(23d) Rapid Generation and Simultaneous Detection of Biomimetic Oxygen Concentration Gradients in vitro

Introduction: Oxygen homeostasis is critical for the functioning of multicellular organisms. Deficiency of oxygen or hypoxia can lead to several pathological conditions such as ischemia, tumorigenesis and drug resistance. Most studies utilize specialized O₂ incubators to generate singular oxygen concentrations that vary significantly from the physiological conditions where hypoxic gradients exist within the tissue. Current microfluidic technology using polydimethylsiloxane-based (PDMS) devices enables generation of such oxygen concentration gradients, but yield low-to-moderate spatial resolution and mostly involve tedious device assembly. We have developed a novel and simplistic approach of reproducibly and rapidly generating stable biomimetic oxygen gradients with high spatial resolution and integrated detection capability. The microfluidic split and recombine strategy utilizing O₂-rich and O₂-depleted media allows generation of prolonged dissolved oxygen (DO) gradients while an underlying sensor layer allows real-time detection of DO gradients generated. A 3-sided glass coating prevents multi-directional diffusion of ambient oxygen. Device functionality for cell based hypoxia studies was demonstrated through viability analysis of immortalized mammary epithelial cells (MCF-12A) in hypoxic environments.

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 as well as underlying sensor layer. Temperature and reaction time of the curing process were characterized to attain an optimal and reproducible glass-coating. Platinum (ii) octaethylporphyrin ketone in polystyrene (PtOEPK/PS) matrix was spin-coated onto a 1x3 inch glass slide and used as the sensor layer for oxygen detection. A thin PDMS-membrane was spin-coated on top of the sensor layer to inhibit PtOEPK mediated cytotoxicity and facilitate cell growth. Calibration for both gaseous and DO were conducted prior to oxygen gradient generation experiments. MCF-12A 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 8 hours incubation period.

Results and Discussion: The average glass coating thickness progressively increases with both temperature and time. Heating at 80⁰C for 60s produced an optimal glass thickness of approximately 7µm with lower frequency of cracks and no unpolymerized spaces. Compared to uncoated devices, the coated devices offer an increased sensitivity by 20%. Linear Stern-Volmer relationship was maintained for both types of devices, the coated devices exhibiting higher I₀:I₁₀₀ ratio in all cases. Stable oxygen concentration gradients with high spatial resolution were generated over various flow rates, ranging from 1µLmin^-1 to 100µLmin^-1. The slope of the oxygen gradient was constant, irrespective of flow rate. Viability analysis in the multi cell-outlet device showed increased mortality of MCF-12A cells with increasing hypoxic stress, with approximately 10% live cells being viable at 0% oxygen conditions. Since the chambers are physically separated, the cells are not able to migrate to oxygen rich areas.

Conclusion: Our proposed technique enables convenient generation and simultaneous detection of biomimetic oxygen gradients in vitro for relatively long periods. The multi cell-outlet design mimics the functionality of several specialized O₂ incubators at once. To further explore wide-scale applicability of this platform, the activity of hypoxia-activated prodrugs (HAP) in cancer cells under hypoxic gradient is currently being investigated.

Acknowledgments: Funding for this project was provided by the National Science Foundation Award # 1642794 and 1645195.