Technique for In vitro Intermittent Hypoxia Exposure

Intermittent hypoxia (IH) occurs in obstructive sleep apnea and other diseases. In vitro IH research is hampered by poor solubility and low diffusion of O 2 in solution. Here, we describe an in vitro system enabling reproducible cell exposure to IH. A unique 24‐well culture plate with a base made of a gas‐permeable fluorocarbon membrane was used. The plate was placed in an airtight box submerged in a heated water bath. An electronically controlled solenoid air‐valve switched inflow between two gas tanks every 3 min. For IH, the box was aerated with control (C; 16% O 2 , 5% CO 2 , 79% N 2 ) or hypoxic (H; 0% O 2 , 5% CO2, 95% N 2 ) gas. In control experiments (IC), 2 C tanks were used. Media oxygen tension and temperature were continuously monitored by submerged sensors. Over 5 hr, oxygen in the media reproducibly cycled between 13.1 ± 0.2 and 0.1 ± 0.02% at 37°C. Experiments were also performed in an O 2 Control Cabinet for In Vitro Studies with a ramp/soak O 2 Controller from Coy Laboratory Products. To test the system, rat insulinoma cells (INS832/13) were grown on the plate and exposed to IH or IC for 5 hr before being subjected to glucose challenge (2 hr; 16 mM). Insulin secretion (ELISA), decreased by 64% (5.5±3.6 vs 2.0±0.6 ng/μg) in IC and IH cell. Thus, this system enables reproducible, sustained IH cell exposure, with phenotype changes such as lower glucose‐stimulated insulin secretion. Grant: NIH RC1 HL099952