Name: anaerobic growth and maintenance of mammalian cell lines
Uploaded: 2018-07-21T15:00:00.000+00:00
Duration: 7 min 15 s
Description: Watch this Scientific Journal Video about Anaerobic Growth and Maintenance of Mammalian Cell Lines at JoVE.com

作者感谢中西部大学的研究和赞助项目的支持。

1. 制备各种哺乳动物细胞系厌氧培养培养基
<ol><li>
为缺氧细胞培养制作完整的 PS-74656 培养基25。
	<ol>
<li>在50毫升蒸馏去离子水中溶解17.25 克亚硝酸根, 使无菌亚硝酸盐库存溶液 (5 米; 100x), 然后过滤消毒。</li>
		<li>添加0.5 毫升的亚硝酸盐库存溶液每50毫升低葡萄糖 DMEM (1 克/升葡萄糖) 与110毫克/升的 l-谷氨酰胺, 584 毫克/升的丙酮酸钠, 10% 胎牛血清 (血清; 热灭活)。 注: 使用血清浓度大于10% 是有毒的。值得注意的是, 一条癌细胞线对亚硝酸盐 (MDA-MB-231, 乳腺癌细胞系) 的培养基耐受性低, 因此在没有亚硝酸盐的情况下生长。此外, 虽然 PS-74656 培养基支持所有经测试的细胞系的生长 (n = 9), 某些细胞系 (例如, 细胞) 表现出较高的细胞浓度和较高的生存能力 PS-74656 高葡萄糖 (4.5 克/升) DMEM25。</li>
		<li>在无菌50毫升圆锥离心管中放置20毫升培养基。不要拧紧盖子;它应该是松散的。</li>
		<li>将管子放在真空钟罐中, 用近似45°角将其脱气, 使介质表面积最大化。</li>
		<li>在室温下使用真空泵或其等效的真空。保持真空, 直到气泡不再被观察 (24–48 h)。</li>
		<li>把管子从罐子里取下, 然后立即关上瓶盖, 封好管子, 尽量减少空气进入介质中的引入。</li>
		<li>把管子放在商业厌氧室里。</li>
		<li>松开管帽, 允许管内的空气与燃烧室中的厌氧气体混合物平衡至少24小时。 注: 介质应留在会议厅内, 直至用完为止。为加快工艺速度, 用缺氧气体混合液将管至少3x 冲洗, 用无菌转移吸管填充缺氧气, 由 H2、CO2和 N2 组成.
</li>
		<li>将所有管 (10–15分钟) 放置在厌氧腔内, 如上文所述, 在密封管和放置在室内。使用前, 使用1.5–2毫升转移吸管或吸管提示, 冲洗它与厌氧气体混合物, 已冲洗的缺氧气体至少3x。</li>
		<li>在厌氧室中, 将介质中的100µL 移除无菌脱气微离心管, 并在腔内使用氧气探针对溶解氧进行测试。 注: 氧电极在使用前按制造商的协议进行校准。正确脱气和平衡厌氧气体混合介质的读数为0。 注意: 如果读数超过 0.3 ppm 的氧气, 继续在厌氧室孵化介质, 因为有更多的时间平衡培养基是必需的。</li>
	</ol>
</li>
</ol>2. 各种哺乳动物细胞系的缺氧培养
<ol><li>
指定的天-1
	<ol>
<li>生长细胞 (37 °c, 5% CO2) 在 T150 瓶到90% 汇合为缺氧和常氧文化控制提供足够数量的细胞为三 24-井板材 (80% 汇合)。 注意: 对于这项研究, 使用了 HeLa 229 和维罗细胞。该议定书也经过测试, 并证明成功的增长和维护七其他细胞线25。</li>
		<li>从烧瓶中取出培养基, 吸入10毫升的 HBSS。</li>
