Name: Mitochondrial Depletion by Rhodamine 6G for Transmitochondrial Cybrid Generation
Uploaded: 2023-03-17T14:20:00
Description: Watch this Scientific Journal Video about Mitochondrial Depletion by Rhodamine 6G for Transmitochondrial Cybrid Generation at JoVE.com

The video outlines a procedure for mitochondrial depletion in a recipient cell line using Rhodamine 6G treatment. It details the seeding of cells, daily treatments, medium supplementation for cell viability, and preparation steps before mitochondrial fusion, highlighting the resulting pink coloration of the treated cells.

mitochondrial depletion by rhodamine 6g for transmitochondrial cybrid generation

יצירת ציבריד טרנסוכונדריאלי מתאי סרטן הגדלים בתרחיף

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).

מחבר זרקור: יצירת ציבריד טרנסוכונדריאלי באמצעות קווי תאים סרטניים  

יצירת ציבריד טרנסוכונדריאלי באמצעות קווי תאים סרטניים

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 Depletion by Rhodamine 6G for Transmitochondrial Cybrid Generation at JoVE.com

Mitochondrial Depletion by Rhodamine 6G for Transmitochondrial Cybrid Generation  

דלדול מיטוכונדריאלי על ידי Rhodamine 6G לייצור ציבריד טרנסוכונדריאלי

כדי לבצע דלדול מיטוכונדריאלי של קו התאים המקבל המיטוכונדריאלי, זרעו מיליון תאים בצלחת בת שש בארות באמצעות מדיום תרבית שלם המכיל 10% FBS ופניצילין-סטרפטומיצין. טפלו בתאים מדי יום עם הריכוז האופטימלי של רודאמין 6G במשך שלושה עד שבעה ימים, בהתאם לסוג קו התאים. כדי לשמור על התאים בחיים, להשלים את מדיום תרבית התא עם 50 מיקרוגרם למיליליטר של אורידין ו 100 מיקרוגרם למיליליטר של פירובט.לאחר הטיפול ולפני האיחוי עם מיטוכונדריה שבודדה מקו תאי התורם, יש להסיר את התווך של תאים שטופלו ברודמין 6G ולהוסיף מדיום תרבית תאים שלם ללא רודאמין 6G. לדגור על התאים בטמפרטורה של 37 מעלות צלזיוס עם 5% פחמן דו חמצני למשך שלוש עד ארבע שעות. לבסוף, כדי להכין את התאים המטופלים מראש של רודאמין 6G לאיחוי, אספו אותם בצינור של 15 מיליליטר, וצנטריפוגו את הצינור בטמפרטורת החדר ו-520G למשך חמש דקות.שימו לב שהגלולה מקבלת צבע ורוד ניאון עקב טיפול רודמין 6G.

Research

JoVE Journal

Cancer Research

Mitochondrial Depletion by Rhodamine 6G 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 ...