Microfluidic system for Caenorhabditis elegans culture and oxygen consumption rate measurements.

Mitochondrial respiration is a key signature for the assessment of mitochondrial functioning and mitochondrial dysfunction is related to many diseases including metabolic syndrome and aging-associated conditions. Here, we present a microfluidic Caenorhabditis elegans culture system with integrated luminescence-based oxygen sensing. The material used for the fabrication of the microfluidic chip is off-stoichiometry dual-cure thiol-ene-epoxy (OSTE+), which is well-suited for reliably recording on-chip oxygen consumption rates (OCR) due to its low gas permeability. With our microfluidic approach, it was possible to confine a single nematode in a culture chamber, starting from the L4 stage and studying it over a time span of up to 6 days. An automated protocol for successive worm feeding and OCR measurements during worm development was applied. We found an increase of OCR values from the L4 larval stage to adulthood, and a continuous decrease as the worm further ages. In addition, we performed a C. elegans metabolic assay in which exposure to the mitochondrial uncoupling agent FCCP increased the OCR by a factor of about two compared to basal respiration rates. Subsequent treatment with sodium azide inhibited completely mitochondrial respiration.

Force microscopy of the Caenorhabditis elegans embryonic eggshell.

Assays focusing on emerging biological phenomena in an animal's life can be performed during embryogenesis. While the embryo of Caenorhabditis elegans has been extensively studied, its biomechanical properties are largely unknown. Here, we demonstrate that cellular force microscopy (CFM), a recently developed technique that combines micro-indentation with high resolution force sensing approaching that of atomic force microscopy, can be successfully applied to C. elegans embryos. We performed, for the first time, a quantitative study of the mechanical properties of the eggshell of living C. elegans embryos and demonstrate the capability of the system to detect alterations of its mechanical parameters and shell defects upon chemical treatments. In addition to investigating natural eggshells, we applied different eggshell treatments, i.e., exposure to sodium hypochlorite and chitinase solutions, respectively, that selectively modified the multilayer eggshell structure, in order to evaluate the impact of the different layers on the mechanical integrity of the embryo. Finite element method simulations based on a simple embryo model were used to extract characteristic eggshell parameters from the experimental micro-indentation force-displacement curves. We found a strong correlation between the severity of the chemical treatment and the rigidity of the shell. Furthermore, our results showed, in contrast to previous assumptions, that short bleach treatments not only selectively remove the outermost vitelline layer of the eggshell, but also significantly degenerate the underlying chitin layer, which is primarily responsible for the mechanical stability of the egg.

Dynamic microfluidic nanocalorimetry system for measuring Caenorhabditis elegans metabolic heat.

Basal heat production is a key phenotype for assessing the metabolic activity of small living organisms. Here, we present a new nanocalorimetric system, based on thin film thermopile sensors combined with microfluidic chips for measuring metabolic heat signals generated by Caenorhabditis elegans larval populations (60 to 220 organisms). In addition to versatile on-chip fluidic manipulation, our microfluidic approach allows confining worm populations close to the sensor surface, thus increasing the sensitivity of the assays. A customized flow protocol for dynamically displacing the worm population on-chip and off-chip was applied. The resulting sequential recordings of heat source and reference signals enabled precise measurements of slow varying heat-generating metabolic processes. We found an increase of the volume-specific basal heat production from the L2 to the L3 larval stage, and a significant decrease from the L3 to the L4 stage. Additionally, we investigated the metabolic heat production of the larval populations during maximal respiratory capacity, i.e. after inducing uncoupled respiration by on-chip treatment with the mitochondrial uncoupling agent carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP). 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.