NemaLife: A structured microfluidic culture device optimized for aging studies in crawlingC. elegans

Abstract

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...

HeALTH: An Automated Platform for Long-term Longitudinal Studies of Whole Organisms under Precise Environmental Control

“…DR altered the level of activity and relative behavioral decline across populations in a non-linear way. Furthermore, the decreased lifespan at higher bacterial concentrations (OD 10) raises the possibility that existing microfluidic lifespan devices inadvertently enact some form of DR or hormetic stress on their cultured populations, due to the lower bacterial concentrations used by these systems on-chip [19][20][21][22]. This in turn could potentially result in confounding effects on longevity.…”

“…The system then alerts the experimenter to intervene. As a result, our platform can reliably culture worms under high bacterial concentrations for over two months, which has not been previously demonstrated in a microfluidic device [19][20][21][22]. This then allows us to robustly perform longterm experiments without introducing artifacts.…”

“…For instance, dietary restriction (DR), an evolutionarily conserved environmental perturbation that modulates aging, cannot be studied as food levels deplete over time within these closed culture systems. Existing microfluidic devices for studying C. elegans lifespans allow for precise spatiotemporal environmental control, but often lack experimental robustness, longitudinal tracking, are limited to a range of low food levels, or have not feasibly demonstrated the ability to scale up for high-throughput studies [19][20][21][22].…”

Phenotypic Screening in C. elegans as a Tool for the Discovery of New Geroprotective Drugs

“…The chips are produced by a soft lithography technique. Food can be added at user defined time points (e.g., once a day) [246,267] or continuously through the use of automated pumps [264]. Various microfluidics chips for lifespan assays have been developed including Lifespan-on-a-chip [246] and WormFarm [264].…”

“…Most recently, a microfluidics device called NemaLife was designed that includes a micropillar arena. This arena allows the worms to crawl as they do on agar plates, rather than swim as in typical microfluidics devices [267]. This device also contains sieve channels, but, because the adults generally remain in the micropillar area, the risk of physically damaging them is reduced.…”