这项工作是支持由国家关节炎，肌肉骨骼及皮肤疾病研究所授予:​​3R01AR054458 - 02S2。

1。第1天 1）板细胞<ol><li>板HMSC T75烧瓶在一个汇合的80％至少18-24小时前标签。备用船只的指令，请参考表1。 </li></ol> 2。第2天 2）准备标签的解决方案。这种准备将标签一（1）T75烧瓶中铁400微克/毫升的浓度在80％汇合。备用船只的指令，请参考表1。 <ol><li>创建一个解决方案（解决方案1），1毫升的无血清培养基和93.1μLferumoxytol（股票:30毫克/毫升）混合在15毫升的锥形管。 </li><li>混合1毫升的无血清培养基和7μL硫酸鱼精蛋白（股票:10毫克/毫升）创建一个第二的解决方案（解决方案2）在第二个15毫升的锥形管。 </li><li>让每一个解决方案，休息5分钟。 </li><li>添加解决方案1和2在一起。轻轻地混合新的标签解决方案。允许休息5分钟的解决方案，允许USPIO - PRO组胺硫酸盐复合物形成。 </li><li>加入5毫升的无血清培养基超顺磁性氧化铁/最终成交量为7毫升的硫酸鱼精蛋白溶液混合2毫升。 </li></ol> 3）准备为标签的细胞<ol><li>从细胞吸媒体。 </li><li>轻轻地用2-3毫升预热毫克/无钙的D - PBS或无血清培养基细胞一次，冲洗残留血清蛋白和其他媒体，或组件可能损害造影剂的吸收和标记效率。吸出的冲洗液。 </li></ol> 4）标签细胞<ol><li>添加完整的标签解决方案，以细胞7毫升。 </li><li>进入孵化器（37 ° C / 5％的CO 2），并允许细胞在4个小时的标签解决方案孵化细胞。 </li><li>加入700μL的FCS细胞的标签解决方案，最终实现10％的血药浓度。血清添加重新建立丰富的环境，其中的细胞是习惯于，并协助在MInimizing细胞死亡的突然转变，可能会导致24小时暴露在无血清的环境。 </li><li>允许细胞孵育一个额外的20小时内的标签解决方案。 </li></ol> 3。第3天 5）准备标记细胞<ol><li>吸从细胞的标签解决方案。 </li><li>用2-3毫升预热镁/钙免费的D - PBS轻轻冲洗细胞。吸出的PBS。重要的是要使用的Mg / Ca无PBS Mg和Ca将损害在下一步的胰蛋白酶的效率。 </li><li>加入2毫升预热的0.05％胰蛋白酶的细胞，来回倾斜烧瓶，烧瓶中，以确保整个表面覆盖着一层薄薄的胰蛋白酶。放置在培养箱（37 ° C / 5％的CO 2）的细胞，让细胞休息5-7分钟，或，直到细胞开始脱离板块。可能有必要在显微镜下确认支队。轻轻拍打的容量瓶中的双方，如果有必要发cilitate细胞表面脱离。 </li><li>预热完整的媒体加入4毫升的容量瓶中的胰蛋白酶。轻轻吹打胰蛋白酶/媒体/细胞溶液上下几次冲洗烧瓶。收集在15毫升的锥形管的整个解决方案。离心5分钟，在400离心力的电池解决方案。 </li><li>小心吸上清，而不会干扰细胞沉淀的。