Introduction

BOOMColi, our engineered probiotic model, targets and releases therapeutics in a controlled manner in the large intestine, specifically the colon. We want to treat Ulcerative Colitis, a type of Inflammatory Bowel Disease affecting the lower part of the colon, and our team has developed a calprotectin-based biosensor system to identify and respond to regions of high inflammation. In theory, this biosensor senses high levels of calprotectin in the intestinal epithelial lining given off at highly inflamed regions while also only triggering when there is a high concentration of cells in the area through a process similar to quorum sensing. As high specificity is an integral part of our engineered probiotic model, we sought to simulate the interior of the colon and characterize how sensitive the cell is to the environments described above.

Microfluidics is a relatively new technology that encompasses all systems that use microchannels of fluid to take advantage of the different dynamics of fluids at small scales. These systems are often used in biological settings to simulate fluidic interactions with enzymes and cells with high specificity. Through an agar-based microfluidic channel, we intend to test and characterize our probiotic’s ability to sense the two environmental conditions described above and get a better understanding of its response to releasing the therapeutic when under these conditions.

Our Goal

Our goal is to simulate the interior of the colon in a manner where the environment can be finely tuned and have a highly accurate readout while also having the microfluidic device platform have a low cost, be easy-to-manufacture, and be reliable. With these parameters in mind, we decided to use a calprotectin-doped agarose-based microfluidic chamber. Calprotectin- our target molecule- will be infused into the agarose used to make this microfluidic chamber; when liquid is passed through the chamber, the calprotectin will diffuse into the fluid, similar to the diffusion of calprotectin from the intestinal epithelium into the colonial space. We wanted to characterize the cell's response to the varying calprotectin concentrations as they move through the folds of the intestine and see if they could effectively lyse to release IL-10 at regions of high calprotectin concentrations.

Our Approach

We first designed our molds on CAD software (OnShape) and 3D printed them. 2% agarose was poured onto the molds to create our chamber, consisting of a bottom layer with microfluidic channels and a thin top layer of agarose. Appropriate tubing was attached to the inlet and outlet that connected the chamber to the syringe pump and Eppendorf tube respectively.


Figure 1. Steps involved in making our microfluidic device

To first characterize the sensitivity of the YKG promoter and the subsequent strength of transcription activation, a mixture of cells with a fluorescent protein regulated by the YKG promoter suspended in nutrient-rich LB media would be flowed through the inlet of the device and its volumetric flow rate would be controlled using a syringe pump. As our engineered E.coli moves through the channels, it will sense the calprotectin that diffuses into the media from the agar. This would lead to the cells expressing the fluorescent protein which would then be assessed for expression through spectrophotometry analysis of the output LB media.


Figure 2. Testing BOOMColi’s sensor system

To test if the final BoomColi cells can function similarly to the former cells, we would conduct the same experiment with a mixture of BOOMColi cells and nutrient-rich LB media. If there is a moderately high calprotectin concentration, the cells will trigger the production of lysis genes and lyse to release IL-10, as shown in the image below.


Figure 3. Experimental plan to test our BOOMColi

The flowthrough collected at the outlet end should consist of nutrient-deficient LB media, cell lysates, and lysed and un-lysed E.coli. A western blot can then be performed with the flowthrough to confirm that our calprotectin-based quorum sensing system worked. The initial lysis step of the western blot can be ignored as our cells are programmed to lyse upon sensing high calprotectin concentrations. Our IL-10 with a periplasmic signal sequence is about 22 kDa, and we expect to see a band in that range.

Creating a functional microfluidic device

Measurement

We first asked Dr. Puchalla how to understand the dynamics of the microfluidic chamber so that we could apply what we learned to the interior of the colon. Dr. Puchalla started by discussing measurements for the pressure of a straight pipe, which can be found using the Hagen–Poiseuille equation. This equation is not robust enough for the microfluidic chamber, however, due to all of its curves and different sizes throughout. Understanding the pressure of a more complicated system needs the use of sophisticated analytical modeling like the ComSol plug-in in Matlab. Rather than dealing with such factors, Dr. Puchalla advised us to not look at pressure but instead look at dwell time since what we are looking at is the amount of time the LB is in contact with the calprotectin agar. Such measurements are much easier to measure, usually being done by using a syringe pump to pump liquid into the system in a volumetric manner and to measure the output flow rate with a mass scale. This cuts down significantly on the complexity of our measurements while still providing enough data to transpose our results to a hypothetical intestine.

Design

Before our discussion, our design consisted of a glass layer, an agar layer with a hollow space running through the entire height of the agar (the microfluidic chamber), and a top glass layer. This design also has two inlets to allow for the mixing of two different LBs if necessary and a chamber width of 1mm.

