哺乳动物细胞系的厌氧生长与维持
Summary
在这里, 我们描述了一种新的方法, 使厌氧长期培养已建立的细胞系。测试的最大生存时间是17天。该方法适用于细胞毒性药物的检测和 anoxically 复制细胞的生理学研究。
Abstract
大多数黏膜表面连同中点在肿瘤和干细胞龛位是身体的地理区域缺氧。先前的研究表明, 在常氧 (5% CO2的空气) 或缺氧 (低氧水平) 条件下, 缺氧孵化 (缺乏自由氧) 的孵化导致生存能力有限 (2–3天)。开发了一种新的方法, 使缺氧细胞培养 (至少17天; 测试的最大时间)。这一方法最关键的方面是确保不将氧气引入系统。这是由介质的脱气, 并通过冲洗管, 盘子, 烧瓶和吸管与厌氧气体混合物 (H2, CO2, N2) 后, 允许材料平衡缺氧 (非氧气) 环境在使用之前。在获取显微照片时必须进行额外的护理, 以确保所获得的显微照片不包含工件。在缺乏氧气的情况下, 细胞形态明显改变。所有厌氧细胞都有两种截然不同的 morphotypes。在缺乏氧气的情况下, 培养和维持哺乳动物细胞的能力可以应用于细胞生理学、polymicrobial 相互作用的分析以及新型癌症药物开发的生物合成途径的表征。
Introduction
细胞从固体肿瘤, 干细胞的利基, 和那些衬里的粘膜表面存在的环境中, 有氧水平降低, 包括缺氧1,2,3,4。在正常的生理状态下, 氧合不同于缺氧的缺氧 (完全没有氧气)5,6。在二十世纪七十年代代初, 发现大气氧对哺乳动物细胞复制产生不利影响, 并且在耗尽的氧气条件下可以优化体外细胞生长。里氏等。7表明, 1–3% 氧水平 (缺氧) 提高电镀效率与大气氧 (20%)。在缺氧培养条件下, 人类双倍细胞寿命也在8。
在体内, 缺氧条件发生时, 氧储存耗尽 (例如, 在激烈的运动期间), 其中 ATP 生产从骨骼肌切换到有氧呼吸到发酵 (厌氧呼吸) 与乳酸的末端产品9。病理上, 在癌性肿瘤中, 肿瘤肿块的内部是缺氧的, 因为血管化10的不良。有限灌注对肿瘤内部的影响是独立的, 由强制厌氧1所殖民的肿瘤内部进行验证。机械化, 在缺氧的肿瘤细胞生存被认为完全依赖于缺氧诱导因子1α基因 (HIF1) 的表达, 这是最初自发反应缺氧4,11,12. HIF1在缺氧条件下诱发的热休克蛋白结合HIF1启动子和 upregulate 基因转录12。这些热休克蛋白被认为有助于诱导的各种表型在肿瘤缺氧微环境。这些表型表现了细胞膜葡萄糖转运体的增加的表示和糖酵解的率 (华宝效应)13。其结果是从线粒体氧化磷酸化转变为乳酸发酵。
缺氧生存也可以利用替代葡萄糖来支持生存现象14,15。最好的研究哺乳动物的例子是鼹鼠, 它可以生存近20分钟没有氧气通过果糖驱动的酵发酵途径14。另一种适应发生在某些鱼类 (如鲤鱼 (鲫鱼 ), 它可以存活明显较长的时间, 使用糖酵解与乙醇作为终端副产品)15。在这两种情况下, 发酵驱动新陈代谢使生存在缺乏氧气。目前的缺氧生存假说是, 只要HIF1在缺氧期间激活, 线粒体呼吸, 不需要氧气, 发生在厌氧条件下16。此外, 据推测, 使用发酵途径的缺氧/缺氧生存, 提高肿瘤生存, 因为细胞避免氧化应激, 可能证明是有害的细胞生存17。最近的一项研究表明, 在心肌细胞中, 缺氧会降低肿瘤细胞17的氧化应激, 这一假说得到了证实。
到目前为止, 缺氧哺乳动物细胞生存的发酵通路的本质是在文献中根深蒂固的, 这在很大程度上是由于无法在完全没有氧气的情况下培养哺乳动物细胞3天以上。然而, 在细菌中, 厌氧生存的一种替代糖酵解。在某些细菌中, 氮或硫酸盐 (在其他化合物中) 可以作为细胞色素氧化酶系统的终端电子受体, 在没有氧18的情况下。虽然细菌和真核进化发生在平行的, 因为从最后一个普遍的共同祖先, 估计线粒体是提供能量的细胞在154.2万年前的氧合的海洋19.由于研究人员已经表明, 孤立的线粒体可以在没有氧气的情况下产生 ATP, 以亚硝酸盐作为终端电子受体, 假设细胞在没有氧气的情况下可以在3天的时间内正常工作。20,21,22,23. 本研究所述的方法对许多细胞系的厌氧哺乳动物细胞生长有效用。
Protocol
Representative Results
Discussion
这种方法代表第一次哺乳动物细胞, 在没有氧气的情况下长时间培养。根据目前的观察,通过非发酵途径的缺氧生长能力似乎是普遍的哺乳动物细胞28, 其中厌氧生长导致表型发散。这是观察到所有细胞系测试。以厌氧耕种, 细胞的增加的比例变得四舍五入, 开发了悬浮象人口, 并且能复制。次级细胞类型仍然附着在基板上;然而, 细胞形态学恢复到主轴 (树突状) 形状。这些发?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢中西部大学的研究和赞助项目的支持。
Materials
