Digital Microfluidic Lab-on-a-Chip Platform for the Culture and Analysis of Three-Dimensional Multicellular Spheroids

Cell spheroids are compact, multicellular aggregates grown in vitro that mimic the three-dimensional morphology of in vivo tissues, thereby providing a more physiologically relevant tissue and disease model than cells cultured in two-dimensional monolayers. Despite the advantages of cell spheroids, their use in routine biomedical research has been limited, in part by the lack of automated, user-friendly, and flexible techniques for cell spheroid culture and analysis. Digital (droplet) microfluidics (DμF) enables the manipulation of discrete drops of liquid through the controlled application of electric fields. Because it allows automated and flexible liquid handling, DμF has the potential to address many of the shortcomings associated with current cell spheroid culture techniques. This dissertation describes the design, fabrication, and operation of a DμF platform that enables the culture and analysis of three-dimensional, multicellular spheroids.To enable spheroid culture on a DμF device, a novel device architecture was developed that incorporates through-holes, or `wells', in the bottom plate of the device that allow for the formation of hanging drops of controlled volume and composition. With the ability to create and address hanging drops in situ, protocols for automated cell spheroid culture were developed. Using DμF liquid handling, spheroids can be initiated and maintained on the device for at least 96 hours, exhibiting good viability (>90%) and size uniformity (~8% CV intra-experiment, ~16% CV inter-experiment). Automated spheroid-based drug screening and migration assays were also performed, demonstrating the ability to create and assay spheroids with higher-order tissue properties as well as stimulate relevant physiological processes. An optimized DμF peptide mass fingerprinting (PMF) sample preparation technique that could be useful for the in situ preparation and analysis of spheroid protein secretions was also developed. The platform described here advances the field of digital microfluidics by introducing a novel functionality, hanging drop formation, which enables the culture of large, three-dimensional micro-tissues on a DμF device. Improvements to the handling of biological solutions on a digital microfluidic device are also presented. This DμF platform also represents an advance in the field of tissue engineering by providing a novel means of automating the culture and analysis of individually addressable cell spheroids. A DμF platform that facilitates the culture and analysis of cell spheroids can potentially lower the barriers to adoption for the use of cell spheroids in routine biomedical research. Ultimately, broader use of spheroids in cell-based assays and screens has the potential to improve pre-clinical drug development efficiency and provide more physiologically relevant insights into our basic understanding of tissues and diseases