		<li>用5毫升的胰蛋白酶取代培养基。搅动烧瓶, 直到细胞从塑料中分离出来。吸入大部分的胰蛋白酶, 然后轻敲烧瓶以去除黏附细胞。添加10–15毫升的高葡萄糖 DMEM (4.5 克/升葡萄糖, 110 毫克 l-谷氨酰胺, 10% 血清, 50 µg/毫升的庆大霉素), 以灭活胰蛋白酶。</li>
		<li>Triturate 细胞使用25毫升的吸管, 直到所有细胞团簇被破坏。</li>
		<li>用标准 hemocytometer 和台盼蓝染料排除法或自动当量测定细胞数并确定其可行性。</li>
		<li>悬浮在高葡萄糖 DMEM 的细胞, 如上所述, 细胞密度为 2.24 x 105细胞/毫升。</li>
		<li>将1毫升的细胞悬浮液添加到24井组织培养板 (2.24 x 105细胞/井; 80% 汇流) 井中。种子1板为缺氧培养和另一个控制常氧文化。</li>
		<li>孵化细胞在37°c 在常氧条件 (5% CO2) 为 24 h。 注: 这些细胞可以在各种大小的板材中电镀。然而, 种子细胞为缺氧生长的烧瓶, 有风险, 引入氧气进入系统, 除非采取谨慎, 以彻底冲洗出大气气体。</li>
	</ol>
</li>
	<li>
指定的天0
	<ol>
<li>常氧孵化24小时后, 将板块用于缺氧生长进入厌氧室 (商业厌氧室;图 1)。</li>
		<li>立即将培养基改为无抗生素的脱气 PS-74656 培养基, 并将其驯化为24小时至厌氧室。将盘子放在一个带有湿巾的容器里, 并松散地关闭以保持湿气。 注: 常氧生长板的处理方式相同, 例外的是所有的治疗都是在大气条件下进行的。</li>
	</ol>
</li>
	<li>
指定的天1
	<ol>
<li>1天的厌氧孵化开始后24小时的孵化在商业厌氧室, 其中包含标准的厌氧气体混合物10% 小时2, 10% CO2, 80% N2。</li>
		<li>在不同的时间段孵化细胞。</li>
		<li>监测厌氧介质的颜色变化随着时间的推移。 注意: 从红橙到洋红色的颜色转换信号的时间改变媒介。这可能需要长达2周的时间才能发生。从红色橙色到黄色的培养基中的颜色变化表明存在细菌污染。</li>
		<li>当介质从红色变为洋红时, 通过吸移去除 runagate 细胞。</li>
		<li>将吸气用过的介质放在脱气和厌氧气体平衡离心管中的 runagate 细胞中, 关闭管, 同时确保密封安全, 然后将其离心 (326 x克; 室温为10分钟)。立即用1毫升新的脱气 PS-74656 培养基替换井中的吸气介质。</li>
		<li>在打开它之前, 将离心管放在厌氧室中的颗粒细胞。吸入所用的培养基, 并用新鲜的脱气 PS-74656 培养基替换离心管中的总容积1毫升。</li>
		<li>将细胞从管中返回到24井组织培养板中的井中。</li>
	</ol>
</li>
</ol>3. 厌氧培养细胞的显微观察对表型细胞分化的评价
<ol><li>
在厌氧室中, 将盘子放在一个密封盒中, 用厌氧气体混合物冲洗, 然后关闭盒子。 注: 盒内必须装有湿纸巾, 以提供防潮, 防止板材干燥。对于显微镜, 三明治袋的工作最好, 因为他们是薄的。<ol>
<li>观察显微镜下的细胞, 完全密封袋并将其从腔内取出。</li>
		<li>仔细拉伸塑料袋在盘子上, 检查细胞使用倒置相对比显微镜与相机附件, 并采取显微照片在各种放大。</li>
		<li>将密封袋中的盘子退回到房间, 并按照步骤2.3.3 中的描述继续孵化。</li>
		<li>要单独分析处于悬浮中的 runagate 细胞, 请按照步骤2.3.5 中概述的步骤进行操作。 注: 这些细胞可以用来进行有氧测试, 也可以被送回厌氧室, 用于测试这个特定的人群。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

