Mammalian cell culture in vertical hood
Author: Lillian Hutchinson | Major: Chemical Engineering | Semester: Fall 2024
My name is Lillian Hutchinson, and I am part of the College of Engineering. I am majoring in chemical engineering with a minor in mathematics. I work within Dr. Younghye Song’s lab in the biomedical engineering department. I have completed my second semester of research on my project titled ‘Protease Expression of Adipose Stem Cells in 3D Scaffold of an Aligned Hydrogel for Disease Modeling’.
My work in Dr. Song’s lab began through a National Science Foundation Research Experience for Undergraduates (REU) program the summer of 2023. When applying for this program, I was interested in the use of hydrogels for tissue engineering applications. This led me to choose Dr. Song as my preferred mentor.
Within this REU, I optimized the procedure for a temperature dependent and aligned collagen I hydrogel. The goal of the project was to develop a hydrogel to mimic the microenvironment of diseased tissue. Increased fiber thickness has been correlated with the development of disease. Fiber alignment can be used to mimic specific tissue. I determined the procedure that resulted in thicker collagen fibers while maintaining fiber alignment. After the program, I continued to work within Dr. Song’s lab and my project developed to model fibrotic tissue.
Fibrosis is characterized by the accumulation of excess collagen which can lead to organ failure. Currently, in vivo models are primarily used to study fibrosis, but a hydrogel disease model would be more cost efficient and replicable. Using the previously optimized disease model seeded with adipose-derived stem cells, I began testing the interactions between cells and their environment.
Matrix metalloproteinases (MMPs) are enzymes secreted by cells capable of naturally degrading collagen. MMP-14 has specifically been found to degrade collagen I and play a role in the regulation of fibrosis. I hypothesized that MMP-14 expression would be increased in the diseased tissue model in comparison to the healthy tissue model. MMP-14 expression is important within the disease model because as the enzyme degrades collagen, it creates a space for cells to attach and grow. The expression of MMP-14 would be increased in a disease model because of the prevalence of thicker collagen fibers. To analyze the concentration of MMP-14 between the two models, I performed western blot and image analysis.
Challenges within this experiment have included the need to retest the optimization of the disease model. Due to equipment malfunction, much of the data taken during my REU had to be redone to ensure accurate results. I developed new hydrogels and measured the fiber thickness via image analysis. I found that 10 minutes at 4°C, 22°C, and 37°C each resulted in thicker fibers for unaligned gels. In contrast, the 10 minutes at 4°C, 15 minutes at 22°C, and 5 minutes at 37°C resulted in thicker fibers for aligned gels. Based off these results, future projects within the lab will use the optimized procedure depending on if the gels are aligned or unaligned. I plan an on performing this experiment an additional time with new equipment to further ensure the accuracy of these results.
Western blot has introduced further challenges in my research. After performing the experiment multiple times, it was determined the procedure needed adjustment. Graduate students in the lab have been assisting with optimizing the protocol. Due to these challenges, more experimentation is needed to determine the concentration of MMP-14 within the model.
To determine if cold-casting or warm-casting has an impact on alignment, I will use a previously established MATLAB code to quantify the degree of alignment. In the future, this model can also be applied to various diseases and damaged tissue.
As I continue to work on this project, I plan to submit an abstract for the annual Biomedical Engineering Society conference. At this conference I will present a poster or podium presentation. I will also have the opportunity to attend other presentations and converse with experts within my field. This will allow me to ask questions to further my research as well as learn about other future avenues for my project.
Funding from the Honors College has allowed me to buy antibodies to identify proteins for western blot analysis. It has also allowed me to focus on my research instead of getting a part time job. This will further assist me in pursuing my future goals and earning my Ph.D.