Commodities

In this section I discuss how experimental requirements shaped the final design of the Mucus Clearance Assay system. In moving through this section it is useful to broadly discuss experiments in the system and break them down into their

constituent parts. This approach allows us to spot commonalities in the different types of experiments and tailor the system and external equipment to that part of the experiment. This assay’s purpose is to explore ASL-cilia interactions. In following that purpose experiments will consist of varying the properties of the ASL or cilia and then observing how that property affects the mucociliary clearance process. Ideally this requires

1. Control over the ASL 2. Control over Cilia 3. Observation of the ASL 4. Observation of the Cilia

The experiments in this dissertation will focus on controlling properties of the ASL. Controlling the cilia is a harder problem to solve, however, the bilayer channel

makes it possible to access both the apical and basolateral compartments of the channel. This access opens up the possibility of future experiments controlling cilia behavior by administering agents such as Pseudomonas aeruginosa II lectin or halothane, which have both been shown to arrest cilia beating (Adam,  Mitchell,   Schumacher,  Grant,  &  Schumacher,  1997;  Manawadu,  Mostow,  &  LaForce,  1979).

Additionally as optical microscopy will be the primary method of

observing/measuring the system, I will condense observation of the ASL and cilia into one section that looks at optimizing the optical properties of the clearance assay and building the optical system to make those measurements.

3.1.3.1 Controlling the Airway Surface Liquid

Controlling the parameters and properties of the airway surface liquid is an essential part of the mucus clearance assay. Specifically in controlling the ASL I want to maintain the ability to remove the endogenous ASL, add exogenous material or simulants, and add tracer particles. The novelty comes with adding the ability to drive fluid over ciliated cultures and to tilt the system to study the effect of drainage. Using a microfluidic bilayer system facilitates most of these abilities: the bilayer system is designed to exchange the fluid in the top compartment and to drive the fluid with a syringe pump. The only real effect that these parameters had on design was in choosing the ports used to access the top compartment. Ports are a

notorious issue in the microfluidics community, however, embedded screw insulators under a layer of PDMS function as robust access ports (Liu  &  Moiseeva,  2008). These ports were mechanically stable in the PDMS, allowed easy connections to

external “plumbing”, and as an added bonus they allowed pipette access for exchanging fluids.

One concern is that the interface between the ASL and chamber sidewalls would change the liquid height due to the influence of wetting. I minimized this issue by making the chambers significantly wider than the anticipated liquid depth (5-20um of ASL depth as mentioned in chapter 2). While wetting would still occur at the walls, this ensured that there was a large region in the middle of the channel to study the drainage phenomenon. I decided to stretch the width of the channel 3mm so that there would be more available space in the “middle” of the channel away from the sidewalls.

3.1.3.2 Optimizing optics: integrating the MCA with a microscope

In addition to designing the mucus clearance assay, I also designed a companion microscope. The primary goal of this scope was to enable tilting experiments: the microscope would make it possible to observe the ASL and cilia while the channel was in a tilted configuration. An added bonus, however, is that I had the opportunity to customize the channel design to the microscope and vice versa. Customizing the channel for optical microscopy was fairly straightforward. PDMS has excellent optical properties, the most important part is making sure that any PDMS in the optical path was level and relatively smooth. A minor issue with PDMS is that it has a tendency to get dirty, so to prevent this and for added mechanical stability I used glass for the bottom surface of the device.

The other factor in optimizing the optics was the working distance between the objectives and the cell layer. I had already chosen to use an inverted

microscope, (in anticipation of the ports and tubing on top of the clearance assay potentially crashing against objectives.) This made the height of the basolateral compartment beneath the cells important in regards to the working distance. Minimizing this height would enable me to

1. Get higher NA objectives with shorter working distances closer to the cell layer to improve the resolution of cilia and tracked particles

2. Look higher in the liquid layer above the cells (an important feature for mapping out flow profiles

I reduced the height of the basolateral compartment by adopting a “dog-bone” design: the basolateral compartment was relatively thin underneath the cell layer, but had two large reservoirs on both ends of the channel.

In addition to modifying the channel, I also modified the microscope so that it was capable of drainage experiments with the clearance assay. A key aspect of the drainage experiments was performing PIV to quantify the fluid behavior while having the ability to measure the cilia beat frequency. This required that the lower optics system (objectives and the fluorescent source), the device, and the upper optics (bright field source, condenser, and phase rings) all had to move to maintain the same relative light path through rotation. To accomplish this a custom built harness was designed so that the entire microscope could rotate in place (Fig. 3.6).

The harness consisted of an elevated board, which attached to external pillars via two axles. The microscope was secured to the board and configured so that the assembly’s center of mass was slightly below the axis of rotation. A large disc on the left axle was included so that the microscope and board could be locked into place at 0, 30, 45, 60 and 90 degrees (Fig. 3.6).

Figure 3.6 Tilting microscope "Ixion". Capable of performing both bright field and epifluorescent imaging while rotated at angles of 0,30,45,60, or 90 degrees.

One final adjustment was to secure the MCA device to the tilting microscope. I accomplished this by locking the normal stage in place and building a custom stage

with a positioner and MCA holder built via 3d printing (Stratasys, Dimension 1200es) (Fig. 3.7).

Figure 3.7 Custom built stage to secure the MCA device to the tilting microscope.