Polak Jan, Studer-Rabeler Karen, McHugh Holly, Hussain Mehboob A, Shimoda Larissa A
Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University, School of Medicine, Baltimore, MD, USA.
Gen Physiol Biophys. 2015 Jul;34(3):235-47. doi: 10.4149/gpb_2014043. Epub 2015 Mar 27.
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.
组织间歇性缺氧（IH）发生于阻塞性睡眠呼吸暂停、镰状细胞贫血、体育锻炼及其他情况。气体在培养基中的溶解性差且扩散缓慢，这阻碍了在体外模拟IH诱导的氧（O₂）转变。我们旨在开发一种系统，使培养的细胞能够暴露于IH，并通过实时O₂测量和细胞反应来验证这种暴露。将标准的24孔培养板和底部由透气膜制成的培养板置于加热箱中。使用可编程螺线管吹扫95% N₂ + 5% CO₂或95% O₂ + 5% CO₂的混合物，以诱导所需的O₂水平循环。在循环过程中测量溶解氧、气压、温度和水分蒸发。通过HEK296细胞中的缺氧诱导因子（HIF）和NF-κB荧光素酶报告基因以及大鼠胰岛素瘤细胞中的胰岛素分泌来评估IH诱导的细胞效应。培养箱中的氧循环在透气培养板的孔底部转化为相同的O₂变化，但在标准培养器皿中则不然。24小时的IH暴露使HIF增加（112%）、NF-κB增加（111%）以及胰岛素分泌增加（44%）。所描述的系统能够在控制重要环境因素的同时，使培养的细胞可重复且长时间地暴露于IH。
