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

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 Fusion and Transmitochondrial Cybrid Generation at JoVE.com

Fusion and Transmitochondrial Cybrid Generation  

융합 및 트랜스미토콘드리아 Cybrid 생성

트랜스미토콘드리아 사이브라이드 생성을 위해, 로다민 6G 처리에 의해 미토콘드리아로 고갈된 미토콘드리아 수용자 세포를 사용하고, 미토콘드리아 공여자 세포로부터 분리된 미토콘드리아와 융합을 수행한다. 소수의 로다민 6G 처리 세포와 분리된 미토콘드리아를 6웰 플레이트의 완전한 배양 배지에 파종하여 수용자 세포에서 적절한 미토콘드리아 기능 폐지와 기증자 세포로부터 세포 소기관 정제를 보장합니다. 배양 1개월 후 웰에 남아 있는 생존 세포가 없는지 확인합니다.융합을 진행하려면 로다민 6G 처리된 세포를 분리된 미토콘드리아 펠릿에 조심스럽게 추가합니다. 그런 다음 세포가 미토콘드리아와 혼합되도록 5분 동안 520g에서 원심분리합니다. 100마이크로리터의 50% 폴리에틸렌 글리콜을 추가하고 펠릿을 30초 동안 부드럽게 재현탁한 다음 현탁액을 30초 동안 그대로 두십시오.마지막으로, 혼합물을 신선한 완전 세포 배양 배지가 있는 6웰 플레이트로 옮기고 5% 이산화탄소가 포함된 섭씨 37도의 인큐베이터에 넣습니다. 일반적으로 약 1주일 후에 트랜스미토콘드리아 사이브리드가 성장하기 시작하여 분석 전에 풀에서 개별적으로 선택하거나 혼합할 수 있는 클론을 생성합니다. 야생형 미토콘드리아의 제한 단편 길이 다형성(RFLP) 분석으로부터 얻어진 제한 단편은 돌연변이체와 달랐다.새로운 트랜스미토콘드리아 세포주에 대한 RFLP 분석은 제한 단편이 각각의 미토콘드리아 기증자에서 얻은 것과 동일하고 수용자 세포주에서 생성된 것과 다르다는 것을 나타냈습니다. 또한, 하이브리드화 공정에 사용된 두 세포주의 전형적인 DNA 핵 유전자형 프로파일의 차이도 핵 DNA 순도를 확인하는 데 사용될 수 있습니다.

fusion-and-transmitochondrial-cybrid-generation

Research

JoVE Journal

Cancer Research

Fusion and Transmitochondrial Cybrid Generation

115 조회수.  University of Zaragoza. 트랜스미토콘드리아 사이브라이드 생성을 위해, 로다민 6G 처리에 의해 미토콘드리아로 고갈된 미토콘드리아 수용자 세포를 사용하고, 미토콘드리아 공여자 세포로부터 분리된 미토콘드리아와 융합을 수행한다. 소수의 로다민 6G 처리 세포와 분리된 미토콘드리아를 6웰 플레이트의 완전한 배양 배지에 파종하여 수용자 세포에서 적절한 미토콘드리아 기능 폐지와 기증자 세?...

비디오: 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 efforts are still needed to fully understand the link involving alterations in mitochondria and tumorigenesis, as well as to identify tumor-associated mitochondrial phenotypes. For instance, to evaluate the contribution of mitochondria in tumorigenesis and metastasis processes, it is essential to understand the influence of mitochondria from tumor cells in different nuclear environments. For this purpose, one possible approach consists of transferring mitochondria into a different nuclear background to obtain the so-called cybrid cells. In the traditional cybridization techniques, a cell line lacking mtDNA (&#961;0, nuclear donor cell) is repopulated with mitochondria derived from either enucleated cells or platelets. However, the enucleation process requires good cell adhesion to the culture plate, a feature that is partially or completely lost in many cases in invasive cells. In addition, another difficulty found in the traditional methods is achieving complete removal of the endogenous mtDNA from the mitochondrial-recipient cell line to obtain pure nuclear and mitochondrial DNA backgrounds, avoiding the presence of two different mtDNA species in the generated cybrid. In this work, we present a mitochondrial exchange protocol applied to suspension-growing cancer cells based on the repopulation of rhodamine 6G-pretreated cells with isolated mitochondria. This methodology allows us to overcome the limitations of the traditional approaches, and thus can be used as a tool to expand the comprehension of the mitochondrial role in cancer progression and metastasis.

This protocol describes a technique for cybrid generation from suspension-growing cancer cells as a tool to study the role of mitochondria in the tumorigenic process.

