In this study, we report a microfluidic device for the whole-life culture of the nematode Caenorhabditis elegans that allows the scoring of animal survival and health measures. This device referred to as the NemaLife chip features: (1) an optimized micropillar arena in which animals can crawl, (2) sieve channels that separate progeny and prevent the loss of adults from the arena during culture maintenance, and (3) ports that allow rapid accessibility for feeding the adult-only population and introducing reagents as needed. The pillar arena geometry was optimized to accommodate the growing body size during culture and emulate the body gait and locomotion of animals reared on agar. Likewise, feeding protocols were optimized to recapitulate longevity outcomes typical of standard plate growth. Key benefits of the NemaLife Chip include eliminating the need to perform repeated manual transfers of adults during survival assays, negating the need for progeny-blocking chemical interventions, and avoiding the swim-induced stress across lifespan in animals reared in liquid. We also show that the culture of animals in pillar-less microfluidic chambers reduces lifespan and introduces physiological stress by increasing the occurrence of age-related vulval integrity disorder. We validated our pillar-based device with longevity analyses of classical aging mutants (daf-2, age-1, eat-2, and daf-16) and animals subjected to RNAi knockdown of age-related genes (age-1 and daf-16). We also showed that healthspan measures such as pharyngeal pumping and tap-induced stimulated reversals can be scored across the lifespan in the NemaLife chip. Overall, the capacity to generate reliable lifespan and physiological data underscores the potential of the NemaLife chip to accelerate healthspan and lifespan investigations in C. elegans.

13Electronic Supplementary Information (ESI) available: Movies demonstrating progeny washing. Worm 14 development and arena optimization information. Protocol for lifespan assay in a microfluidic device. Abstract 34Caenorhabditis elegans is a powerful animal model in aging research. Standard longevity assays 35 on agar plates involve the tedious task of picking and transferring animals to prevent younger 36 progeny from contaminating age-synchronized adult populations. Large-scale studies employ 37 progeny-blocking drugs or sterile mutants to avoid progeny contamination, but such 38 manipulations change adult physiology and alter the influence of reproduction on normal aging. 39Moreover, for some agar growth-based technology platforms, such as automated lifespan 40 machines, reagents such as food or drugs cannot be readily added/removed after initiation of 41 the study. Current microfluidic approaches are well-suited to address these limitations, but in 42 their liquid-based environments animals swim rather than crawl, introducing swim-induced 43 stress in the lifespan analysis. Here we report a simple microfluidic device that we call NemaLife 44 that features: 1) an optimized micropillar arena in which animals can crawl, 2) sieve channels 45 that separate progeny and prevent the loss of adults from the arena during culture 46 maintenance, and 3) ports which allow rapid accessibility to feed the adult-only population and 47 emulate the body gait, locomotion, and lifespan of animals reared on agar. We validated our 51 approach with longevity analyses of classical aging mutants (daf-2, age-1, eat-2, and daf-16) 52 and animals subjected to RNAi knockdown of age-related genes (age-1 and daf-16). We also 53 showed that healthspan measures such as pharyngeal pumping and tap-induced stimulated 54 reversals can be scored across the lifespan. Overall, the capacity to generate reliable lifespan 55 and physiological data from the NemaLife chip underscores the potential of this device to 56 accelerate healthspan and lifespan investigations in C. elegans. 57 58 59 60 61 62 63 64 65Aging is a significant risk factor for a broad range of diseases including neurodegenerative 66 disorders, diabetes and cancer 1-5 . With the growing aging population, the socioeconomic 67 burden attributed with age-associated diseases is staggering and development of therapies that 68 promote healthy aging is imperative. C. elegans is a powerful model organism for aging 69 investigations with a short lifespan (3-5 weeks), remarkable genetic similarity with humans (~ 70 38 % orthologs 6 ) and conserved signaling pathways 7 . Additionally, a fully mapped genome 8 and 71 incredible genetic plasticity 9,10 makes C. elegans an attractive tool for aging studies. Advances in 72 fluorescent microscopy 11 and genomic technology (RNAi, CRISPR) 12,13 have further expanded 73 the number of possible ways in which C. elegans can be used to study healthy aging. 74Lifespan analysis has become a classic method for evaluating the effects of a wide variety of 75 gene...