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

Transmitokondriell Cybrid-generering fra suspensjonsvoksende kreftceller

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).

Forfatter Spotlight: Transmitokondriell cybridgenerering ved bruk av kreftcellelinjer  

Transmitochondrial cybrid generasjon ved hjelp av kreftcellelinjer

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  

Mitokondriell isolasjon for transmitochondrial cybrid generasjon

Når rundt 25 millioner mitokondriedonorceller er oppnådd på dag syv, høst de eksponentielt dyrkede cellene i et 50 milliliters rør og samle dem ved sentrifugering ved romtemperatur og 520g i fem minutter. Vask cellene med kaldt fosfatbufret saltvann og sedimenter dem ved sentrifugering ved romtemperatur i 520g i fem minutter. Nå utfør hele mitokondrieekstraksjonen ved fire grader Celsius ved hjelp av kalde reagenser og hold cellene eller mitokondriene på is.Etter den tredje sentrifugeringen, kast supernatanten ved aspirasjon ved hjelp av en glasspipette koblet til en vakuumpumpe og resuspender de pakkede cellene i et volum hypoton buffer lik syv ganger cellepelletsvolumet. Overfør deretter cellesuspensjonen til et homogeniseringsrør og la cellene svulme ved å inkubere dem på is i to minutter. Bryt cellemembranene ved å utføre 8 til 10 slag i homogenisatoren koblet til en motordrevet pestle som roterer ved 600 rotasjoner per minutt.Inn i cellehomogenatet, tilsett den hypotoniske bufferen lik syv ganger volumet av den opprinnelige pelleten for å generere et isotonisk miljø. Overfør homogenatet til et 15 milliliter rør og sentrifuge det i en fast rotor ved 1.000g og fire grader Celsius i fem minutter. Deretter samler du bare 3/4 av supernatanten, og etterlater en stor margin fra pelleten for å unngå forurensning med kjerner eller intakte celler og overfør den til et annet rør.Etter å ha gjentatt prosessen to ganger, overfør supernatanten som inneholder mitokondriell fraksjon i 1,5 milliliter rør. Sentrifuge rørene med maksimal hastighet på 18.000g i to minutter ved fire grader Celsius. Kast supernatanten og vask den mitokondrieberikede pelleten med buffer A.Kombiner innholdet i de to rørene i ett og sentrifuge som vist tidligere.Gjenta prosessen til alt materialet er i bare ett rør. Vask pelleten oppnådd fra den siste sentrifugeringen ved bruk av 300 mikroliter buffer A.Kvantifiser mitokondrieproteinkonsentrasjonen ved hjelp av Bradford-analysen. Før fusjon med mitokondriell mottakercellelinje, vurder fraværet av kjernekontaminanter i mitokondriefraksjonen ved immunodeteksjon av nukleære proteiner.Alternativt utføre kvantitativ polymerasekjedereaksjon eller QPCR-amplifisering av et nukleært gen.

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 ...