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.

Stem cell technology and tissue engineering offer exciting opportunities for improving medical therapies. Success in these endeavors will depend on advances in our basic understanding of tissue culture and the development of supporting technologies. Digital microfluidics (DµF) is one technology that could offer important and unique advantages for automating stem cell culture and cell-based assays to support tissue culture, drug discovery and basic biomedical research.

Digital microfluidics refers to a miniaturized lab-on-a-chip platform that enables the automation of a wide array of laboratory procedures by handling liquids as droplets rather than streams. It has been applied to many chemical and biochemical protocols and assays. Advantages include reduced reaction times and reduced reagent volumes, and the ability to process multiple samples in an automated way: in series or in parallel, identically or uniquely.

This dissertation describes technological advances and applications of DµF for tissue engineering. Vertical dimensionality was developed by stacking multiple layers and incorporating a protocol for transferring droplets between layers. This added functionality enables new applications, exemplified by the demonstration of three previously un-achievable applications: creating a calcium alginate hydrogel with a radial crosslink density; creating a hydrogel based particle sieve; and the ability to retrieve 3D embryoid bodies on-chip. Protocols for growing three-dimensional tissue structures were established by encapsulating cells within hydrogel matrices. This protocol was used to demonstrate invasion assays for modeling tumor growth. Stem cell microenvironments were investigated by developing a protocol for the long-term growth and differentiation of embryoid bodies for cardiac tissue engineering. Non-invasive impedance assays were demonstrated for observing phenotypic behavior, maturation, and responses to chronotropic and inotropic agents. Finally, preliminary experiments were carried out to explore the feasibility of integrating piezoelectric PZT-based materials into DµF devices for added functionality. The voltage change generated via the pyroelectric effect was measured for exothermic chemical reactions, and strain induced in the substrate produced from contracting cardiomyocytes was monitored via piezoelectric effect.

The innovations presented here will provide the DµF and tissue engineering communities with design parameters and processing protocols necessary for manipulating collagen, producing 3D cell-ECM constructs, and creating a stem cell microenvironment for cardiomyogenesis.

This article describes the equilibrium shape of a liquid drop under applied fields such as gravity and electrical fields, taking into account material properties such as dielectric constants, resistivities, and surface tension coefficients. The analysis is based on an energy minimization framework. A rigorous and exact link is provided between the energy function corresponding to any given physical phenomena, and the resulting shape and size dependent force term in Young's equation. In particular, the framework shows that a physical effect, such as capacitive energy storage in the liquid, will lead to 1/R "line-tension"-type terms if and only if the energy of the effect is proportional to the radius of the liquid drop: Eproportional toR. The effect of applied electric fields on shape change is analyzed. It is shown that a dielectric solid and a perfectly conducting liquid are all that is needed to exactly recover the Young-Lippmann equation. A dielectric liquid on a conducting solid gives rise to line tension terms. Finally, a slightly resistive liquid on top of a dielectric, highly resistive solid gives rise to contact angle saturation and accurately matches the experimental data that we observe in our electro-wetting-on-dielectric devices. (C) 2003 American Institute of Physics.