This thesis presents the concept and applications of microfluidic hanging-drop platforms for the culture and analysis of 3D microtissues – an emerging in vitro cell culture model. The motivation of this work was to bridge the gap between advances in 3D cell cultures and the commonly used cell culture platforms, which are designed prevalently for static and 2D cell cultures, and to demonstrate the potential of dedicated 3D cell culture platforms in providing suitable and reliable experimental conditions.
Hanging drops are used for the scaffold-free formation and culture of spherical 3D microtissues. These microtissues are easy to handle and feature many organotypic tissue functions, which renders them a suitable in vitro model system for both, basic research, and pharmaceutical industry. Despite the biological relevance and advantages of these microtissue model systems, the lack of optimized platforms for culturing and analysis still limits the widespread use and application of 3D microtissues in biomedical testing and pharmaceutical compound screening.
Microfabrication techniques offer a great toolbox to build next-generation cell culturing and analysis systems. The combination of microfluidics to realize physiologically-relevant culture conditions and microfabricated sensor units enables the realization of integrated systems for organ-, and body-on-a-chip applications.
This thesis presents the fabrication, working principle and operation of microfluidic hanging-drop networks for the culturing and analysis of 3D microtissues, and introduces three dedicated platforms for interrogating 3D models.
i. Biosensing in hanging-drop networks. Enzyme-based biosensors were integrated in hanging-drop networks for real-time in situ multi-analyte monitoring of 3D microtissue metabolism. The device enabled online detection of lactate secretion and glucose consumption of human colon cancer microtissues.
ii. High-resolution imaging in hanging-drop networks. Integration of hydrogels in hanging-drop networks enabled both, immobilization of 3D microtissues for high-resolution and long-term imaging, as well as providing fine control over the microenvironment of the 3D microtissues. The system allows for investigating complex biological processes down to single-cell level and for observation of physiologically events at subcellular scale.
iii. FlowGSIS in hanging drops. A hanging-drop perfusion system was developed for studying the dynamics of glucose-stimulated insulin secretion (GSIS) of human endocrine pancreas islets. The device enabled high-temporal-resolution sampling to resolve the bi-phasic and pulsatile insulin release of single islets to study the effects of anti-diabetic medication.
The presented platforms represent a set of novel tools for culturing and interrogating 3D microtissues. The design and operation of each platform has been optimized for the respective application, ranging from precise stimulation of microtissues and subsequent metabolite detection, to long-term and high-resolution imaging. The broad range of applications served by these platforms and the platform complementarity greatly improve our capability of taking full advantage of 3D microtissues as organotypic in vitro model systems
