(432j) Rapid Screening of Active Small Animals Enabled By Open-Surface on-Demand Selection through Digital Mapping

Phenotype-based mutant selection of in vivo small animal models is critical to uncover the genetic mechanism that underlies many fundamental biological questions, such as neurodevelopment, cell lineage and aging. However, whole-animal level mutant screening and enrichment is challenging due to the difficulty to isolate, characterize, and select small, active animals in a high-throughput manner. Many different microfluidic techniques have been developed to meet this need. Taking one of the most-studied model organisms in genetics, Caenorhabditis elegans, as an example, continuous-flow based microfluidic systems have been designed to sequentially isolate C. elegans in microchannels, and then interrogate and sort them by active flow control coupled with automated microscopy. However, flow-based microfluidic techniques usually need sophisticated off-chip controllers and user expertise to operate, which impedes their widespread adaptation. In addition, phenotype-based mutant sorting using flow-based microfluidics require users to estimate the selection threshold with a small-scale pilot test. This, as a result, may introduce selection bias and increase false positives, especially in the case of rare mutant enrichment. Therefore, a new mutant screening and selection method is needed to allow for simple isolation of active small animals without the need of complex off-chip controls, and to select animals on-demand without prior knowledge of phenotypical distribution.

To address this challenge, we develop an open-surface microfluidic device combined with a “digital mapping” strategy. We demonstrate rapid isolation, phenotyping, and parallel mutant selection of small model organisms, using C. elegans as an example. Our device consists of an array of PEG-based microgel pads on which active C. elegans can be isolated and imaged. The hydrophilic PEG-microgel pads are surrounded by a less hydrophilic plastic surface made of polyimide Kapton tape. This wettability contrast between the microgel pad and the surrounding area enables a novel capillary-driven separation mechanism, “microswimmer combing”; it uses the moving contact line on the patterned 2D surface to isolate individual C. elegans from a mixed population in suspension. A few hundred of live animals can be loaded in an addressable on-chip array within 1 minute; image-based mutant phenotyping and selection can ensue subsequently. The parallel array format allows us to first perform un-biased phenotyping of the entire population. Multiple phenotypes, including whole-animal behavioral phenotypes and fluorescent cellular features, can be assessed to precisely determine the mutant selection criteria. We show that we can then identify the in-array location of mutant animals with a high degree of accuracy. To achieve rapid mutant selection, we take advantage of the unique open-accessibility and parallel addressability of our array device. Using fast blade-cutting, we “print” a digital selection map on a thin plastic film on which through-holes are opened at the mutant in-array locations. By covering the digital selection map on the animal loaded array to only expose mutant animals of interest, we can retrieve all mutant animals at once by a simple wash. This on-demand digital map printing and parallel mutant collection can be completed within 10 minutes, which is important to maintain the viability of live organisms. To demonstrate the utility of our method, we select mutant C. elegans that show synaptic transmission defect in cholinergic neurons from wild-type animals based on their behavioral response to aldicarb treatment, an acetylcholinesterase inhibitor. The high selection accuracy is then validated by mutant-specific fluorescent markers. In summary, our open-surface microfluidic method provides an ultra-simple, high-accuracy, and rapid mutant enrichment technique for bioparticles such as small model organisms. Due to its simplicity, we envision this technique can be easily adopted in various biological applications, such as forward genetic screening and experimental evolution. With proper scaling, this device can also be tailored for other model biological systems, such as D. rerio and Ciona larvae, or model bacteria, with simple modifications.