Long-term imaging, automated high-content phenotyping and sorting of C. elegans larvae and embryos using microfluidic platforms

C. elegans is frequently studied to address fundamental biological questions motivated by its short life cycle, small size, hermaphrodite behavior and well-annotated genome. In this work, we present novel microfluidic platforms that support long-term culture, automated high-content phenotyping, high-resolution imaging and multiplexing, as well as a hand-held device for sorting of larvae and embryos of C. elegans. In our first study, we present a multiplexed polydimethylsiloxane (PDMS)-based microfluidic platform for the rapid high-content phenotyping of populations of C. elegans. We reversibly and repeatedly confined nematodes during their life cycle inside tapered channels for stable fluorescence imaging and extraction of growth parameters, in combination with a determination of motility phenotypes obtained from video-recording of freely moving nematodes. We extracted 19 phenotypic parameters and proved that dietary restriction (DR) plays a crucial role in the disease regression of a worm model of Huntington's disease. In a second work, we demonstrate a multiplexed, high-throughput and automated embryo phenotyping microfluidic platform to analyze embryogenesis of C. elegans under application of different chemical compounds. We executed up to 800-embryo experiments in 35 minutes and performed 12 hours of imaging, which was followed by an automated phenotyping using machine learning and image processing approaches. Our validation study of osmosensation on the embryos indicated a developmental lag and an induction of mitochondrial stress in embryos exposed to high doses (200 mM) of glucose and NaCl, while small doses of sucrose and glucose accelerated development. We have realized a multiplexed, potentially high-throughput, and high-resolution microfluidic platform to culture C. elegans from embryo to the adult stage at single-animal resolution. As a proof-of-concept, we exposed the nematodes to 8 different concentrations, from 1 nM to 1 mM, of the anthelmintic drug tetramisole diluted in an E. coli feeding solution, and we observed from our automated phenotyping results that, towards the late stages of the nematodes' life, tetramisole's influence on the development and motility became more prominent. We have also realized an epigenetics-oriented microfluidic platform to observe mother-to-progeny heritable transmission in the nematode C. elegans at high-resolution, under significant automation and enabling a high degree of parallelization. After a 24 hours culture of nematodes starting from L4 larvae under application of various concentrations and durations of the drug doxycycline, we quantified the average green fluorescent protein (GFP) intensity as an indicator to mitochondrial stress inheritance. We noticed that a minimum doxycycline concentration of 30 ug/mL and a drug exposure time of 15 hours applied to the mothers could induce mitochondrial stress in embryos. In the last part of this thesis, we demonstrate a PDMS microfluidic device for high-throughput, efficient and extremely rapid sorting of C. elegans at various life stages. In parallel, we could retain the remaining population in the chip, enabling collection of two sorted larval stages at both outputs of the device. In addition, we employed the equivalent of a standard bleaching procedure for embryo harvesting on-chip and showed that egg extraction and a synchronized L1 population could also be easily obtained.

Gijs, Martinus
Lausanne, EPFL

 Record created 2019-10-29, last modified 2019-11-04

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