(69c) Investigation of Drug Efficacy Under in Vitro Hypoxic Gradients in Glioblastoma Multiforme
Materials and Methods: PDMS devices were fabricated using standard lithography techniques. A 3-sided glass coating (cured sol-gel) was deposited on the inner walls of the microfluidic channels to prevent diffusion of ambient oxygen into the media. Temperature and reaction time of the curing process were characterized to attain an optimal and reproducible glass-coating. Calibration for both gaseous and DO were conducted prior to oxygen gradient generation experiments. SF-539 cells were then seeded in a multi cell-outlet device and subjected to hypoxic gradient by flowing regular (21% O2) and deoxygenated (0% O2) media at 1.5µLmin-1 for 24 hours incubation period. Prior to seeding, the cells were stained with 10µM Cell Tracker Green CMFDA dye for live cell staining.
Results and Discussion: The average glass coating thickness progressively increases with both temperature and time. Heating at 800C for 60s or 1000C for 20s produced optimal glass thickness of approximately 7µm with lower frequency of cracks and no unpolymerized spaces. Stable oxygen concentration gradients with high spatial resolution were generated over various flow rates, ranging from 1µLmin-1 to 100µLmin-1. Linear Stern-Volmer relationship (I0:I100) was maintained throughout. Viability analysis in the multiple-outlet device showed that SF-539 cells perished under oxygen levels below 2%, confirming the activation of the prodrug tirapazamine under the hypoxic concentrations generated by the device. Viability reduced to 40% under these conditions whereas tirapazamine had no effect on the SF-539 cells seeded in the chambers containing higher pO2 levels. The cellular proliferation profile was comparable to controls conducted in separate hypoxia chambers using the WST-1 assay.
Conclusion: Our proposed technique enables convenient generation of in vitro DO gradients and allows us to properly investigate effect of localized hypoxia on chemotherapeutic efficacy on GBM cells. This is one of the few such studies capable of doing so. Modifications to design of the device can be done to investigate potential cellular phenomenon, such as epithelial-to-mesenchymal transition in GBM cell lines, a feat that has yet to be achieved.
Acknowledgments: Funding for this project was provided by the National Science Foundation Award # 1642794 and 1645195.