这项研究由RSA，JMB和AA的授权号PID2019-105128RB-I00以及PFS和RML的PGC2018-095795-B-I00资助，均由MCIN / AEI / 10.13039 / 501100011033和授权号B31_20R（RSA，JMA和AA）和E35_17R（PFS和RML）资助，并由Gobierno de Aragón资助。RSA的工作得到了西班牙反对协会PRDAR21487SOLE的赠款支持。作者要感谢萨拉戈萨大学使用高级调查总局。

注意： 表1中指定了所有培养基和缓冲液组合物。在cybrid生成之前，必须对来自供体和受体细胞的线粒体和核DNA图谱进行分型，以确认细胞系之间两个基因组中存在遗传差异。在这项研究中，使用了市售的L929细胞系及其衍生的细胞系L929dt，该细胞系是我们实验室自发产生的（更多信息见13 ）。这些细胞系在其 mt-Nd2 基因序列中存在两种差异，一旦细胞化过程完成，可用于确认mtDNA的纯度13。在这种情况下，核背景的纯度通过抗生素敏感性得到证实，因为与L929dt细胞相反，L929对遗传素具有抗性。
1.罗丹明6G处理（受体细胞）耗竭线粒体
注意：成功生成cybrid的第一步是完全和不可逆地消除受体细胞中的线粒体功能。为此，有必要事先确定每个细胞系的适当浓度和罗丹明6G的处理持续时间。这个足够的浓度应该略低于药物诱导的细胞死亡（在治疗过程中不会杀死细胞的最高浓度）。定义最佳条件后，执行以下操作。
<ol><li>使用完整的培养基在6孔板中接种106 个细胞（详见 表1 ），并根据细胞系每天用最佳浓度的罗丹明6G处理3-10天。传统浓度范围为2至5μg/mL，持续3-10天20，21。在这项研究中，对于L929来源的细胞，选择2.5μg/mL罗丹明6G7天治疗13，22。</li>
	<li>为了保持细胞存活，用50μg/ mL尿苷和100μg/ mL丙酮酸补充细胞培养基，并每24小时更新一次。</li>
	<li>处理后和融合前，将罗丹明6G处理的细胞的培养基更换为不含罗丹明6G的完全细胞培养基，并将它们在该培养基中在37°C的培养箱中放置3-4小时，CO2。 注意：罗丹明6G毒性作用是不可逆的，具有无功能线粒体的细胞不应恢复20。因此，处理后，即使在补充尿苷的培养基中，不接受功能性线粒体的细胞也应死亡。因此，没有必要进行核选择。然而，建议使用不含线粒体的罗丹明6G处理细胞的对照融合。</li>
</ol>2. 扩增和线粒体分离（供体细胞）
<ol><li>在罗丹明6G治疗的时间间隔内扩增线粒体供体细胞，以获得约25 x 106 细胞。</li>
	<li>在第 7 天，收获 50 mL 管中指数生长的细胞，并在室温 （RT） 下以 520 x g 离心 5 分钟收集它们。用冷磷酸盐缓冲盐水（PBS）洗涤细胞3倍，并在室温下以520× g 离心5分钟沉淀。从现在开始，使用冷试剂在4°C下执行线粒体提取的所有步骤，并将带有细胞或线粒体的管保持在冰上。</li>
	<li>第三次离心后，使用与真空泵耦合的玻璃移液管抽吸丢弃上清液，并将填充的细胞重悬于等于细胞沉淀体积7倍的低渗缓冲液中。然后，将细胞悬液转移到均质管中，并通过在冰上孵育2分钟让细胞膨胀。</li>
