Microfluidic system for on-chip high-throughput whole-animal sorting and screening at subcellular resolution

Abstract

We report a suite of key microfluidic devices for complex highthroughput whole-animal genetic and drug screens. We demonstrate a high-speed microfluidic sorter that can isolate and immobilize Caenorhabditis elegans in a well defined geometry for screening phenotypic features at subcellular resolution in physiologically active animals. We show an integrated chip containing individually addressable screening-chamber devices for incubation and exposure of individual animals to biochemical compounds and high-resolution time-lapse imaging of many animals on a single chip without the need for anesthesia. We describe a design for delivery of compound libraries in standard multiwell plates to microfluidic devices and also for rapid dispensing of screened animals into multiwell plates. When used in various combinations, these devices will facilitate a variety of high-throughput assays using whole animals, including mutagenesis and RNAi and drug screens at subcellular resolution, as well as high-throughput high-precision manipulations such as femtosecond laser microsurgery for large-scale in vivo neural degeneration and regeneration studies.Caenorhabditis elegans ͉ femtosecond laser microsurgery ͉ immobilization and time-lapse imaging ͉ mutagenesis ͉ RNAi and drug screening E xisting large vertebrate animal models currently cannot be used in high-throughput assays for rapid identification of new genes and drug targets because of the size and complexity of the instrumentation with which these models are studied. In recent years, the advantages of using small invertebrate animals as model systems for human disease have become increasingly apparent and have resulted in two Nobel Prizes in physiology and medicine during the last five years for studies conducted on the nematode Caenorhabditis elegans. The availability of a wide array of species-specific genetic techniques, along with the transparency of the worm and its ability to grow in minute volumes make C. elegans an extremely powerful model organism.However, since the first studies on C. elegans in the early 1960s, little has changed in how scientists manipulate this tiny organism by manually picking, sorting, and transferring individual animals. As a result, large-scale assays such as mutagenesis and RNAi screens (1-3) can take months or even years to complete manually. Currently, high-throughput C. elegans assays are performed by adapting techniques developed for screening cell lines, such as flow-through sorters and microplate readers (4-6). Because of the significant limitations of these methods, highthroughput small-animal studies either have to be dramatically simplified before they can be automated or cannot be conducted at all.Here, we report key components of an integrated, wholeanimal, high-throughput sorting and large-scale screening platform for drug and genetic assays with subcellular resolution using microfluidic devices. Although microfluidics have previously been used to perform novel assays on C. elegans, so far research has been limited to specific appl...

Durable spatiotemporal surveillance of Caenorhabditis elegans response to environmental cues

“…Microfluidics has been used for multiple applications in worm biology in recent years 10,11 . These applications include preparing and immobilizing worms for imaging and microsurgery 12 ; rapid control of changes in chemical environment for studies of the chemosensory system 13 ; trapping of worms for quantification of undulatory dynamics 14 ; as well as animal sorting 15 and screening 16 . In particular, several approaches to long-term imaging have been proposed [17][18][19] .…”

On-chip manipulation of single microparticles, cells, and organisms using surface acoustic waves

“…For example, the ability to precisely control the physical location of a cell facilitates the investigation of cell-cell and cell-environment interactions (1). Manipulation techniques could also provide tools to help researchers observe the behavior of entire organisms such as Caenorhabditis elegans (2,3). Additionally, these techniques might also aid in molecular dynamics/mechanics studies by allowing researchers to precisely monitor and control the interactions between biomolecules.…”

“…C. elegans is an attractive model organism for many biological and medical studies, mainly because of its relatively small size (approximately 1 mm long), optical transparency, well-mapped neuronal system, diverse repertoire of behavioral outputs, and genetic similarities to vertebrates (2). However, trapping and manipulating C. elegans has proven to be difficult and generally involves anesthetics, vacuum, cooling, or direct-contact mechanical procedures (2,3,44). To our knowledge, our acoustic tweezers are the first to achieve contact-free, noninvasive, precise manipulation of C. elegans.…”

Scalable electrophysiology in intact small animals with nanoscale suspended electrode arrays

“…This approach provides a scalable method to study extracellular electrophysiology in intact C. elegans and screen for drugs that affect this activity 21 ; however, these measurements are limited to the pharynx in nematode-like organisms. A scalable method for electrophysiology in a variety of small animals combined with the suite of high-throughput technologies to measure anatomical 22,23 and behavioral 13,24 properties would provide unprecedented opportunities to study fundamental neurobiology and neurological disease models.…”