MICRORAFT ARRAY PLATFORMS FOR SORTING LIVE-CELL COLONIES BASED ON INTRACELLULAR PROTEIN FLUORESCENCE

Characterization of intracellular protein states in live cells is necessary to monitor changes in cellular physiology. Protein labeling methods, such as gene-engineered fluorescent proteins or fluorescently tagged antibodies are extensively used to measure intracellular protein abundance, co-localization, and post-translational modification within cells. However, the development of clonal fluorescent reporter cell lines is limited by the low efficiency of gene-engineering, laborious sorting methods, and cell sensitivities; and intracellular immunofluorescence assays are typically end-point assays without straightforward options to relate cell fluorescence measurements to downstream biological studies. Microraft array microdevices may address these methodological issues by miniaturizing cell culture sites, enabling gentle manipulation of cells, and being compatible with automation. Still, no robust microtechnologies. including microraft arrays, have combined pipelines for cell cloning and culture with colony sampling, collection, and assay in parallel. Parallelization of these processes has the potential to stream-line colony screening pipelines, leading to cutting-edge research discoveries and more rapid development of cell lines for biomedical applications. In this dissertation, microraft array-based platforms were advanced to miniaturize cell sampling, bioassay, and banking pipelines for stem and cancer cells. Specifically, the platforms were optimized for the culture, characterization, and sorting of human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs) and basophilic leukemia cells. First, microraft arrays were applied to the cloning and sorting of hESCs gene-engineered to express endogenous reporters of pluripotency, resulting in clonal reporter lines that will enable the complex networks involved in pluripotency and differentiation to be investigated. Next, quad microraft arrays were optimized for hiPSC colony maintenance and subculture, and the reprogramming of peripheral blood cells into hiPSCs, enabling preservation and generation of pluripotency in cells, essential for personalized medicine applications. Finally, a semi-automated colony sorting pipeline involving a novel magnetic microwell-based immunostaining plate was developed, enabling the collection of colony fragments, intracellular protein level quantification, and retrieval of viable mother colonies matched to the daughter fragment. Together, these microraft array-based approaches have the potential to advance colony screening pipelines by increasing bioassay throughput and enabling cell sorting by a large number of criteria not currently suitable for viable cell sorting.Doctor of Philosoph