Engineering PhD

After working as an engineer for eight years and managing to make the transition from medical device design to aerospace, I started getting the itch to apply to a graduate program. I was well positioned professionally, but I’d often wondered if I could do science and engineering in the rigorous academic setting of a Ph.D. program, and felt a need to prove to myself that I could do it.

Having seen first-hand the maturity of vehicle design at SpaceX, I chose to pursue a graduate degree focused on what I saw then as the next step—the human element of space exploration. I applied to several schools with programs in bioastronautics, visiting each campus with my husband. Depsite the distance from home and the challenges of a cross-country move, he and I both agreed that Dartmouth was clearly the best program for my goals, and in the Fall of 2019, just before the start of the pandemic, we packed up our two cats and drove across the country to New England. Thankfully I had a few months to get to know people and get a foothold before the miasma of disease, lockdowns, and remote learning descended.

During my time at Dartmouth, I’ve had the unique and valuable experience of working under two fantastic advisors in two different laboratories: Ryan Halter’s Bioengineering/Bioimpedance Lab and Jay Buckey’s Space Medicine Innovations Lab.

Space Physiology Research

Urinary Calcium

On the space side, I have served as Principal Investigator on two NASA grants. In the first grant we developed a urinary calcium measurement device for use in spaceflight. We used a compact fluorimetric reader to detect the intensity of green light emitted by calcein-calcium complexes in sampled urine. In initial laboratory studies we refined the reagent recipe and prototyped a functional optrode. We then tested device accuracy on 100 human urine samples, enrolling participants and collecting urine per an IRB approved protocol. Each urine sample was tested by both the hospital clinical laboratory and our prototype device and agreement between the two methods was assessed with a Bland-Altman plot.

Numerical Modeling of the Human Cardiovascular System in Weightlessness

In addition to this NASA grant funded work, I’ve utilized a novel cranio-vascular numerical model built in MATLAB with features specific for microgravity research to study how weightlessness effects the human cardiovascular system. This modeling work has produced novel hypotheses on how the newly discovered risk of jugular venous flow thrombosis may occur in spaceflight. The model has suggested that intracranial pressure (ICP) in spaceflight is reduced rather than elevated relative to the supine position on Earth, leading our group to a novel theory of how spaceflight associated neuro-ocular syndrome (SANS) develops based solely on hydrostatic forces rather than predicated on pathologically elevated ICP. Additionally, decreased arterial pressure simulated by the model, combined with analysis of past spaceflight experiments suggests a second novel theory that could explain the paradoxical behavior of the arterial baroreflex system in microgravity, where sympathetic nerve activity is elevated while total peripheral resistance is reduced.

EIT & Cell Biology

With Ryan Halter, my work has centered around a project beginning in 2019, to build a non-invasive, real-time system to track the progression of 3D tissue cultures using electrical impedance. This was funded by The Advanced Regenerative Manufacturing Institute (ARMI) in collaboration with EpiBone, a company developing technology to create bone tissue from a patient's mesenchymal stem cells in vitro, for use in autologous bone grafts. I came into this project at the start of the pandemic with almost no cell-biology or cell-culture experience and without any cell-bio expert in our lab to learn under. In the past, when finding myself in deep and unfamiliar water, I’ve found it useful to start from first principles. I took that approach here, digging through all the manuals I could find in the lab, slowly, methodically working my way through each step, designing for myself the necessary hardware (custom circuit boards, 3-D printed six well plate apparatus, bioreactors, etc.) and adapting software (algorithmic cell counters, MATLAB image reconstruction scripts, etc.).

A word here on scope: EIT is a fairly complex, technical imaging modality. Instead of getting into those details here, I’ve tried to provide a broad overview of how things evolved with this project during the last four years, focusing more on experimental design and problem solving. If you happen to be a researcher reading this and would like more detail on the specifics of my protocol or the like, please contact me—I’m more than happy to share what I have learned.

Initial Design

My first design was a simple six-well plate with electrode arrays affixed directly to the bottom surface of each well. This seemed like the most straightforward approach and this arrangement allowed me to culture a cellular monolayer directly on top of the electrode array, keeping them in close proximity to the current-driving and voltage-sensing electrodes. I began by testing which AC current frequencies and amplitudes were most acceptable to cell line I was using.

As I carried out this initial set of experiments, a few significant problems became apparent:

• Fixing the electrodes to the base of the well obscured the view from below, which meant using an upright microscope to visualize and photograph activity in the well was my only option. However, from above, the objective lens would run into the edges of the six-well plate limiting me to a view of only a small circular area in the center of the well.

• A variety of factors combined to make algorithmic cell counting difficult (electrodes in the background, cell dye/stain choice, and limitations resulting from the aforementioned upright microscopy).

• Cells seemed to have trouble adhering to the flexible PCB material and the gold plated surface, making it difficult to judge if the cells were unhappy with the injected current or were absent from a region because of poor initial attachment.

Second Iteration

To address these problems, I designed and 3D printed an apparatus to suspend the electrodes above the cell monolayer in each well. Because these could be easily removed from the well at the end of an experiment, I could now use the inverted microscope to image from below, allowing the objective lens to move freely from edge to edge underneath the six well plate, with neither obstruction nor background artifact.

Moving to 3D Tissue Cultures

This design was a significant improvement, but as my experimental needs grew more complex and I began experimenting on 3D tissue cultures, I needed to access the well to change media twice a week. Unfortunately, removing and replacing the electrode arrays to replace the media created small positional differences that interfered with the generation of high quality time difference impedance data, and this issue proved difficult to mitigate. Additionally, this process would inadvertently create air bubbles that collected on the electrode surface, creating high-impedance artifacts in the data. I needed a repeatable, reliable, but still not-too-tedious way to lock everything in at the start of an experiment and perform the media change without disturbing the electrodes and cell culture scaffold. After some literature review and careful planning, I set my sights on creating a bioreactor with good visual and physical access.

Once I had the bioreactor chamber designed, I had initially planned to slowly push cell media through with a syringe pump, but switched to a peristaltic pump with an air permeable media bag, after learning the importance of gas exchange in closed loop culture systems.

After many iterations I now have a fully-functioning custom designed and fabricated bio-reactor system, which integrates our lab’s specialized electrical impedance tomography hardware. I am continuing to iterate on this design, working toward a system that can non-destructively characterize human cell-growth and differentiation in real time.