来自国家癌症研究所通过奖号码U54CA143876微环境及转移工作的过程中得到了康奈尔大学的中心。内容完全是作者的责任，并不代表官方意见的国家癌症研究所或国家卫生研究院。额外资助的这项工作是由国家科学基金会研究生研究奖学金（ADH）的部分。

下面的协议是一个单一的微管设备的生产。 1。埃洛石纳米管溶液的制备<ol><li>超声粉碎（10-13 W（RMS））250μL，埃洛石纳米管溶液（6.6 wt％的水）30秒。用冷水重复超声一次冷静的解决方案。降温再次。 </li><li>绘制到一个注射器超声波处理解决方案，将用0.45μm注射器过滤器，过滤成干净的离心管的解决方案。涡的解决方案偶尔保持同质。 </li></ol> 2。埃洛石纳米管与微管内表面涂层<ol><li>获得50厘米长的路段（300微米内径35.3μL积）微Renathane microtubing的。在对角线切割微管的一端插入小艺达思适配器一块。 29G的3/10毫升注射器针头插入另一端的微管。 </li><li>要清理的微管，放置微管的开口端（结束适配器）到70％的乙醇和抽奖〜50μL注射器进入乙醇来填补微管。 </li><li>乙醇冲洗出来的微管抽入注射器大方量的蒸馏水。 </li><li>脱离微管的注射器，注射器清空，然后重新连接到微管。 </li><li>准备一个0.02％W / V聚L赖氨酸在水中的解决方案。成微管吸取50微升，并允许在室温（RT）5分钟坐。 </li><li>微管吸引到100μL的过滤碳纳米管的解决方案，允许3分钟在室温下孵育。 </li><li>冲洗出来的碳纳米管溶液通过微管吸取100μL水。允许涂层微管坐，装满水，在室温下过夜。 </li></ol> 3。微管细胞分离制备<ol><li>准备解决的10微克/毫升杜尔贝科1X的磷酸盐G蛋白缓冲液（PBS，pH值7.0 - 7.2）。通过微管绘制为50μLPBS和然后吸取50μL的蛋白-G的解决方案，并允许在室温下为1.5小时孵化。 </li><li>含5μg/ mL的E - 选择素IgG和50μg/ ml的抗体（抗-EpCAM的大多数癌症样本，抗PSMA的前列腺癌样本）在PBS准备的解决方案。吸引到了微管的E-选择素和抗体溶液50μL，并允许在室温下为2小时孵化。 </li><li>非特异性细胞粘附与5％的牛奶蛋白被封锁。准备一个5％（W / V）牛奶蛋白在PBS的解决方案。微管吸引到50μL牛奶蛋白解决方案，并允许在室温下为1小时孵化。 </li><li>绘制成管50μL的PBS及在室温下离开，直到血或白膜样品准备处理。 </li><li> 10分钟前使用隔离的微管，吸取50微升PBS已经饱和，以激活选择的分子与Ca 2 +（PBS +“）。 </li></ol> 4。样品制备，细胞分离<ol><li>绘制或索取10毫升的血液，从病人到肝素管。 </li><li>地方10毫升到50 mL离心管Ficoll分离 - Paque淋巴细胞分离的解决方案。轻轻层Ficoll分离顶部10毫升全血，以免混合血液和Ficoll。 </li><li> 2000XĞ离心15分钟在4°C间最小减速。 </li><li>除去白膜层和地点在新的管。 </li><li>用PBS洗白膜（230XĞ离心10分钟，弃上清）。 </li><li> 1毫升红细胞裂解液轻轻悬浮细胞孵育10分钟在室温下溶解红细胞。 </li><li>加入10毫升的PBS，轻轻混匀，离心10分钟230XĞ。弃上清，轻轻悬浮颗粒在3至4毫升PBS +。 </li></ol> 5。细胞分离<ol><li>附加功能化微管5毫升注射器，使用艺达思适配器的一端。 </li><li>插入到注射泵的注射器。 </li><li>淹没成细胞suspensio的官能微管的开口端N。 </li><li>通过在1至4 mL / h的微管处理的细胞悬液。 </li><li>转让的微管的开口端管PBS +和300μL，PBS +到注射器吸取0.016毫升/分钟，从松散的绑定和细胞的微管。 </li><li>放入一个干净的管的微管的开口端。从微管断开注射器和附加Accutase预填充注射器。轻轻地灌注到微管填补（〜50μL）足够的Accutase和允许在室温下孵育10分钟。 </li><li>将注射器1毫升培养基（79％，20％胎牛血清的RPMI 1％青霉素链霉素）和微管灌注预先填写，收集到组织文化的污水处理孔板。 </li><li>培养细胞在37℃，湿条件下CO 2 5％。 </li></ol> 6。代表结果这项技术的目标是可行的癌细胞，癌症患者的血液从隔离。几种方法存在的文化，以确定癌细胞的设备成功的必要的核查。我们选择了除了到DAPI染色与针对上皮基，如EpCAM的上皮细胞粘附分子（）或PSMA的前列腺特异性膜抗原（）的抗体细胞培养，以确定完整的细胞核（图2和3）。夺取癌细胞的数量，使用这种技术必然是一个CTCs的数量开始样品中的功能，病人可变性高。在处理IV期癌症患者确诊样本中，我们经常捕获100和500之间每管血癌细胞，纯度&gt; 50％。紧随隔离，污染白细胞更大的可能是存在的。然而，这些数字将被耗尽以下潜伏期长达5天，在培养基。 <img alt="图1" src="/files/ftp_upload/4248/4248fig1.jpg" /> <st荣&gt;图1示意图反恐显示选择素介导的滚动抗体介导的静态粘附的隔离，微管的功能。 <img alt="图2" src="/files/ftp_upload/4248/4248fig2.jpg" /> 图2。反恐隔离的代表从乳腺癌患者和肺癌患者的血液样本得出的数据。验明正身CTC EpCAM的染色的活细胞的数量是由坚实的酒吧和涉及到的左坐标和鉴定细胞的百分比表示，作为反恐相比，捕获的细胞总数的代表是由开放的酒吧和测量正确的坐标。结果培养5天。 <img alt="图3" src="/files/ftp_upload/4248/4248fig3.jpg" /> 从单独的捐助者（A和B）在文化CTC随后的5天到isolati代表显微图3。从一个癌症病人​​的血液样本。细胞荧光染色与AlexaFluor 488（绿色）和4&#39;,6-diamidino-2-苯基吲哚（DAPI）的可视化的核（蓝色）EpCAM的。

