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

Anal Chem. 2021 Feb 2;93(4):2570-2577. doi: 10.1021/acs.analchem.0c04627. Epub 2021 Jan 18.

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 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 O2 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • A549 Cells
  • Cell Culture Techniques / instrumentation
  • Equipment Design / instrumentation*
  • Equipment Design / methods*
  • Humans
  • Microfluidic Analytical Techniques / instrumentation*
  • Oxygen*
  • Printing, Three-Dimensional / instrumentation*

Substances

  • Oxygen