Open AccessIn search of an effective cell disruption method to isolate intact mitochondria from C hinese hamster ovary cells
Author(s) -
Bahnemann Janina,
Kayo Sabrina,
Wahrheit Judith,
Heinzle Elmar,
Pörtner Ralf,
Zeng AnPing
Publication year - 2014
Publication title -
engineering in life sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.547
H-Index - 57
eISSN - 1618-2863
pISSN - 1618-0240
DOI - 10.1002/elsc.201200182
Subject(s) - mitochondrion , digitonin , cell disruption , chinese hamster ovary cell , microbiology and biotechnology , homogenization (climate) , biology , cell , biochemistry , membrane , chemistry , biodiversity , ecology , receptor
An 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 C hinese 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 D ounce 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 D ounce 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.