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	<li>Allard, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cells+circulate+in+the+peripheral+blood+of+all+major+carcinomas+but+not+in+healthy+subjects+or+patients+with+nonmalignant+diseases.">Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases.</a> Clin. Cancer Res. 10, 6897-6904 (2004).</li><li>Hughes, A. D., King, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanobiotechnology+for+the+capture+and+manipulation+of+circulating+tumor+cells.">Nanobiotechnology for the capture and manipulation of circulating tumor cells.</a> Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology. , (2011).</li><li>Hughes, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtube+Device+for+Selectin-Mediated+Capture+of+Viable+Circulating+Tumor+Cells+from+Blood.">Microtube Device for Selectin-Mediated Capture of Viable Circulating Tumor Cells from Blood.</a> Clinical Chemistry. 58, 846-853 (2012).</li><li>Hughes, A. D., King, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+naturally+occurring+halloysite+nanotubes+for+enhanced+capture+of+flowing+cells.">Use of naturally occurring halloysite nanotubes for enhanced capture of flowing cells.</a> Langmuir. 26, 12155-12164 (2010).</li><li>Al-Mehdi, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+origin+of+metastasis+from+the+proliferation+of+endothelium-attached+tumor+cells:+a+new+model+for+metastasis.">Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis.</a> Nat. Med. 6, 100-102 (2000).</li><li>Gupta, G. P., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+metastasis:+Building+a+framework.">Cancer metastasis: Building a framework.</a> Cell. 127, 679-695 (2006).</li><li>Lee, D., King, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcontact+Printing+of+P-Selectin+Increases+the+Rate+of+Neutrophil+Recruitment+Under+Shear+Flow.">Microcontact Printing of P-Selectin Increases the Rate of Neutrophil Recruitment Under Shear Flow.</a> Biotechnology Progress. 24, 1052-1059 (2008).</li><li>Gout, S., Tremblay, P. L., Huot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selectins+and+selectin+ligands+in+extravasation+of+cancer+cells+and+organ+selectivity+of+metastasis.">Selectins and selectin ligands in extravasation of cancer cells and organ selectivity of metastasis.</a> Clin. Exp. Metastasis. 25, 335-344 (2008).</li><li>Geng, Y., Marshall, J., King, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycomechanics+of+the+Metastatic+Cascade:+Tumor+Cell-Endothelial+Cell+Interactions+in+the+Circulation.">Glycomechanics of the Metastatic Cascade: Tumor Cell-Endothelial Cell Interactions in the Circulation.</a> Annals of Biomedical Engineering. , 1-16 .</li><li>Stott, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+circulating+tumor+cells+using+a+microvortex-generating+herringbone-chip.">Isolation of circulating tumor cells using a microvortex-generating herringbone-chip.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 18392-18397 (2010).</li></ol>

