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

Geração de cíbridos transmitocondriais a partir de células cancerígenas em suspensão

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).

Autor em destaque: Geração de cíbridos transmitocondriais usando linhas de células cancerígenas  

Geração de Cybrid Transmitochondrial Usando Linhas de Células Cancerígenas

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 Mitocondrial para Geração de Cibridas Transmitocondrial

Uma vez que cerca de 25 milhões de células doadoras de mitocôndrias são obtidas no sétimo dia, colher as células exponencialmente cultivadas em um tubo de 50 mililitros e coletá-las por centrifugação à temperatura ambiente e 520g por cinco minutos. Lavar as células com solução salina tamponada com fosfato frio e sedimentá-las por centrifugação à temperatura ambiente em 520g por cinco minutos. Agora em diante, realizar toda a extração mitocondrial a quatro graus Celsius usando reagentes frios e manter as células ou mitocôndrias no gelo.Após a terceira centrifugação, descartar o sobrenadante por aspiração com pipeta de vidro acoplada a bomba de vácuo e ressuspender as células do empacotamento em um volume de tampão hipotônico igual a sete vezes o volume da pastilha celular. Em seguida, transfira a suspensão celular para um tubo homogeneizador e deixe as células incharem incubando-as no gelo por dois minutos. Quebrar as membranas celulares realizando 8 a 10 golpes no homogeneizador acoplado a um pilão acionado por motor girando a 600 rotações por minuto.No homogeneizado celular, adicionar o tampão hipotônico igual a sete vezes o volume da pastilha inicial para gerar um ambiente isotônico. Transfira o homogeneizado para um tubo de 15 mililitros e centrifuga-o em um rotor fixo a 1.000g e quatro graus Celsius por cinco minutos. Em seguida, colete apenas 3/4 do sobrenadante, deixando uma grande margem do pellet para evitar contaminação com núcleos ou células intactas e transfira-o para outro tubo.Depois de repetir o processo duas vezes, transfira o sobrenadante contendo a fração mitocondrial para tubos de 1,5 mililitro. Centrifugar os tubos à velocidade máxima de 18.000g durante dois minutos a quatro graus Celsius. Descarte o sobrenadante e lave o pellet enriquecido com mitocôndrias com tampão A.Combine o conteúdo dos dois tubos em um e centrífugo como demonstrado anteriormente.Repita o processo até que todo o material esteja em apenas um tubo. Lavar o pellet obtido da última centrifugação usando 300 microlitros de tampão A.Quantificar a concentração de proteína mitocondrial usando o ensaio de Bradford. Antes da fusão com a linhagem celular receptora mitocondrial, avaliar-se a ausência de núcleos contaminantes na fração mitocondrial por imunodetecção de proteínas nucleares.Alternativamente, realizar reação em cadeia da polimerase quantitativa ou amplificação por QPCR de um gene nuclear.

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 ...