An integrated micro-electro-fluidic and protein arraying system for parallel analysis of cell responses to controlled microenvironments

Abstract

Living cells have evolved sophisticated signaling networks allowing them to respond to a wide array of external stimuli. Microfluidic devices, facilitating the analysis of signaling networks through precise definition of the cellular microenvironment often lack the capacity of delivering multiple combinations of different signaling cues, thus limiting the throughput of the analysis. To address this limitation, we developed a microfabricated platform combining microfluidic definition of the cell medium composition with dielectrophoretic definition of cell positions and protein microarray-based presentation of diverse signaling inputs. Ligands combined at different concentrations were spotted along with an extracellular matrix protein onto a glass substratum in alignment with an electrode array. This substratum was combined with a polydimethylsiloxane chip for microfluidic control of the soluble medium components, in alignment with the electrode and protein arrays. Endothelial cells were captured by dielectrophoretic force, allowed to attach and spread on the protein spots; and the signaling output of the NF-kappaB pathway in response to diverse combinations of IGF1 and TNF was investigated, in the absence and presence of variable dose of the pathway inhibitor. The results suggested that cells can be potently activated by immobilized TNF with IGF1 having a modulating effect, and the response could be abolished to different degrees by the inhibitor. This study demonstrates considerable potential of combining precise cell patterning and liquid medium control with protein microarray technology for complex cell signaling studies in a high-throughput manner.

Figure 1

A schematic flow chart of the experimental platform. Electrode array was formed by etching the Indium-Tin-Oxide (ITO) layer on a glass slide. The slide was then derivatized with N-hydroxysuccinimide (NHS) to enable protein immobilization (A). The center of each printed protein spot coincided with the mid-point between the tips of the two paired triangular electrodes. After protein array printing, a PDMS microchannel chip was bonded onto the slide by gentle bonding to form cell culture channels containing the protein spots aligned with electrodes for cell capture and culture in the presence of immobilized signaling ligands (B, C).

Figure 2

Combining dielectrophoresis-assisted microfluidics with protein micro-arraying for cell signaling studies. A: Four columns of Alexa 488 conjugated goat-anti-mouse antibody printed onto the glass substratum part of the device, with concentration increasing from left to right, 0.001, 0.01, 0.1 and 1 mg/ml, respectively; B: A photograph of a fully assembled functional device, highlighting the electrical wiring access to the on-glass ITO electrodes (parts not readily visible indicated in white broken lines), the PDMS microfluidic chips with channels filled with a food dye and inlets - annotated; C: Demonstration of the alignment of a part of the in-chip protein array (green spots indicating the printed antibody as in (A)) with the triangular electrode array inside one of the fluidic channels; D: One Oregon Green 488 conjugated collagen IV array spot (25 μg/ml coating concentration) with several iHUVECs captured and cultured on it. Cells were stained within the chip to indicate the location of the nucleus (stained with DAPI, blue) and anti-p65 antibody (red).

Figure 3

Cell patterning onto the printed protein array spots within a device using DEP. The protein spotted, the cells and staining are as in Fig. 2D. A: DEP cell patterning of single cells, cell pairs and a small group of cells around each electrode pair. B: Protein spots (green) with a single cell, cell pair and a small group of cells. The flow rate and electric field controlling DEP were decreased in (B) to allow an increased number of cells to be captured.

Figure 4

Stimulation of the NF-κB pathway by printed TNFα and IGF1 in iHUVECs. A: Time courses of NF-κB stimulation in iHUVECs by a 10 ng/ml TNFα (filled circles) and 10 ng/ml IGF1 added to the cell medium. The fluorescence intensity values represent in arbitrary units the amount of NF-κB in cell nuclei measured by immunostaining using an anti-p65 antibody (approximately 60 cells were measured for each data point). Error bars represent the standard error of the mean; B: Stimulation of NF-κB by TNFα and IGF1 printed onto the glass cell adhesion substratum. In groups 1 to 4, the coating TNFα concentration changes 0, 2, 10 to 50 μg/ml, while within each group, the coating IGF1 concentration increases 0, 2, 10 to 50 μg/ml from left to right (approximately 30 cells were measured for each data point). Florescence intensities are as in (A). Error bars indicate the standard error of the mean. The insets show typical images of the p65 staining in cells captured on the protein spots corresponding to the indicated conditions (minimal and maximal coating TNFα and IGF1 concentrations). C. Stimulation of NF-κB by TNFα and IGF1 dissolved in the cell medium. From group 1 to 3, TNFα increased as 0,0.1 to 1 ng/ml, while within each group, IGF1 concentration increased from 0 (left) to 10 ng/ml (right).

Figure 5

Inhibition of TNFα stimulated NF-κB activation by a mcirofluidically delivered IκB Kinase (IKK) inhibitor Sc-514. A: iHUVECs stimulated with 10 ng/ml TNFα added to the medium for 1 hour and exposed to different concentrations of Sc-514 into the medium for 2 hours. Approximately 80 cells were measured for each data point. Error bars represent the standard error of the mean. Insets show typical images corresponding to 100 (left) and 0 (right) μM of Sc514; B: iHUVECs stimulated by printed TNFα (50 μg/ml coating concentration) for 1 hour were exposed to a Sc-514 gradient within a microfluidic channel for subsequent 2 hours, with concentrations ranging from 0 to 100 μM. The gradient visualized within the device and one of the channels using Alexa 594 and the typical cell groups captured on the protein spots are shown in the upper part of the panel. The results were binned according to the Sc-514 concentration ranges: ‘low’ (0–33 μM), ‘medium’ (33–66 μM) and ‘high’ (66–100 μM). Approximately 50 cells were measured for each data point, with error bars representing the standard error of the mean. In both (A) and (B) T-test significance is represented by one asterisk, P< 0.05 to 0.01; two asterisks, P< 0.01 to 0.001, three asterisks, P< 0.001.