MRK的是一个CellTraffix公司（纽约州罗切斯特市）的科学顾问。

这是经常发生的情况，在发现新的癌症疗法的早期步骤，利用肿瘤细胞株，其中承担问题的原发肿瘤细胞相似，但仍然在使用，因为他们在实验室中使用的方便。将加快发展进入新的癌症疗法的研究，如果早在新的研究主要利用人类癌细胞。反恐委员会是最容易进入癌细胞的类型，由于其存在于血液和易用性的标准抽血。此外，循环肿瘤细胞代表了在5,6转移过程中的必要步骤，因此其相关的疾病，并针对新药物的效用，是明确的。反恐血液在体外分离是复杂的，其低浓度：每百万亿红细胞7％的白细胞或秩序。目前的方法检测血液中的四氯化碳，包括唯一获得FDA批准的技术细胞中的大多数<html>搜索（Veridex）的，在检测过程中损坏或破坏细胞，排除使用超出枚举。上面介绍的方法，不破坏细胞活力，从而打开了未来癌症临床研究的大门。作为完整的细胞恢复是这项技术的目标，这是势在必行，细胞被小心处理，特别是在血液中分离步骤。 有许多可调参数，在这个系统可以修改，以达到提高产量的关键特征，如癌症类型而定。在上述步骤，我们已经选择使用EpCAM的的CTC具体处理前列腺癌样本时除外抗体作为一个为所有类型的癌症，其中抗PSMA的使用。癌症特异性抗体的进一步换人当然是可以用于改善个别病人的性能。此外，选择和抗体浓度可以改变，以提高捕捉8。 “设备的基本特征是E-选择素分子在表面上成立。E-选择通常招募快速移动的白细胞向炎症部位的内皮细胞功能的壁面上表示。流动的白细胞结合瞬时到选择的分子，在较慢的，滚动的行为，有利于慢，通过整合内皮细胞结合。令人信服的实验证据存在的情况下，反恐委员会可以渗出9,10由一个类似的机制。选择列入使该器件能够在更大的流速经营，价格，否则将阻止细胞结合的抗体，11。因此我们的设备生理模仿小静脉，以biomimetically捕捉流动反恐不造成细胞损伤。 增强设备性能，可以归结为埃洛石纳米管涂层除了壁面该设备。以往的研究表明，有三个主要组成部分的碳纳米管涂层，可以改进的功能。首先，碳纳米管涂层提供表面积增加，从而在表面上4蛋白沉积更大。其次，在表演上的碳纳米管涂层的原子力显微镜，我们已经决定从表面到流，单个碳纳米管突出。这使选择分子将高达1微米以上的表面，使细胞可以被捕获，并通过管4招募表面早在他们的轨迹。最后，埃洛石纳米管涂层能够防止白细胞粘附和蔓延，表面上允许的捕获与CTC沿白细胞数量减少，从而更大的后续反恐纯度3。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> 300微米内径管</td><td>布伦特里科学</td><td> MRE025 </td></tr><tr><td>更大的油管连接注射器</td><td>艺达思健康及科学</td><td> 1507L </td></tr><tr><td> 5ml注射器泵</td><td>屋宇署</td><td> 309603 </td></tr><tr><td>连接器，小到大油管</td><td>艺达思健康及科学</td><td>的P-770 </td></tr><tr><td>大油管连接到注射器</td><td>艺达思健康及科学</td><td>架F-120和P-659 </td></tr><tr><td>微管注射器（3/10cc胰岛素注射器的U-100 29G 1/2“） </td><td>屋宇署</td><td> 309301 </td></tr><tr><td> accutase </td><td> MP Biomedicals有限责任公司</td><td> 1000449 </td></tr><tr><td> ficol-Paque加</td><td> GE的治​​愈thcare生物科学AB </td><td> 17-1440-03 </td></tr><tr><td>红细胞裂解液（缓冲的EL，1000ML） </td><td> Qiagen公司</td><td> 1014614 </td></tr><tr><td> G蛋白，5毫克</td><td> Calbiochem公司（calbiochem.com） </td><td> 539303 </td></tr><tr><td>人类EpCAM/TROP-1抗体</td><td> R＆D系统</td><td> MAB960 </td></tr><tr><td> PSMA的抗体（GCP-04） </td><td> abcam </td><td> ab66911 </td></tr><tr><td> 96孔板（微孔96） </td><td>屋宇署猎鹰</td><td> 35-3072 </td></tr><tr><td>杂交级拦截非脂肪干奶，300G </td><td> BIO-RAD实验室</td><td> 216005508 </td></tr><tr><td>埃洛石纳米管</td><td> NaturalNano的</td><td> NN-HNT200 </td></tr><tr><td>碳酸钙，5G </td><td>奥尔德里奇化学</td><td> 481807 </td></tr><tr><td> rhE-Selectin/Fc奇美拉，100微克</td><td> R＆D系统</td><td> 724-ES </td></tr><tr><td>培养基中，1640 </td><td> Gibco公司</td><td> 22400-089 </td></tr><tr><td> DPBS </td><td> Gibco公司</td><td> 14190-095 </td></tr><tr><td>聚-L赖氨酸0.1％W /水中v </td><td> Sigma-Aldrich公司</td><td> P8920-100ML </td></tr><tr><td>索尼克Dismembrator </td><td> Fisher Scientific则</td><td>型号100 </td></tr><tr><td>胎牛血清</td><td>亚特兰大生化</td><td> S11056H </td></tr><tr><td>青霉素链霉素</td><td>西格玛生命Siiences </td><td> P0781 </td></tr></tbody></table>

