Live-Dead analysis of 3D stem cell samples using Immunofluorescence

Author: Hyunseo Seok | Major: Biomedical Engineering | Semester: Spring 2024

Since my freshman year in the Fall of 2021, I have been working with Dr. Younghye Song in Biomedical Engineering research. My research journey with Dr. Song began during my First-Year Engineering “Honors Research Experience” (GNEG 131H1) course, where I gained the opportunity to embark on a two-semester project in a laboratory setting. Drawn by our shared interests in regenerative medicine and disease modeling, I chose to work with Dr. Song. During this time, I gained extensive experience and knowledge about cell culturing and protein-protein interactions in the extracellular matrix. I then focused on utilizing this knowledge to fabricate and test neural-regenerative collagen scaffolds embedded with adipose-derived stem cells. The excitement and novelty of this project solidified my decision to dedicate my undergraduate research to exploring collagen scaffolds.

After expressing my desire to further work with her, Dr. Song and I discussed different projects we could work on. Throughout my sophomore year, I spent my time exploring different incorporations in collagen scaffolds that could potentially improve treatment prognosis in the clinical setting. One of these incorporations included the differentiation of the adipose-derived stem cells. When adipose-derived stem cells are introduced to specific growth factors, their morphology and behavior can be differentiated to act like Schwann-like cells. Schwann cells are cells that provide assistance in natural regeneration of neural injury, so incorporating these differentiated cells into the scaffold will assist the regeneration of neural injuries. Alongside stem cell differentiation, I explored the addition of cellulose nanocrystals (CNC) under the guidance of PhD student Patrick Kuczwara. Since collagen scaffolds would be implanted within the site of peripheral nerve injury, it would be pertinent for the scaffold to have high flexibility and mechanical integrity. CNC would help maintain the gels mechanical integrity while also limiting the immunological response from the host.

With much experience from previous projects related to neural-regenerative collagen scaffold design, I decided to dedicate my honors project on a related field of disease modeling. My project now is involved with utilizing collagen scaffolds to mimic the diseased state of spinal cords under the guidance of PhD student Niko Ala-Kokko. Now, instead of adipose-derived stem cells, I am using human astrocytes due to their activity in spinal cords. Additionally, decellularized rat spinal cords are composited with collagen gels to provide the extracellular protein of spinal cords into the scaffold. The final part that remains is, how do I mimic the injury state? When there is injury within the spinal cord, the body’s natural response is to create fibrotic lesion/scarring in the area by depositing a higher amount of extracellular protein. We can mimic this denser/fibrous environment by gelling the collagen in a cold environment. We have found that this method holds a lot of promise since we found an upregulation of specific proteins (that is upregulated in injury) in the cold gels compared to the control. Now, I am focused on analyzing the glucose metabolic behaviors of the astrocytes in the disease state. This project has been providing me with even more experience in different procedures such as Western Blotting, decellularization procedure, and Multi-Photon Microscopy. With all of the results I obtain from the project, I hope to present my findings at this year’s Biomedical Engineering Society Annual Meeting at Baltimore, MD.

However, claiming that this experience has been smooth and steady would be me being dishonest. I encountered numerous roadblocks along the way, and I expect to go through a lot more in the future. For example, the process of culturing cells and incorporating them into a 3D model is lengthy and prone to complications at any stage. It took me many attempts at the start to even get the gels ready for imaging, but over time, I improved significantly. Additionally, there were countless times that I spent weeks building up an experiment just for the results to not match my expectations. From these many cases, I would have to think introspectively to figure out what mistakes I might have made, and many times there were more than one! I learned from research that much of the process is optimizing. Things rarely go right from the first try, it has to be refined by repeated testing alongside an introspective attitude to further improve the protocol. I gained valuable experience from the various procedures that I have learned, but more importantly, I developed greater patience, tenacity, and self-analysis.

However, I was never truly alone during this journey! Much of my progress is due to the invaluable guidance and support from Dr. Song and the graduate students who assisted me along the way. The environment of the laboratory is very supportive; even though I work under one graduate student at a time, I have received countless of advice and guidance from the other graduate students who are there. Their collective support has been essential to my growth and success in this journey.

Currently, I am funded by the Honors College Research Grant to conduct this research for the Fall and Spring of 2024. This grant provides me with financial support throughout the semester, enabling me to focus fully on my lab work without financial concerns. Additionally, I plan to save a portion of this stipend to attend additional conferences. I believe that attending these conferences will enhance my scientific presentation skills and expand my knowledge of the field.

I am very grateful for this grant and hope to repay this generosity with hard work and meaningful results.