媒体5毫升（含药血清或无血清）重悬细胞。离心5分钟，在400离心力的电池解决方案。 </li><li>重复步骤5.5中所描述的细胞洗。在总额中，细胞会被冲洗三次，以消除媒体和细胞表面上的残留，无造影剂。 </li><li>细胞计数在这一点上达到最准确的细胞计数，细胞将失去在以前的洗衣机。执行细胞的活力测试。现在准备后续分析，或实验应用细胞。可以进行磁共振成像T1 - W<html> T2 - W参数，因为虽然ferumoxytol是T2 - W的造影剂，ferumoxytol也表现出T1的效果。代表T2 - W MRI，可用于图像ferumoxytol标记细胞的参数包括:法兰克福或SE_Multislice 2000-2500 MS和TE 60-80毫秒的TR序列。要获得一个T1 - W的形象，降低TE值或一个FSE SE_Multislice序列或使用一个GRE序列具有较高的TR和低的TE。图4演示了标记干细胞成像在体内的应用潜力。 </li></ol> 4。代表性的成果: 标记细胞表现出显著变暗或负上T2加权MR图像的对比效果和对T1加权磁共振图像增亮或阳性对比效果（图2）。纵向磁共振成像和可行性测试后的标签进行了5天没有表现出显着的MR信号，并没有显著的可行性的影响相比，无标签控制（数据未显示）。 jove_content"&gt;标记的干细胞随后可以分化成各种细胞类型或静脉注射注入体内调查。 <img alt="图1" src="/files/ftp_upload/3482/3482fig1.jpg" /> 图1。时间线，ferumoxytol氧化铁纳米粒子标记干细胞的示意图。 <img alt="图2" src="/files/ftp_upload/3482/3482fig2.jpg" /> 图2。MR图像矢状截面thrrough的Eppendorf管细胞在细胞沉淀。一）未标记的控制。 B）标记的细胞表现出显著的T2或T2加权FSE或阴性造影剂效果SE_Multislice成像和T1或T1加权GRE序列的阳性对照剂的作用。 <img alt="图3" src="/files/ftp_upload/3482/3482fig3.jpg" /> 跨矢状Eppendorf管中的部分细胞PEL 细胞图3 。让。通过T2 - W磁共振成像可检测低至万ferumoxytol标记细胞。 <img alt="图4" src="/files/ftp_upload/3482/3482fig4.jpg" /> 图4。议员可视化ferumoxytol标记的干细胞注入小鼠脑室。 a）轴向，没有USPIO标记的干细胞注入小鼠barin MR图像。二）轴向，C）冠状，和D）矢状位磁共振图像描绘USPIO标记的干细胞在小鼠的大脑注入（白色箭头）T2，消极的对比效果。 <img alt="表1" src="/files/ftp_upload/3482/3482table1.jpg" /> 表1。标签替代血管细胞的数量adaptions 。

