Living cells are remarkably complex. To unravel this complexity, living-cell assays have been developed that allow delivery of experimental stimuli and measurement of the resulting cellular responses. High-throughput adaptations of these assays, known as living-cell microarrays, which are based on microtiter plates, high-density spotting, microfabrication, and microﬂuidics technologies, are being developed for two general applications: (1) to screen large-scale chemical and genomic libraries and (2) to systematically investigate the local cellular microenvironment. These emerging experimental platforms offer exciting opportunities to rapidly identify genetic determinants of disease, to discover modulators of cellular function, and to probe the complex and dynamic relationships between cells and their local environment.
Microfluidic Living Cell Array
We have developed a scalable experimental platform that combines microfluidic addressability with quantitative live cell imaging of fluorescent protein transcriptional reporters to achieve real-time characterization of gene expression programs in living cells. Integrated microvalve arrays control row-seeding and column-stimulation of 256 nanoliter-scale bioreactors to create a high density matrix of stimulus–response experiments. We demonstrate the approach in the context of hepatic inflammation by acquiring 5000 single-time-point measurements in each automated and unattended experiment. Experiments can be assembled in hours and perform the equivalent of months of conventional experiments. By enabling efficient investigation of dynamic gene expression programs, this technology has the potential to make significant impacts in basic science, drug development, and clinical medicine.
Microfluidic living cell array: Layer 1 (yellow) consists of a 16 × 16 array of circular “cell visualization chambers” (50 µm height and 420 µm diameter). Each 2 × 2 subarray in layer 1 is isolated from the others by 2 sets of reversible PDMS barriers. These barriers are controlled by two valve control manifolds (green and purple) in layer 2. Cell lines are drawn from separate inlets (left) through a common outlet (right) to seed the device with rows of different reporters. Similarly, each stimulus is drawn from separate inlets (top) through a common stimulation outlet (bottom). Layer 2 seeding valves (green) are dead-end channels controlled by the pressure in a single inlet (top right) and stimulation valves (purple) are controlled by a single control line (bottom right).
- 50 micrometer deep channels
- two integrated 72-valve arrays
- 64 unique stimulus-reporter combinations
- 256 chambers (<10 nL each), 4 replicates per stimulus-reporter combination
Cell Culture in the Living Cell Array
Cell culture in the microfluidic array—(a) Phase contrast images of 8 representative wells in the array. (b) Enlarged phase contrast image of a single confluent cell-visualization chamber with cells exhibiting morphologies similar to those observed on conventional tissue culture plastic. (c) Fluorescence overlay of red- and green-labeled cells seeded in adjacent rows. Valves are closed to separate rows and columns during cell attachment. (d) Fluorescence overlay of calcein red and green being delivered through adjacent columns while the array is in stimulation configuration.
Profiling Hepatocyte Inflammatory Gene Expression Dynamics
Profiling hepatocyte inflammatory gene expression dynamics (a) Heat map of a single microfluidic living cell array experiment. Each reporter was stimulated with bacterial endotoxin (LPS—25 μg ml−1), inflammatory cytokines (TNF-α—25 ng ml−1, IL-1—25 ng ml−1, IL-6—25 ng ml−1, and IFNγ—10 ng ml−1), a synthetic glucocorticoid hormone (dexamethasone, 4 μM), and combinations thereof (Cyts = TNF-α/IL-1/IL-6) or (Cyts+Dex = TNF-α/IL-1/IL-6/Dex). Cellular fluorescence was measured from 3 cell chambers for each of the 64 stimulus–response pairs every 90 min for 36 h to create the 192 time series comprised of 4608 single-time-point measurements. Data was normalized to initial and maximum levels to highlight the time course of the responses. (b) Responses of NFκB reporters to TNF-α, IL-1, (TNF-α/IL-1/IL-6), and (TNF-α/IL-1/IL-6/Dex). (c) Responses of NFκB, STAT3, and HSE reporters to TNF-α stimulation.
GFP Reporter Cell Line Dynamics
GFP reporter cell line dynamics—(a) Comparison of NFκB and GRE reporter dynamics quantification using FACS and microscopy with image analysis of cells in microfluidic channels. FACS analysis of (b) NFκB and (c) GRE reporter cell populations at 0, 5, 8, and 18 h after stimulation with 25 ng ml−1 TNF-α and 4 μM dexamethasone, respectively. Fluorescence time lapse images of (d) NFκB and (e) GRE reporters in microfluidic cell visualization chambers 2, 5, 8, 11, 14, and 17 h after stimulation.