Optimization of an IDO Potency Assay for Mesenchymal Stem Cells
Discipline: Biological Sciences
Subcategory: Biomedical Engineering
Session: 2
Room: Exhibit Hall A
Richard Wagner - Athens Technical College
Co-Author(s): Ty Maughon, University of Georgia, Athens GA; Dr. Steven Stice, University of Georgia, Athens GA
Standardized potency testing is crucial for the manufacturing process of mesenchymal stem cells (MSC). Indoleamine 2,3 dioxygenase (IDO) is an intracellular enzyme of interest to measure because it has been shown to be one mechanism by which MSCs suppress the immune response. Interferon-gamma stimulates MSCs to express IDO, at which point this enzyme removes tryptophan from the extracellular environment. Tryptophan is crucial in T-cell proliferation, thus decreasing the concentration of tryptophan will decrease immune response. As tryptophan is degraded inside the cell, a metabolite is secreted into the media called L. kynurenine. The goal of this experiment was to contribute to the standardization effort and determine the optimal storage conditions of spent media when measuring IDO activity.
This experiment was performed at two different population doubling levels (PDL), that being 6.21 and 9.03. Human bone marrow-derived MSCs were seeded at 10,000 cells/cm2 in a 12 well plate and stimulated with interferon-gamma for 24 hours. The spent media was then collected and stored at temperatures of 4? Celsius, – 20? Celsius, and – 80? Celsius. These samples were transferred to a 96 well plate where excess proteins were removed using trichloroacetic acid. The samples were then stained with Ehrlich?s Reagent and analyzed at 490 nanometers of visible spectrum light. A standard curve of known concentrations of L. kynurenine to media was also analyzed at the same spectrum.
From the standard curve, the slope of the line was extrapolated. The absorbance readings were then plugged into the equation of the line and the micrograms of L. kynurenine per milliliter was determined. The L. kynurenine per milliliter was then normalized to cell number. Statistical analysis was then evaluated on the amount of L. kynurenine per cell at the three different temperatures and at each PDL.
A significant difference was found between the amount of L. kynurenine between 4? Celsius and – 20? Celsius, at their respected PDLs (p<0.05). Media that was stored at -20? Celsius conserved much more L. kynurenine, leading us to conclude that 4? Celsius is not the best temperature to store spent media. No other significant differences were found when comparing the results, which lead us to a few conclusions. The first being there was no loss in potency between the two PDLs. Additionally, absorbance readings on the standard curve were at the lower end of the curve. This makes it hard to distinguish differences in groups because the variance causes an overlap in the standards at this low end. Similar experiments should be performed in the future with a greater difference in PDLs and an increased concentration of L. kynurenine in the media. References: I. Sarisozen, C., & Bilir, C. (2017). Indoleamine 2,3-dioxygenase (IDO): Only an enzyme or a checkpoint controller? Journal of Ontological Sciences, 3(2), 52-56. doi: 10.1016-j.jons.2017.04.001 This work is supported by the NSF engineering research center for Cell Manufacturing Technologies.
Funder Acknowledgement(s): NSF engineering research for Cell Manufacturing Technologies.
Faculty Advisor: Dr. Steven Stice, sstice@uga.edu
Role: Most of this experiment was performed by my mentor showing me how to work in a cell manufacturing lab and me taking over when I understood. I performed the techniques in PDL 6.21 while my mentor verified volumes and best practices. At PDL 9.03 I performed all of the techniques by myself. As the summer progressed it became my responsibility to maintain the health of our MSCs. It was my responsibility to know when and how to passage the cells as well as changing the media. Finally, much of the statistical analysis was new to me. Ty walked me through where to find the equations and I did the math and documented the results.