High-throughput microfluidic living cell arrays for spatiotemporal gene expression profiling

Abstract

The cellular microenvironment is remarkably complex. In the small space near each cell, growth factors are liberated from extracellular matrix, cytokines are secreted from neighboring cells, and hormones arrive from distant organs. These spatially and temporally diverse cues are integrated by signal transduction cascades to modulate the activity of transcription factors, the principle regulators of gene expression. To date, experimental investigation of spatial and temporal transcription factor activation patterns has been limited by the use of destructive measurement techniques that require averaging responses over large cell populations. Similarly, control of complex microenvironments has been limited by the use of static tissue culture platforms. This thesis describes development of a high-throughput experimental platform called the microfluidic living cell array (mLCA) that combines fluidically-addressable cell arrays with a library of GFP reporter cells to enable nondestructive spatiotemporal gene expression profiling in living cells. The first section describes construction of the GFP reporter library and the development of methodologies for performing routine seeding and culture of cells in microfluidic channels. Microfluidic circuits are then designed to achieve parallel control of soluble stimulus concentration and timing for delivery to downstream cells. A novel "Flow-encoded Switching" (FES) design strategy is introduced to control simultaneous delivery of temporally distinct stimulus patterns using a single input. These circuits are demonstrated by profiling dynamic transcriptional responses to cytokine stimulation, and in each case, cell responses are found to depend quantitatively and qualitatively on the timing of the stimulus.

(cont.) The second section describes development of a two-dimensional valve-controlled mLCA for simultaneously profiling the entire transcriptional reporter library in response to a panel of stimuli. Integrated microvalve arrays control row-seeding and column-stimulation of 256 nanoliter-scale bioreactors, creating a high density matrix of stimulus-response experiments. The platform is demonstrated in the context of the hepatocyte stress response by collecting -5000 single-time-point measurements in each automated and unattended experiment. Results from these studies revealed a novel relationship between TNF-alpha and heat shock response activation, and more generally, illustrated that a single cytokine can activate multiple transcription factors with distinct dynamics. The third section transitions from temporal to spatial profiling and describes discovery and exploration of a spatially heterogeneous gene expression pattern in the innate immune system. Using a stable monoclonal ISRE-GFP reporter, double-stranded DNA (dsDNA) stimulation is found to result in 'colonylike' patterns of reporter activity in an otherwise confluent monolayer. Cell sorting and expression profiling reveal that activated reporter colonies are functionally distinct from their non-activated neighbors, and that colonies are responsible for the majority of cytokine and chemokine expression, including the potent antiviral interferon-beta. Using a novel transplant co-culture experiment, colonies are shown to form by contact-dependent intercellular communication and furthermore, this communication is found to depend on gap junctions. In summary, this thesis introduces promising new tools for conducting high-throughput investigations of spatiotemporal gene expression patterns in living cells, and it provides evidence for a novel dsDNA-induced intercellular communication mechanism that amplifies innate immune responses.