Slipstreaming Mother Machine: A Microfluidic Device for Single-Cell Dynamic Imaging of Yeast

Abstract

The yeast Saccharomyces cerevisiae is one of the most basic model organisms for studies of aging and other phenomena such as division strategies. These organisms have been typically studied with the use of microfluidic devices to keep cells trapped while under a flow of fresh media. However, all of the existing devices trap cells mechanically, subjecting them to pressures that may affect cell physiology. There is evidence mechanical pressure affects growth rate and the movement of intracellular components, so it is quite possible that it affects other physiological aspects such as aging. To allow studies with the lowest influence of mechanical pressure, we designed and fabricated a device that takes advantage of the slipstreaming effect. In slipstreaming, moving fluids that encounter a barrier flow around it forming a pressure gradient behind it. We trap mother cells in this region and force daughter cells to be in the negative pressure gradient region so that they are taken away by the flow. Additionally, this device can be fabricated using low resolution lithography techniques, which makes it less expensive than devices that require photolithography masks with resolution under 5 µm. With this device, it is possible to measure some of the most interesting aspects of yeast dynamics such as growth rates and Replicative Life Span. This device should allow future studies to eliminate pressure bias as well as extending the range of labs that can do these types of measurements.

Phenomenology and dynamics of competitive ecosystems beyond the niche-neutral regimes

“…Although it may be difficult to experimentally determine and control the immigration rate, the competition strength, and the carrying capacity, practical proxies for these parameters exist. By way of an example, the flow rate carrying bacteria into a chamber of a microfluidic device is a well-controlled quantity that approximates well the immigration rate for populations encased in the chamber ( 99 ). Another commonly used and robustly estimated experimental observable is the SRA, which can be used to infer the SAD to which it is closely mathematically related ( SI Appendix , section 7 ), although in practice the conversion might be constrained by limitations of noise and quantity of the experimental data.…”

Platforms for Optogenetic Stimulation and Feedback Control

“…In ( Kumar et al, 2021 ), the authors cultured yeast cells in a monolayer for up to 6 h by sandwiching them between a solid nutrient agarose pad and a glass coverslip. Microfluidic chips provide another way for achieving constrained cell proliferation under the microscope as shown in several studies for yeast ( Rullan et al, 2018 ; Durán et al, 2020 ), bacteria ( Chait et al, 2017 ; Lugagne et al, 2017 ), and mammalian cells ( Woodruff and Maerkl, 2016 ). These chips are suitable for long-term experiments as they facilitate constant replenishment of nutrients and removal of dead cells via medium flow.…”

“…This makes tracking, stimulating and imaging a single cell challenging in long-term optogenetic control experiments. One can use a microfluidic “mother machine” device ( Chait et al, 2017 ; Durán et al, 2020 ) under the microscope to ensure single cell tracking in long-term experiments, but these special cell culturing devices are only compatible with bacterial or yeast cells. Additionally, while designing synthetic constructs in cells, one is restricted to using fluorescent proteins as a reporter system because observation on these optogenetic platforms is mostly carried out via fluorescence imaging.…”

Microfluidics for long-term single-cell time-lapse microscopy: Advances and applications

“…Each jail is composed of three PDMS posts (among other designs) that behave as “jail bars” to retain a single mother cell, while daughter cells produced by budding are washed away with the flow of media. Similarly, A Long-term Culturing And TRApping System (ALCATRAS) ( Crane et al, 2014 ), High-throughput Yeast Aging Analysis chip (HYAA) ( Jo et al, 2015 ), and slipstreaming MM ( Durán et al, 2020 ) devices comprise an array of PDMS trapping units, many of which may fit into a single field of view during imaging, to enable tracking of individual mother cells over their lifespan. In the latter, S. cerevisiae cells are loaded through the outlet of the device such that media flow reversal enables the trapping of mother cells in a low-pressure zone behind PDMS pillars.…”