Integrated microfluidic devices for combinatorial cell-based assays

Abstract

The development of miniaturized cell culture platforms for performing parallel cultures and combinatorial assays is important in cell biology from the single-cell level to the system level. In this paper we developed an integrated microfluidic cell-culture platform, Cell-microChip (Cell-microChip), for parallel analyses of the effects of microenvironmental cues (i.e., culture scaffolds) on different mammalian cells and their cellular responses to external stimuli. As a model study, we demonstrated the ability of culturing and assaying several mammalian cells, such as NIH 3T3 fibroblast, B16 melanoma and HeLa cell lines, in a parallel way. For functional assays, first we tested drug-induced apoptotic responses from different cell lines. As a second functional assay, we performed "on-chip" transfection of a reporter gene encoding an enhanced green fluorescent protein (EGFP) followed by live-cell imaging of transcriptional activation of cyclooxygenase 2 (Cox-2) expression. Collectively, our Cell-microChip approach demonstrated the capability to carry out parallel operations and the potential to further integrate advanced functions and applications in the broader space of combinatorial chemistry and biology.

Fig. 1

(a) Schematic representation of an integrated Cell-microChip (Cell-μChip) for performing multiple cell culture and assays under a digitally controlled interface. Three pairs of parallel-oriented cell culture chambers are incorporated in a Cell-μChip, where multiple cell types can be cultured under two different modes of medium supply, i.e., circulatory (channels i, iii and v) and direct feeding (channels ii, iv and vi). The operation of this microchip is controlled by pressure driven valves with their delegated functions indicated by their colors: red for regular valve (for isolation and gating) and yellow for pumping valve (for fluid transport and circulation). (b) Optical image of the actual device. The microchip was loaded with various colors of food dyes to enhance the visualization of different parts in the entire system: red and yellow as in (a); blue indicates the flow channel and the medium reservoir

Fig. 2

Fibronectin coating efficiency on the PDMS surface in a Cell-μChip determined by immunofluorescence assay. (a) Fluorescence images of immunostained FN on the PDMS surface. (b) Quantitative analysis of FN coating efficiency determined with fluorescence images shown in (a)

Fig. 3

Long term culture of NIH 3T3 cells in a Cell-μChip. (a) Time lapse images of the NIH 3T3 cell proliferation in the microchip in a duration of 8 days. (b)–(e) Dead (PI)/live (AO) staining of NIH 3T3 cells cultured in a Cell-μChip for 4 days. (b) A bright field micrograph of NIH 3T3 cell morphologies. (c) A green fluorescence micrograph of the live-stained cells. (d) A red fluorescence micrograph of the dead-stained cells. (e) A merged fluorescence image of (b) and (c)

Fig. 4

Demonstration of six parallel cell cultures in a closely related microenvironment. After 3 days culture, the cell morphologies were shown in (a) NIH 3T3, (b) HeLa and (c) B16. (d) Growth curves of chip-cultured NIH 3T3, HeLa and B16 cells were quantified by monitoring the number of cells inside the cell culture chambers over time. After 5 days culture, we could not count cell number precisely due to cell confluence and multiple cell layers in the cell culture chambers

Fig. 5

Multiparametric apoptosis assays performed in the Cell-μChips. NIH 3T3, HeLa and B16 cells were treated with either staurosporine or actinomycin D to induce apoptosis. Apoptotic cells were stained with Annexin V conjugated with Alexa488 (green), and living cells were stained with MitoTracker (red)

Fig. 6

On-chip transfection and EGFP induction in NIH 3T3 cells. The plasmid vector which encodes EGFP driven by a Cox-2 promoter was transfected with NIH 3T3 cells. (a) Bright field and (b) fluorescence images of NIH 3T3 cells stimulated with TPA for 7 h. (c) Bright field and (d) fluorescence images of NIH 3T3 cells without stimulation