Microfluidic and micromechanical in vivo studies of Caenorhabditis elegans

Caenorhabditis elegans is one of the most attractive model organisms in biomedical research for understanding human diseases and for drug testing at a whole-organism level, since many biological pathways and genes have been conserved between itself and humans. The nematode is sufficiently complex so that complex biological questions can be addressed, yet the organism is still simple enough to be used in high-content in vivo assays. In this work, we present new microfluidic and MEMS-based tools enabling the characterization of C. elegans worms and embryos in vivo.

In a first part of this thesis, we measured the metabolic heat production of small C. elegans larval populations using a custom nanocalorimetric system. Our system is based on microfabricated Si thermopile sensors which are integrated with PDMS microfluidic chips. In addition to versatile fluidic manipulation of samples on-chip, our microfluidic approach allows confining worm populations close to the Si sensor surface, thus enabling high sensitivity of the assays. Our results indicate an increase of the heat production per worm body volume from the L2 to the L3 larval stages, and a significant decrease from the L3 to the L4 stages. Additionally, we investigated the metabolic heat production of the larval populations during maximal respiratory capacity after treatment with a mitochondrial uncoupling agent. Depending on the larval stage, inducing uncoupled respiration causes an increase of the metabolic heat production ranging from 55% up to 95% with respect to untreated worms. Our experiments demonstrated that nanocalorimetry is a direct and highly-sensitive technique that can be combined with other analytical methods to open the way to a more holistic understanding of fundamental biological processes.

In a second study, we present a new microfluidic worm culture system with integrated luminescence-based sensing of the on-chip oxygen concentration for measuring the oxygen uptake of C. elegans worms. The microfluidic chip was fabricated in OSTE+ to allow reliable measurement of the dissolved oxygen and the fabrication of high-resolution microstructures down to the &m-range. Our microfluidic approach allows confining a single nematode from the L4 stage in a culture chamber over a time span of up to 7 days. An automated protocol for feeding and for performing oxygen consumption rate (OCR) measurements during the worm development was applied. We found an increase of OCR values from the L4 stage to the adult worm, and a continuous decrease as the worm ages. In addition, we performed a C. elegans metabolic assay using a mitochondrial uncoupling agent, which increased OCR by a factor of ¿ 2 when compared to basal respiration rates. Treatment with sodium azide inhibited mitochondrial respiration and returned OCR values to zero.

The biomechanical properties of the C. elegans embryonic eggshell are largely unknown. In a third study, we developed a new method to study its mechanical properties. In our work, we use cellular force microscopy (CFM), a technique that combines micro-indentation with high-resolution force sensing approaching that of atomic force microscopy, to quantitatively study the mechanical properties of the eggshell of living C. elegans embryos.

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