-defined, submillimeter-scale channels with any orientation. These submillimeter channels allow for bulk liquid flow through the hydrogel to improve nutrient and oxygen transport. In the second part of this thesis, we describe a simple, reversible, plug-based connector designed to couple tubing to a hydrogel-based fluidic device to allow for pressurized liquid flow through the system. The resulting connection can withstand liquid pressures significantly greater than traditional, connector-free approaches, enabling long-term flow through 3D-printed hydrogels. In the third part of this thesis, we characterize the printability of photopolymerizable resins containing particles that slowly dissolve to release oxygen and thereby improve cell viability. The light-based 3D bioprinting technologies we describe in this thesis will improve material transport through 3D printed hydrogels and enable a wide variety of applications in 3D bioprinting and hydrogel fluidics.Item Freeze foaming: a novel process for the synthesis of foam ceramics(Montana State University - Bozeman, College of Engineering, 2018) Johnson, Nathaniel Peyton; Chairperson, Graduate Committee: Stephen W. SofieFoam is a class of materials that was developed only after World War II and ceramic foams are still in development. Many of the processes for synthesizing ceramic foam require the burning out of a polymer scaffold or the use of chemical reactions to generate pores. This thesis investigates the development of a novel synthesis approach called freeze foaming. In the freeze foaming process, pores are made by putting an aqueous solution under vacuum. The reduced pressure causes the air within the slurry to expand and form bubbles. Then once the foam is formed, it is frozen into place. Then the water is removed from the system through sublimation. Finally, the foam is densified by traditional sintering. After successfully creating ceramic foam samples, the parameters in the freeze foaming process were identified and investigated. Foam samples were characterized by taking density measurements, examining the macrostructure and microstructure with light microscopy, and determining mechanical properties through compression testing. In the end, highly porous foam samples with adjustable properties were synthesized using a novel manufacturing process.Item Counterion influence on micelle size(Montana State University - Bozeman, College of Letters & Science, 1961) Ghose, Hirendra M.Item Colloidal suspension flow and transport behavior in small channels by magnetic resonance microscopy(Montana State University - Bozeman, College of Engineering, 2007) Brown, Jennifer Ruth; Chairperson, Graduate Committee: Joseph D. Seymour; Sarah Codd (co-chair)The research presented addresses colloidal transport issues in small channel systems using Magnetic Resonance Microscopy techniques. In transport phenomena, the interaction between convection or deterministic motions and diffusion or random motions is important in many engineering and natural applications, especially relating to multiphase flows. Magnetic Resonance methods have the ability to separate coherent from incoherent motion, as well as measure spatially resolved velocity, probability distributions of displacement, and microstructure on the pore scale, even within a multiphase colloidal system. A dilute (f < 0.10) suspension of ~2.5 mm Brownian particles under shear flow in a 1 mm diameter glass capillary was investigated using spectrally resolved Pulsed Gradient Spin Echo techniques. The results indicate particle migration inward towards the capillary center. In addition, dispersion coefficients measured via flow-compensated Pulsed Gradient Spin Echo techniques as a function of observation time indicate the onset of irreversible dynamics with increasing total strain. Particle migration and irreversible dynamics are generally not expected to occur in dilute Brownian suspensions and are therefore not considered in the modeling of flow systems.