Cryopreservation of brain mitochondria: a novel methodology for functional studies.
Often, comparative studies involving large number of animals or human post-mortem tissue samples are precluded, especially those requiring structurally and functionally intact cells and/or organelles. The ability to 'bank' such samples for storage and restore or 'reanimate' them at a later time without causing damage to the structure and/or function becomes imperative. However, to date, such attempts have produced conflicting results. We here demonstrate for the first time that isolated rat brain mitochondria can be successfully cryopreserved and restored for later use. We added a well characterized cryoprotectant 10% (v/v) dimethyl sulfoxide (DMSO) to purified rat cortical mitochondria and allowed them to cool at a uniform rate of approximately 1 degree C/min and stored them at -80 degrees C. Freshly isolated as well as reanimated brain mitochondria were analyzed for respiration. Structural integrity of cryopreserved mitochondria was also verified by electron microscopy. Mitochondrial membrane marker levels were assessed along with cytochrome c levels. Intact structure and function of the cryopreserved brain mitochondria observed allows us the opportunity to store mitochondria for longer periods of time as well as perform metabolic studies as needed. This will considerably expand the time-frame required for carrying out functional analysis in large comparative studies.
Nukala
Vidya N
VN
Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, KY 40536, USA.
Singh
Indrapal N
IN
Davis
Laurie M
LM
Sullivan
Patrick G
PG
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000502:physiology* / Q000648:ultrastructure*; D002540:Cerebral Cortex / Q000502:physiology / Q000648:ultrastructure; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D045304:Cytochromes c / Q000378:metabolism; D004121:Dimethyl Sulfoxide; D004734:Energy Metabolism / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D008854:Microscopy, Electron; D008928:Mitochondria / Q000502:physiology*; D010101:Oxygen Consumption / Q000502:physiology; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D050994:Voltage-Dependent Anion Channels / Q000378:metabolism
D003451:Cryoprotective Agents; D050994:Voltage-Dependent Anion Channels; D045304:Cytochromes c; D011451:Prostaglandin-Endoperoxide Synthases; D004121:Dimethyl Sulfoxide