Cell treatment media change
Author: Neel Patel | Major: Biomedical Engineering
During the Fall of 2020, I joined the Samsonraj Stem cell research lab. Over the course of the fall 2021 semester, I have been working on my mesenchymal stem cell (MSC) muscle differentiation research project. MSCs are a type of adult stem cell that can differentiate, or “transform”, into bone cells, cartilage cells, and fat cells. But MSCs can also be differentiated into muscle cells. While I was discussing the topic of muscle differentiation with my mentor, we were both intrigued on the ways we could improve musculoskeletal disorders in the clinical setting using MSCs. We hypothesized that priming MSCs toward myogenic differentiation could be useful in muscle regeneration. However, the problem with muscle differentiation is that it requires supraphysiological amounts of growth factors to maintain myogenic potential efficacy, which can ultimately result in complicated side effects. So my project focused on finding, optimizing and comparing muscle differentiation methods that resulted in high upregulation of precursor muscle regulatory factors, such as Myf5, MyoD, myogenin (MyoG) and MRF4.
In my experiment there were two muscle differentiation protocols. Method one was the Hydrocortisone + Dexamethasone induction media and method two was the Azacytidine + FGF-2 induction media. After applying both treatment groups to two different cell lines, I had to analyze the gene expression of the muscle regulatory factors with qPCR. Conducting qPCR takes a lot of precision, patience, and time. Because I couldn’t buy ready made primers for the genes I needed, I had Kadek, a graduate student, design the necessary primers. He also assisted me throughout the qPCR process.
There were times where we had to test these primers multiple times to ensure accuracy and reliability. If the primers did not work consistently, it could jeopardize the reliability of the results. To get to the gene analysis step, it took about three weeks of cell culture and another week to do the qPCR. Along the way, there were some unexpected contaminations in my data set, which meant I had to restart the qPCR process. In addition, I had a calculation error and applied the wrong dosage on one of my treatment groups leading to total cell death for that treatment group. My research mentor told me that it was okay to make mistakes like these and that undergraduate research is a cocktail mix of attempts, failures, and reevaluations with a splash of success mixed in. Throughout this grant funding period, I realized that failing as an undergraduate, where the stakes are relatively low, can help prevent repetitive failures when I go to graduate school. This never ending cycle of failures and refinements is the reason I love research so much. Research forces me to develop my critical thinking and calluses my mind to push through and complete an objective. After analyzing my results and discussing them with my mentor, we came up with a plan that focuses on optimizing the myogenic protocols we currently have. It is definitely clear that I have a long way to go to get my research published, but I am making slow and steady refinements to my experiments to achieve the best results possible.
This research project also helped me narrow down my career path to pursue graduate research in stem cell engineering. One of my long term college goals is to present my research at the BMES annual conference next fall. I love the idea of sharing knowledge as it can spark discussion amongst brilliant scientists across the world. Presenting my research is a great way for me to practice my scientific presentation skills in a setting with top level researchers and a great way to gather constructive feedback. As I continue to optimize my experiments, I would also like to publish my research. Without the honors college research grant, I would not have been able to grow my research skills and discover my passion for research.