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Micro-respirometry of whole cells and isolated mitochondria

机译：全细胞和分离线粒体的微呼吸测定

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Oxygen consumption is a key metric of metabolism in aerobic organisms. Current respirometric methods led to seminal discoveries despite limitations such as high sample demand, exchange with atmospheric O-2, and cumulative titration protocols leading to limited choice of useable tissue, complex data interpretation, and restricted experimental design. We developed a sensitive and customizable method of measuring O-2 consumption rates by a variety of biological samples in microliter volumes without interference from the aerobic environment. We demonstrate that O-2 permeability of the photopolymer, VeroClear, is comparable to that of polyetheretherketone (0.125 vs. 0.143 barrer, respectively) providing an efficient barrier to oxygen ingress. Optical transparency of VeroClear, combined with high resolution 3D printing, allows for optode-based oxygen detection in enclosed samples. These properties yield a microrespirometer with over 100x dynamic range for O-2 consumption rates. Importantly, the enclosed respirometer configuration and very low oxygen permeability of materials makes it suitable, with resin pre-conditioning, for quantitative assessment of O-2 consumption rates at any desired [O-2], including hyperbaric, physiological or hypoxic conditions as necessary for each cell type. We characterized two configurations to study soluble enzymes, isolated mitochondria, cells in suspension, and adherent cells cultured on-chip. Improved sensitivity allows for routine quantitative detection of respiration by as few as several hundred cells. Specific activity of cell suspensions in the microrespirometer was in close agreement with that obtained by high-resolution polarographic respirometry. Adherent cell protocols allowed for physiologically relevant assessment of respiration in retinal pigment epithelial cells, ARPE-19, which displayed lower metabolic rates compared with those in suspension. By exchanging medium composition, we demonstrate that cells can be transiently inhibited by cyanide and that 99.6% of basal O-2 uptake is recovered upon its removal. This approach is amenable to new experimental designs and precision measurements on limited sample quantities across basic research and applied fields.
机译：耗氧量是代谢的需氧生物的一个关键指标。导致了开创性的发现，尽管目前的限制呼吸速率的方法，如高采样需求，与大气中的O-2交换，和累积滴定协议，导致可用的组织，复杂的数据解释的有限的选择，并限制实验设计。我们开发了多种在微升体积的生物样品的测量O-2的消耗速率，而从有氧环境干扰的敏感性和可定制的方法。我们证明光聚合物，VeroClear，那O-2渗透性是比得上聚醚醚酮（0.125与0.143巴勒，分别地）的提供有效阻隔氧气进入。 VeroClear的光学透明性，具有很高的分辨率3D印刷相结合，允许封闭样品中基于光极 - 氧检测。这些性质产生具有超过100倍的动态范围的microrespirometer为O-2的消耗率。重要的是，材料的封闭呼吸配置和非常低的氧渗透性使得它适合与树脂预调节，为的O-2的消耗率定量评估在任何所需的[O-2]，包括高压，生理条件或低氧条件作为必要对于每个细胞类型。我们其特征在于两种结构来研究可溶性酶，分离的线粒体，在悬浮液中的细胞，并贴壁细胞片上培养。改进的灵敏度允许呼吸的常规定量检测由少至几百个细胞。在microrespirometer细胞悬液的具体活动是在通过高清晰度极谱呼吸运动获得了接近于达成协议。允许用于在视网膜色素上皮细胞的呼吸生理相关的评估的贴壁细胞的协议，ARPE-19，其显示低代谢率与在悬浮液中进行比较。通过交换介质组合物，我们表明，细胞可以通过氰化物和基底O-2摄取的99.6％时将其移除回收瞬时抑制。这种方法适合于在有限的样品量新的实验设计和精确测量的跨越基础研究和应用领域。

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    《RSC Advances》 |2019年第57期|共11页
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