The development of a new technology to measure single-cell mitochondrial respiration

在过去的20年里，人们对线粒体在健康和疾病中的作用进行了大量的研究。曾经被认为仅仅是细胞的“发电站”，我们现在知道线粒体参与了许多细胞过程，包括炎症、氧化应激和程序性细胞死亡。由于这些不同的作用，线粒体功能障碍可以影响细胞功能的多个方面，经常导致病理状况，如神经退行性疾病、癌症和心血管疾病1。另一方面，线粒体途径的改变可以是适应性的，使人类和其他动物能够生活在恶劣的栖息地，如高海拔或高温环境2。因此，了解线粒体生理学对包括医学，生态学和保护在内的各种学科具有深远的意义。研究线粒体生理学的金标准是使用克拉克型电极或荧光探针的微呼吸测定法。在这项经典技术中，线粒体呼吸是用药理学方法测量的，以研究电子传递系统的不同方面3。然而，市售的微呼吸测量系统只能测量大量细胞的总体耗氧量，而不能测量单个细胞。这个警告极大地限制了我们测量的诊断价值，并减少了我们的实验控制。此外，多细胞制剂限制了我们对其他细胞内变量（如钙和pH）进行平行综合测量的能力，而且目前的系统不适合从事机械工作的动态细胞类型，如心脏细胞。由于这些原因，人们对开发一种测量单细胞线粒体呼吸作用的新系统非常感兴趣。新技术的成功开发需要广泛的跨学科和机构间的研究努力。为此，该项目汇集了一个由细胞生理学家、生物化学家和电化学家组成的多元化团队，开发了一种用于测量单细胞呼吸的新型微电极系统。电极将在扫描电化学显微镜最新进展的基础上与化学系合作开发。该系统将在心血管科学部的心肌细胞中进行优化，以诊断心脏病中的代谢功能障碍。一旦进行了优化，我们的行业合作伙伴凯恩研究所的安德鲁·艾伦博士和我们的生化顾问哈维尔·伊格莱西亚斯博士将把学术设计转化为可用于标准实验室显微镜的功能性商业原型。学生将接受各种交叉研究技能的培训，包括；微电极制造，细胞分离，荧光显微镜，产品设计和快速原型。

Over the last 20 years there has been an explosion of research into the role of the mitochondria in health and disease. Once regarded as merely the "powerhouse" of the cell, we now know that mitochondria are involved in numerous cellular processes beyond energy production, including inflammation, oxidative stress, and programmed cell death. Due to these diverse roles, mitochondrial dysfunction can impact multiple aspects of cellular function, often leading to pathological conditions, such as neurodegenerative disorders, cancer, and cardiovascular disease1. On the other hand, modifications in mitochondrial pathways can be adaptive, allowing humans and other animals to inhabit hostile habitats, such as high altitude or high temperature environments2. Consequently, understanding mitochondrial physiology has far-reaching implications for a variety of disciplines, including medicine, ecology and conservation. The gold standard for investigating mitochondrial physiology is microrespirometry using a Clark-type electrode or fluorescent probe. In this classic technique, mitochondrial respiration is measured with pharmacological protocols to investigate different aspects of the electron transfer system3. However, commercially available microrespirometry systems are only capable of measuring bulk oxygen consumption in large populations of cells, rather than single cells. This caveat greatly limits the diagnostic value of our measurements and reduces our experimental control. Furthermore, multicellular preparations limit our ability to make parallel integrative measurements of other intracellular variables (e.g. calcium and pH), and current systems are inappropriate for dynamic cell types that engage in mechanical work, such as heart cells. For these reasons, there is great interest in developing a new system for measuring mitochondrial respiration in single cells4.The successful development of new technologies requires a broad cross-disciplinary and inter-institutional research effort. To this end, this project brings together a diverse team of cellular physiologists, biochemists and electrochemists to develop a new microelectrode system for measuring single-cell respiration. The electrodes will be developed in collaboration with the Department of Chemistry on the basis of recent advances in scanning electrochemical microscopy5. The system will be optimised in cardiomyocytes at The Division of Cardiovascular Sciences with a view to diagnosing metabolic dysfunction in cardiac diseases. Once optimised, our industry partner Dr Andrew Allan at Cairn Research, and our biochemical consultant, Dr Javier Iglesias, will translate the academic design into a functional commercial prototype that can be used on standard laboratory microscopes. The student will be trained in a wide variety of cross-cutting research skills including; microelectrode fabrication, cell isolation, epifluorescent microscopy, product design and rapid prototyping.