A droplet microchip with substance exchange capability for the developmental study of C. elegans

Abstract

The nematode Caenorhabditis elegans (C. elegans) has been widely used as a multicellular organism in developmental research due to its simplicity, short lifecycle, and its relevance to human genetics and biology. Droplet microfluidics is an attractive platform for the study of C. elegans in integrated mode with flexibility at the single animal resolution. However, it is still challenging to conduct the developmental study of worms within droplets initiating at the L1 larval stage, due to the small size, active movement, and the difficulty in achieving effective substance exchange within the droplets. Here, we present a multifunctional droplet microchip to address these issues and demonstrate the usefulness of this device for investigating post-embryonic development in individual C. elegans initiating at the larval L1 stage. The key components of this device consist of multiple functional units that enable parallel worm loading, droplet formation/trapping, and worm encapsulation in parallel. In particular, it exhibits superior functions in encapsulating and trapping individual larval L1 worms into droplets in a controlled way. Continuous food addition and expulsion of waste by mixing the static worm-in-droplet with moving medium plugs allows for the long-term culture of worms under a variety of conditions. We used this device to investigate the development processes of C. elegans in transgenic strains with deletion and overexpression of the hypoxia-inducible factor (HIF-1), a highly conserved transcript factor in regulating an organism's response to hypoxia. This microdevice may be a useful tool for the high throughput analysis of individual worms starting at the larval stage, and facilitates the study of developmental worms in response to multiple drugs or environmental toxins.

A size threshold governsCaenorhabditis elegansdevelopmental progression

“…Worm growth has also been studied using an array of chambers in which L4 worms were individually loaded to monitor development to adulthood [26], or using a device developed to observe collections of 30-40 adults worms [27]. A device with multiple observation chambers for adult worms was exploited to study chemical effects on worm behaviour [28] and oil-inwater emulsions were used to encapsulate individual worm embryos [29,30] or L1s [31] which could develop to adulthood. While encapsulation allows the study of even early larval stages which are otherwise challenging, proper feeding and the physiological effect of surfactant required for droplet production limit the applicability of this method [32].…”

From single-molecule detection to next-generation sequencing: microfluidic droplets for high-throughput nucleic acid analysis

“…Subsequent to their generation, droplets need to be manipulated in ways that mimic the standard analytical procedures used on the bench top. Fortunately, a wide range of (both passive and active) functional components have been presented for operations that include droplet merging (Niu et al 2008;Deng et al 2013;Mazutis and Griffiths 2012;Akartuna et al 2015), dilution (Niu et al 2011;Sun and Vanapalli 2013), dosing (Abate et al 2010;Chen et al 2008), splitting (Link et al 2004;Gao et al 2016), pairing (Ahn et al 2011;Bai et al 2010), sorting (Baret et al 2009;Nam et al 2012;Cao et al 2013), trapping/releasing Korczyk et al 2013;Courtney et al 2017), counting (Boybay et al 2013;Yesiloz et al 2015;) and incubation (Huebner et al 2009;Wen et al 2015). An instructive example in this respect was reported by Hatch et al (2011), who used successive bifurcations to split single droplets into 256 daughter droplets in a rapid and passive fashion ( Fig.…”

High-throughput screening in the C. elegans nervous system

“…Devices capable of automatically generating large arrays of animal-containing droplets have been described for assessing neurotoxin effects in high throughput [Shi et al, 2010, 2008]. A version of these devices has been outfitted for substance exchange between the droplets and inflow of media, allowing for parallel long-term culture of 160 newly hatched animals to adulthood [Wen et al, 2015]. The Caenorhabditis -in-Drop method (CID) is a microdroplet-based system developed for studying quiescence in larval animals but capable of culturing animals for up to five days.…”