	<li>通过在均质器中执行8至10次冲程来破坏细胞膜，该均质器与以600rpm旋转的电机驱动的杵耦合。 注意：细胞膜破碎的步骤可能因细胞类型而异;因此，必须针对每种细胞类型进行优化。</li>
	<li>向细胞悬液中加入相同体积的高渗缓冲液（细胞沉淀体积的 7 倍）以产生等渗环境。</li>
	<li>将匀浆转移到15mL管中，并在固定转子中以1，000× g 在4°C下离心5分钟。 然后，仅收集3/4的上清液，从沉淀中留下大量边缘，以避免被细胞核或完整细胞污染，并将其转移到另一个管中。重复相同的过程两次。请注意，必须保留上清液并丢弃沉淀。</li>
	<li>保存线粒体部分（上清液）。将其转移到1.5mL管中，并在4°C下以最大速度（18，000× g）离心2分钟。</li>
	<li>弃去上清液并用缓冲液A洗涤富含线粒体的沉淀，将两个管的内容物合并为一个，并在与步骤2.7中所述的相同条件下离心。重复相同的过程，直到所有材料都在一个管子中。</li>
	<li>用 300 μL 缓冲液 A 进行额外洗涤，并使用Bradford 测定法 23 定量线粒体蛋白浓度。对于每个细胞化测定，必须确定转移过程的最佳线粒体量（在我们的例子中，每106 个细胞的线粒体蛋白浓度在10-40μg之间）。</li>
	<li>同时，通过将罗丹明 6G 预处理的细胞收集在 15 mL 管中并在室温下以 520 x g 离心 5 分钟来制备用于融合的罗丹明 6G 预处理细胞。 请注意，由于罗丹明 6G 处理，颗粒获得霓虹粉色。</li>
	<li>为了确保正确执行受体细胞中的线粒体功能消除以及来自供体的细胞器纯化，使用完整的培养基在 6 孔板中接种少量罗丹明 6G 处理的细胞和分离的线粒体并培养一个月以检查任何孔中没有存活细胞残留（图 2）。</li>
	<li>同时，通过免疫检测核蛋白（即层粘连蛋白 β、组蛋白 H3 等）或通过核基因（即 SDH、18S rRNA 等）的定量聚合酶链反应 （qPCR） 扩增来评估线粒体部分中是否存在细胞核污染物。 注意：为避免污染，建议在无菌条件下在层流罩下执行所有步骤。</li>
</ol>3. 聚变和赛布里德生成
<ol><li>为了进行融合，小心地将106个罗丹明6G处理的细胞添加到分离的线粒体沉淀（10-40μg线粒体蛋白）中，并以520× g 离心5分钟，以使细胞与线粒体混合。</li>
	<li>加入 100 μL 聚乙二醇 （PEG， 50%），轻轻重悬沉淀 30 秒。然后，让原封不动地再静置 30 秒。</li>
	<li>最后，将混合物转移到带有新鲜完全细胞培养基的6孔板中，并置于37°C的培养箱中，并用5%CO2。几天后（通常为1周），透射软骨体cybris应该开始生长（图2），从而产生可以在分析之前单独选择或在池中混合的克隆。</li>
</ol>4. 线粒体和核背景的验证
注意：一旦建立了新的细胞系并且细胞开始呈指数级增长，就必须验证其线粒体和核DNA的纯度。因此，原始细胞系应该在其基因组中具有不同的突变或多态性，以使它们可识别。
<ol><li>总脱氧核糖核酸分离<ol><li>通过使用商业基因组DNA提取试剂盒（参见材料表）或使用苯酚-氯仿-异戊醇提取和醇沉淀执行标准方案，分离用于cybrid生成的所有细胞系的基因组DNA。</li>
