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Micro-respirometry of whole cells and isolated mitochondria

机译：全细胞和分离的线粒体的微呼吸测定法

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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 100× 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：耗氧量是有氧生物代谢的关键指标。尽管存在诸如高样本需求，与大气O _（2）交换以及局限性累积滴定规程等局限性，当前的呼吸测定法仍导致开创性发现，导致可用组织的选择受限，复杂的数据解释和受限的实验设计。我们开发了一种灵敏且可自定义的方法，可以测量微升体积中各种生物样品的O _（2）消耗速率，而不会受到有氧环境的干扰。我们证明，光聚合物VeroClear的O _（2）渗透性可与聚醚醚酮（分别为0.125与0.143 barrer）相媲美，从而为氧气的进入提供了有效的屏障。 VeroClear的光学透明性与高分辨率3D打印相结合，可以在封闭的样品中进行基于光电二极管的氧气检测。这些特性可为O _（2）消耗速率提供具有超过100倍动态范围的微呼吸仪。重要的是，封闭的呼吸仪配置和极低的材料透氧性使其适合使用树脂预处理，用于定量评估任何期望的[O _（2）]的O _（2）消耗率，包括高压，生理或每种细胞类型所需的缺氧条件。我们表征了两种配置来研究可溶性酶，分离的线粒体，悬浮细胞和芯片上培养的贴壁细胞。更高的灵敏度允许对多达数百个细胞的呼吸进行常规定量检测。微呼吸计中细胞悬浮液的比活与通过高分辨率极谱呼吸测定法获得的活度非常一致。粘附细胞方案允许对视网膜色素上皮细胞ARPE-19进行生理相关的呼吸评估，与悬浮状态相比，其代谢率较低。通过交换培养基组成，我们证明了氰化物可以瞬时抑制细胞，并且去除后可回收99.6％的基础O _（2）摄取。这种方法适用于基础研究和应用领域中的有限样本量的新实验设计和精确测量。

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