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

Abstract

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 which 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 twelve 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 O2 levels below 9% caused a stepwise increase in cell death for cells treated with the hypoxia-activated anti-cancer drug Tirapirizamine (TPZ). Additionally, the 96-well plate was further leveraged to simultaneously test multiple TPZ concentrations over an oxygen gradient and generate a 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.

Emulating clinical pressure waveforms in cell culture using an Arduino-controlled 3D-printed platform for 96-well plates

“…In recent years, methods for cell culture within multiwell plates have grown increasingly complex and more accurate at mimicking in-vivo conditions. Insert devices and custom-made plates now can incorporate a growing number of physiological variables such as gas tension, [22][23][24][25] perfusion, 26 matrix-stiffness, 27 mechanical confinement, 28,29 and cell-cell interaction. 30 Multiwell plates are a powerful tool to investigate many conditions simultaneously and are the workhorse for high-throughput screening.…”

“…8,10 which do not resemble those experienced by cells in-vivo. However, re-cent advances in open-source microfluidic pressure control 12 and methods developed by our laboratory to control gaseous environments within the headspace of 96-well plates 13 have set the stage for precise control of complex waveforms within cell culture. Building on the strengths of the gas pressure method and overcoming its weakness, we introduce a high-throughput, workflow-compatible, precise-waveform device for in-vitro hydrostatic pressure control in 96-well plates.…”

Influence of the Hypercapnic Tumor Microenvironment on the Viability of Hela Cells Screened by a CO2-Gradient-Generating Device

“…Eddington and co-workers recently developed a 3D-printed gas delivery device that adapts to the standard 96-well plate. 17 In their design, a linear oxygen (O 2 ) profile was produced by mixing two gases through distribution networks, and O 2 of 12 quantized concentrations was equally delivered to the wells of each column. Without the need for specialized gas mixing network fabrication, we previously developed a modular device that can generate gradient CO 2 at tunable concentrations for the full-factorial evaluation of the effects of multiple interacting environmental stressors on microalgae.…”