	</ol></li>
	<li>mtDNA评估（图3） 注意：可以执行多种技术，例如测序，限制性片段长度多态性（RFLP）分析或等位基因特异性qPCR，以分析mtDNA的纯度。要通过RFLP确认mtDNA序列变异的存在，请遵循后续协议步骤。<ol><li>通过 PCR 扩增含有核苷酸变化的 mtDNA 片段。<ol><li>在这里，L929dt细胞在L929细胞中不存在的 mt-Nd2 基因（m.4206C>T）内在4206位置呈现mtDNA突变。为了确认透射软骨蠹骨中存在这种取代，请使用标准方案和以下引物通过PCR扩增397 bp片段：1）5'-AAGCTATCGGG CCCATACCCCG-3'（位置3862-3884）和2）5'-TAATCAGAAGTGGAATGGGGGG-3'（位置4236-4258）。</li>
		</ol></li>
		<li>使用识别序列变化并为两种细胞系生成不同切割模式的特定核酸内切酶，分析RFLP所需核苷酸取代的存在。 注意：当存在L929dt mtDNA变体（4206T）时，在步骤4.2.1中获得的扩增子包含两个SspI限制性位点（参见 材料表），可产生306 bp，52 bp和39 bp的三个DNA片段。当存在野生型 （WT） 版本 C4206 时，生成 52 和 39 波段的限制性位点被破坏，并且出现 91 bp 的新波段。因此，分析中包括用于完全消化SspI的内部对照。按照制造商的说明在37°C下进行消化反应。</li>
		<li>通过电泳分离限制性内切酶片段并比较条带模式。<ol><li>进行酶解后，在10%聚丙烯酰胺-三硼酸盐-EDTA（TBE）凝胶中通过电泳分析获得的限制性内切性片段（参见 表1 的1x TBE组成）。在室温下以80V运行电泳1小时。 在室温下在1x TBE的溴化乙锭溶液中凝胶染色15分钟后可视化DNA片段（凝胶染色溶液组成参见 表1 ）。</li>
		</ol></li>
	</ol></li>
	<li>核DNA基因分型 注意：核DNA基因分型必须使用先前用于RFLP分析的DNA提取样品进行。<ol><li>通过使用商业特异性寡核苷酸池扩增16个位点（D21S11，CSF1PO，vWA，D8S1179，TH01，D18S51，D5S818，D16S539，D3S1358，D2S1338，TPOX，FGA，D7S820，D13S317，D19S433和AMEL）（参见 材料表）。</li>
		<li>使用基因分析仪进行电泳，以分离先前获得的片段。 注意：扩增后，使用capilar系统通过电泳分析不同的位点（参见 材料表）。碎片在填充有商业聚合物的50厘米长的帽状物中分离。阴极和阳极缓冲液也可市售，如 材料表所示。在60°C下在19.5kV下进行电泳20分钟。</li>
		<li>使用生物信息学工具确定对应于每个扩增位点的等位基因（参见 材料表）。</li>
		<li>将步骤4.3.3中获得的数据与核DNA谱数据库（表1）进行比较，以检查核DNA图谱是否与线粒体受体细胞系谱匹配（图4）。</li>
	</ol></li>
</ol>

