1。主机细胞转染<ol><li>种子2 × 10 6 CHO - DXB11细胞在T75康宁的细胞培养瓶15毫升生长培养基（MEMA核苷酸和核苷（GIBCO，目录编号为12571），辅以10％的γ-辐照特点胎牛血清（cFBS）（Hyclone公司，目录编号SH30071.03）和10毫升/大号45％葡萄糖溶液（Sigma公司，目录号G8769））转染前一天。 </li><li>在转染时，准备转染复合物如下: <ol class="lalpha"><li>在800μL培养液稀释，在无菌的离心管血清8微克的DNA。轻轻混匀。 </li><li>无血清中的聚苯乙烯管，800μL培养液稀释40μL脂质体™（Invitrogen公司18324012）。 </li><li>将稀释后的DNA稀释的脂质体™。轻轻混匀，30分钟，在室温下孵育。 </li></ol></li><li>删除从细胞的培养基中，在T75和无血清6.4毫升培养液取代。加入转染复合物1.6毫升的容量瓶中。摇板，轻轻混匀。 </li><li> 6个小时，在CO 2培养箱孵育的细胞在37 ° C。 </li><li>加入8毫升培养液的烧瓶中，在37 °的CO 2培养箱中过夜彗星。 </li><li>取出吸液并添加新鲜生长培养基的烧瓶中。的CO 2培养箱中过夜培养的细胞在37 ° C。 </li><li>选择培养基更换生长培养基（MEMA介质无核苷酸或核苷（GIBCO，目录编号为12561），辅以10％（DFB）的γ辐照透析胎牛血清（Hyclone公司，产品目录号SH30079.03）和10 mL / L的45 ％葡萄糖溶液（Sigma公司，目录号G8769））。 </li><li>在37 ° C的细胞中的CO 2培养箱培养2-3周。 </li></ol> 2。流式细胞仪检测激活细胞分选的克隆<ol><li>撞出5分钟从烧瓶中，在800转的离心机细胞在4 ° C。 </li><li>辅以2％牛血清白蛋白，荧光标记的抗人IgG抗体在1:20稀释的选择培养基重悬细胞。 </li><li>孵育冰浴15-30分钟，然后用冷PBS洗两次。在1X PBS重悬在聚丙烯管细胞。 </li><li>准备在BD FACSAria™无菌排序。准备96孔细胞培养板，包含在每孔200μL选择培养基。 </li><li>无菌负载管与细胞和BD FACSAria™分析，记录的结果，分别染色和未染色细胞。 </li><li>设置的大门，设置分类到96孔板（S）的单细胞所需的荧光染色轮廓。 <ol class="lalpha"><li>转染细胞中首先分析了基于前向散射对侧向散射。一个活细胞的大门，是基于前向散射，测量细胞的大小，侧向散射，是衡量一个细胞的复杂性或粒度。 </li><li>属于这扇大门内的细胞，然后用于PE染色检查。 PE负栅极设置为不染转染的细胞或染的宿主细胞。 "高PE"门染色流式细胞仪检测转染细胞，然后划定包括所有用于PE染色阳性的细胞，前5％。 </li></ol></li><li>两个星期，在37 °彗星孵化器孵育的板块。 </li></ol> 3。集落形成的文件CloneSelect™成像仪 0天，第3天，7天，排序后流式细胞仪第13天CloneSelect成像™96孔板的文件集落形成。图像文件将确保，从一个单细胞产生的克隆选择。在本文档中的一个关键的步骤是让焦平面正确调整为0天测量，因为在这一点上只有一个细胞在每口井。这是非常重要的，这些单个细胞的图像的重点和试图调整对他们的重点，充其量可能会产生误导。焦平面可以显著不同板块之间的品牌，甚至可能从很多很多同一品牌。因此需要使用预先确定的焦平面。这可以通过重点测试微珠或存入板所使用的同一地段的高密度细胞。另外，同一地段，完全成长板可用于这一目的。流式细胞仪进行排序后，它也是重要的是让细胞成像前至少2小时定居，让他们将被"固定"在上板的位置。 4。高生产力的克隆筛选和选择<ol><li>在接收的96孔检测板的文件样本的位置。 </li><li> 20μL上清（如果有必要稀释）到96孔检测板TECAN自由EVO ®液体处理系统的传输等份。这种灵活的和有能力的机器人IC设计平台已经适应和其他自动细胞培养工作。特别节目的书面最好地利用和增强细胞系开发的机器人功能的Freedom EVO。这样的一个例子是设置TECAN公司解释CloneSelect™热像仪的数据采样与单菌落井。其他节目都必须执行额外的细胞，如文化的任务，使所采取的样品适当稀释，解释，目录和手动标记的样品板，如果需要，扩大规模生产克隆高，等这些方案中有许多可以很容易地适用于384孔板以及原来的96孔格式。 </li><li>分析样品中的抗体生产三明治人类检测IgG抗体酶联免疫吸附（皮尔斯，罗克福德，IL）TECAN自由EVO ®液体处理系统。 <ol><li>样品和标准（人类IgG1的多发性骨髓瘤，KAPPA）（Sigma公司，圣路易斯，密苏里州）预涂层的生物涂层的抗人IgG含量的板（BD公司，加利福尼亚州圣何塞，）加载。 </li><li>室温孵育两个小时，三个PBS洗涤板。 </li><li> ImmunoPure山羊抗人IgG（FC -γ）- HRP（皮尔斯，罗克福德，IL）被添加到该板块在1:2000稀释，并在室温下1小时孵育。 </li><li>板，用PBS洗三次之前加入ABTS底物（皮尔斯，罗克福德，IL），在盘子上使用的检测在405 nm处的吸光度读者读。 </li></ol></li><li>选择高生产力的克隆和评估中的悬浮培养的生产力。补料分批培养，细胞接种0.3X106细胞/ mL蛋白的培养基JRH直接的好处中，65578（SAFC Biosciences公司，列涅萨，KS）与5 g / L的大豆蛋白水解物（DMV国际，目录号SE50MAF - UF，很多补充7.5％的CO 2号）和孵育在37 ° C。文化的继续和补充为2 g / L葡萄糖（Sigma公司，目录编号为G8769，批号098K2329）当葡萄糖加文化中的乳酸浓度低于2 g / L的达成，以及美联储2毫米谷氨酰胺​​（Gibco公司，目录编号25030，批号568177），谷氨酰胺水平时达到低于200毫克/升（YSI生物分析仪测得的代谢产物）。补料分批培养第14天终止和抗体的产生反相高效液相色谱法测定。 </li><li>荧光原位杂交（FISH）5候选克隆的遗传特性。 </li></ol> 5。代表性的成果: 例如开发抗体生产克隆与高通量方法是在图1和图2所示。转染细胞池染色与荧光标记的抗人IgG抗体（图1A），分析和BD FACSAria™（图1B）排序。菌落形成在96孔板上成像CloneSelect成像™（图1C）。细胞培养上清中包含单个菌落从井采样，并用ELISA法测定TECAN自由EVO ®液体处理系统（图1D）。高生产率的克隆细胞表现出较高的表面抗体水平与荧光标记的抗人IgG抗体染色反映。当比较两个种群的初始转染细胞池，在生产力的巨大差异在补料分批，文化观察（图2）产生的克隆。从表面抗体高表达的细胞（PE高）人口产生的前两名克隆，具体生产力介于50-70皮克/细胞/天。而抗体生产力〜20皮克/细胞/天的前两名从低表面抗体的表达（PE低）的细胞群产生的克隆。此外，通过流式细胞仪排序的细胞株更稳定，比那些通过人工黎明稀释（图3A）克隆抗体生产。顶级克隆特定的生产力，"克隆"，由手动黎明稀释生成的下降〜30〜10培养80细胞倍增后PCD PCD。另一方面，顶部的亚克隆进行排序，"克隆2"流式细胞仪产生的，是能够保持至少100个细胞倍增约20 PCD的具体生产力。荧光原位杂交（FISH），我们证明克隆1的表型的混合人口显示一个（85％）和表型B（15％）。相比之下，流式细胞仪排序克隆（克隆2）显示，99.5％的细胞要更均匀的人口，被证明是表型（图3B）。 <img src="/files/ftp_upload/3010/3010fig1.jpg" alt="图1" /> 图1。CHO细胞生产单克隆抗体制造细胞系的发展。一）初始转染细胞池的表面染色，用荧光标记的抗人IgG抗体。二）到96孔板细胞具有较高的荧光强度（PE高）和荧光强度低（低PE）的人口排序。三）记录殖民地从第一天开始形成0至13天CloneSelect成像™后播种。四）通过ELISA筛选克隆。 <img src="/files/ftp_upload/3010/3010fig2.jpg" alt="图2" /> 图2表面抗体水平相关抗体细胞的生产力。高和低的荧光染色的克隆均发展成细胞株和补料分批培养进行评估。体积滴度和前2每个种群的克隆之间进行了比较具体的生产力。 <img src="/files/ftp_upload/3010/3010fig3.jpg" alt="图3" /> 图3。流式细胞仪排序表现出较高的遗传的同质性和克隆稳定性产生的克隆。一）抗体在分批培养生产的稳定〜80文化经过时间的细胞倍增。 Δ，克隆通过有限稀释，细胞接种在2000细胞/孔，1000个细胞/孔和500个细胞/孔产生。 <img src="/files/ftp_upload/3010/3010triangle.jpg" alt="黑三角" /> ，克隆是通过1 /细胞流式细胞仪生成。二）转基因整合位点在CHO细胞染色体荧光原位杂交鉴定。

