Yeast glycolysis is one of the best studied metabolic pathways and is a particularly good model system to study oscillatory behaviour, due to the tendency of yeast populations to synchronise their oscillations1. To resolve the question whether isolated yeast cells can oscillate, we studied yeast in micro-fluidic cells, under conditions that prevent cell-cell communication (low cell density, high flow rate). Thus, we could separate oscillations from synchronisation, which is not possible in typical population studies where a population average is monitored (i.e. where only synchronised cultures can be studied). After characterising the yeast oscillations in isolated cells, it is now important to allow cell-cell communication in the system to study the synchronisation characteristics.
A setup, consisting of an optical tweezers system and microfluidic devices coupled with fluorescence imaging was designed to perform a time dependent observation during artificially induced glycolytic oscillations. Multi-channel flow devices and diffusion chambers were fabricated using soft lithography. Automatized pumps controlled specific flow rates of infused glucose and cyanide solutions, used to induce the oscillations. Flow and diffusion in the microfluidic devices were simulated to assure experimentally the desired coverage of the solutions across the yeast cells, a requirement for time dependent measurements.
Using near infrared optical tweezers, yeast cells were trapped and positioned in array configurations, ranging from a single cell to clusters of various symmetries, in order to obtain information about cell-cell communications during the metabolic cycles.
Single cell analysis techniques provide a unique opportunity of determining the intercellular heterogeneity in a cell population, which due to genotype variations and different physiological states of the cells i.e. size, shape and age, cannot be retrieved from averaged cell population values. In order to obtain high-value quantitative data from single-cell experiments it is important to have experimental platforms enabling high-throughput studies. Here, we present a microfluidic chip, which is capable of capturing individual cells in suspension inside separate traps. The device consists of three adjacent microchannels with separate inlets and outlets, laterally connected through the V-shaped traps. Vshaped traps, with openings smaller than the size of a single cell, are fabricated in the middle (main) channel perpendicular to the flow direction. Cells are guided into the wells by streamlines of the flows and are kept still at the bottom of the traps. Cells can then be exposed to extracellular stimuli either in the main or the side channels. Microchannels and traps of different sizes can be fabricated in polydimethylsiloxane (PDMS), offering the possibility of independent studies on cellular responses with different cell types and different extracellular environmental changes. We believe that this versatile high-throughput cell trapping approach will contribute to further development of the current knowledge and information acquired from single-cell studies and provide valuable statistical experimental data required for systems biology.
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