Schematic representation of the continuous media perfusion setup.

<p>A syringe pump is used to pump media through gas-permeable tubing to adjust gaseous tension levels before entering the culture device.</p

Modelling of flow conditions in the microfluidic chip.

<p>(<b>a</b>) represents the velocity field at half the height of the inlet channel. (<b>b</b>) represents the velocity field 15 um above the culture plane (ACP). (<b>c</b>) shows velocity profiles at x₀ along the z-axis.</p

Quantification of hESC colony size.

<p>Due to the time required to image larger culture areas, only the central area of the control dish, which contained the majority of colonies, was imaged. The numbers of distinct colonies shrinks as nearby colonies grow into each other.</p

Staining of hESC colonies following perfusion culture.

<p>Representative images of the feeder cells and hESC colonies in the culture device after 2 days of continuous perfusion culture. Each row shows the phase contrast images (<b>a</b>, <b>e</b>) of the feeder-attached hESC colonies and the corresponding results from DAPI (<b>b</b>, <b>f</b>,) and pluripotency marker staining for Oct-4 (<b>c</b>), Tra-1-81 (<b>d</b>) and SSEA-3 (<b>g</b>). All images were taken with a 20× objective, scale bar is 200 µm.</p

Co-cultured hESCs in microfabricated culture device and control dish.

<p>Representative phase contrast images of iMEF feeder cells and individual hESC colonies cultured in the microfabricated culture device (<b>a-c</b>) and in the control dishes (<b>d-f</b>). The same two colonies are shown at each of three time points; after 1 day of static culture (a, d), after 1 day of the perfused culture (b, e), and at the end of the 2 days of perfused culture (c, f). All images were taken with a 4× objective, scale bar is 500 µm.</p

Monitoring a hESC colony in the microfabricated culture device during the course of an experiment.

<p>The same colony is shown after (a) 1 day static culture, (b) 1 day perfused culture and (c) 2 days perfused culture. The columns show, from left to right, the raw phase contrast image taken with a 4× objective, an overlay of the automated detection using the image processing algorithm, and the detected area. The scale bars are 500 µm.</p

Design of the microfabricated culture device.

<p>(<b>a</b>) Exploded view showing all parts of the modular microfabricated culture device. (<b>b</b>) Schematic representation of a longitudinal section of the interconnect assembly, showing compression of the PDMS chip around the inlet/outlet ports (dashed rectangle), by the interconnect. (<b>c</b>) Top view of the microfluidic chip with dashed lines showing the footprints of the lid and interconnect bosses. (<b>d</b>) Cross-sectional view showing the two PDMS layers of the microfluidic chip. The lower ‘spacer’ layer elevates the flow equalisation barriers of the top layer and thus reduces the hydrodynamic shear exposure for the cells.</p

Photograph of the assembled modular culture device with the re-sealable lid attached.

<p>Photograph of the assembled modular culture device with the re-sealable lid attached.</p