<ol>
	<li>Narsinh, K. H., Plews, J., Wu, 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=Comparison+of+human+induced+pluripotent+and+embryonic+stem+cells:+fraternal+or+identical+twins?.">Comparison of human induced pluripotent and embryonic stem cells: fraternal or identical twins?.</a> Mol Ther. 19, 635-638 (2011).</li><li>Bulte, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+MRI+cell+tracking:+clinical+studies.">In vivo MRI cell tracking: clinical studies.</a> AJR. Am. J. Roentgenol. 193, 314-325 (2009).</li><li>Henning, T. D., Boddington, S., Daldrup-Link, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+hESCs+and+hMSCs+with+Iron+Oxide+Nanoparticles+for+Non-Invasive+in+vivo+Tracking+with+MR+Imaging.">Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging.</a> J. Vis. Exp. (13), e685-e685 (2008).</li><li>Tallheden, T., Nannmark, U., Lorentzon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+MR+imaging+of+magnetically+labeled+human+embryonic+stem+cells.">In vivo MR imaging of magnetically labeled human embryonic stem cells.</a> Life. Sci. 79, 999-1006 (2006).</li><li>Jung, C. W., Jacobs, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+chemical+properties+of+superparamagnetic+iron+oxide+MR+contrast+agents:+ferumoxides,+ferumoxtran,+ferumoxsil.">Physical and chemical properties of superparamagnetic iron oxide MR contrast agents: ferumoxides, ferumoxtran, ferumoxsil.</a> Magn. Reson. Imaging. 13, 661-674 (1995).</li><li>Coyne, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferumoxytol+for+treatment+of+iron+deficiency+anemia+in+patients+with+chronic+kidney+disease.">Ferumoxytol for treatment of iron deficiency anemia in patients with chronic kidney disease.</a> Expert. Opin. Pharmacother. 10, 2563-2568 (2009).</li><li>Li, Z., Suzuki, Y., Huang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+reporter+gene+and+iron+particle+labeling+for+tracking+fate+of+human+embryonic+stem+cells+and+differentiated+endothelial+cells+in+living+subjects.">Comparison of reporter gene and iron particle labeling for tracking fate of human embryonic stem cells and differentiated endothelial cells in living subjects.</a> Stem Cells. 26, 864-873 (2008).</li><li>Metz, S., Bonaterra, G., Rudelius, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacity+of+human+monocytes+to+phagocytose+approved+iron+oxide+MR+contrast+agents+in+vitro.">Capacity of human monocytes to phagocytose approved iron oxide MR contrast agents in vitro.</a> Eur. Radiol. 14, 1851-1858 (2004).</li><li>Nedopil, A., Klenk, C., Kim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR+signal+characteristics+of+viable+and+apoptotic+human+mesenchymal+stem+cells+in+matrix-associated+stem+cell+implants+for+treatment+of+osteoarthritis.">MR signal characteristics of viable and apoptotic human mesenchymal stem cells in matrix-associated stem cell implants for treatment of osteoarthritis.</a> Invest. Radiol. 45, 634-640 (2010).</li><li>Kraitchman, D. L., Heldman, A. W., Atalar, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+magnetic+resonance+imaging+of+mesenchymal+stem+cells+in+myocardial+infarction.">In vivo magnetic resonance imaging of mesenchymal stem cells in myocardial infarction.</a> Circulation. 107, 2290-2293 (2003).</li><li>Stuckey, D. J., Carr, C. A., Martin-Rendon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+particles+for+noninvasive+monitoring+of+bone+marrow+stromal+cell+engraftment+into,+and+isolation+of+viable+engrafted+donor+cells+from,+the+heart.">Iron particles for noninvasive monitoring of bone marrow stromal cell engraftment into, and isolation of viable engrafted donor cells from, the heart.</a> Stem Cells. 24, 1968-1975 (2006).</li><li>Henning, T. D., Sutton, E. J., Kim, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+ferucarbotran+on+the+chondrogenesis+of+human+mesenchymal+stem+cells.">The influence of ferucarbotran on the chondrogenesis of human mesenchymal stem cells.</a> Contrast. Media. Mol. Imaging. 4, 165-173 (2009).</li><li>Arbab, A. S., Yocum, G. T., Kalish, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+magnetic+cell+labeling+with+protamine+sulfate+complexed+to+ferumoxides+for+cellular+MRI.">Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI.</a> Blood. 104, 1217-1223 (2004).</li><li>Nedopil, A. J., Mandrussow, L. G., Daldrup-Link, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+Ferumoxides+Labeled+Human+Mesenchymal+Stem+Cells+in+Cartilage+Defects.">Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects.</a> J. Vis. Exp. (38), e1793-e1793 (2010).</li><li>Arbab, A. S., Yocum, G. T., Wilson, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+transfection+agents+in+forming+complexes+with+ferumoxides,+cell+labeling+efficiency,+and+cellular+viability.">Comparison of transfection agents in forming complexes with ferumoxides, cell labeling efficiency, and cellular viability.</a> Mol Imaging. 3, 24-32 (2004).</li><li>Babic, M., Horak, D., Trchova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(L-lysine)-modified+iron+oxide+nanoparticles+for+stem+cell+labeling.">Poly(L-lysine)-modified iron oxide nanoparticles for stem cell labeling.</a> Bioconjug Chem. 19, 740-750 (2008).</li><li>Golovko, D. M., T, H. e. n. n. i. n. g., Bauer, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+stem+cell+labeling+with+ferucarbotran+and+protamine.">Accelerated stem cell labeling with ferucarbotran and protamine.</a> Eur. Radiol. 20, 640-648 (2010).</li><li>Lu, M., Cohen, M. H., Rieves, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FDA+report:+Ferumoxytol+for+intravenous+iron+therapy+in+adult+patients+with+chronic+kidney+disease.">FDA report: Ferumoxytol for intravenous iron therapy in adult patients with chronic kidney disease.</a> Am. J. Hematol. 85, 315-319 (2010).</li></ol>