rapid isolation of viable circulating tumor cells from patient blood samples

循环肿瘤细胞（CTC）的传播从整个循环系统的原发肿瘤的细胞，可以最终形成二次肿瘤，在遥远的地方。 CTC数量可以用来追踪病情恶化的基础上，四氯化碳浓度之间的相关血液和疾病的严重程度1。作为一种治疗工具，CTC可以研究在实验室中，制定个性化治疗。为此，反恐委员会的隔离必须不会引起细胞损伤，与其他类型的细胞，尤其是白细胞的污染，必须避免尽可能多尽可能2。许多目前的技术，包括唯一获得FDA批准用于反恐枚举设备，破坏隔离进程的一部分CTC（更多信息请参见参考文献2）。一个微流体装置，捕捉可行的反恐委员会介绍，除了对3上皮标记的抗体与E-选择素糖蛋白功能表面组成。为了提高设备perfor曼斯纳米粒子涂层组成的埃洛石纳米管，铝纳米粘土4收获。 E-选择素分子提供了一种手段来捕捉快速移动的反恐委员会通过泵装置，贷款比替代微流体器件的优势，其中较长的处理时间是必要的，以提供足够的时间与表面相互作用的靶细胞。上皮目标的抗体提供反恐特异性设备，以及提供一个现成的可调参数来调整隔离。最后，埃洛石纳米管涂层可显着增强的隔离相比其他技术，以帮助捕捉快速移动的细胞，提供蛋白质的吸附表面积增加，并击退污染白细胞3,4。该装置是由一个简单的技术使用的现成材料，并已成功地用于捕捉癌细胞从血液转移癌症患者。捕获细胞维持至15天的文化隔离后，这些样品通常&gt; 50％可行的每个病人的原发肿瘤细胞组成。此设备已被用于捕捉可行的四氯化碳稀释的全血和白膜样品。最终，我们提出在临床上的功能来开发个性化的癌症治疗技术。

