Development of Modular, Droplet Microfluidic Workflows to Investigate Cellular Heterogeneity
Cook, Claire
2023
Abstract
Cells exist in unique contexts within a population, but miniscule differences in their environment, epigenome, and cell cycle produce an assortment of observable characteristics in cells. Conventional cellular analysis tools routinely combine millions of cells within samples to ascertain “averaged” cellular states. Many methods have been developed to isolate, process, and analyze small and even single-cell samples, but they require long experiments, large reagent volumes, and expensive equipment. Microfluidic technologies have been developed as cheaper, automated, high-throughput routes to study cells in a miniaturized context. Droplet microfluidic platforms encapsulate individual cells in sub-microliter droplets for isolated processing. However, most of these devices adapt singular steps from benchtop protocols, requiring pre-purification of cells or post-processing to generate results. This dissertation describes the development of multi-device, droplet microfluidic workflows as wholistic, automated alternatives for single-cell assays. Chapter 2 introduces CellMag-CARWash–a workflow that combines positive, magnetic selection with droplet microfluidic devices to isolate desired cells from a mixture and incorporate specific biochemical cues within individual droplets. We demonstrate CellMag-CARWash’s abilities by isolating single cells from multi-cell mixtures with two different cell types from equal prevalence to >93% purity. Cells require a minimum of 4 – 5 beads to be recaptured, depending on size, which prevents non-specific, bead-bound contaminating cells from entering the product stream. Molecular treatments can be delivered to cells at the single-cell level through CellMag-CARWash’s washing buffer. We leveraged this feature to study heterogeneity in extracellular (EV) secretion dynamics from MCF7 cells. A magnetic droplet splitter enabled isolation and analysis of secreted EVs from single cells within droplets, revealing the underlying distribution of secretion rates. This chapter reports the first measurement of β-estradiol’s effect on EV secretion from single MCF7 cells. Chapter 3 combines two previously isolated droplet microfluidic devices into a cohesive workflow to study nucleosomal organization within cellular subpopulations. Nucleosome positioning throughout chromatin controls DNA accessibility so cells can respond to environmental cues. Our workflow employs fluorescence activated droplet sorting (FADS) and a micrococcal nuclease (MNase) digestion device to gently enrich cells within droplet populations and produce mononucleosomal DNA fragments. Diversion of droplets via FADS is demonstrated for pure and mixed samples containing fluorescent solutions and cells. Successful enrichment of fluorescent cells is demonstrated for multiple cell mixture compositions. Simultaneous cell wall digestion, lysis, and DNA fragmentation occur via the MNase digestion device, generating ~70% mononucleosome-length fragments and demonstrating improved reaction efficiencies relative to benchtop controls. The full FADS-MNase workflow is applied to generate mononucleosomal DNA fragments from enriched droplet samples produced by both FADS outputs. The final chapter summarizes major findings for each workflow, proposes technical improvements and interesting new concepts to explore, and places them into the broader context of tools enabling study of cellular heterogeneity. These technologies automate multiple portions of cellular protocols, and all devices are input independent, making implementation easier, faster, and more flexible for the user. Further work to integrate them with next generation sequencing analysis would produce all-in-one, high-throughput technologies for studying heterogeneity in cell samples. Scaling to larger numbers of cells is as simple as increasing the number of droplets generated, which positions these workflows for rapid deployment into clinical spheres and patient sample analysis. Overall, this dissertation demonstrates the capability and potential of modular, droplet microfluidic workflows to revolutionize cellular heterogeneity studies.Deep Blue DOI
Subjects
Droplet microfluidics Cellular heterogeneity Single-cell Extracellular vesicles (EVs) Epigenetics Nucleosome positioning
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