Schematic of the polyester μ-assay chip. (a) The device cross-section shows four layers (a top PDMS layer, a polyester layer containing fluidic channels, a polyester layer containing microwells, and a bottom PDMS layer), which were aligned and bonded utilizing the silicon glue on the polyester film. (b) Schematic of the channels and the well layers (top view). The branches 1–10 were superposed onto different rows of microwells. (c) Photograph of the assembled device.
Comparison between results of the comsol simulation ((a)–(c)) and the experimental setup ((d)–(f)) showing the normalized FITC concentration profiles for different input flow rates: 200 μl/h, 50 μl/h, 10 μl/h (per stream). These values translate to a maximum average flow velocity of 350 μm/s per channel. A top view representation of the microfluidic device is also shown in (a)–(c), indicating the normalized concentration of FITC in each branch for the respective flow rates.
Colorimetric map indicating the shear stress acting on the cells after the initial cell loading (a) and later in the culture, when the cell aggregate almost completely filled the microwell (b). Both images show the cross-section of a microwell. The direction of flow is from left to right. The total applied flow rate in the model was 400 μl/h. This translates to an average velocity per channel branch of 350 μm/s.
(a) Representative phase contrast and live/dead fluorescence images of mESC aggregates growing inside the microwells over the course of 5 days (at 2× and 10× magnification). Live cells were labeled green, while dead cells were labeled red. (b) Fraction of microwell area occupied by cell aggregates and (c) fraction of fully occupied wells as a function of culture time. Scale bars: 400 μm.
Expression of GFP driven by Oct4 promoter ((a)–(d)) in representative microwells from branches 1 (no LIF) and 10 (1000 U/ml LIF), on days 1 and 5 of culture. The numerically predicted LIF concentration gradient is shown in (e), giving rise to a gradient in GFP (and hence Oct4) expression across the ten branches on days 1 and 5 (f). Images from the control experiment (static culture) are shown in (g), and the corresponding data in (h), relating the GFP intensity to the predicted LIF concentration. Scale bars: 400 μm.
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