The first thing that Jason advised was to significantly decrease the chamber width since a width of 1mm would cause movement that is too fast. Instead, he told us to redesign the chamber based on previous chambers for biological material flowthrough. Secondly, he noted that the seal between agar and glass cannot be made watertight, therefore if any significant pressure is added to the system, there is going to be loss due to leakage between this boundary layer. Rather than have the design be an agar layer with a channel running through the entire height of the gel, have the microfluidic chamber be enclosed in the agar completely, only having tiny inlet and outlet holes which would be punched through with a biopsy punch rather than through molding. To create this hollow chamber within the agar, a more complex process than a simple mold would be necessary. Dr. Puchalla described a 2 step process where the initial piece of agar would be molded, flipped, and then set on another piece of hot agar in order to close the chamber. This process was quickly implemented into the new design of the agar chamber.


Figure 4. Designs

Equipment

Dr. Puchalla not only helped us in the design and measurement aspects of the project but also gave us access to equipment which brought our microfluidic device to another level of precision and testing ability. This included a syringe pump to regulate the flow rate to an extremely high level of volumetric precision when compared to a normal gravitational device, a PBMS block to use as the top layer of the system which has the same clear properties as glass but is much easier to punch inlet and outlet holes through, a biopsy punch to make the holes

Steps involved in creating our functional microfluidic device:

1. Designing our molds

Our molds were designed using Onshape- a CAD software. We determined our microfluidic chamber dimensions after reviewing previous research in microfluidics

Figure 5. Onshape mold design.

2. 3D printing our molds

We used Creality Ender 3 3D printer with Creality PLA Filament 1.75mm to print all of our designs. We used Ultimaker Cura to splice all of the molds with standard “Super Quality” settings and “Raft” build plate adhesion.

3. Making our microfluidic chamber

Our microfluidic chamber was made using a 2% agarose solution in a 1:10 diluted TAE buffer. The serpentine mold was placed in a container and 2% agarose at 65°C was poured until it covered the top. The top layer of the microfluidic chamber was made by placing the agarose strip face down on top of thin agarose gel supports and pouring 2% agarose at 65°C on the sides.

Figure 6. Steps to make our microfluidic chamber

4. Assembling a functional microfluidic device

A syringe pump was used to regulate the flow rate. Appropriate tubing was attached to a 10ml syringe and connected to the inlet of the microfluidic device. Another tube attached to the outlet end led to an empty Eppendorf tube.

Figure 7. Assembling our microfluidic device

Figure 8. Our assembled microfluidic device

Testing our microfluidic device

We mixed water with a blue dye (Gel Loading Dye Blue(6x) NEB, 1:1000 concentration in media. ) and flowed it through our system. This is a short video of the liquid moving through the microfluidic channels.

Determining the fluid velocity/ volumetric flowrate to be used

The fluid velocity through the intestine varies and depends on factors like – meal intake, diameter, and the volumetric flowrate through each section of the intestine . On average, the fluid velocity through the human gut can be taken as 0.4mm/s . To calculate the pumping rate our syringe pump must be set to, we need to calculate the volumetric flowrate . As our focus is a treatment for ulcerative colitis, we will stimulate the epithelium of the descending colon. For this purpose, we will dispense our cell mixture into the microfluidic chamber at the same average velocity as the colon. Velocity is chosen as the constant parameter over the volumetric flow rate, as our focus is on the interaction of the cell mixture with the intestinal epithelium. Using a greater fluid velocity in the microfluidic chamber than that of the colon will result in the inaccurate characterization of our sensor system.

Figure 11. Estimating the volumetric flowrate (Q)
Therefore, the volume of liquid that we need to pass through the microfluidic chamber per second is 0.16 ml.

Conclusion

Due to time constraints and issues with funding, we were unable to run cell based experiments using the microfluidic chamber however we were able to show that using our method, we can produce a functioning microfluidic chamber that models the intestinal epithelium of the colon.

Our Engineering Success:

References

1. J. Cremer et al., Effect of flow and peristaltic mixing on bacterial growth in a gut-like channel. Proc. Natl. Acad. Sci. U.S.A. 113, 11414–11419 (2016).
2. Sidar, B., Jenkins, B. R., Huang, S., Spence, J. R., Walk, S. T., & Wilking, J. N. (2019). Long-term flow through human intestinal organoids with the gut organoid flow chip (GOFlowChip). Lab on a chip, 19(20), 3552–3562. https://doi.org/10.1039/c9lc00653b
3. Tiwari, S.K., Bhat, S. & Mahato, K.K. Design and Fabrication of Low-cost Microfluidic Channel for Biomedical Application. Sci Rep 10, 9215 (2020). https://doi.org/10.1038/s41598-020-65995-x