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

현탁액 성장 암세포에서 투과연골 Cybrid 생성

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).

저자 스포트라이트: 암 세포주를 사용한 투과연골 Cybrid 생성  

암 세포주를 이용한 트랜스미토콘드리아 Cybrid 생성

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  

Transmitochondrial Cybrid 생성을 위한 미토콘드리아 분리

7일째에 약 2,500만 개의 미토콘드리아 기증자 세포가 확보되면 기하급수적으로 성장한 세포를 50밀리리터 튜브에서 수확하고 실온에서 520g을 5분 동안 원심분리하여 수집합니다. 차가운 인산완충식염수로 세포를 세척하고 5분 동안 실온에서 5분간 원심분리하여 침전시킨다. 이제 차가운 시약을 사용하여 섭씨 4도에서 전체 미토콘드리아 추출을 수행하고 세포 또는 미토콘드리아를 얼음 위에 보관하십시오.3차 원심분리 후, 진공 펌프에 결합된 유리 피펫을 사용하여 흡인에 의해 상층액을 버리고, 세포 펠릿 부피의 7배에 해당하는 부피의 저장성 완충액에 충진된 세포를 재현탁한다. 그런 다음 세포 현탁액을 균질화 튜브로 옮기고 얼음에서 2분 동안 배양하여 세포가 부풀어 오르도록 합니다. 분당 600회 회전하는 모터 구동 유봉에 연결된 균질화기에서 8 내지 10번의 스트로크를 수행하여 세포막을 파괴합니다.세포 균질액에 초기 펠릿 부피의 7배에 해당하는 저삼투압 완충액을 첨가하여 등장성 환경을 생성합니다. 균질액을 15 밀리리터 튜브로 옮기고 1, 000g 및 섭씨 4도에서 5 분 동안 고정 로터에서 원심 분리합니다. 그런 다음 상청액의 3/4 만 수집하고 펠릿에서 큰 여백을 남겨 핵 또는 손상되지 않은 세포로 오염되는 것을 방지하고 다른 튜브로 옮깁니다.이 과정을 2회 반복한 후, 미토콘드리아 분획을 함유하는 상청액을 1.5-밀리리터 튜브로 옮긴다. 튜브를 섭씨 4도에서 2분 동안 최대 속도 18, 000g으로 원심분리합니다. 상등액을 버리고 미토콘드리아 농축 펠릿을 완충액으로 세척하십시오. A. 두 튜브의 내용물을 하나로 결합하고 이전에 시연 된대로 원심 분리합니다.모든 재료가 하나의 튜브에 들어갈 때까지 이 과정을 반복합니다. 300 마이크로리터의 완충액을 사용하여 마지막 원심분리로부터 얻은 펠릿을 세척한다. A.Bradford 분석을 사용하여 미토콘드리아 단백질 농도를 정량화합니다. 미토콘드리아 수용자 세포주와 융합하기 전에 핵 단백질의 면역검출을 통해 미토콘드리아 분획에 핵 오염 물질이 없는지 평가합니다.또는 핵 유전자의 정량적 중합효소 연쇄 반응 또는 QPCR 증폭을 수행합니다.

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 ...