Microfluidic Approaches for Manipulating, Imaging, and Screening C. elegans

Abstract

The nematode C. elegans (worm) is a small invertebrate animal widely used in studies related to fundamental biological processes, disease modelling, and drug discovery. Due to their small size and transparent body, these worms are highly suitable for experimental manipulations. In recent years several microfluidic devices and platforms have been developed to accelerate worm handling, phenotypic studies and screens. Here we review major tools and briefly discuss their usage in C. elegans research.

Keywords: C. elegans; drug discovery; electrotaxis; high-throughput screening; live imaging; microfluidics; neurobiology.

Figure 1

Microfluidic devices for C. elegans sorting using (A) electrotaxis (Rezai et al. [10], (B) mechanical microstructures (Solvas et al. [12], (C) deflectable membranes (Dong et al. [17], and (D) fiber-based fluorescent detection (Yan et al. [15]). Reproduced with permission from The Royal Society of Chemistry. Panel (A) shows the electric trap-based sorting device. Loading chamber contains mixed stage worms. Sorted worms accumulate in the separation chamber and are recovered via unload channels. The smart maze concept is shown in panel (B). The four insets show worm orientation (inset 1); flushing of small larvae (inset 2); dimensions of the successful design (inset 3); and successful recovery of adults in an experiment (inset 4). The deflectable membrane device in panel (C) shows eight individual worm selection units (one of these connected with tubes). The fluidic and valve control channels are enlarged to show details. The fluidic path is squeezed upon activation of the control valve. The device in panel (D) contains inlets and outlets for worms and buffer. The optical fiber channels (LED 625 and 375 nm) are used to differentiate between wild-type and fluorescing worms. Refer to respective references for more details.

Figure 2

Microfluidic devices for culturing and long-term studies of worms inside cultivation chambers while immobilization and imaging is performed by (A) tapered microchannels (Hulme et al. [23] or (B) responsive hydrogels (Krajniak et al. [29]. Reproduced with permission from The Royal Society of Chemistry. The tapered microchannels connected to growth chambers (panel (A)) allow single worms (early L4 stage) to enter into each chamber. Arrows indicate the direction of liquid flow. Once the worm has grown it is unable to escape the chamber. For imaging purposes, the worm is temporarily immobilized in the tapered region. Panel (B) The two sub-panels B-i and B-ii show the device that contains valves (red) to control fluid flow, channel for flowing heating liquid (light blue), and eight worm culturing chambers (two sets of four) and a central waste outlet tube connected to a loading channel (green). Refer to respective references for more details.

Figure 3

Microfluidic devices to immobilize C. elegans using (A) deflectable membrane (Gilleland et al. [33]; (B) tapered microchannels (Kopito and Levine [34]); or (C) CO₂ exposure (Chokshi et al. [35]. Reproduced with permissions from The Royal Society of Chemistry and Macmillan Publishers Ltd. Nature Protocols. Panel (A) shows the chip containing an array of narrow channels to apply suction pressure. Worm is loaded/removed through port-B and restrained by the narrow channel array. Pressure through port-A causes the compression layer to move downwards and immobilize the worm (explained on the right). Releasing the pressure allows the worm to be recovered. The WormSpa device, in panel (B), contains four regions for worm loading and distribution (1), egg chambers (2), egg collection (3), and outflow (4). The device for CO₂ based immobilization is shown in panel (C). It contains modules for behaviour assay (first row of pictures) and immobilization (second row of pictures). Refer to respective references for more details.

Figure 4

Microfluidic devices for microinjection in (A) closed microchannels (Ghaemi [41] and (B) open chambers (Song et al. [43]. Panel (B) reproduced with permission from American Institute of Physics Publishing. The device in panel (A) contains worm loading and washing channels (on the right) and an outlet for collecting injected worms. Worm is immobilized in the middle region for injection. The image frames in panel (B) show a sequence of worm loading, injection, and flushing. Refer to respective references for more details.

Figure 5

Microfluidic devices for multidirectional orientation and imaging of C. elegans using (A) rotatable glass capillaries (Ardeshiri et al. [54] and (B) acoustofluidic rotational manipulation (ARM) (Ahmed et al. [55]. Panel (B) reproduced with permission from Adapted by permission from Macmillan Publishers Ltd. Nature Communications. Panel (A) shows an adult worm inside the channel with the region of interest (ROI) in the middle. The worm is held by the negative pressure in the glass capillary. The two sets of brightfield and fluorescent images below show pre- and post-rotated views of specific neuronal processes (VC). Schematic view of the ARM device (B). It contains a piezoelectric transducer to generate acoustic waves. Air bubbles within sidewall cavities cause worms to rotate. The image below shows a mid-L4 worm trapped by oscillating bubbles. Refer to respective references for device details.

Figure 6

Microfluidic devices to investigate C. elegans behavior in response to (A) chemicals and heat (McCormick et al. [59]); (B) chemical gradients (schematic drawing of the device used by Hu et al. [60]); and (C) electric field (Rezai et al. [9]). Panel (C) reproduced with permissions from the Royal Society of Chemistry. Panel (A) shows head swinging of the worm in response to chemical exposure. In panel (B), the circular channel pattern used to generate the chemical gradient is shown. Worms enter into channels 1–8, which are 300 μm wide, 80 μm high, and 10 μm long depending upon their attractive responses to the NaCl gradient. The electrotaxis device in panel (C) contains electrodes to apply a DC electric field and a long channel for worm swimming. Refer to respective references for more details.

Figure 7

Microfluidic devices to investigate C. elegans (A) egg-laying (Li et al. [83]) and (B) development (Wen et al. [30]), by isolating worms inside microchambers with renewable chemical environment. Reproduced with permission from the Royal Society of Chemistry. The left-hand diagram in panel (A) shows eight chambers on each side (one of which is enlarged on the right). Inlets are used to load worms and outlets for bacterial flow. The middle counting region (2 mm × 2 mm), indicated by the red rectangle, is monitored by camera. Panel (B) shows the droplet chip. Schematics of worm encapsulation and substrate exchange in each droplet are shown in three steps on the right side. The amount of substrate exchange is indicated by the color change. Refer to respective references for more details.

Figure 8

Microfluidic devices for neuromuscular electrophysiological studies on C. elegans (Lockery et al. and Hu et al. [92,94]). Panel (A) reproduced with permission from the Royal Society of Chemistry. The EPG recording device (panel (A)) contains a worm channel and a funnel-shaped trap region. Fluid flows through the side-arm channel. Panel (B) shows the neurochip. Blue indicates the layer containing the microfluidic region (for worms and chemicals) and white shows the pneumatic control layer. The red circle contains the trapped worm’s head. The red square contains micropillars to correctly orient the worm. V1–4 are valves and the solid black squares are microelectrodes. Refer to respective references for more details.

Figure 9

A microfluidic device for (A) laser nanoaxotomy of the ALM neuron in C. elegans and (B) investigation of time-lapse nerve regeneration (Guo et al. [97]). Reproduced with permission from Adapted by permission from Macmillan Publishers Ltd. Nature Methods. On the left (A-i) the trap system (yellow rectangle) and the three recovery chambers (blue rectangle) are indicated. The right panel (A-ii) shows a magnified view of the trapping system. The small yellow dotted rectangles show four valves to control worms. Panels (B-i to B-iv) show axonal recovery. Branching is visible several minutes after axotomy. By 70 min the nerve has regrown and appears to be reconnected. Refer to the references for more details.