来自悬浮生长癌细胞的传输软骨 Cybrid 生成

作者声明不存在利益冲突。

自从Otto Warburg报道癌细胞改变其新陈代谢并增强“有氧糖酵解”3，4，同时减少线粒体呼吸以来，对线粒体在癌症转化和进展中的作用的兴趣呈指数级增长。近年来，mtDNA突变和线粒体功能障碍已被假定为许多癌症类型的标志25。迄今为止，许多研究已经分析了特定肿瘤6，26，27，28，29</...

重编程能量代谢是癌症1的标志，这是奥托·沃伯格（Otto Warburg）在1930年代首次观察到的2。在有氧条件下，正常细胞将葡萄糖转化为丙酮酸，然后产生乙酰辅酶A，为线粒体机器提供燃料并促进细胞呼吸。然而，Warburg证明，即使在常氧条件下，大多数癌细胞将从糖酵解过程中获得的丙酮酸转化为乳酸，从而改变其获取能量的方式。这种代谢调节被称为“Warburg效应”，使一些癌细胞能够提供其快速生长和分裂的能量需求，尽管产生ATP的效率低于有氧过程3，4，5。近几十年来，许多工作支持代谢重编程在癌症进展中的含义。因此，肿瘤能量学被认为是对抗癌症的有趣靶标1。作为能量代谢和必需前体供应的中心枢纽，线粒体在这些细胞适应中起着关键作用，迄今为止，我们只部分了解这些细胞。
根据上述情况，线粒体DNA（mtDNA）突变已被提出为这种代谢重编程的可能原因之一，这可能导致电子传递链（ETC）性能受损6，并解释为什么一些癌细胞增强其糖酵解代谢以存活。事实上，据报道mtDNA在癌细胞内积累突变，至少存在于50%的肿瘤中7。例如，Yuan等人最近进行的一项研究报告称，肾脏癌，结直肠癌和甲状腺癌中存在超突变和截短的mtDNA分子8。此外，许多研究表明，某些mtDNA突变与更具侵袭性的肿瘤表型和癌细胞转移潜力的增加有关9，10，11，12，13，14，15，16。
尽管线粒体基因组与癌症进展有明显的相关性，但由于目前可用的实验模型和技术的局限性，对这些突变及其对疾病的贡献的研究一直具有挑战性17。因此，需要新技术来了解线粒体DNA对癌症疾病发展和进展的真正影响。在这项工作中，我们介绍了一种从悬浮生长的癌细胞中传输软骨细胞生成的协议，该协议基于罗丹明6G预处理细胞与分离线粒体的重新繁殖，克服了传统胞化方法的主要挑战18，19。该方法允许使用任何细胞核供体，无论其相应的ρ0 细胞系的可用性如何，以及线粒体从遵循传统技术难以去核的细胞（即非贴壁细胞系）转移。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3500XL Genetic Analyzer&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>4406016</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Corning</td>
			<td>08-772-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>AmpFlSTR Identifiler Plus PCR Amplification Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>4427368</td>
			<td></td>
		</tr>
		<tr>
			<td>Anode Buffer Container 3500 Series</td>
			<td>Applied Biosystems</td>
			<td>4393927</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>PanReac</td>
			<td>131015</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford assay</td>
			<td>Biorad</td>
			<td>5000002</td>
			<td></td>
		</tr>
		<tr>
			<td>Cathode Buffer Container 3500 Series</td>
			<td>Applied Biosystems</td>
			<td>4408256</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks</td>
			<td>TPP</td>
			<td>90076</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>PanReac</td>
			<td>131026</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Sigma-Aldrich</td>
			<td>E8751</td>
			<td></td>
		</tr>
		<tr>
			<td>Geneticin</td>
			<td>Gibco</td>
			<td>10131027</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer Teflon pestle</td>
			<td>Deltalab</td>
			<td>196102</td>
			<td></td>
		</tr>
		<tr>
			<td>L929 cell line</td>
			<td>ATCC</td>
			<td>CCL-1</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniProtean Tetra4 Gel System</td>
			<td>BioRad</td>
			<td>1658004</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma-Aldrich</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Sigma-Aldrich</td>
			<td>Custom products</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyacrylamide Solution 30%</td>
			<td>PanReac</td>
			<td>A3626</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol</td>
			<td>Sigma-Aldrich</td>
			<td>P7181</td>
			<td></td>
		</tr>
		<tr>
			<td>POP-7</td>
			<td>Applied Biosystems</td>
			<td>4393714</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P5280</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAmp DNA Mini Kit</td>
			<td>Qiagen</td>
			<td>51306</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine-6G</td>
			<td>Sigma-Aldrich</td>
			<td>R4127</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum Fetal Bovine</td>
			<td>Sigma-Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>SspI</td>
			<td>New England Biolabs</td>
			<td>R3132</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin/penicillin</td>
			<td>PAN biotech</td>
			<td>P06-07100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S3089</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Sigma-Aldrich</td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>PanReac</td>
			<td>P14030b</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma-Aldrich</td>
			<td>U3750</td>
			<td></td>
		</tr>
	</tbody>
</table>