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?...

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。

<table><tbody><tr><td>Whitney A35 anaerobic chamber</td><td>Don Whitley Scientific&amp;nbsp;</td><td>Microbiology International</td><td>The ability to remove the front of the chamber makes it easy to put instruments inside without concern about getting them through the portals.</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Fisher Scientific</td><td>3531</td><td></td></tr><tr><td>Tissue Culture Hood</td><td>Labconco</td><td>DO55735</td><td></td></tr><tr><td>pipet aid</td><td>Drummond</td><td>4-000-100</td><td></td></tr><tr><td>sterile transfer pipets</td><td>Santa Cruz</td><td>sc-358867</td><td></td></tr><tr><td>50 cc sterile conical centrifuge tubes</td><td>DOT</td><td>451-PG</td><td></td></tr><tr><td>vaccum jar</td><td>Nalgene 111410862889</td><td>BTA-Mall 5311-0250</td><td></td></tr><tr><td>DMEM high glucose (4.5 g/L)</td><td>CellGro</td><td>10-017-CM</td><td></td></tr><tr><td>DMEM low glucose (1 g/L)</td><td>CellGro</td><td>10-014-CV</td><td></td></tr><tr><td>FBS</td><td>VWR</td><td>1500-500</td><td></td></tr><tr><td>HBSS</td><td>VWR</td><td>20-021-CV</td><td></td></tr><tr><td>trypsin</td><td>VWR</td><td>25-052-CI</td><td></td></tr><tr><td>gentamicin</td><td>Gibco</td><td>15750-060</td><td></td></tr><tr><td>sterile pipets</td><td>CellTreat</td><td>229210B, 229205B</td><td></td></tr><tr><td>Tissue culture flask (T75 or T150)</td><td>Santa Cruz</td><td>sc-200263, sc-200264</td><td></td></tr><tr><td>24 well tissue culture treated dishes</td><td>DOT</td><td>667124</td><td></td></tr><tr><td>glad ziplock sandwich bags</td><td>Ziploc</td><td>Costco</td><td></td></tr><tr><td>inverted phase microscope (10, 20 40x objectives with camera mont)</td><td>Nikon Eclipse</td><td>TS100</td><td></td></tr><tr><td>trypan blue</td><td>Invitrogen</td><td>T10282</td><td></td></tr><tr><td>hemocytometer</td><td>Invitrogen</td><td>C10283</td><td></td></tr><tr><td>Countess Automated Cell Counter</td><td>Life Technologies</td><td>AMQAF1000</td><td></td></tr><tr><td>Rainin microtiter pipets</td><td>Mettler Toledo</td><td>Rainin Classic Pipette PR-100</td><td></td></tr><tr><td>microtiter tips</td><td>Santa Cruz Biotechnology</td><td>sc-213233 &amp;amp; sc-201717&amp;nbsp;</td><td></td></tr><tr><td>test tube rack (50cc tubes)</td><td>The Lab Depot</td><td>HS29050A</td><td></td></tr><tr><td>sodium nitrite</td><td>Sigma</td><td>https://www.sigmaaldrich.com/catalog/product/sigald/237213?lang=en&amp;amp;region=US</td><td></td></tr><tr><td>clear boxes with lids</td><td>Rosti Mepal</td><td>Rubber maid</td><td></td></tr><tr><td>paper towels</td><td>In House</td><td></td><td></td></tr><tr><td>cell scraper</td><td>CellTreat</td><td>229310</td><td></td></tr><tr><td>PBS</td><td>In house prepared</td><td></td><td></td></tr><tr><td>70% Ethanol</td><td>Fisher Scientific</td><td>64-17-5</td><td></td></tr><tr><td>microcentrifuge tubes sterile</td><td>Santa Cruz</td><td>sc-200273</td><td></td></tr><tr><td>biohazard bags with holder (desktop)</td><td>Heathrow Scientific</td><td>HS10320</td><td></td></tr><tr><td>Nitrile Gloves</td><td>VWR</td><td>89428</td><td></td></tr><tr><td>oxygen electrode</td><td>eDAQ</td><td>EPO354</td><td></td></tr><tr><td>pH meter</td><td>Jenway</td><td>3510</td><td></td></tr><tr><td>pH paper/ pHydrion</td><td>Sigma Aldich</td><td>Z111813</td><td></td></tr></tbody></table>

anaerobic growth and maintenance of mammalian cell lines

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。

这个协议的力量在于它支持的长寿和多细胞系的生长, 并承认有深刻的变化和分化的细胞形态学25。这项研究最关键的因素是在严格的 anaerobiosis 下细胞的转移和维持。这就需要组织一个厌氧室来最大化该协议 (图 1), 并保证在密封的环境中保存用于显微镜或其他测试的细胞。到目前为止, 缺氧生存的范围是15–17天为不朽的癌细胞系和...

