Rogers Zachary J, Colombani Thibault, Khan Saad, Bhatt Khushbu, Nukovic Alexandra, Zhou Guanyu, Woolston Benjamin M, Taylor Cormac T, Gilkes Daniele M, Slavov Nikolai, Bencherif Sidi A
Department of Chemical Engineering, Northeastern University, Boston, MA 02115, USA.
Department of Bioengineering, Northeastern University, Boston, MA 02115, USA.
bioRxiv. 2023 Oct 3:2023.10.02.560369. doi: 10.1101/2023.10.02.560369.
Oxygen (O) tension plays a key role in tissue function and pathophysiology. O-controlled cell culture, in which the O concentration in an incubator's gas phase is controlled, is an indispensable tool to study the role of O . For this technique, it is presumed that the incubator setpoint is equal to the O tension that cells experience (., pericellular O). We discovered that physioxic (5% O) and hypoxic (1% O) setpoints regularly induce anoxic (0.0% O) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O consumption is the driving factor. RNA-seq revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to anoxic exposure followed by rapid reoxygenation. To better understand the relationship between incubator gas phase and pericellular O tensions, we developed a reaction-diffusion model that predicts pericellular O tension . This model revealed that the effect of cellular O consumption is greatest in smaller volume culture vessels ( 96-well plate). By controlling pericellular O tension in cell culture, we discovered that MCF7 cells have stronger glycolytic and glutamine metabolism responses in anoxia . hypoxia. MCF7 also expressed higher levels of , , , . and lower levels of , , . in response to hypoxia . anoxia. Proteomics revealed that 4T1 cells had an upregulated epithelial-to-mesenchymal transition (EMT) response and downregulated reactive oxygen species (ROS) management, glycolysis, and fatty acid metabolism pathways in hypoxia . anoxia. Collectively, these results reveal that breast cancer cells respond non-monotonically to low O, suggesting that anoxic cell culture is not suitable to model hypoxia. We demonstrate that controlling atmospheric O tension in cell culture incubators is insufficient to control O in cell culture and introduce the concept of .
氧（O）张力在组织功能和病理生理学中起着关键作用。氧控制细胞培养是研究氧作用的不可或缺的工具，在这种培养方式中，培养箱气相中的氧浓度是可控的。对于这项技术，假定培养箱设定值等于细胞所经历的氧张力（即细胞周围的氧）。我们发现，在贴壁细胞培养和悬浮细胞培养中，生理氧浓度（5% O）和低氧浓度（1% O）的设定值经常会诱导出无氧（0.0% O）的细胞周围张力。电子传递链抑制消除了这种效应，表明细胞耗氧是驱动因素。RNA测序显示，在生理氧条件下培养的原代人肝细胞因缺氧暴露后快速复氧而经历缺血再灌注损伤。为了更好地理解培养箱气相与细胞周围氧张力之间的关系，我们开发了一个预测细胞周围氧张力的反应扩散模型。该模型表明，细胞耗氧在较小体积的培养容器（96孔板）中的影响最大。通过控制细胞培养中的细胞周围氧张力，我们发现MCF7细胞在缺氧和低氧条件下具有更强的糖酵解和谷氨酰胺代谢反应。MCF7在低氧和缺氧条件下还表达了更高水平的……和更低水平的……。蛋白质组学显示，4T1细胞在低氧和缺氧条件下上皮-间质转化（EMT）反应上调，活性氧（ROS）管理、糖酵解和脂肪酸代谢途径下调。总的来说，这些结果表明乳腺癌细胞对低氧的反应是非单调的，这表明无氧细胞培养不适用于模拟低氧。我们证明，控制细胞培养箱中的大气氧张力不足以控制细胞培养中的氧，并引入了……的概念。
