Microfluidic Chips For Culture And Stimulation Of Caenorhabditis Elegans

The nematode Caenorhabditis elegans(C.elegans)has been widely used as a primary model organism for studying basic mechanisms in fields of genetics,development,neuroscience and behavior physiology etc.for decades,which benefits from its versatility in behavior,simple nervous system,short life cycle,and availability of powerful genetic tools.However,despite of the unique and ideal advantages of the worms,they are difficult to be exploited using the conventional labour-consuming and time-consuming manual methods.In recent years,microfluidic chip has been recognized as a novel platform for fundamental studies in C.elegans due to its good transparency,biocompatibility and highthrough,especially its comparable microscale dimension to the worms.The microfluidic “Worm-chips” have emerged for phenotyping,stage screening,nerve system imaging,behavioral dynamics,microsurgery and microinjection in the C.elegans research,which further benefit developmental biology,neuroscience,drug screening and other fields involving C.elegans study.In this dissertation,several advanced microfluidic platforms were developed for culturing and stimulation analysis of C.elegans.To systematically analyze the short-term and long-term effects of bacterial food over a wide range of concentrations,a microfluidic platform was developed for establishing on-chip logarithmic concentration gradient of bacteria,long-term worm culture,and parallel live imaging.Gradient of bacterial food supplements with four orders of magnitudes were automatically generated by simply perfusing two streams into our device.This platform was employed to evaluate the effects of a large range of bacterial concentration on the lifespan of C.elegans.In addition,DAF-16::GFP nuclear localization induced by various concentrations of bacterial food were also investigated to better understand the mechanism of dietary restriction's effect on the worms.The motility of organisms directing in response to fluid velocity gradients,which is called rheotaxis,is important in C.elegans life cycles,enabling them to navigate their environment and to maintain their positions in the presence of adverse flow.To study which velocity is most favorite in C.elegans and the mechanism underlying this behavior,we present a rapid and robust microfluidic approach to realize quantitatively analyzing the worms' rheotaxis behavior to velocity gradients.The flow-based microfluidic chip contained six helical spline microchannels for generating six different flow streams.By using this microfluidic chip,the distribution of worms was successfully monitored for quantitative analysis of the rheotaxis behavior.To study the process of electrical stimulation in C.elegans intestinal cells,a microfluidic chip was demonstrated for quantitatively investigating the calcium signal of the worm in responses to different voltage.Programmable solenoid valve was utilized to control the stimulation time exactly.By using this device,obvious intestions calcium signals were initiated by continuous or instantaneous stimulation.In addition,the intestine would be broken with the increasing voltage,which could be used in anatomy.This simple device could be further used to explore the mechanism of calcium signal transduction.The C.elegans has a compact nervous system with only 302 neurons.However,the dynamics of signaling through C.elegans neural circuits is largely unknown.Optogenetics have been used to infer neuronal fuctions in recent years.Therefore,we developed a new strategy combining optogenetics and optofluidics based on microfluidic chip technology.This microdevice could realize optical stimulation,observe its corresponding neurons and the dynamic response of the downstream circuits.A laser beam crosses an optical fiber channel fabricated with 3D printing technology,to stimulus target light sensitive proteins Ch R-2.Once it activated the related neurons in nematodes,signals could transmit to downstream neuron and lead to corresponding behavioral changes.This device puts forward a simple and rapid low cost chip that could be further used to study the nematodes neural circuits.In summary,we developed microfluidic toolkits to investigate the food restriction,rheotaxis,electrical stimulation,optogenetics and optofluidics in C.elegans,realizing long-term culture and stimulation analysis.The microfluidic chips enabled high-throughput collection,real-time stimulation,and statistical data from the worms.