High-throughput analysis of yeast replicative aging using a microfluidic system

Abstract

Saccharomyces cerevisiae has been an important model for studying the molecular mechanisms of aging in eukaryotic cells. However, the laborious and low-throughput methods of current yeast replicative lifespan assays limit their usefulness as a broad genetic screening platform for research on aging. We address this limitation by developing an efficient, high-throughput microfluidic single-cell analysis chip in combination with high-resolution time-lapse microscopy. This innovative design enables, to our knowledge for the first time, the determination of the yeast replicative lifespan in a highthroughput manner. Morphological and phenotypical changes during aging can also be monitored automatically with a much higher throughput than previous microfluidic designs. We demonstrate highly efficient trapping and retention of mother cells, determination of the replicative lifespan, and tracking of yeast cells throughout their entire lifespan. Using the high-resolution and large-scale data generated from the high-throughput yeast aging analysis (HYAA) chips, we investigated particular longevity-related changes in cell morphology and characteristics, including critical cell size, terminal morphology, and protein subcellular localization. In addition, because of the significantly improved retention rate of yeast mother cell, the HYAA-Chip was capable of demonstrating replicative lifespan extension by calorie restriction.microfluidics | high-throughput | replicative aging | calorie restriction | Saccharomyces cerevisiae

Multigenerational silencing dynamics control cell aging

“…The conventional method for studying yeast aging requires laborious manual separation of daughter cells from mother cells after each division and does not allow tracking of molecular processes over multiple generations during aging (7). Recent advances in microfluidics technology have automated cell separation and enabled continuous single-cell measurements during aging (8)(9)(10)(11)(12)(13)(14). Building on these efforts, we developed a microfluidic aging device.…”

“…Supplying media through ∼20-μm-tall main channels readily allowed excess cells to be washed away and prevented clogging. Therefore, a critical feature of our device is its two-layer design, making it extremely robust over the course of our experiments, which takes more than 80 h. This is a unique feature compared with recently published devices that are all single-layer (10,13,14). The device was optimized for using continuous gravity-driven flow during operation, with the three-inlet design also facilitating media switching experiments.…”

“…2B, color map). These two distinct types of age-associated phenotypic changes suggest different molecular causes of aging in isogenic cells (8,9,14). Previous studies showed that the aging phenotype with small round daughters could be related to an age-dependent mitochondrial dysfunction (9,12), but the molecular mechanisms underlying the other aging type characterized by elongated daughters remain largely unclear.…”

A High-Throughput Screen for Yeast Replicative Lifespan Identifies Lifespan-Extending Compounds

“…The lifespan of model organisms can range from years in mammals to several days in the yeast Saccharomyces cerevisiae . Throughput limitations have been partially addressed with massive parallel studies in the moderately long-lived organism Caenorhabditis elegans (Petrascheck, Ye, and Buck 2009) or technology that enables rapid, but not scalable, experiments in short-lived models (Liu, Young, and Acar 2015; Jo et al 2015). These approaches are constrained in that they permit either large-scale or quick turn-around, but not both.…”

Creating Isolated Liquid Compartments Using Photopatterned Obstacles in Microfluidics

“…In the literature, there are two broad categories of studies related to liquid entrapment. The first focuses on the design of microfabricated traps such as depressions and other lithographic features for entrapment of individual droplets [7][8][9][10][11][12][13][14][15]. In contrast, the second focuses on the design of a complex porous network for larger-scale liquid entrapment [16][17][18][19][20][21][22][23][24][25][26].…”