A system for exposing molecules and cells to biologically relevant and accurately controlled steady-state concentrations of nitric oxide and oxygen

Nitric oxide (NO) plays key roles in cell signaling and physiology, with diverse functions mediated by NO concentrations varying over three orders-of-magnitude. In spite of this critical concentration dependence, current approaches to NO delivery in vitro result in biologically irrelevant and poorly controlled levels, with hyperoxic conditions imposed by ambient air. To solve these problems, we developed a system for controlled delivery of NO and O2 over large concentration ranges to mimic biological conditions. Here we describe the fabrication, operation and calibration of the delivery system. We then describe applications for delivery of NO and O2 into cell culture media, with a comparison of experimental results and predictions from mass transfer models that predict the steady-state levels of various NO-derived reactive species. We also determined that components of culture media do not affect the steady-state levels of NO or O2 in the device. This system provides critical control of NO delivery for in vitro models of NO biology and chemistry.

► We developed a system for controlled delivery of biological relevant levels of NO and O2. ► The fabrication, operation and calibration of the delivery system are described. ► The system was applied to deliver controlled steady-state NO and O2 levels into cell culture media. ► Components of culture media do not to affect the steady-state levels of NO or O2 in the device.