In search of an effective cell disruption method to isolate intact mitochondria from Chinese hamster ovary cellsAn efficient isolation of mitochondria from cells under physiological conditions is crucial for many studies in life sciences but still challenging in many cases such as in metabolic characterization of mitochondria. In this work, four methods for the disruption of Chinese hamster ovary cells were evaluated regarding their influence on mitochondrial integrity and yield. After cell disruption, mitochondria released from cells were separated from the remaining cell homogenate by differential centrifugation. Sonication was shown to be a rapid and sensitive isolation method. Yields of 14.0 ± 0.3 mg raw mitochondrial protein per 10 8 cells were obtained. The mitochondria were morphologically intact, with membrane integrities of 67% (outer membrane) to 94% (inner membrane). Compared with the methods using Dounce homogenization, digitonin permeabilization, or electroporation for cell disruption the ultrasound method provided the highest yield of isolated mitochondria. Furthermore, this method is rapid (≈ 45 s for disruption), more robust than Dounce homogenization regarding their influence on mitochondrial integrity and especially suitable for preparing a relatively large amount of mitochondria. The results of this work can be helpful for quantitative and dynamic studies of molecular processes related to mitochondria under physiological conditions for many questions in both biomedicine and biotechnology.

In this work, we present a method to isolate mitochondria of mammalian cells after cell disruption on microscale. The device is composed of linear microchannels cast in PDMS (polydimethylsiloxane). Specific antibodies against the translocase outer membrane protein of the mitochondria are immobilized on the surface of the substrate using an avidin-biotin sandwich construct. The mitochondria can be captured in the channel, whereas the remains of the cell lysate flow out the chip unhindered. The captured mitochondria can be observed directly on chip. A successful immobilization of pre-isolated mitochondria was shown at a flow rate between 0 and 5 μl min(-1) (≈0-2.5 mm s(-1)). After fluorescence staining, we demonstrated that the mitochondria covered around 3% of the channel surface. The mitochondria appeared in a distinct spherical shape with a diameter of around 0.8-1.2 μm. Further validation of the microfluidic device using non-treated cell lysate was done at 2 μl min(-1). The immobilized mitochondria were smaller with a diameter of around ≈490 nm. We observed a surface coverage of around 4%. The immobilized mitochondria were active and stable for over 2 h without cooling and were shown to be able to produce ATP under stage 3 respiration on chip.