这项研究的所有出资者没有披露。

提高疗效的干细胞engraftments是再生医学的进步是至关重要的。一个在体内的干细胞的非侵入性可视化技术，大大增强了我们的理解能力成功植入成果的机制，导致。磁性标签的议员，如我们已经证明过程的可视化，可在体内的干细胞与磁共振成像跟踪。以前的磁标记干细胞被移植到靶组织，已可视化与磁共振成像 7周。长期的标签和纵向成像是可能的， 因为 8的氧化?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> D - MEM高糖</td><td>西格玛</td><td> D5648 </td><td>或其他所需的干细胞系用于基础培养基</td></tr><tr><td> D - PBS（ 钙离子，镁+免费） </td><td> GIBCO公司</td><td> 14190-144 </td><td></td></tr><tr><td>胰蛋白酶EDTA 0.05％ </td><td> Invitrogen公司</td><td> 25300-120 </td><td></td></tr><tr><td>胎牛血清（FBS） </td><td> Hyclone公司</td><td> SH30071.03 </td><td></td></tr><tr><td> Ferumoxytol （Feraheme） </td><td> AMAG </td><td> 59338-0775-​​01 </td><td></td></tr><tr><td>硫酸鱼精蛋白</td><td> APP的医药。 </td><td> 22930 </td><td></td></tr>
</tbody>
</table>

labeling stem cells with ferumoxytol, an fda-approved iron oxide nanoparticle

干细胞为基础的疗法提供了显著的再生医学领域潜力。然而，仍有许多工作需要了解有关在体内移植的细胞动力学。一种非侵入性的方法来重复监视体内移植的干细胞，将允许调查人员直接监控干细胞移植，并确定成功或不成功的植入结果。 广泛的干细胞仍然是无数的应用研究。此协议侧重于3种不同的干细胞群:人胚肾293（HEK293）细胞，人骨髓间充质干细胞（HMSC）和诱导多能干细胞（iPS）细胞。 HEK 293细胞来自人类胚胎肾细​​胞在培养与剪切的腺病毒5 DNA。这些细胞被广泛用于研究，因为它们很容易培养，生长迅速，而且很容易转。成人骨髓中发现的hMSCs。这些细胞的CAn是未分化的细胞复制，同时保持多能或潜在的分化成的细胞命运的数量有限。的hMSCs可以分化为间充质组织，包括成骨细胞，脂肪细胞，软骨，肌腱，肌肉，骨髓基质谱系。 iPS细胞的基因重新编​​程成年细胞已被修改，以表达基因和类似胚胎干细胞的定义属性的因素。这些细胞是多能干细胞的意义，他们有能力分化成所有细胞谱系1。双方的hMSCs和iPS细胞已经证明体内组织的再生能力。 磁共振（MR）成像与超顺磁性氧化铁（SPIO）纳米颗粒细胞标签的使用已被证明有效的，在体内的干细胞，由于附近的显微解剖决议跟踪，更长的血液半衰期，允许纵向成像和高灵敏度Ÿ纳米粒子的超顺磁性氧化铁MR成像提供细胞检测2-4。此外，SPIOs使用MR成像是临床上的翻译。与右旋糖酐，carboxydextran或淀粉表面涂层服务包含从血浆成分的生物活性铁芯的氧化铁核心组成SPIOs。这些代理商创造局部磁场的不均匀性，导致一个下降T2加权MR图像5信号。不幸的是，SPIOs不再生产。第二代超小型SPIOs（USPIO），但是，提供一个可行的选择。 Ferumoxytol（FerahemeTM）是一种非化学计量的磁铁矿由缩合葡萄糖山梨醇carboxymethylether大衣包围的核心组成之一USPIO。 ferumoxytol胶体，粒径17-30纳米光散射确定。分子量为750 kDa的，2T磁共振成像领域的弛豫常数为58.609毫米1秒1的实力 4 。 Ferumoxytol最近被FDA批准一个是一个在与肾功能衰竭6患者的补铁治疗缺铁。本集团已应用在"关闭标签"细胞标记应用程序的使用此代理。我们的技术演示ferumoxytol显著MR MR图像标记细胞信号的影响，导致干细胞的有效标签。这种技术可应用于非侵入性的干细胞疗法在临床前和临床设置监测。

Watch this Scientific Journal Video about Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle at JoVE.com