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	<li>Hung, F., Deng, L., Ravnikar, P., Condon, R., Li, B., Do, L., Saha, D., Tsao, Y. S., Merchant, A., Liu, Z., Shi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+stability+and+antibody+production+in+CHO+cells:+improvement+through+gene+optimization.">mRNA stability and antibody production in CHO cells: improvement through gene optimization.</a> Biotechnol. J. 5, 393-401 (2010).</li><li>Brezinsky, S. C., Chiang, G. G., Szilvasi, A., Mohan, S., Shapiro, R. I., MacLean, A., Sisk, W., Thill, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+enriching+populations+of+transfected+CHO+cells+for+cells+of+higher+specific+productivity.">A simple method for enriching populations of transfected CHO cells for cells of higher specific productivity.</a> J. Immunol. Methods. 277, 141-155 (2003).</li><li>Kromenaker, S. J., Srienc, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+producer+hybridoma+cell+lines+after+cell+sorting:+a+case+study.">Stability of producer hybridoma cell lines after cell sorting: a case study.</a> Biotechnol. Prog. 10, 299-307 (1994).</li><li>Marder, P., Maciak, R. S., Fouts, R. L., Baker, R. S., Starling, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+cloning+of+hybridoma+cells+for+enhanced+immunoglobulin+production+using+flow+cytometric+cell+sorting+and+automated+laser+nephelometry.">Selective cloning of hybridoma cells for enhanced immunoglobulin production using flow cytometric cell sorting and automated laser nephelometry.</a> Cytometry. 11, 498-505 (1990).</li><li>Henegariu, O., Heerema, N., Wright, L. L., Bray-Ward, P., Ward, D. C., Vance, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvements+in+cytogenetic+slide+preparation:+controlled+chromosome+spreading+chemical+aging+and+gradual+denaturing.">Improvements in cytogenetic slide preparation: controlled chromosome spreading chemical aging and gradual denaturing.</a> Cytomery. 43, 101-109 (2001).</li><li>Meng, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grenn+fluorescent+protein+(GFP)+as+a+second+selectable+marker+for+selection+of+high+producing+clones+from+transfected+CHO+cells.">Grenn fluorescent protein (GFP) as a second selectable marker for selection of high producing clones from transfected CHO cells.</a> Gene. 242, 201-207 (2000).</li><li>DeMaria, C. T., Cairns, V., Schwarz, C., Zhang, J., Guerin, M., Zuena, E., Estes, S., Karey, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+clone+selection+for+recombinant+CHO+cells+using+a+FACS-based+high-throughput+screen.">Accelerated clone selection for recombinant CHO cells using a FACS-based high-throughput screen.</a> Biotechnol. Prog. 23, 465-472 (2007).</li><li>Weaver, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel+microdrop+technology+for+rapid+isolation+of+rare+an+high+producer+cells.">Gel microdrop technology for rapid isolation of rare an high producer cells.</a> Nat. Med. 3, 583-585 (1997).</li></ol>

