Ultrasound as an effective method to isolate intact mitochondria from CHO cells for physiological studies

Mitochondria play key roles in energy metabolism and cell physiology. The properties and metabolic capacity of mitochondria in suspension cultures of mammalian cells have been seldom studied despite their importance for the production of recombinant proteins. Current metabolic studies of mammalian cells generally examine the cells as a whole, regardless of the compartmentalization of metabolism. Within the frame of a systems biology study of mammalian cell culture we are making efforts to expand our knowledge of in vivo dynamics of key metabolic reactions in the conjunction of glycolysis and tricarboxylic acid cycle which take place in both, cytosol and mitochondria. For this purpose, a fast and sensitive isolation of mitochondria is needed. In this work, four methods for the disruption of Chinese hamster ovary (CHO-K1) cells are evaluated regarding their influence on mitochondrial integrity and yield. Sonication has been found to be a rapid and sensitive isolation procedure of mitochondria which resulted in high yields with CHO cells being disrupted using an ultrasound bar. Mitochondria released from the cells are then separated from the remaining cell homogenate by differential centrifugation in a sucrose containing medium. Yields of 14.0 ± 0.3 mg raw mitochondrial protein per 1 • 10 8 cells were obtained. These mitochondria are morphologically intact, with membrane integrities of 67% (outer membrane) to 94% (inner membrane). In addition, the functionality of isolated mitochondria was proven by measurement of oxygen consumption rate. By comparing this approach to mitochondria isolation methods using Dounce homogenization, digitonin permeabilization, or electroporation for cell disruption we demonstrated that the ultrasound approach provides the highest yield of isolated mitochondria. Furthermore, this procedure is relatively rapid (≈ 45 s for disruption), provides highly efficient cell disruption, and is more sensitive regarding mitochondrial integrity. Thus, ultrasound is well suitable for the preparation and investigation of mitochondria of suspension cell culture.