SRIG 25-03: Characterization of Vesicles Expression in Human Splenic Fibroblasts During Calcium Phosphate-Based Transfection

What was the issue being addressed?

The project aimed to investigate the formation and molecular characteristics of vesicles produced by human splenic fibroblast cells in response to transfection with the SARS-CoV-2 RBD-spike protein. Although previous work has described the morphological changes and vesicle emergence following spike protein transfection, the contents and potential function of these vesicles remained unknown. This study specifically addressed that gap by isolating the vesicles and analyzing their protein composition to better understand their biological significance, particularly in the context of cellular stress, immune signaling, or intercellular communication triggered by viral protein gene expression. 

Provide a brief, lay description of the work undertaken/initiative.

In this project, we explored how human spleen cells respond when introduced to the spike protein gene from the COVID-19 virus. In earlier work, we observed that these cells release small bubble-like structures called vesicles soon after receiving the gene. This time, our goal was to find out what those vesicles contain and why they form. We began by growing human splenic fibroblast cells in the lab, and introduced the spike protein gene using a method called calcium phosphate transfection, which allows exogenous DNA to enter cells. After eight hours, at the point at which vesicles are known to form, we collected the samples and used differential centrifugation to isolate the vesicles while removing unwanted cell debris. Finally, we analyzed the protein contents of the vesicles using Western blotting. 

What is the expected impact this project will have on the community?

This study adds to our understanding of how human cells respond at the microscopic level to viral components like the COVID-19 RBD-spike protein. By studying the vesicles released by the cells, we are now understanding how they might be sending signals, managing stress, or activating immune pathways. In the long run, this kind of work can contribute to broader biomedical research, including studies on immune responses, viral infections, and even gene therapy. It’s a small but important step in understanding how cells behave under stress and how we might use that knowledge to develop future treatments.