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

The video details the process of generating transmitochondrial cybrids by fusing Rhodamine 6G-treated mitochondrial recipient cells with isolated mitochondria from donor cells. It outlines steps for cell culture, fusion techniques, and analyzing the resulting cell lines using RFLP and nuclear DNA profiling to confirm mitochondrial and nuclear DNA purity.

fusion and transmitochondrial cybrid generation

懸濁増殖がん細胞からのトランスフォコンドリアサイブリッドの作製

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).

著者スポットライト:がん細胞株を使用したトランスミトコンドリアサイブリッドの生成  

がん細胞株を用いた伝達性サイブリッドの作製

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 Fusion and Transmitochondrial Cybrid Generation at JoVE.com

Fusion and Transmitochondrial Cybrid Generation  

融合と透過性サイブリッドの生成

伝達性サイブリッドの生成には、ローダミン6G処理によりミトコンドリアに枯渇したミトコンドリアレシピエント細胞を用い、ミトコンドリアドナー細胞から単離したミトコンドリアとの融合を行う。少数のローダミン6G処理細胞と単離されたミトコンドリアを6ウェルプレートの完全培養培地に播種することにより、レシピエント細胞における適切なミトコンドリア機能消失およびドナー細胞からの細胞小器官精製を確実にします。1ヶ月の培養後にウェルに生き残った細胞が残っていないことを確認します。融合を進めるには、ローダミン6G処理細胞を単離したミトコンドリアペレットに注意深く加えます。次に、520gで5分間遠心分離し、細胞をミトコンドリアと混合させます。100マイクロリットルの50%ポリエチレングリコールを加え、ペレットを30秒間静かに再懸濁します次に、懸濁液をそのままさらに30秒間放置します。最後に、ミックスを新鮮な完全細胞培養培地を含む6ウェルプレートに移し、5%二酸化炭素を含む摂氏37度のインキュベーターに入れます。通常、約1週間後、伝達軟骨サイブリッドが成長し始め、分析前に個別に選択またはプール内で混合できるクローンが生成されます。野生型ミトコンドリアの制限断片長多型(RFLP)分析から得られた制限断片は、変異型ミトコンドリアの分析とは異なりました。新しい伝達軟骨細胞株のRFLP分析は、制限断片がそれぞれのミトコンドリアドナーで得られたものと同一であり、レシピエント細胞株で生成されたものとは異なることを示しました。さらに、ハイブリダイゼーションプロセスで使用された2つの細胞株の典型的なDNA核ジェノタイピングプロファイルの違いも、核DNA純度を確認するために使用することができる。

Research

JoVE Journal

Cancer Research

Fusion and 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 ...