Details
| Original language | English |
|---|---|
| Pages (from-to) | 161-169 |
| Number of pages | 9 |
| Journal | Engineering in Life Sciences |
| Volume | 14 |
| Issue number | 2 |
| Publication status | Published - 28 Aug 2013 |
| Externally published | Yes |
Abstract
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 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 108 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.
Keywords
- Cell disruption, CHO cells, Compartment metabolism, Mitochondria isolation, Ultrasound
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Environmental Science(all)
- Environmental Engineering
- Chemical Engineering(all)
- Bioengineering
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In: Engineering in Life Sciences, Vol. 14, No. 2, 28.08.2013, p. 161-169.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - In search of an effective cell disruption method to isolate intact mitochondria from Chinese hamster ovary cells
AU - Bahnemann, Janina
AU - Kayo, Sabrina
AU - Wahrheit, Judith
AU - Heinzle, Elmar
AU - Pörtner, Ralf
AU - Zeng, An Ping
PY - 2013/8/28
Y1 - 2013/8/28
N2 - 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 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 108 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.
AB - 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 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 108 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.
KW - Cell disruption
KW - CHO cells
KW - Compartment metabolism
KW - Mitochondria isolation
KW - Ultrasound
UR - http://www.scopus.com/inward/record.url?scp=84895927011&partnerID=8YFLogxK
U2 - 10.1002/elsc.201200182
DO - 10.1002/elsc.201200182
M3 - Article
AN - SCOPUS:84895927011
VL - 14
SP - 161
EP - 169
JO - Engineering in Life Sciences
JF - Engineering in Life Sciences
SN - 1618-0240
IS - 2
ER -