transmitochondrial cybrid generation using cancer cell lines

近年来，致力于确定线粒体与癌症之间联系的研究数量显着增加。然而，仍然需要做出更多的努力来充分了解涉及线粒体改变和肿瘤发生的联系，以及识别与肿瘤相关的线粒体表型。例如，为了评估线粒体在肿瘤发生和转移过程中的贡献，必须了解线粒体在不同核环境中肿瘤细胞的影响。为此，一种可能的方法是将线粒体转移到不同的核背景中以获得所谓的cybrid细胞。在传统的细胞化技术中，缺乏mtDNA（ρ0，核供体细胞）的细胞系被来自去核细胞或血小板的线粒体重新填充。然而，剜除过程需要良好的细胞粘附在培养板上，在许多情况下，侵袭性细胞会部分或完全失去这一特征。此外，传统方法中发现的另一个困难是从线粒体受体细胞系中完全去除内源性mtDNA，以获得纯核和线粒体DNA背景，避免在生成的cybrid中存在两种不同的mtDNA物种。在这项工作中，我们提出了一种应用于悬浮生长癌细胞的线粒体交换协议，该方案基于罗丹明6G预处理细胞与分离线粒体的重新繁殖。这种方法使我们能够克服传统方法的局限性，因此可以用作扩展对线粒体在癌症进展和转移中的作用的理解的工具。

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).

作者聚焦：使用癌细胞系进行传递软骨 Cybrid 生成  

使用癌细胞系的传递软骨Cybrid生成

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.

遵循上述方案后，应获得具有保守核背景但具有新线粒体基因型的同质cybrid细胞系，如图1和图2中的示意图所示。存在于cybrid中的线粒体和核DNA的纯度可以通过RFLP和核DNA基因分型分析（如图4所示）来确认。
如果线粒体转移成功完成，则通过RFLP分析获得的cybrid细胞系的结果必须显示与受...

Watch this Scientific Journal Video about Transmitochondrial Cybrid Generation Using Cancer Cell Lines at JoVE.com

该协议描述了一种从悬浮生长的癌细胞生成cybrid的技术，作为研究线粒体在致瘤过程中作用的工具。

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

这项研究旨在更好地了解线粒体在癌症进展和转移中的作用。传递软骨胚的产生可以帮助我们阐明线粒体基因型如何以及在什么水平上参与肿瘤细胞的侵袭性。获取线粒体在癌症中的作用信息的技术之一是cybrid生成。我们将该工具与各种其他方法相结合，包括供体电泳、呼吸分析和其他线粒体功能测定、流式细胞术、蛋白质组学等。癌细胞的可塑性以及核基因和线粒体基因之间的相互作用网络增加了我们实验和数据解释的复杂性。这就是为什么有必要开发新的实验方法的原因之一。我们研究中最令人兴奋的发现之一是发现了两种线粒体DNA突变，这些突变与细胞从游戏培养皿中分离有关，类似于转移的产生，并使细胞完全依赖于有氧糖酵解，并且还使它们在体内模型中更具肿瘤性和转移性。这种线粒体交换方案可以应用于悬浮生长的癌细胞。年表可以克服传统方法的局限性，其中成核过程和从线粒体受体细胞系中完全去除线粒体DNA通常成为真正的挑战。

transmitochondrial-cybrid-generation-using-cancer-cell-lines

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JoVE Journal

Cancer Research

Transmitochondrial Cybrid Generation Using Cancer Cell Lines

1.6K 次观看.  University of Zaragoza. 这项研究旨在更好地了解线粒体在癌症进展和转移中的作用。传递软骨胚的产生可以帮助我们阐明线粒体基因型如何以及在什么水平上参与肿瘤细胞的侵袭性。获取线粒体在癌症中的作用信息的技术之一是cybrid生成。我们将该工具与各种其他方法相结合，包括供体电泳、呼吸分析和其他线粒体功能测定、流式细胞术、蛋白质组学等。癌细胞的可塑性以及核基因和线粒体?...

视频: 作者聚焦：使用癌细胞系进行传递软骨 Cybrid 生成  

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.

科研

癌症研究

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.

罗丹明6G对线粒体消耗的影响

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.

线粒体分离用于传递软骨蜗牛的生成

Fusion and Transmitochondrial Cybrid Generation

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.