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

我们描述一个标签和跟踪与FDA批准的，超顺磁性氧化铁（SPIO），ferumoxytol（Feraheme）的干细胞的技术。这种细胞成像技术，利用磁共振（MR）成像的可视化，易于成功或不成功的患者干细胞engraftments的长期监测和诊断。

Ferumoxytol，一个FDA批准的氧化铁纳米粒子标记干细胞

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

labeling-stem-cells-with-ferumoxytol-an-fda-approved-iron-oxide

Research

JoVE Journal

Medicine

20.8K 次观看.  Molecular Imaging Program at Stanford (MIPS). 我们描述一个标签和跟踪与FDA批准的，超顺磁性氧化铁（SPIO），ferumoxytol（Feraheme）的干细胞的技术。这种细胞成像技术，利用磁共振（MR）成像的可视化，易于成功或不成功的患者干细胞engraftments的长期监测和诊断。

视频: Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

科研

医学

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

与器官型脑片和癌细胞的3D球体共培养系统

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

毛喉素诱导在肠组织体溶胀:一个<em&gt;体外</em&gt;检测方法的评估囊性纤维化患者对药物的反应

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. Repeated transfusions of packed red blood cells (PRBCs) is currently the simplest and most used technique for chronic transfusion programs. However, iron overload is one of the major side effects of this therapy. More developed methods exist, notably the apheresis of RBC (erythrapheresis), which is currently the safest and most efficient method. However, it is costly, complicated, and cannot be implemented everywhere, nor is it suitable for all patients. Manual exchange transfusions combine one or more manual phlebotomies with a PRBC transfusion.
At the Reference Center of Sickle Cell Disease, we set up a continuous method of manual exchange transfusion that is feasible for all hospital settings, demands no specific equipment, and is widely applicable. In terms of HbS decrease, stroke prevention, and iron overload prevention, this method showed comparable efficiency to erythrapheresis. In cases where erythrapheresis is not available, this method can be a good alternative for patients and care centers.

We have outlined a method of continuous manual exchange transfusion for the treatment of sickle cell disease in patients. This safe protocol was designed to effectively limit iron overload in patients in need of chronic transfusions and can be used extensively without any special equipment.

与镰状细胞疾病患者连续手动换血:的有效方法避免铁超负荷

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

超超磁氧化铁纳米探针在肺外结核检测的合成、表征及应用

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

聚乙胺包覆氧化铁纳米粒子作为载体, 在体外和体内向巨噬细胞输送小干扰 rna

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

一种分离和扩增人类脂肪衍生的美源性干细胞的无酶方法

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

跟踪超副磁性氧化铁标记的美感血干细胞使用MRI后，创伤性脑损伤Murine模型内伤分娩

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) crystals. Crystalluria occurs when urine becomes supersaturated with minerals (e.g., calcium, oxalate, phosphate) and precedes kidney stone formation. Standard methods to assess crystalluria in stone formers include microscopy, filtration, and centrifugation. However, these methods primarily detect microcrystals and not nanocrystals. Nanocrystals have been suggested to be more harmful to kidney epithelial cells than microcrystals in vitro. Here, we describe the ability of Nanoparticle Tracking analysis (NTA) to detect human urinary nanocrystals. Healthy adults were fed a controlled oxalate diet prior to drinking an oxalate load to stimulate urinary nanocrystals. Urine was collected for 24 hours before and after the oxalate load. Samples were processed and washed with ethanol to purify samples. Urinary nanocrystals were stained with the calcium binding fluorophore, Fluo-4 AM. After staining, the size and count of nanocrystals were determined using NTA. The findings from this study show NTA can efficiently detect nanocrystalluria in healthy adults. These findings suggest NTA could be a valuable early detection method of nanocrystalluria in patients with kidney stone disease.

The objective of this study was to determine whether nanoparticle tracking analysis (NTA) could detect and quantify urinary calcium containing nanocrystals from healthy adults. The findings from the current study suggest NTA could be a potential tool to estimate urinary nanocrystals during kidney stone disease.

使用氟磷钙标签和纳米粒子跟踪分析估计人类泌尿纳米晶体

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) ...