| Whitney A35 anaerobic chamber | Don Whitley Scientific | Microbiology International | The ability to remove the front of the chamber makes it easy to put instruments inside without concern about getting them through the portals. |
| CO2 incubator | Fisher Scientific | 3531 | |
| Tissue Culture Hood | Labconco | DO55735 | |
| pipet aid | Drummond | 4-000-100 | |
| sterile transfer pipets | Santa Cruz | sc-358867 | |
| 50cc sterile conical centrifuge tubes | DOT | 451-PG | |
| vaccum jar | Nalgene 111410862889 | BTA-Mall 5311-0250 | |
| DMEM high glucose (4.5 g/L) | CellGro | 10-017-CM | |
| DMEM low glucose (1 g/L) | CellGro | 10-014-CV | |
| FBS | VWR | 1500-500 | |
| HBSS | VWR | 20-021-CV | |
| trypsin | VWR | 25-052-CI | |
| gentamicin | Gibco | 15750-060 | |
| sterile pipets | CellTreat | 229210B, 229205B | |
| Tissue culture flask (T75 or T150) | Santa Cruz | sc-200263, sc-200264 | |
| 24 well tissue culture treated dishes | DOT | 667124 | |
| glad ziplock sandwich bags | Ziploc | Costco | |
| inverted phase microscope (10, 20 40x objectives with camera mont) | Nikon Eclipse | TS100 | |
| trypan blue | Invitrogen | T10282 | |
| hemocytometer | Invitrogen | C10283 | |
| Countess Automated Cell Counter | Life Technologies | AMQAF1000 | |
| Rainin microtiter pipets | Mettler Toledo | Rainin Classic Pipette PR-100 | |
| microtiter tips | Santa Cruz Biotechnology | sc-213233 & sc-201717 | |
| test tube rack (50cc tubes) | The Lab Depot | HS29050A | |
| sodium nitrite | Sigma | https://www.sigmaaldrich.com/catalog/product/sigald/237213?lang=en®ion=US | |
| clear boxes with lids | Rosti Mepal | Rubber maid | |
| paper towels | In House | ||
| cell scraper | CellTreat | 229310 | |
| PBS | In house prepared | ||
| 70% Ethanol | Fisher Scientific | 64-17-5 | |
| microcentrifuge tubes sterile | Santa Cruz | sc-200273 | |
| biohazard bags with holder (desktop) | Heathrow Scientific | HS10320 | |
| Nitrile Gloves | VWR | 89428 | |
| oxygen electrode | eDAQ | EPO354 | |
| pH meter | Jenway | 3510 | |
| pH paper/ pHydrion | Sigma Aldich | Z111813 |
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