An Novel Open—microfluidic—chip And Its Application To Investigating Cellular Ca~2+Signaling Of C.Elegans

The genetically tractable model organism-Caenorhabditis elegans (C. elegans) provided a number of powerful experimental advantages in the studies of neurobiology and developmental biology for its small size, simple organization, short life cycle, easy generation, a fully sequenced and well-annotated genome as well as a well characterized genetic analysis. However, it is difficult to operate these small organisms using the conventional methods. In recent years, microfluidic-based analytical approaches have been widely employed to facilitate fundamental studies of C. elegans thanks to its several unique properties, such as compatible sizes with the worms and convenient manipulation of worms through fluidic control. Microfluidic chips have been widely employed for the worm immobilization during the imaging, drug stimulation, high-throughout screening and so on. For instance, by utilizing tapered microchannel, microvalves, side-wall suction microchannels, CO2anesthesia, cooling and thermo-sensitive gel, live worms could be effectively immobilized on microfluidic chips for imaging and laser ablation. Microfluidic-based methods are playing a developing role in the research of C. elegans.In the research of C. elegans, microinjection is an important tool. The worm immobilization is the primary task for microinjection. Currently, microfluidic-suction methods could act as feasible tools to get the worm immobilized. However, it is difficult to couple microinjection with on-chip immobilization of worms, since the PDMS layer prevents direct contact of external devices with the immobilized animals. In our work, we developed a semi-open PDMS microfluidic chip for C. elegans microinjection assays. A piece of PDMS was abscised off the microfluidic chip to form an open chamber, in which worms could be immobilized by suction channels in the chip. This would be feasible for the operation of the external micro-manipulator, such as microinjection. Further applications, such as micro-anatomy, microelectronic assay could be applied to C. elegans by using other external devices. In our experiments, pseudocoelom, gonads and even a single intestinal cell were successfully injected with fluorescence dye using this method. Worm could be long-term immobilized with a considerable activity using this device, which was ideal for the long-term imaging analysis. Moreover, this semi-open device could integrate with others microfluidic devices to form a complex microfluidic system for more functions.In the intestine of C.elegans, spontaneous Ca2+oscillations could initiate in the pacemaker cells-the last two intestinal cells. These rhythmic Ca2+oscillations could anteriorly propagate to generate the intercellular Ca2+waves. However, the mechanisms of this spontaneous Ca2+oscillations and the propagation of Ca2+waves are still not well understood. In our assay, chemical agonists (thapsigargin, TG) were emerged to microinject into the intestine of the worm to stimulate the calcium signaling in the intestine simultaneously with the calcium imaging using our device. Obvious intestinal calcium signals are initiated upon the TG injection. In addition, repetitive microinjections of TG could induce calcium oscillations in the intestine cell with a similar frequency. As the TG was injected again, Ca2+in the most posterior intestinal cells, which regulated the rhythm defecation behaviours, would oscillate one by one without any intervals. Calcium waves could initiate from the last two cells and anteriorly propagated in the intestine. Moreover, during the Ca2+wave propagation, some weak Ca2+waves were ’abortive’ and it seemed that only large Ca2+spike could initiate an anteriorly propagated Ca2+wave. The posterior body wall muscles contraction (pBoc) occurred immediately as the TG injected into the posterior intestinal cells. Furthermore, chemical agonists could be directly used to stimulate the dissected intestine with our device. Stimulation of the intestine by light could also be conducted with different light intensity. Additionally, electric stimulations to the intestinal cells could be carried out using the microclectrode technology on our device.Thus, this new microfluidic method could provide a customized tool for in vivo studying the mechanisms underlying calcium signaling, such as spontaneous calcium signaling, calcium oscillations and calcium propagations in C. elegans. Moreover, this device could be further developed for more applications in future.