A high-throughput microfluidic diploid yeast long-term culturing (DYLC) chip capable of bud reorientation and concerted daughter dissection for replicative lifespan determination

Abstract

Background Budding yeast, Saccharomyces cerevisiae, has been extensively favored as a model organism in aging and age-related studies, thanks to versatile microfluidic chips for cell dynamics assay and replicative lifespan (RLS) determination at single-cell resolution. However, previous microfluidic structures aiming to immobilize haploid yeast may impose excessive spatial constraint and mechanical stress on cells, especially for larger diploid cells that sprout in a bipolar pattern. Results We developed a high-throughput microfluidic chip for diploid yeast long-term culturing (DYLC), optical inspection and cell-aging analysis. The DYLC chip features 1100 “leaky bowl”-shaped traps formatted in an array to dock single cells under laminar-perfused medium and effectively remove daughter cells by hydraulic shear forces. The delicate microstructures of cell traps enable hydrodynamic rotation of newborn buds, so as to ensure bud reorientation towards downstream and concerted daughter dissection thereafter. The traps provide sufficient space for cell-volume enlargement during aging, and thus properly alleviate structural compression and external stress on budding yeast. Trapping efficiency and long-term maintenance of single cells were optimized according to computational fluid dynamics simulations and experimental characterization in terms of critical parameters of the trap and array geometries. Owing to the self-filling of daughter cells dissected from traps upstream, an initial trapping efficiency of about 70% can rapidly reach a high value of over 92% after 4-hour cell culturing. During yeast proliferation and aging, cellular processes of growth, budding and daughter dissection were continuously tracked for over 60 h by time-lapse imaging. Yeast RLS and budding time interval (BTI) were directly calculated by the sequential two-digit codes indicating the budding status in images. With the employed diploid yeast strain, we obtained an RLS of 24.29 ± 3.65 generations, and verified the extension of BTI in the first couple of generations after birth and the last several generations approaching death, as well as cell de-synchronization along diploid yeast aging. Conclusions The DYLC chip offers a promising platform for reliable capture and culturing of diploid yeast cells and for life-long tracking of cell dynamics and replicative aging processes so that grasping comprehensive insights of aging mechanism in complex eukaryotic cells. Graphical Abstract

Integrating microfluidics and synthetic biology: advancements and diverse applications across organisms

“…They have optimized the microfluidic trapping structure which allows newborn buds to rotate in the downstream direction. Wang et al , 75 have increased the number of traps and optimized the platform for long-term culturing for cell-aging analysis and studying replicative lifespan (RLS) of diploid yeast. The device was designed with 1100 traps arranged in an array to hold single yeast cells and remove daughter cells under a laminar-perfused medium.…”

Efficient Capture and Separation of Cancer Cells Using Hyaluronic Acid-Modified Magnetic Beads in a Microfluidic Chip

“…Recently, microfluidic chip technology has aroused great interest due to its low sample cost, high speed, good flexibility, high-throughput performance, and suitability for integrating multiple functional units for cell manipulation. − Various types of microfluidic chips have been designed based on optical trapping, , dielectrophoresis, − inertial focusing, , magnetic tweezers, or acoustophoresis. , Although each technique has its own limitations because of the heterogeneity of tumor cells, it performs well in the enrichment of CTCs under certain conditions. Among these techniques, magnetic separation is a promising tool for CTC enrichment due to its easy manipulation, high capture efficiency, and convenient coupling with immunocytochemistry and the polymerase chain reaction assay. − …”