A microfluidic device for automated, high-speed microinjection of Caenorhabditis elegans

Abstract

The nematode worm Caenorhabditis elegans has been widely used as a model organism in biological studies because of its short and prolific life cycle, relatively simple body structure, significant genetic overlap with human, and facile/inexpensive cultivation. Microinjection, as an established and versatile tool for delivering liquid substances into cellular/organismal objects, plays an important role in C. elegans research. However, the conventional manual procedure of C. elegans microinjection is labor-intensive and time-consuming and thus hinders large-scale C. elegans studies involving microinjection of a large number of C. elegans on a daily basis. In this paper, we report a novel microfluidic device that enables, for the first time, fully automated, high-speed microinjection of C. elegans. The device is automatically regulated by on-chip pneumatic valves and allows rapid loading, immobilization, injection, and downstream sorting of single C. elegans. For demonstration, we performed microinjection experiments on 200 C. elegans worms and demonstrated an average injection speed of 6.6 worm/min (average worm handling time: 9.45 s/worm) and a success rate of 77.5% (post-sorting success rate: 100%), both much higher than the performance of manual operation (speed: 1 worm/4 min and success rate: 30%). We conducted typical viability tests on the injected C. elegans and confirmed that the automated injection system does not impose significant adverse effect on the physiological condition of the injected C. elegans. We believe that the developed microfluidic device holds great potential to become a useful tool for facilitating high-throughput, large-scale worm biology research.

FIG. 1.

Microfluidic device for high-speed microinjection of C. elegans. (a) Schematic layout of the microfluidic device. (b) Blow-up of the injection area on the device. (c) Photograph of an injection device during fluorescence imaging of the injected worm.

FIG. 2.

Finite element simulation result of the pressure profile of the immobilization channel with one worm loaded and another worm at the channel inlet. The simulation was performed in ANSYS 13.0 (Canonsburg, USA). The pressure drop along the worm trapped at the inlet of the immobilization channel is too small to push the animal into the immobilization channel. The 3D model of the immobilization channel and worm and the associated mesh were developed by the ANSYS Workbench 13.0. The worm body was represented by a cylinder with cone-shaped head and tail. Incompressible steady-state Navier-Stokes equations and no-slip boundary conditions were used in the simulation. The pressure difference between the inlet and outlet of the channel was set to be 30 kPa.

FIG. 3.

System setup for automated C. elegans injection. (a) Schematic layout and (b) photograph of the system.

FIG. 4.

Image frame sequence of the injection area of the microfluidic device during worm injection. (a) Before worm loading. (b) A worm entering the immobilization channel. (c) The worm being injected. (d) The worm being flushed away.

FIG. 5.

Experimental validation of the worm body expansion as a reliable indication for successful injection. The dashed lines show the boundaries of the worm and the PDMS channel. (a) An immobilized worm before injection of fluorescence dye. No obvious fluorescence signal was observed in the worm body. (b) The worm after injection of fluorescence dye. The injected fluorescence dye was observed in the proximity of the injection site, which correlates the worm body expansion observed during injection.

FIG. 6.

The average worm handling time obtained in worm injection experiments on five different microfluidic devices, showing repeatable injection speed of the system using different devices.

FIG. 7.

Experimental results of post-injection viability tests. (a) Pharyngeal pumping rates of the control and experimental (injected) groups (n = 15). (b) Number of eggs laid per worm at 24 h post injection from the control and experimental groups (n = 10). (c) The daily survival rates of the control and experimental groups (n = 10). Worms hatched from eggs on day 0 and were loaded into the microfluidic device for injection experiments on day 3.