The video provides a detailed protocol for isolating mitochondrial donor cells, including techniques for cell harvesting, centrifugation, homogenization, and purification to obtain a clean mitochondrial fraction. It emphasizes maintaining cold temperatures, avoiding nuclear contamination, and assessing mitochondrial protein concentration before the fusion with recipient cells.

mitochondrial isolation for transmitochondrial cybrid generation

Transmitochondrial Cybrid Generation From Suspension-Growing Cancer Cells

Reprogramming energy metabolism is a hallmark of cancer1 that was observed for the first time by Otto Warburg in the 1930s2. Under aerobic conditions, normal cells convert glucose into pyruvate, that then generates acetyl-coA, fuelling the mitochondrial machinery and promoting cellular respiration. Nevertheless, Warburg demonstrated that, even under normoxic conditions, most cancer cells convert pyruvate obtained from the glycolysis process into lactate, shifting their way to obtain energy. This metabolic adjustment is known as the &#34;Warburg effect&#34; and enables some cancer cells to supply their energetic demands for rapid growth and division, despite generating ATP less efficiently than the aerobic process3,4,5. In recent decades, numerous works have supported the implication of metabolism reprogramming in cancer progression. Hence, tumor energetics is considered an interesting target against cancer1. As a central hub in energetic metabolism and in the supply of essential precursors, mitochondria play a key role in these cell adaptations that, to date, we only partially understand.
In line with the above, mitochondrial DNA (mtDNA) mutations have been proposed as one of the possible causes of this metabolic reprogramming, which could lead to an impaired electron transport chain (ETC) performance6 and would explain why some cancer cells enhance their glycolytic metabolism to survive. Indeed, it has been reported that mtDNA accumulates mutations within cancer cells, being present in at least 50% of tumors7. For example, a recent study carried out by Yuan et al. reported the presence of hypermutated and truncated mtDNA molecules in kidney, colorectal, and thyroid cancers8.&#160;Moreover, many works have demonstrated that certain mtDNA mutations are associated with a more aggressive tumor phenotype and with an increase in the metastatic potential of cancer cells9,10,11,12,13,14,15,16.
Despite the apparent relevance of the mitochondrial genome in cancer progression, the study of these mutations and their contribution to the disease have been challenging due to limitations in the experimental models and technologies currently available17. Thus, new techniques to understand the real impact of mitochondria DNA in cancer disease development and progression are needed. In this work, we introduce a protocol for transmitochondrial cybrid generation from suspension-growing cancer cells, based on the repopulation of rhodamine 6G-pretreated cells with isolated mitochondria, that overcomes the main challenges of traditional cybridization methods18,19. This methodology allows the use of any nuclei donor regardless of the availability of their corresponding &#961;0 cell line and the transfer of mitochondria from cells that, following the traditional techniques, would be difficult to enucleate (i.e., non-adherent cell lines).

Author Spotlight: Transmitochondrial Cybrid Generation Using Cancer Cell Lines  

Transmitochondrial Cybrid Generation Using Cancer Cell Lines

This research aims to better understand the contribution of mitochondrion in cancer progression and metastasis. The transmitochondrial cybrid generation can help us to elucidate how and at what level mitochondrial genotypes are involved in the aggressiveness of a tumor cell. One of the technologies to obtain information on mitochondrial role in cancer is cybrid generation.We combine this tool with a variety of other methods that include donative electrophoresis, respiration analysis and other mitochondrial functional assays, flow cytometry, proteomics, et cetera. The plasticity of cancer cells together with the interaction networks between nuclear and mitochondrial genes, increases the complexity of our experiments and data interpretation. This is one of the reasons why is necessary to develop a new experimental approaches.One of the most exciting findings from our research has been the discovery of two mitochondrial DNA mutations associated with detachment of cells from the play dish resembling metastasis generation and making the cells completely dependent on aerobic glycolysis, and also making them more tumorigenic and metastatic in in vivo models. This mitochondria exchange protocol can be applied to suspension growing cancer cells. The chronology can overcome the limitations of traditional approaches where the nucleation process and the complete removal of mitochondrial DNA from mitochondrial recipient cell lines often become a real challenge.

Watch this Scientific Journal Video about Mitochondrial Isolation for Transmitochondrial Cybrid Generation at JoVE.com

Mitochondrial Isolation for Transmitochondrial Cybrid Generation  

Once around 25 million mitochondria donor cells are obtained on day seven, harvest the exponentially grown cells in a 50-milliliter tube and collect them by centrifuging at room temperature and 520g for five minutes. Wash the cells with cold phosphate-buffered saline and sediment them by centrifugation at room temperature in 520g for five minutes. Now onwards, perform the whole mitochondrial extraction at four degrees Celsius using cold reagents and keep the cells or mitochondria on ice.After the third centrifugation, discard the supernatant by aspiration using a glass pipette coupled to a vacuum pump and resuspend the packed cells in a volume of hypotonic buffer equal to seven times the cell pellet volume. Then transfer the cell suspension into a homogenizer tube and let the cells swell by incubating them on ice for two minutes. Break the cell membranes by performing 8 to 10 strokes in the homogenizer coupled to a motor-driven pestle rotating at 600 rotations per minute.Into the cell homogenate, add the hypotonic buffer equal to seven times the volume of the initial pellet to generate an isotonic environment. Transfer the homogenate into a 15-milliliter tube and centrifuge it in a fixed rotor at 1, 000g and four degrees Celsius for five minutes. Then collect only 3/4 of the supernatant, leaving a large margin from the pellet to avoid contamination with nuclei or intact cells and transfer it to another tube.After repeating the process twice, transfer the supernatant containing the mitochondrial fraction into 1.5-milliliter tubes. Centrifuge the tubes at the maximum speed of 18, 000g for two minutes at four degrees Celsius. Discard the supernatant and wash the mitochondria-enriched pellet with buffer A.Combine the content of the two tubes into one and centrifuge as demonstrated before.Repeat the process until all the material is in only one tube. Wash the pellet obtained from the last centrifugation using 300 microliters of buffer A.Quantify the mitochondrial protein concentration using the Bradford assay. Before fusion with the mitochondrial recipient cell line, evaluate the absence of nuclei contaminants in the mitochondrial fraction by immunodetection of nuclear proteins.Alternatively perform quantitative polymerase chain reaction or QPCR amplification of a nuclear gene.

mitochondrial-isolation-for-transmitochondrial-cybrid-generation

Research

JoVE Journal

Cancer Research

155 Views.  University of Zaragoza. Once around 25 million mitochondria donor cells are obtained on day seven, harvest the exponentially grown cells in a 50-milliliter tube and collect them by centrifuging at room temperature and 520g for five minutes. Wash the cells with cold phosphate-buffered saline and sediment them by centrifugation at room temperature in 520g for five minutes. Now onwards, perform the whole mitochondrial extraction at four degrees Celsius using cold reagents and keep the cells or mitochondria on ice.