Watch this Scientific Journal Video about Anaerobic Growth and Maintenance of Mammalian Cell Lines at JoVE.com

Anaerobic Growth and Maintenance of Mammalian Cell Lines

哺乳动物细胞系的厌氧生长与维持

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。

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...

anaerobic-growth-and-maintenance-of-mammalian-cell-lines

Research

JoVE Journal

Biology

10.0K 次观看.  Midwestern University. 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。

视频: Anaerobic Growth and Maintenance of Mammalian Cell Lines

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

一个<em在体外系统来研究肿瘤休眠和转移性增长

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

科研

医学

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

FIBS功能的无创性代谢轮廓

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

生物工程

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

原子吸纳光谱测量哺乳动物细胞细胞内锌池

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

生物化学

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

培养哺乳动物细胞使用一次性使用气动生物反应器系统

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

在球体哺乳动物细胞大规模扩张营养调控连续通

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These insects are the predominant vectors of Xylella fastidiosa (X. fastidiosa), a xylem-limited bacterium that is the causal agent of Pierce&#39;s disease (PD) of grapevine. Pierce&#8217;s disease is economically damaging; thus, H. vitripennis have become a target for pathogen management strategies. A dicistrovirus identified as Homalodisca coagulata virus-01 (HoCV-01) has been associated with an increased mortality in H. vitripennis populations. Because a host cell is required for HoCV-01 replication, cell culture provides a uniform environment for targeted replication that is logistically and economically valuable for biopesticide production. In this study, a system for large-scale propagation of H. vitripennis cells via tissue culture was developed, providing a viral replication mechanism. HoCV-01 was extracted from whole body insects and used to inoculate cultured H. vitripennis cells at varying levels. The culture medium was removed every 24 hr for 168 hr, RNA extracted and analyzed with qRT-PCR. Cells were stained with trypan blue and counted to quantify cell survivability using light microscopy. Whole virus particles were extracted up to 96 hr after infection, which was the time point determined to be before total cell culture collapse occurred. Cells were also subjected to fluorescent staining and viewed using confocal microscopy to investigate viral activity on F-actin attachment and nuclei integrity. The conclusion of this study is that H. vitripennis cells are capable of being cultured and used for mass production of HoCV-01 at a suitable level to allow production of a biopesticide.

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

Here we present a protocol to propagate Homalodisca vitripennis cells and HoCV-1 in vitro. Medium was removed from HoCV-1 positive cultures and RNA extracted every 24 hr for 168 hr. Cell survivability was quantified by trypan blue staining. Whole virus particles were extracted post-infection. Extracted RNA was quantified by qRT-PCR.

传播<em&gt; Homalodisca coagulata病毒01</em&gt;通过<em&gt; Homalodisca vitripennis</em&gt;细胞培养

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These ...

免疫与感染

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

结合表观遗传修饰和生物力学线索的两步策略，以产生哺乳动物多能细胞

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

生物学

采集小鼠骨骼肌以培养干细胞及其生态位

Unfractionated Bulk Culture of Mouse Skeletal Muscle to Recapitulate Niche and Stem Cell Quiescence

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and recovery from injuries depend on a multitude of cell types and on molecular signals between the core muscle cells (myofibers, muscle stem cells) and their niche. Most experimental settings do not preserve this complex physiological microenvironment, and neither do they allow the ex vivo study of muscle stem cells in quiescence, a cell state that is crucial for them. Here, a protocol is outlined for the ex vivo culture of muscle stem cells with cellular components of their niche. Through the mechanical and enzymatic breakdown of muscles, a mixture of cell types is obtained, which is put in 2D culture. Immunostaining shows that within 1 week, multiple niche cells are present in culture alongside myofibers and, importantly, Pax7-positive cells that display the characteristics of quiescent muscle stem cells. These unique properties make this protocol a powerful tool for cell amplification and the generation of quiescent-like stem cells that can be used to address fundamental and translational questions.

作者聚焦:使用生态位组分培养静止肌肉干细胞的离体方案 

Skeletal muscle comprises multiple cell types, including resident stem cells, each with a special contribution to muscle homeostasis and regeneration. Here, the 2D culture of muscle stem cells and the muscle cell niche in an ex vivo setting that preserves many of the physiological, in vivo, and environmental characteristics are described.

小鼠骨骼肌的普通散装培养物，以概括生态位和干细胞静止

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and ...

哺乳动物细胞系的厌氧生长与维持

摘要

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一个系统来研究肿瘤休眠和转移性增长

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培养哺乳动物细胞使用一次性使用气动生物反应器系统

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传播

结合表观遗传修饰和生物力学线索的两步策略，以产生哺乳动物多能细胞

小鼠骨骼肌的普通散装培养物，以概括生态位和干细胞静止

小鼠骨骼肌的普通散装培养物，以概括生态位和干细胞静止

小鼠骨骼肌的普通散装培养物，以概括生态位和干细胞静止