Four different device designs were obtained from the original publishers in order to eval- uate their suitability for the study of p-bodies. The design principles of each of these de- vices can be seen in Figure 20 to Figure 23.
The microfluidic dissection device of S. Lee et al., 2012, is a multilayer single channel device that retains cells below multiple columns attached to the roof of the channel (imagine a stalactite). This column ends approximately 4 μm from the glass coverslip, while the chan- nel roof is significantly higher. Mother cells are loaded below the columns by flowing cells into the device above the normal operating pressure, pushing the columns upwards and trapping cells when the pressure is released. Media is then flowed through at a reduced pressure, cells begin to grow, and daughter cells are removed in the media flow as they grow beyond the area of the base of the column.
The ALCATRAS (Crane et al., 2014) and HYAA (Jo et al., 2015) devices use similar concepts in order to trap the mother yeast cells. Either V shape or cup shaped traps are placed in an array within a microfluidic channel, and cells are flowed through the channel in a uni- directional manner. Mother cells become trapped against/within the trapping structures due to the pressure of the media flow and daughter cells, budding either above or through
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the gaps in the trap structure, are washed away. One of the main advantages of these devices is that they are single layer of 5 - 6 μm, making the fabrication process significantly
Figure 21: The High throughput Yeast Ageing Array device can accommodate up to 16 strains simultaneously via separate loading and media flow channels. Cells are held in place by the force of media flow and buds are removed in this flow by budding either through or above the traps. The distance between the 2 trap elements is optimised to allow budding of cells while retaining mother cells.
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simpler and less time consuming. The HYAA device also allows multiple strains to be pro- cessed on the same device, greatly increasing throughput.
The final device considered was the CliC (Cell loaded into cavity) device (Fehrmann et al., 2013). While this device was originally published as a single channel, low throughput de- vice, the designers of the chip have recently developed a high throughput 10-channel ver- sion that was used in this study. This device works in a similar way to the single plane microfluidic devices mentioned previously, with a few key differences. In the CliC device the imaging plane is a 3.3 μm tall, in order to trap the yeast cells when sufficient pressure is applied to raise the channel roof during loading. Around this single plane imaging area there is a much deeper channel for media flow, which also allows for cells growing beyond the imaging area to be washed away and prevent clogging of the device. Finally, inside the imaging plane, there are a series of long cavity structures, formed by a layer of PDMS (the construction material) that contacts the glass coverslip. As cells grow across the imag- ing plane, they fill these cavities. When a cell begins to bud in the direction of the cavity opening, it pushes all subsequent daughter cells out of the cavity, and they are eventually washed away into the deeper media channel. As most wild type yeast strains bud in a
Figure 22: The ALCATRAS microfluidic device uses the same trapping concept as the HYAA chip, although a single channel is used rather than multiple channels, and a higher number of inlet channels allows for variation of media conditions during the experiment with aster switching times than external control methods.
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unipolar manner for their replicative lifespan, this allows the mother cell to be trapped within the cavity while all daughter cells are removed. The advantage of this chip design is
Figure 23: The CliC High throughput ageing device uses cavities within a PDMS trapping area to trap cells on a single focal plane. Mother cells are then immobilised within the cavities as daughter cells bud towards the entrance putting pressure on the mother cells. This design has the advantage of multiple independent channels allowing several strains to be analysed in one imaging cycle, as well as a reduced possibility of clogging due to the variation in chamber height between the trapping and flow areas.
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that it is not dependent on the media flow to trap the cell, and once within the cavity, the mother cell is trapped by the presence of other dividing yeast cells. This allows the media flow rate to be adjusted across a much larger range of values for various studies and make the CliC device the most similar to those used in the past for p-body analysis.
For the Lee et al device, the ALCATRAS and the HYAA, CAD design files were obtained from the original authors. The CAD files were used to create Su-8 moulds for PDMS casting as described previously. For the CliC device, an unbonded PDMS chip was obtained from the authors and reverse moulded as described previously.