Dynamic Gene Expression Profiling Using a Microfabricated Living Cell Array

Abstract

We describe the development of a microfluidic platform for continuous monitoring of gene expression in live cells. This optically transparent microfluidic device integrates high-throughput molecular stimulation with nondestructive monitoring of expression events in individual living cells, hence, a living cell array (LCA). Several concentrations of a soluble molecular stimulus are generated in an upstream microfluidic network and used to stimulate downstream reporter cells, each containing a green fluorescence reporter plasmid for a gene of interest. Cellular fluorescence is continuously monitored and quantified to infer the expression dynamics of the gene being studied. We demonstrate this approach by profiling the activation of the transcription factor NF-kappaB in HeLa S3 cells in response to varying doses of the inflammatory cytokine TNF-alpha. The LCA platform offers a unique opportunity to simultaneously control dynamic inputs and measure dynamic outputs from adherent mammalian cells in a high-throughput fashion. This approach to profiling expression dynamics, in conjunction with complementary techniques such as DNA microarrays, will help provide a more complete picture of the dynamic cellular response to diverse soluble stimuli.

Microfluidics-based systems biology

“…If the bottomlevel output channels are not converged into a single outlet, the device could be used in order to create and isolate several differently concentrated mixtures of the input streams. 40,41 The gradient shape was adjustable in width and shifted from side to side by changing the individual input stream flow rates, which can be performed dynamically. With little modification, linear and nonlinear concentration gradients that could be controlled temporally and spatially were generated.…”

“…The Living Cell Array (LCA) is an exquisite representation of the capabilities of 41 microfluidic devices for systems biology; it is a device capable of high-throughput, dynamic, whole cell gene expression profiling. 40 LCA consists of the pyramidal gradient generator 33 that flows different concentrations of stimulus in medium through each column of an array of cell culture chambers. By seeding the device with cells containing a green fluorescence protein (GFP) reporter attached to a target gene, fluorescence levels representative of gene expression were dynamically recorded (Fig.…”

“…As shown in the top right, the fluorescence intensity in each chamber would be a dynamically measurable level of specific gene expression. 40 Fig . 11 Diagram of the microfluidic H-filter.…”

High-throughput study of alpha-synuclein expression in yeast using microfluidics for control of local cellular microenvironment

“…The expression dynamics of the gene of interest tagged with green fluorescent protein (GFP) can be extracted using fluorescence imaging. [1][2][3][4] This platform is of particular relevance since a variety of conditions can be screened simultaneously and thus affords efficient exploration of a wide parameter space for molecular stimulation of cells.…”

Nanoliter scale microbioreactor array for quantitative cell biology

“…In recent years, the development of the soft lithography molding technology has greatly improved the flexibility of designing microscale devices for cell biology research (Walker et al, 2004;Whitesides et al, 2001). This method has been used to demonstrate mammalian cell patterning in an enclosed array (Chiu et al, 2000;Takayama et al, 1999), computerized cell culture (Gu et al, 2004), cellular responses to chemical gradients (Li Jeon et al, 2002), investigation of cellular differentiation (Tourovskaia et al, 2005), tissue bioreactors (Leclerc et al, 2004;Powers et al, 2002a), and observation of dynamic gene expression (Thompson et al, 2004). In order to scale to larger arrays, it is necessary to selectively control fluid transport through the system while maintaining a favorable cell culture environment.…”