The goal of this thesis is to design, optimize and implement a device that can measure the oxygen consumption rate (OCR) of live single cells. A microfluidic device has been designed with the ability to rapidly seal and unseal microchambers containing individual cells and an extracellular optical oxygen sensor for measuring the OCR of live single cells. The device consists of two parts, one with the sensor in microwells (top half) and the other with channels and cells trapped in Pachinko-type micro-traps (bottom half). When the two parts of the device are placed together the wells enclose each cell. Oil is flown in through the channels of the device to produce isolated and sealed microchamber around each cell. Different fluids can be flowed in and out of the device, alternating with oil, to rapidly switch between sealed and unsealed microenvironment around each cell. A fluorescent ratiometric dual pH and oxygen sensor is placed in each well. The thesis focuses on measuring changes in the oxygen consumption rate of each cell within a well. Live and dead cells are identified using a fluorescent live/dead cell assay. Finally, the technology is designed to be scalable for high-throughput applications by controlling the flow rate of the system and increasing the cell array density.
Inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, is an immune disorder that causes chronic inflammation of the gastrointestinal tract. There is an unmet need for effective pharmacological treatments as current standard therapies including aspirin derivatives and corticosteroids often fail to control the disease. For a significant portion, 30% or more, of patients, surgical removal of the affected bowel is required at some point in their disease course to address complications of bleeding, infections with sepsis, toxic megacolon among many others. There are also associated severe complications involving many other organs due to the underlying immune mediated reactions. In this study, PEGylated Serp-1 (PEGSerp-1) a modified Myxomavirus-derived serine protease inhibitor that binds and inhibits serine proteases in the coagulation and complement cascades, is evaluated in a pre-clinical model of IBD. Experimental colitis was induced in male C57BL/6J mice by oral administration of dextran sulfate sodium (DSS). In mice with acute colitis induced by exposure to 5% DSS for 6 days, daily treatment with PEGSerp-1 significantly improved survival. When initiation of treatment was delayed by 7 days in this acute colitis model, PEGSerp-1 treatment improved markers of disease severity and significantly reduced inflammation in the colon. PEGSerp-1 is an effective treatment of acute DSS-induced colitis when used as a preventative or delayed treatment.
The glucose metabolism level reflects cell proliferative status. A polymeric glucose ratiometric sensor comprising poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) and poly[2-(methacryloyloxy)ethyl]trimethylammonium chloride (PMAETMA) was synthesized. Cellular internalization and glucose response of the polymer within HeLa cells were investigated.
Functional and molecular cell-to-cell variability is pivotal at the cellular, tissue and whole-organism levels. Yet, the ultimate goal of directly correlating the function of the individual cell with its biomolecular profile remains elusive. We present a platform for integrated analysis of functional and transcriptional phenotypes in the same single cells. We investigated changes in the cellular respiration and gene expression diversity resulting from adaptation to repeated episodes of acute hypoxia in a premalignant progression model. We find differential, progression stage-specific alterations in phenotypic heterogeneity and identify cells with aberrant phenotypes. To our knowledge, this study is the first demonstration of an integrated approach to elucidate how heterogeneity at the transcriptional level manifests in the physiologic profile of individual cells in the context of disease progression.
Driven by an increasing number of studies demonstrating its relevance to a broad variety of disease states, the bioenergy production phenotype has been widely characterized at the bulk sample level. Its cell-to-cell variability, a key player associated with cancer cell survival and recurrence, however, remains poorly understood due to ensemble averaging of the current approaches. We present a technology platform for performing oxygen consumption and extracellular acidification measurements of several hundreds to 1,000 individual cells per assay, while offering simultaneous analysis of cellular communication effects on the energy production phenotype. The platform comprises two major components: a tandem optical sensor for combined oxygen and pH detection, and a microwell device for isolation and analysis of single and few cells in hermetically sealed sub-nanoliter chambers. Our approach revealed subpopulations of cells with aberrant energy production profiles and enables determination of cellular response variability to electron transfer chain inhibitors and ion uncouplers.