A method for isolating and cryopreserving intact mitochondria with improved integrity and functionality
Mitochondria isolated from cells are essential tools in biological research. However, many mitochondria are often damaged during the isolation process. Although cryopreservation can greatly improve the usability of isolated mitochondria, it typically leads to significant loss of activity following f...
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| Format: | Article |
| Language: | English |
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The Biophysical Society of Japan
2025-06-01
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| Series: | Biophysics and Physicobiology |
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| Online Access: | https://doi.org/10.2142/biophysico.bppb-v22.0012 |
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| author | Arima Okutani Jannatul Naima Asaka Ogihara Taihei Motoichi Ikuroh Ohsawa Yoshihiro Ohta |
| author_facet | Arima Okutani Jannatul Naima Asaka Ogihara Taihei Motoichi Ikuroh Ohsawa Yoshihiro Ohta |
| author_sort | Arima Okutani |
| collection | DOAJ |
| description | Mitochondria isolated from cells are essential tools in biological research. However, many mitochondria are often damaged during the isolation process. Although cryopreservation can greatly improve the usability of isolated mitochondria, it typically leads to significant loss of activity following freezing and thawing. In this study, we present our own techniques for mitochondrial isolation and cryopreservation to overcome these challenges. Our isolation method begins by selectively weakening the plasma membrane through the incorporation of digitonin, under conditions that do not increase membrane permeability. The plasma membrane is then selectively ruptured to release mitochondria. Notably, mitochondria contract within the cell before the plasma membrane ruptures, a process that facilitates their extraction. The isolated mitochondria showed polarized inner membranes in approximately 90% of the population. Compared to mitochondria isolated by homogenization, they retained more intermembrane space proteins and exhibited greater outer membrane integrity. For cryopreservation, rapid thawing was critical to maintaining mitochondrial activity after freeze-thaw cycles. When thawing was completed in under 1.5 minutes, the proportion of polarized mitochondria decreased by only about 10%. These findings suggest that our isolation and cryopreservation protocols are promising for applications requiring intact, functional mitochondria. |
| format | Article |
| id | doaj-art-15c12957f2a540b0a4306bb48f97d136 |
| institution | OA Journals |
| issn | 2189-4779 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | The Biophysical Society of Japan |
| record_format | Article |
| series | Biophysics and Physicobiology |
| spelling | doaj-art-15c12957f2a540b0a4306bb48f97d1362025-08-20T02:37:42ZengThe Biophysical Society of JapanBiophysics and Physicobiology2189-47792025-06-012210.2142/biophysico.bppb-v22.0012A method for isolating and cryopreserving intact mitochondria with improved integrity and functionalityArima Okutani0Jannatul Naima1Asaka Ogihara2Taihei Motoichi3Ikuroh Ohsawa4Yoshihiro Ohta5Division of Biotechnology and Life Sciences, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, JapanDivision of Biotechnology and Life Sciences, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, JapanDivision of Biotechnology and Life Sciences, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, JapanDivision of Biotechnology and Life Sciences, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, JapanBiological Process of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo, 173-0015, JapanDivision of Biotechnology and Life Sciences, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, JapanMitochondria isolated from cells are essential tools in biological research. However, many mitochondria are often damaged during the isolation process. Although cryopreservation can greatly improve the usability of isolated mitochondria, it typically leads to significant loss of activity following freezing and thawing. In this study, we present our own techniques for mitochondrial isolation and cryopreservation to overcome these challenges. Our isolation method begins by selectively weakening the plasma membrane through the incorporation of digitonin, under conditions that do not increase membrane permeability. The plasma membrane is then selectively ruptured to release mitochondria. Notably, mitochondria contract within the cell before the plasma membrane ruptures, a process that facilitates their extraction. The isolated mitochondria showed polarized inner membranes in approximately 90% of the population. Compared to mitochondria isolated by homogenization, they retained more intermembrane space proteins and exhibited greater outer membrane integrity. For cryopreservation, rapid thawing was critical to maintaining mitochondrial activity after freeze-thaw cycles. When thawing was completed in under 1.5 minutes, the proportion of polarized mitochondria decreased by only about 10%. These findings suggest that our isolation and cryopreservation protocols are promising for applications requiring intact, functional mitochondria.https://doi.org/10.2142/biophysico.bppb-v22.0012mitochondriaisolationcryopreservation |
| spellingShingle | Arima Okutani Jannatul Naima Asaka Ogihara Taihei Motoichi Ikuroh Ohsawa Yoshihiro Ohta A method for isolating and cryopreserving intact mitochondria with improved integrity and functionality Biophysics and Physicobiology mitochondria isolation cryopreservation |
| title | A method for isolating and cryopreserving intact mitochondria with improved integrity and functionality |
| title_full | A method for isolating and cryopreserving intact mitochondria with improved integrity and functionality |
| title_fullStr | A method for isolating and cryopreserving intact mitochondria with improved integrity and functionality |
| title_full_unstemmed | A method for isolating and cryopreserving intact mitochondria with improved integrity and functionality |
| title_short | A method for isolating and cryopreserving intact mitochondria with improved integrity and functionality |
| title_sort | method for isolating and cryopreserving intact mitochondria with improved integrity and functionality |
| topic | mitochondria isolation cryopreservation |
| url | https://doi.org/10.2142/biophysico.bppb-v22.0012 |
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