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

Generazione di cibridi trasmettitocondriali da cellule tumorali in sospensione

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).

Autore in primo piano: Generazione di cibridi trasmissocondriali utilizzando linee cellulari tumorali  

Generazione di cibridi transmitocondriali utilizzando linee cellulari tumorali

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  

Isolamento mitocondriale per la generazione di cibridi transmitocondriali

Una volta ottenuti circa 25 milioni di cellule donatrici di mitocondri il settimo giorno, raccogliere le cellule cresciute esponenzialmente in un tubo da 50 millilitri e raccoglierle centrifugando a temperatura ambiente e 520 g per cinque minuti. Lavare le cellule con soluzione salina tamponata con fosfato freddo e sedimentarle mediante centrifugazione a temperatura ambiente in 520g per cinque minuti. Ora in poi, eseguire l'intera estrazione mitocondriale a quattro gradi Celsius usando reagenti freddi e mantenere le cellule o i mitocondri sul ghiaccio.Dopo la terza centrifugazione, scartare il surnatante mediante aspirazione utilizzando una pipetta di vetro accoppiata ad una pompa per vuoto e risospendere le celle imballate in un volume di tampone ipotonico pari a sette volte il volume del pellet cellulare. Quindi trasferire la sospensione cellulare in un tubo omogeneizzatore e lasciare che le cellule si gonfino incubandole sul ghiaccio per due minuti. Rompere le membrane cellulari eseguendo da 8 a 10 colpi nell'omogeneizzatore accoppiato a un pestello motorizzato che ruota a 600 rotazioni al minuto.Nell'omogenato cellulare, aggiungere il tampone ipotonico pari a sette volte il volume del pellet iniziale per generare un ambiente isotonico. Trasferire l'omogenato in un tubo da 15 millilitri e centrifugarlo in un rotore fisso a 1.000 g e quattro gradi Celsius per cinque minuti. Quindi raccogliere solo 3/4 del surnatante, lasciando un ampio margine dal pellet per evitare la contaminazione con nuclei o cellule intatte e trasferirlo in un altro tubo.Dopo aver ripetuto il processo due volte, trasferire il surnatante contenente la frazione mitocondriale in tubi da 1,5 millilitri. Centrifugare i tubi alla velocità massima di 18.000 g per due minuti a quattro gradi Celsius. Scartare il surnatante e lavare il pellet arricchito di mitocondri con tampone A.Unire il contenuto dei due tubi in uno e centrifugare come dimostrato in precedenza.Ripetere il processo fino a quando tutto il materiale è in un solo tubo. Lavare il pellet ottenuto dall'ultima centrifugazione utilizzando 300 microlitri di tampone A.Quantificare la concentrazione proteica mitocondriale utilizzando il saggio Bradford. Prima della fusione con la linea cellulare ricevente mitocondriale, valutare l'assenza di contaminanti nucleici nella frazione mitocondriale mediante immunorilevazione di proteine nucleari.In alternativa eseguire la reazione a catena della polimerasi quantitativa o l'amplificazione QPCR di un gene nucleare.

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 ...