title: System for exposing cultured cells to intermittent hypoxia utilizing gas permeable cultureware.
abstract: Tissue intermittent hypoxia (IH) occurs in obstructive sleep apnea, sickle cell anemia, physical exercise and other conditions. Poor gas solubility and slow diffusion through culture media hampers mimicking IH-induced transitions of O(2) in vitro. We aimed to develop a system enabling exposure of cultured cells to IH and to validate such exposure by real-time O(2) measurements and cellular responses. Standard 24-well culture plates and plates with bottoms made from a gas permeable film were placed in a heated cabinet. Desired cycling of O(2) levels was induced using programmable solenoids to purge mixtures of 95% N(2) + 5% CO(2) or 95% O(2) + 5% CO(2). Dissolved oxygen, gas pressure, temperature, and water evaporation were measured during cycling. IH-induced cellular effects were evaluated by hypoxia inducible factor (HIF) and NF-κB luciferase reporters in HEK296 cells and by insulin secretion in rat insulinoma cells. Oxygen cycling in the cabinet was translated into identical changes of O(2) at the well bottom in gas permeable, but not in standard cultureware. Twenty-four hours of IH exposure increased HIF (112%), NF-κB (111%) and insulin secretion (44%). Described system enables reproducible and prolonged IH exposure in cultured cells while controlling for important environmental factors.
authors: Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University, School of Medicine, Baltimore, MD, USA. jan.polak@lf3.cuni.cz.
authors: Jan
authors: J
authors: Polak
authors: Karen
authors: K
authors: Studer-Rabeler
authors: Holly
authors: H
authors: McHugh
authors: Mehboob A
authors: MA
authors: Hussain
authors: Larissa A
authors: LA
authors: Shimoda
mesh_terms: D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004867:Equipment Design; D019544:Equipment Failure Analysis; D017022:Flow Injection Analysis / Q000295:instrumentation*; D057809:HEK293 Cells; D006361:Heating / Q000295:instrumentation; D006801:Humans; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D010100:Oxygen / Q000008:administration & dosage* / Q000378:metabolism*; D051381:Rats
chemical_list: D010100:Oxygen