Automated Microraft Array Platform for Immune Cell Assays and Cell Sorting

Immunology research and cell therapies have provided great advancements in recent years and have highlighted the need to understand the effects of single cell heterogeneity within immune cell populations. Single cell platforms currently used in immune cell analysis often include tedious manual work, are not able to measure immune cell function in a time-dependent manner, or are not selective. The following work describes the development of an automated microraft array platform for analyzing the function of individual immune cells, including helper T cells and chimeric antigen receptor T cells (CAR-T cells), and for the sorting of cells of interest. In this dissertation, an automated microraft array platform was developed and adapted to address the challenges seen in immune cell research. In Chapter 2, an automated microraft array platform was developed to assay thousands of single cells in parallel and sort individual cells of interest. The platform was used to assay single CD4+ T cells, isolate cells displaying proliferation in response to allogeneic cell stimulation, and sequence their T cell receptor genes. In Chapter 3, a next-generation magnetic microbead-based microraft array was developed as an alternative to nanoparticle-based microraft arrays. The microbead-based arrays were shown to substantially reduce fabrication time compared to nanoparticle-based microraft arrays and improve performance in imaging of fluorescently labeled cells. Chapter 4 focused on the development and application of the automated microraft array platform to assay CD19 CAR-T cells for cell-mediated cytotoxicity and isolate T cells of interest for gene expression analysis. CAR-T cells were shown to participate in serial-killing of target cells and T cells demonstrating high cytotoxicity were isolated for future gene expression analysis using single-cell multiplex qPCR. The findings presented in this dissertation demonstrate the capabilities of an automated microraft array platform and its uses in immunology research. The studies described in each chapter provided valuable insight into the behavior and phenotype of immune cells at the single cell level.Doctor of Philosoph