Watch this Scientific Journal Video about Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples at JoVE.com

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

癌症患者的血液循环肿瘤细胞的分离，而造成细胞损伤。肿瘤细胞的分离，实现使用的E-选择双分子表面，除了对上皮标记的抗体。碳纳米管涂层，特别是促进癌细胞粘附造成捕获纯度高。

从病人的血液样本可行的循环肿瘤细胞的快速分离

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

rapid-isolation-viable-circulating-tumor-cells-from-patient-blood

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26.5K 次观看.  Cornell University. 癌症患者的血液循环肿瘤细胞的分离，而造成细胞损伤。肿瘤细胞的分离，实现使用的E-选择双分子表面，除了对上皮标记的抗体。碳纳米管涂层，特别是促进癌细胞粘附造成捕获纯度高。

视频: Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

使用光声血流循环黑色素瘤细胞的检测和隔离

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

科研

生物工程

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation -&#160;is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.

In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.

In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.

The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

隔离细胞脂滴：两个纯化技术从酵母细胞与人类的胎盘开始

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

从手术组织小学的人结肠癌细胞的分离和培养直接它们柔软的弹性基底牵引术

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

人肺泡骨膜间充质干细胞的分离及维生素 D 对骨膜衍生细胞成骨活性的影响

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

透明质酸基水凝胶对3维患者源性胶质瘤细胞培养的研究

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Patient-Specific Polyvinyl Alcohol Phantom Fabrication with Ultrasound and X-Ray Contrast for Brain Tumor Surgery Planning

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create anatomically accurate head phantoms with realistic brain imaging properties because standard fabrication methods are not optimized to replicate any patient-specific anatomical detail and 3D printing materials are not optimized for imaging properties. In order to test and validate a novel navigation system for use during brain tumor surgery, an anatomically accurate phantom with realistic imaging and mechanical properties was required. Therefore, a phantom was developed using real patient data as input and 3D printing of molds to fabricate a patient-specific head phantom comprising the skull, brain and tumor with both ultrasound and X-ray contrast. The phantom also had mechanical properties that allowed the phantom tissue to be manipulated in a similar manner to how human brain tissue is handled during surgery. The phantom was successfully tested during a surgical simulation in a virtual operating room.
The phantom fabrication method uses commercially available materials and is easy to reproduce. The 3D printing files can be readily shared, and the technique can be adapted to encompass many different types of tumor.

This protocol describes the fabrication of a patient specific skull, brain and tumor phantom. It uses 3D printing to create molds, and polyvinyl alcohol (PVA-c) is used as the tissue mimicking material.

患者特异性聚乙烯酒精幻影制造与超声波和X射线对比脑肿瘤手术规划

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

成纤维细胞衍生的人类工程结缔组织用于筛查应用

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Characterization of Blood Outgrowth Endothelial Cells (BOEC) from Porcine Peripheral Blood

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, inflammation, and homeostasis. The endothelium also plays an important role in pathophysiologies such as atherosclerosis, hypertension, and diabetes. Endothelial cells form the inner lining of blood and lymphatic vessels and display heterogeneity in structure and function. Various groups have evaluated the functionality of endothelial cells derived from human peripheral blood with a focus on endothelial progenitor cells derived from hematopoietic stem cells or mature blood outgrowth endothelial cells (or endothelial colony-forming cells). These cells provide an autologous resource for therapeutics and disease modeling. Xenogeneic cells may provide an alternative source of therapeutics due to their availability and homogeneity achieved by using genetically similar animals raised in similar conditions. Hence, a robust protocol for the isolation and expansion of highly proliferative blood outgrowth endothelial cells from porcine peripheral blood has been presented. These cells can be used for numerous applications such as cardiovascular tissue engineering, cell therapy, disease modeling, drug screening, studying endothelial cell biology, and in vitro co-cultures to investigate inflammatory and coagulation responses in xenotransplantation.