A Generalizable, Tunable Microfluidic Platform for Delivering Fast Temporally Varying Chemical Signals to Probe Single-Cell Response Dynamics

Abstract

Understanding how biological systems transduce dynamic, soluble chemical cues into physiological processes requires robust experimental tools for generating diverse temporal chemical patterns. The advent of microfluidics has seen the development of platforms for rapid fluid exchange allowing ease of changes in the cellular microenvironment and precise cell handling. Rapid exchange is important for exposing systems to temporally varying signals. However, direct coupling of macroscale fluid flow with microstructures is potentially problematic due to the high shear stresses that inevitably add confounding mechanical perturbation effects to the biological system of interest. Here, we have devised a method of translating fast and precise macroscale flows to microscale flows using a monolithically integrated perforated membrane. We integrated a high-density cell trap array for nonadherent cells that are challenging to handle under flow conditions with a soluble chemical signal generator module. The platform enables fast and repeatable switching of stimulus and buffer at low shear stresses for quantitative live, single-cell fluorescent studies. This modular design allows facile integration of any cell-handling chip design with any chemical delivery module. We demonstrate the utility of this device by characterizing heterogeneity of oscillatory response for cells exposed to alternating Ca2+ waveforms at various periodicities. This platform enables the analysis of cell responses to chemical perturbations at a single-cell resolution that is necessary in understanding signal transduction pathways.

Single-cell resolution of intracellular T cell Ca2+dynamics in response to frequency-based H2O2stimulation

“…Recent microfluidic devices have also enabled delivery of robust, time-varying chemical signals, in contrast to conventional experimental techniques which measure the response of cells to a single perturbation of step increase or bolus of stimulus 33, 34 . The enhanced experimental capability can be combined with frequency response analysis such that the underlying complex signalling networks can be discerned more easily.…”

Microfluidic techniques for high throughput single cell analysis

“…Cell traps have been used to individually confine large numbers of single cells for long term observation[16], and a variety of methods are used to array cells including passive hydrodynamics[17], dielectrophoresis[18], and optical gradients[19]. The open configuration of cell trap arrays enables the monitoring of cell-cell signaling[14] and has been used to induce and analyze temporal biochemical patterns over time[20]. …”

Spatially and temporally controlled immune cell interactions using microscale tools

“…The open array format is particularly amenable to different types of measurements, and facilitates scalability (up to 250 000 wells). Although not yet demonstrated with microwell arrays, reversible attachment of microfluidic modules with underlying perforated membrane (for solutions to diffuse) on top of the microwells could further enable their use in closed format, allowing dynamic modulation of the environment when desired [19]. One disadvantage of microwell arrays, however, is the probabilistic (Poisson-limited) loading of wells, which is exacerbated when generating cell pairs or clusters.…”