MITOCHONDRIAL RESPIROMETRY IN FROZEN BIOLOGICAL SAMPLES

摘要 线粒体呼吸受损在代谢、心血管和衰老相关疾病中起着关键作用 疾病。然而，线粒体呼吸测量分析目前需要对活组织进行处理 在从病人身上取下样本后一小时内。这一要求使得呼吸测量分析变得非常重要 对于标准的临床实践和临床研究是不可行的。我们发明了一种新技术来评估最大 之前冷冻的生物样本中的线粒体呼吸能力，到目前为止还被认为是 科学界认为这是不可能的。并列比对证实了我们的分析准确地反映了 结果在新鲜样品中测量。因此，我们的技术克服了标准的根本限制 需要活组织的方法。此外，我们的方法包括简化的样品制备，该方法 使用的生物材料显著减少。 Enspire Bio将进一步开发这项获得专利的冷冻Mitos技术，以实现呼吸测量 临床应用和冷冻样本的测量，目前无法获得。我们将实现 我们的目标1：建立冰冻组织耗氧量测量的标准规程 从人类和小鼠组织样本中提取的样本。我们将通过以下方式进一步提高吞吐量 测量组织匀浆的耗氧量，而不是分离的线粒体。目标2： 建立专有套件并将其交付给五个Beta测试站点，其中包括意见领先的学术网站 实验室、线粒体疾病中心以及一家生物技术和制药公司进行测试并提供反馈。 为了向没有能力进行湿法实验室分析的客户提供冷冻样本分析，我们将开发一种CRO- 在内部服务类型中，样品将被运送到EnspirreBio进行分析。与一组飞行员一起工作 学术、临床和行业客户将提供反馈，以制定程序和经济 冰冻组织呼吸测量的CRO模型方法。

Abstract Impaired mitochondrial respiration plays a key role in metabolic, cardiovascular, and aging-related diseases. However, mitochondrial respirometry analysis currently requires processing of the living tissue sample within an hour after being taken from the patient. This requirement makes respirometry analysis largely unfeasible to standard clinical practice and clinical studies. We invented a new technology to assess maximal mitochondrial respiratory capacity in previously-frozen biological samples, which thus far been considered impossible by the scientific community. Side-by-side comparison confirmed that our assay accurately reflects results measured in fresh samples. Our technology thus overcomes the fundamental limitation of standard approaches that required living tissue. Moreover, our approach consists of simplified sample preparation which uses significantly less biological material. Enspire Bio will further develop this patented Frozen Mitos technology to enable respirometry measurements for clinical applications and frozen samples, which currently is not available. We will achieve our goals by Aim 1: Establishing standard protocols for oxygen consumption measurements from frozen tissue samples derived from human and mouse tissue samples. We will further improve throughput by enabling measurements of oxygen consumption from tissue homogenate instead of isolated mitochondria. Aim 2: Establishing proprietary kits and delivering those to five beta testing sites including opinion-leading academic labs, mitochondrial disease centers and a biotech and pharmaceutical company to test and provide feedback. To provide frozen sample analysis to clients that have no capacity for wet lab assays, we will develop a CRO- type service in-house where samples will be shipped to EnspireBio for analysis. Working with a set of pilot academic, clinical and industry clients will provide feedback to establish procedures and the economics of the CRO model approach for the frozen tissue respirometry.