Logarithmic bacterial gradient chip for analyzing the effects of dietary restriction on C. elegans growth

Abstract

a b s t r a c t C. elegans is a commonly used model organism for studying the basic mechanisms of lifespan extension by dietary restriction (DR). Progress has been limited, however, by the lack of an automated system for quantitative analysis of this effect of bacterial diet. In this work, multifunctional logarithmic gradientcustomizing microfluidic device, which takes advantage of hydrodynamics, was developed to long-term maintain worm culture on the chip with parallel live imaging and generate logarithmic concentration gradient of bacteria. We successfully employed this platform to investigate the longevity and stress response of the worms in response to various concentrations of bacterial food. The results indicated that the lifespan extension of the worms was induced by DR when bacteria density was 10 8 cells mLand further dilutions would reduce the lifespan, which proposed a new dietary restriction regime in C. elegans. In addition, imaging analysis showed that DR regulated the subcellular localization DAF-16 in a way different from starvation that reduced lifespan of the worms. The platform allows not only horizontal studies of C. elegans in response to various environments over a wide range of concentrations, but also multiparameter evaluation of longevity in the worms with high temporal resolution, providing clues to better understand the aging process and help age-related drug screen.

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].…”

How do C. elegans worms survive in highly viscous habitats?

“…Caenorhabditis elegans has been intensively used for studies of calorie restriction associated with aging and longevity (Klass, 1977(Klass, , 1983Johnson and Wood, 1982;Kenyon et al, 1993;Lakowski and Hekimi, 1998;Lenaerts et al, 2008;Greer and Brunet, 2009;Ge et al, 2018) because it is easy to handle and observe as a result of its small size (about 1 mm in length), optical transparency, short life cycle and genetic tractability (Wood, 1988;The C. elegans Sequencing Consortium, 1998). Previous studies have reported that calorie restriction without starvation or malnutrition increases the life span of C. elegans worms (Klass, 1977(Klass, , 1983Johnson and Wood, 1982;Kenyon et al, 1993;Lakowski and Hekimi, 1998;Lenaerts et al, 2008;Greer and Brunet, 2009;Ge et al, 2018). However, the lifespan of malnourished or starved worms can be increased by increasing the concentration of food (<10 8 -10 9 cells ml −1 ) (Klass, 1977;Greer and Brunet, 2009;Ge et al, 2018).…”

“…Previous studies have reported that calorie restriction without starvation or malnutrition increases the life span of C. elegans worms (Klass, 1977(Klass, , 1983Johnson and Wood, 1982;Kenyon et al, 1993;Lakowski and Hekimi, 1998;Lenaerts et al, 2008;Greer and Brunet, 2009;Ge et al, 2018). However, the lifespan of malnourished or starved worms can be increased by increasing the concentration of food (<10 8 -10 9 cells ml −1 ) (Klass, 1977;Greer and Brunet, 2009;Ge et al, 2018). To investigate the lifespan of worms, survival assays have been performed with both solid and liquid culture media.…”