这篇文章的作者是默克公司，公司的员工，使用这种方法大规模生产哺乳动物细胞株的蛋白质疗法。

我们提出了一个自动化的制造单元线的开发平台，使生产力最旺盛的克隆的隔离，并在平均同时确保克隆和克隆稳定。接触表面抗体染色FACSAria™细胞分选的是获得单克隆抗体生产力的关键。我们的研究结果报告和其他报告2-4表明，瞬时显示细胞表面的抗体水平与细胞的生产力。因此，荧光标记抗体可以用来识别膜相关的产品和援助高生产者的孤立。此外，报告基因的共表达和凝胶microdrop技术已被用于促进流式细胞仪 6-8高产无性系的选择。除了高产无性系通过流式细胞仪分离，其它技术已演变为实现同一目标。这些措施包括格子的生物科学CellSpot技术，使用microspere抗体检测系统和激光筛选技术，以确定高生产力的克隆和消除不必要的细胞，Genetix ClonePix FL和干细胞技术ClonaCell EasyPick平台的细胞，用荧光抗体检测系统的生产线进行高通量筛选。我们选择实施基于表面相关抗体水平的流式细胞仪克隆方法，因为它结合了易于操作，对生产力和克隆选择性。 TECAN自由EVO ®液体处理系统的就业，显著提高了筛选的吞吐量，它提供从一个更大的细胞池的高产无性系选择的机会。其他如HTRF，BioForte字节，高效液相色谱仪等，检测仪器，是选择高生产者和筛选出低生产者一切可能的手段。通过跟踪与CloneSelect成像™克隆形成，我们产生必要的文件，数字化细胞系的历史证明克隆。克隆是进一步证实了单基因在染色体上的整合位点的基因特征。一个亚克隆步骤，这是通常采取以确保克隆，可能会因此被淘汰。细胞系的发展，这将节省4-8周的时间。综上所述，高通量自动化平台的质量细胞产生的线表现出较高的生产力，生产稳定和记录细胞系的历史，应满足大规模生产的需求和目前的监管要求。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td></tr><tr><td> BD流式细胞仪咏叹调™细胞分选</td><td> BD生物科学</td></tr><tr><td> CloneSelect™成像仪</td><td> Genetix </td></tr><tr><td> TECAN自由EVO ®液体处理系统</td><td> TECAN集团</td></tr></tbody></table>

a high-throughput automated platform for the development of manufacturing cell lines for protein therapeutics

快速增长的生物制药行业的迅速发展要求的高效，可靠的生产系统，药品供应，以满足不断增长的需求。生产细胞株的产生历来涉及手工操作，是劳动密集型，低吞吐量和易受人为错误。我们在这里报告为发展生产蛋白质药物的生产细胞系的综合的高通​​量和自动化平台。 BD流式细胞仪咏叹调的组合™细胞分选，CloneSelect™热像仪和TECAN自由EVO ®液体处理系统，使一个高吞吐量和更有效的细胞系的发展过程。在这次行动中，生产的宿主细胞转染与表达，治疗选择代理的利益1基因的载体。稳定转染的细胞，然后用荧光标记的抗人IgG抗体染色，流式细胞仪分析 2-4，其后受。根据荧光强度和高生产率的细胞是由孤立的单细胞在BD FACSAria™分拣。从单细胞阶段的殖民地形成显微镜检测和一系列时间圈的数字图像CloneSelect™成像细胞系的历史文件。后已经形成了单一的克隆，这些克隆进行了筛选，生产力ELISA法上TECAN自由EVO ®液体处理系统。约2,000 - 10,000克隆可以筛选每个运行周期与当前的系统设置。 这种集成方法已被用来产生高产的中国仓鼠卵巢（CHO）细胞为治疗的单克隆抗体（mAb）以及他们的融合蛋白的生产线。随着不同类型的检测探头的援助，该方法可用于开发其他的蛋白质疗法，或应用到其他生产主机系统。比较传统的手工程序，这个自动化的平台，表现出显着增加的能力，确保克隆，细胞行历史与电子文档的可追溯性，大大减少操作员错误的机会的优势。

Watch this Scientific Journal Video about A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics at JoVE.com

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

描述的是一种高通量，自动化平台生产蛋白质药物的制造细胞系的发展。 BD流式细胞仪咏叹调细胞分选的实施，CloneSelect成像和TECAN自由EVO液体处理系统已显示出显着增加细胞系的发展与提高细胞的质量和高重现性的加工能力。

一种高通量自动化的平台，制造蛋白质治疗的细胞系发展

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

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22.2K 次观看.  Merck & Co., Inc. 描述的是一种高通量，自动化平台生产蛋白质药物的制造细胞系的发展。 BD流式细胞仪咏叹调细胞分选的实施，CloneSelect成像和TECAN自由EVO液体处理系统已显示出显着增加细胞系的发展与提高细胞的质量和高重现性的加工能力。

视频: A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

老鼠的眼睛剜除术的远程高通量的表型

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

科研

医学

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

的的膀胱测压小老鼠:A膀胱Chemosensation研究的使用

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

清醒小鼠滴鼻的CNS药物治疗管理

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

原发性肿瘤和/或转移相关的临床前和治疗的临床开发的离体处理响应

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

对专为递送治疗因素的神经保护策略成人干细胞高通量表征

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

从肌肉活检患者来源的细胞系疾病建模隔离和永生化

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...