Miniaturised Microwell-based Cell Assays

Abstract: Cell heterogeneity in genetically identical cell populations is becoming a well-known and important phenomenon in cell biology. Current methods commonly utilise population-based analysis founded on averaged result. Hence there is a need for high-throughput cell assays on the single-cell level. By using miniaturised devices it is possible to enhance spatial and temporal control of the individual cells, increase the potential throughput and minimise the needed sample and reagent volume while enabling a wide range of biological applications.This thesis is based on the results generated with a miniaturised microwell slide for cell assays. The microwell slide’s high-throughput compartmentalised configuration enables several hundred isolated experiments to be run simultaneously. The bottom of the wells is made out of a thin glass slide, which supports high-resolution imaging. The slide has a standardised format and its’ compatibility with conventional instruments is used extensively throughout the thesis. The presented papers all contribute to the development of the microwell slide by adding technical value or increasing the number of potential applications. For example, the slide was success-fully implemented as a chip-to-world output format for single microfluidic droplets in Paper I, by interfacing two miniaturised systems with fluorescence-activated cell sorting. In Paper II and III, microfluidic channels were integrated to increase spatial and temporal control of the added samples and reagents. In Paper II an automated stepwise concentration gradient generator was developed delivering a drug gradient to adherent mammalian cells in designated wells. In Paper III fluidic-imposed shear stress on endothelial cells was studied. In Paper IV, the slide was functionalised by coating the surfaces of the wells with several antibiotics at a defined concentration range. The coated slide was used for multiplex antibiotic susceptibility testing of bacterial pathogens, using an algorithm-based identification of the point defining lag to exponential phase transition. It successfully determined the pathogens susceptibility profile in 3-5 hours. Finally, in Paper V, a method to retrieve bacteria colonies with a desired phenotype from the wells for downstream genetic analysis was developed. In summary, the presented work has furthered the development of miniaturised high-throughput tools for various cell heterogeneity assays.