96-Well Oxygen Control Using a 3D-Printed Device

Oxygen concentration varies tremendously within the body and has proven to be a critical variable in cell differentiation, proliferation, and drug metabolism among many other physiological processes. Currently, researchers study the gas's role in biology using low-throughput gas control incubators or hypoxia chambers in which all cells in a vessel are exposed to a single oxygen concentration. Here, we introduce a device that can simultaneously deliver 12 unique oxygen concentrations to cells in a 96-well plate and seamlessly integrate into biomedical research workflows. The device inserts into 96-well plates and delivers gas to the headspace, thus avoiding undesirable contact with media. This simple approach isolates each well using gas-tight pressure-resistant gaskets effectively creating 96 mini-incubators. Each of the 12 columns of the plate is supplied by a distinct oxygen concentration from a gas-mixing gradient generator supplied by two feed gases. The wells within each column are then supplied by an equal flow-splitting distribution network. Using equal feed flow rates, concentrations ranging from 0.6 to 20.5% were generated within a single plate. A549 lung carcinoma cells were then used to show that O-2 levels below 9% caused a stepwise increase in cell death for cells treated with the hypoxia-activated anticancer drug tirapirizamine (TPZ). Additionally, the 96-well plate was further leveraged to simultaneously test multiple TPZ concentrations over an oxygen gradient and generate a three-dimensional (3D) dose-response landscape. The results presented here show how microfluidic technologies can be integrated into, rather than replace, ubiquitous biomedical labware allowing for increased throughput oxygen studies.

Automated summary

Researchers have developed a device that can deliver 12 unique oxygen concentrations to cells in a 96-well plate, allowing for in-depth studies on the effects of varying oxygen concentrations on cell responses. This technology seamlessly integrates into biomedical research workflows, demonstrating the potential for increased throughput in oxygen studies.