MITOCHONDRIAL RESPIROMETRY IN FROZEN BIOLOGICAL SAMPLES

摘要 线粒体呼吸功能受损在代谢、心血管和衰老相关疾病中起着关键作用。 疾病然而，线粒体呼吸测定分析目前需要处理活组织 在从患者身上采集样本后一小时内。这一要求使得呼吸计量分析在很大程度上 不适用于标准临床实践和临床研究。我们发明了一种新技术来评估 线粒体呼吸能力在以前冷冻的生物样品，迄今为止被认为是 这是科学界不可能的。并排比较证实，我们的分析准确地反映了 在新鲜样品中测量的结果。因此，我们的技术克服了标准 需要活体组织的方法此外，我们的方法包括简化的样品制备， 使用的生物材料要少得多。 Enspire Bio将进一步开发这项专利的冷冻Mitos技术， 用于临床应用和冷冻样品的测量，目前尚不可用。我们将实现 我们的目标1：建立冷冻组织耗氧量测量的标准方案 来自人和小鼠组织样品的样品。我们将进一步提高吞吐量， 测量来自组织匀浆而不是分离的线粒体的氧消耗。目标二： 建立专有的工具包，并将其提供给五个beta测试网站，包括意见领先的学术 实验室、线粒体疾病中心和一家生物技术和制药公司进行测试并提供反馈。 为了向没有湿实验室分析能力的客户提供冷冻样本分析，我们将开发一个CRO- 内部类型服务，样本将运送至EnspireBio进行分析。与一组试点合作 学术，临床和行业客户将提供反馈，以建立程序和经济的 冷冻组织呼吸测定的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.