Name: Mitochondrial Isolation for Transmitochondrial Cybrid Generation
Uploaded: 2023-03-17T14:20:00
Description: Watch this Scientific Journal Video about Mitochondrial Isolation for Transmitochondrial Cybrid Generation at JoVE.com

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

Génération de cybrides transmissibles à partir de cellules cancéreuses en suspension

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).

Pleins feux sur l’auteur : Génération de cybrides transmissibles à l’aide de lignées cellulaires cancéreuses  

Génération transmitochondriale de Cybrides à l’aide de lignées cellulaires cancéreuses

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  

Isolement mitochondrial pour la génération transmitochondriale de Cybrides

Une fois qu’environ 25 millions de cellules donneuses de mitochondries sont obtenues le septième jour, récoltez les cellules cultivées exponentiellement dans un tube de 50 millilitres et recueillez-les par centrifugation à température ambiante et 520g pendant cinq minutes. Lavez les cellules avec une solution saline froide tamponnée au phosphate et sédimentez-les par centrifugation à température ambiante dans 520g pendant cinq minutes. Maintenant, effectuez l’extraction mitochondriale complète à quatre degrés Celsius en utilisant des réactifs froids et gardez les cellules ou les mitochondries sur la glace.Après la troisième centrifugation, éliminer le surnageant par aspiration à l’aide d’une pipette en verre couplée à une pompe à vide et remettre en suspension les cellules emballées dans un volume de tampon hypotonique égal à sept fois le volume de granulés de cellule. Ensuite, transférez la suspension cellulaire dans un tube homogénéisateur et laissez les cellules gonfler en les incubant sur de la glace pendant deux minutes. Casser les membranes cellulaires en effectuant 8 à 10 coups dans l’homogénéisateur couplé à un pilon motorisé tournant à 600 rotations par minute.Dans l’homogénat cellulaire, ajouter le tampon hypotonique égal à sept fois le volume de la pastille initiale pour générer un environnement isotonique. Transférer l’homogénat dans un tube de 15 millilitres et le centrifuger dans un rotor fixe à 1 000 g et quatre degrés Celsius pendant cinq minutes. Ensuite, prélevez seulement les 3/4 du surnageant, en laissant une grande marge de la pastille pour éviter la contamination par des noyaux ou des cellules intactes et transférez-le dans un autre tube.Après avoir répété le processus deux fois, transférez le surnageant contenant la fraction mitochondriale dans des tubes de 1,5 millilitre. Centrifuger les tubes à la vitesse maximale de 18 000 g pendant deux minutes à quatre degrés Celsius. Jeter le surnageant et laver la pastille enrichie en mitochondries avec un tampon A.Combinez le contenu des deux tubes en un seul et centrifugez comme démontré précédemment.Répétez le processus jusqu’à ce que tout le matériau soit dans un seul tube. Laver la pastille obtenue à partir de la dernière centrifugation en utilisant 300 microlitres de tampon A.Quantifier la concentration en protéines mitochondriales à l’aide du test de Bradford. Avant la fusion avec la lignée cellulaire réceptrice mitochondriale, évaluer l’absence de contaminants nucléiaux dans la fraction mitochondriale par immunodétection des protéines nucléaires.Vous pouvez également effectuer une réaction en chaîne par polymérase quantitative ou une amplification QPCR d’un gène nucléaire.

Research

JoVE Journal

Cancer Research

Mitochondrial Isolation for Transmitochondrial Cybrid Generation

Transmitochondrial Cybrid Generation Using Cancer Cell Lines

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more ...