Isolation of functional mitochondria from rat kidney and skeletal muscle without manual homogenization.

Isolation of functional and intact mitochondria from solid tissue is crucial for studies that focus on the elucidation of normal mitochondrial physiology and/or mitochondrial dysfunction in conditions such as aging, diabetes, and cancer. There is growing recognition of the importance of mitochondria both as targets for drug development and as off-target mediators of drug side effects. Unfortunately, mitochondrial isolation from tissue is generally carried out using homogenizer-based methods that require extensive operator experience to obtain reproducible high-quality preparations. These methods limit dissemination, impede scale-up, and contribute to difficulties in reproducing experimental results over time and across laboratories. Here we describe semiautomated methods to disrupt tissue using kidney and muscle mitochondria preparations as exemplars. These methods use the Barocycler, the PCT Shredder, or both. The PCT Shredder is a mechanical grinder that quickly breaks up tissue without significant risk of overhomogenization. Mitochondria isolated using the PCT Shredder are shown to be comparable to controls. The Barocycler generates controlled pressure pulses that can be adjusted to lyse cells and release organelles. The mitochondria subjected to pressure cycling-mediated tissue disruption are shown to retain functionality, enabling combinations of the PCT Shredder and the Barocycler to be used to purify mitochondrial preparations.