Automated longitudinal monitoring of in vivo protein aggregation in neurodegenerative disease C. elegans models

Background: While many biological studies can be performed on cell-based systems, the investigation of molecular pathways related to complex human dysfunctions - e.g. neurodegenerative diseases - often requires long-term studies in animal models. The nematode Caenorhabditis elegans represents one of the best model organisms for many of these tests and, therefore, versatile and automated systems for accurate time-resolved analyses on C. elegans are becoming highly desirable tools in the field. Results: We describe a new multi-functional platform for C. elegans analytical research, enabling automated worm isolation and culture, reversible worm immobilization and long-term high-resolution imaging, and this under active control of the main culture parameters, including temperature. We employ our platform for in vivo observation of biomolecules and automated analysis of protein aggregation in a C. elegans model for amyotrophic lateral sclerosis (ALS). Our device allows monitoring the growth rate and development of each worm, at single animal resolution, within a matrix of microfluidic chambers. We demonstrate the progression of individual protein aggregates, i.e. mutated human superoxide dismutase 1 - Yellow Fluorescent Protein (SOD1-YFP) fusion proteins in the body wall muscles, for each worm and over several days. Moreover, by combining reversible worm immobilization and on-chip high-resolution imaging, our method allows precisely localizing the expression of biomolecules within the worms' tissues, as well as monitoring the evolution of single aggregates over consecutive days at the sub-cellular level. We also show the suitability of our system for protein aggregation monitoring in a C. elegans Huntington disease (HD) model, and demonstrate the system's ability to study long-term doxycycline treatment-linked modification of protein aggregation profiles in the ALS model. Conclusion: Our microfluidic-based method allows analyzing in vivo the long-term dynamics of protein aggregation phenomena in C. elegans at unprecedented resolution. Pharmacological screenings on neurodegenerative disease C. elegans models may strongly benefit from this method in the near future, because of its full automation and high-throughput potential.

Long-term culture and high-resolution imaging of C. elegans using an automated microfluidic platform

Johan Auwerx, Matteo Cornaglia, Martinus Gijs, Govindan Narayanan Krishnamani, Laurent Mouchiroud

The nematode Caernohabditis elegans is one of the most employed small model organisms in biology. Studies on transgenic animals often require the accurate observation of highly localized fluorescent signals inside the worms at high magnification, hence demanding the full immobilization of the animals. Moreover, in order to observe the dynamics of biological processes over the lifespan of a worm, the same worm has to be immobilized repeatedly, in a reversible manner and under normal physiological conditions. We describe a microfluidic platform for the automated culture, treatment and long-term high-resolution imaging of C. elegans. Our device features: (i) a microfluidic design tailored for the isolation of L4 larvae from a mixed larval population and for their successive culture and treatment; (ii) a worm immobilization method, based on the thermoreversible sol-gel transition of the biocompatible triblock copolymer Pluronic F127 inside the microfluidic chip, thereby enabling high-resolution imaging; (iii) an integrated temperature control system, both to ensure viable environmental conditions for C. elegans culture and to steer the worm immobilization/release process. We apply this device to observe mitochondrial dynamics in muscle cells during aging at single worm resolution. We expect our platform to enable the simultaneous study and repeated observation of multiple phenotypes in single worms or specific worm populations, such as lifespan and motility assays, in addition to high-resolution imaging.

2015

A microfluidic device for longitudinal studies of C.elegans neurodegenerative disease models

Johan Auwerx, Matteo Cornaglia, Martinus Gijs, Govindan Narayanan Krishnamani, Thomas Lehnert, Laurent Mouchiroud

We describe a microfluidic device for automated culture and long-term high resolution imaging of Caenorhabditis elegans nematodes, which we specifically employed as model organisms for the analysis of neurodegenerative disease progression. In this platform, we integrated: (i) a microfluidic design tailored for the confinement of worms at the L2 larval stage in separate culture chambers by means of passive hydrodynamics; (ii) an optimized protocol for worm feeding and progeny removal at desired rate, allowing follow-up of the same worms over long-term studies; (iii) a technique for reversible C.elegans immobilization, based on the thermoreversible gelation of Pluronic F127 (PF127) inside the microfluidic chip; (iv) active control of the device temperature; (v) a compact device assembly, suitable for automated multi-dimensional imaging on any upright or inverted microscope.

2015

Microfluidic systems for high-throughput and high-content screening using the nematode Caenorhabditis elegans

Matteo Cornaglia, Martinus Gijs, Thomas Lehnert

In a typical high-throughput drug screening (HTS) process, up to millions of chemical compounds are applied to cells cultured in well plates, aiming to find molecules that exhibit a robust dose-response, as evidenced for example by a fluorescence signal. In high-content screening (HCS), one goes a step further by linking the tested compounds to phenotypic information, obtained, for instance, from microscopic cell images, thereby creating richer data sets that also require more advanced analysis methods. The nematode Caenorhabditis elegans came into the screening picture due to the wide availability of its mutants and human disease models, its relatively easy culture and short life cycle. Being a whole-organism model, it allows drug testing under physiological conditions at multi-tissue levels and provides additional observable phenotypes with respect to cell models, related, for instance, to development, aging, behavior or motility. Worm-based HTS studies in liquid environments on microwell plates have been demonstrated, while microfluidic devices allowed surpassing the performance of plates by enabling more versatile and accurate assays, precise and dynamic dosing of compounds, and readouts down to single-animal resolution. In this review, we discuss microfluidic devices for C. elegans analysis and related studies, published in the period from 2012 to 2017. After an introduction to the different screening approaches, we first focus on microfluidic systems with potential for screening applications. Various enabling technologies, e.g. electrophysiological on-chip recordings or laser axotomy, have been implemented, as well as techniques for reversible worm immobilization and high-resolution imaging, combined with algorithms for automated experimentation and analysis. Several devices for developmental or behavioral assays, and worm sorting based on different phenotypes, have been proposed too. In a subsequent section, we review the application of microfluidic-based systems for medium-and high-throughput screens, including neurobiology and neurodegeneration studies, aging and developmental assays, toxicity and pathogenesis screens, as well as behavioral and motility assays. A thorough analysis of this work reveals a trend towards microfluidic systems more and more capable of offering high-quality analyses of large worm populations, based on multiphenotypic and/or longitudinal readouts, with clear potential for their application in larger HTS/HCS contexts.

Royal Soc Chemistry2017

Microfluidic systems for high-throughput and high-content screening using the nematode Caenorhabditis elegans

Matteo Cornaglia, Martinus Gijs, Thomas Lehnert

Royal Soc Chemistry2017