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

연구

암 연구학

Mitochondrial Depletion by Rhodamine 6G for Transmitochondrial Cybrid Generation

To perform mitochondrial depletion of the mitochondrial recipient cell line, seed 1 million cells in a six-well plate using complete culture medium containing 10%FBS and Penicillin-Streptomycin. Treat the cells daily with the optimal concentration of Rhodamine 6G for three to seven days, depending on the type of cell line. To keep the cells alive, supplement the cell culture medium with 50 micrograms per milliliter of uridine and 100 micrograms per milliliter of pyruvate.After treatment and prior to fusion with a mitochondria isolated from the donor cell line, remove the medium of Rhodamine 6G treated cells and add complete cell culture medium without Rhodamine 6G. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide for three to four hours. Finally, to prepare the Rhodamine 6G pretreated cells for the fusion, collect them in a 15-milliliter tube, and centrifuge the tube at room temperature and 520G for five minutes.Note that the pellet acquires a neon pink color due to Rhodamine 6G treatment.

트랜스미토콘드리아 Cybrid 생성을 위한 Rhodamine 6G에 의한 미토콘드리아 고갈

Mitochondrial Isolation for Transmitochondrial Cybrid Generation

Once around 25 million mitochondria donor cells are obtained on day seven, harvest the exponentially grown cells in a 50-milliliter tube and collect them by centrifuging at room temperature and 520g for five minutes. Wash the cells with cold phosphate-buffered saline and sediment them by centrifugation at room temperature in 520g for five minutes. Now onwards, perform the whole mitochondrial extraction at four degrees Celsius using cold reagents and keep the cells or mitochondria on ice.After the third centrifugation, discard the supernatant by aspiration using a glass pipette coupled to a vacuum pump and resuspend the packed cells in a volume of hypotonic buffer equal to seven times the cell pellet volume. Then transfer the cell suspension into a homogenizer tube and let the cells swell by incubating them on ice for two minutes. Break the cell membranes by performing 8 to 10 strokes in the homogenizer coupled to a motor-driven pestle rotating at 600 rotations per minute.Into the cell homogenate, add the hypotonic buffer equal to seven times the volume of the initial pellet to generate an isotonic environment. Transfer the homogenate into a 15-milliliter tube and centrifuge it in a fixed rotor at 1, 000g and four degrees Celsius for five minutes. Then collect only 3/4 of the supernatant, leaving a large margin from the pellet to avoid contamination with nuclei or intact cells and transfer it to another tube.After repeating the process twice, transfer the supernatant containing the mitochondrial fraction into 1.5-milliliter tubes. Centrifuge the tubes at the maximum speed of 18, 000g for two minutes at four degrees Celsius. Discard the supernatant and wash the mitochondria-enriched pellet with buffer A.Combine the content of the two tubes into one and centrifuge as demonstrated before.Repeat the process until all the material is in only one tube. Wash the pellet obtained from the last centrifugation using 300 microliters of buffer A.Quantify the mitochondrial protein concentration using the Bradford assay. Before fusion with the mitochondrial recipient cell line, evaluate the absence of nuclei contaminants in the mitochondrial fraction by immunodetection of nuclear proteins.Alternatively perform quantitative polymerase chain reaction or QPCR amplification of a nuclear gene.

Transmitochondrial Cybrid 생성을 위한 미토콘드리아 분리

For transmitochondrial cybrid generation, use the mitochondrial recipient cells that have been depleted to mitochondria by Rhodamine 6G treatment, and perform fusion with mitochondria isolated from the mitochondria donor cells. Ensure proper mitochondrial function abolishment in recipient cells and organelle purification from donor cells by seeding a small number of Rhodamine 6G-treated cells and isolated mitochondria in complete culture medium in a six-well plate. Check for no surviving cells remaining in the wells after one month of culture.To proceed with the fusion, carefully add the Rhodamine 6G-treated cells to the isolated mitochondria pellet. Then centrifuge at 520g for five minutes to allow the cells to mix with the mitochondria. Add 100 microliters of 50%polyethylene glycol and gently resuspend the pellet for 30 seconds Then allow the suspension to rest untouched for another 30 seconds.Finally, transfer the mix into a six-well plate with fresh complete cell culture medium, and place it in the incubator at 37 degrees Celsius with 5%carbon dioxide. Usually, after about one week, transmitochondrial cybrids should start growing, giving rise to clones that can be individually selected or mixed in a pool prior to their analysis. The restriction fragments obtained from the restriction fragment length polymorphism, or RFLP, analysis of wild-type mitochondria differed from the mutant one.The RFLP analysis of the new transmitochondrial cell lines indicated that restriction fragments were identical to those obtained in their respective mitochondrial donors and different from those generated with the recipient cell lines. Further, the difference in a typical DNA nuclear genotyping profile of the two cell lines used in the hybridization process could also be used to confirm nuclear DNA purity.