Tracking lineages of single cells in lines using a microfluidic device

Abstract

Cells within a genetically identical population exhibit phenotypic variation that in some cases can persist across multiple generations. However, information about the temporal variation and familial dependence of protein levels remains hidden when studying the population as an ensemble. To correlate phenotypes with the age and genealogy of single cells over time, we developed a microfluidic device that enables us to track multiple lineages in parallel by trapping single cells and constraining them to grow in lines for as many as 8 divisions. To illustrate the utility of this method, we investigate lineages of cells expressing one of 3 naturally regulated proteins, each with a different representative expression behavior. Within lineages deriving from single cells, we observe genealogically related clusters of cells with similar phenotype; cluster sizes vary markedly among the 3 proteins, suggesting that the time scale of phenotypic persistence is protein-specific. Growing lines of cells also allows us to dynamically track temporal fluctuations in protein levels at the same time as pedigree relationships among the cells as they divide in the chambers. We observe bursts in expression levels of the heat shock protein Hsp12-GFP that occur simultaneously in mother and daughter cells. In contrast, the ribosomal protein Rps8b-GFP shows relatively constant levels of expression over time. This method is an essential step toward understanding the time scales of phenotypic variation and correlations in phenotype among single cells within a population.epigenetics ͉ protein expression ͉ single cell assay ͉ yeast

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

“…The designs reported previously can track trapped mother cells for only up to 18 h (24) and for up to eight divisions (25). Although another system could track the whole lifespan of trapped mother cells, the retention rate of these cells in the original traps over the time course was ∼40-60% (26).…”

“…Microfluidic system designs for studying yeast aging have been reported previously (24)(25)(26); however, they did not permit assays of yeast replicative lifespan because they are unable to track the entire lifespan of mother cells due to the low efficiency in removing daughter cells. The designs reported previously can track trapped mother cells for only up to 18 h (24) and for up to eight divisions (25).…”

Applications of Microfluidics in Quantitative Biology

“…For example, the Weitz group trapped yeast cells in lines, and analyzed the fluctuations and patterns in protein expression within single cells over multiple generations. [17] The Li group developed a microfluidic device that retained mother yeast cells by PDMS (Polydimethylsiloxane) pillars. [18] They attached the yeast cells on a wall coated by avidin to track individual mother cells throughout their whole lifespan, and found that the aging of cells was characterized by an increased general stress and a progressive lengthening of cell cycle for the last few cell divisions.…”

A microfluidic array with cellular valving for single cell co-culture

“…18 Importantly, particles are arrayed with a perfect yield, making it a prime candidate technology for research with rare and precious cells, such as those harvested from biopsy samples. More recently, parallel arrays of trap structures and bypass channels have been used for tracking yeast 19 and human cell lineages. 20 In this contribution we introduce a cellular valving principle for use with a Tan and Takeuchi microfluidic architecture for highly efficient and reliable single cell coupling.…”