我们要感谢G·富尔达（电子显微镜中心，罗斯托克大学，德国）用于收购过滤顺磁纳米颗粒的TEM图像，并履行其X射线分析技术支持。在RTC罗斯托克进行的工作是由联邦教育与研究部德国（FKZ 0312138A，FKZ 316159和VIP + 03VP00241）和国家梅克伦堡 - 前波莫瑞州与欧盟结构基金（ESF / IV-WM-B34-支持0030/10和ESF / IV-BM-B35-0010 / 12）和由DFG（DA 1296-1），则湿热基金会和德国心脏基金会（F / 01/12）。弗兰克·威克霍斯特由欧盟第七框架计划的研究项目 "Nanomag" FP7-NMP-2013-大7的支持。

我们有没有竞争经济利益透露。

生产装载有超顺磁性纳米颗粒和其进一步磁控制的指导基因工程细胞的呈现在当前协议。这种策略的成功应用允许的细胞治疗的一些困难，例如在受伤区域2，3，4低保留和植入差分辨率，通过对移植提供靶向细胞产物。此外，同时引入适当选择的遗传物质的可促进细胞特性，从而可以增加功能性益处41。 目前的协议已经开发了血管内皮细胞的一种模式。这些细胞被称为是难以转染和易受影响有毒18，19，20。该DEM典型地用基于PEI的载体获得的PEI / MNP转染后的荧光团标记的miR摄取onstrated水平超过95％，并且通常使用的基于阳离子脂质的转染试剂（ 例如，脂质体）42。对于NP 40 - 此外，25的最佳NP比率为20先前公布的值相对应。然而，测试的荧光团标记的miR的高效摄取并不一定保证递送43经过适当的miR处理和功能。这一点是在PEI的情况下特别相关的，与NA的其紧静电冷凝。因此，递送的miR的得到的功能容量也必须进行测试。这里，功能性内源的miR在HUVEC中表达，的miR-92A 31，选择，和针对其抗miR使用PEI和PEI / MNP递送。其结果，靶的miR-92a的几乎完全敲低证明了抗miR f的有效释放ROM中的转染复合物。 重要的是，在以前的工作中，PEI / MNP矢量的成功应用被证明用于递送质粒DNA（pDNA的）和miR的其它细胞类型，无论是贴壁（ 例如，COS7，人间质干细胞15，16）和在悬浮液（ 例如，骨髓衍生的CD133 +造血干细胞44）。所有这些实验都证实了众所周知的事实，转染效率和毒性是细胞类型相关的45。因此，最佳载体组合物的选择（ 即，量的miR，NP比，和MNP铁的量）应当为每一个特定的细胞类型中进行，使用作为基准点以前建立最佳条件。 此外，所获得的遗传修饰的瞬态字符应占。在一方面，我t是非常适合于某些功能，如生长因子的短期交货。在另一方面，表达的持续时间可能不会持续足够长的时间，适用于各种需要长期表达的目的。然而，证明了PEI / MNP矢量的安全支持其可以重复施用一旦条件进一步优化。 PEI及其衍生物由于常用于它们的高效率相比于其他非病毒载体和它们的柔性变形例7。然而，尽管在体外和体内 PEI的成功，其在临床试验中应用是非常有限的（ 即，几个阶段1和2的试验或癌症患者）7，14由于PEI毒性7,13。由于我们组16，以及该协议的早期作品中，已经证明，的MNP能够显著降低PEI的毒性。特别是，的miR / PEI的转染已损坏的HUVEC以在它们的基质管的能力，因而不能主体外功能试验这些细胞。与此相反，的miR / PEI / MNP-HUVEC的管形成性能与未处理的细胞。值得注意的是，这种降低PEI毒性的，通过引入组分，例如氧化铁，其是生物相容的和常规用作在人受试者40的造影剂的低含量来实现。 除了维持功能特性，重要的是，经修饰的细胞是能够建立细胞间通讯的，因为这个功能是必要的细胞治疗移植后46的适当整合。此外，经修饰的细胞可被利用作为用于递送治疗的miR的到受伤组织47的载体。在这种情况下，他们的卡帕奇TY与邻近细胞交换分子定义它们的作为车辆的成功。由于PEI / MNP转允许GJIC在修改内皮细胞，它们在体内的整合潜力和提供的miR运送到受灾地区。 值得注意的是，对细胞磁化先前发表的作品用于随后的定位已经报道〜4的小区负载和铁/小区48,49，50，51的30微克。这些数字显著超过值〜0.16-0.7 pg /细胞，这是足够用于静态和动态模拟条件的体外靶向NA / PEI / MNP-HUVEC的。这样低的铁的量是在安全性方面是有益的:（ 即，活性氧类别（ROS））是已知的铁氧化物纳米颗粒相关的毒性的一个公知的机构与INTE的量直接相关联rnalized纳米颗粒40。此外，几个研究已经证明，氧化铁纳米颗粒的高细胞内水平可导致剂量依赖的细胞骨架结构破坏和损伤52,53。重要的是，大多数报告的小区磁化的情况下，为了定位不包括遗传细胞操作。与此相反，所述miR / PEI / MNP向量提供了一个机会同时修改细胞和添加的磁特性。 然而，低负荷MNP可能是磁靶向随血流动荡的环境不够。为了更接近体内情况的具有挑战性的，动态条件进行了模拟在体外:在悬浮液与磁化的miR / PEI / MNP-的HUVEC培养板放置在旋转振荡器34。这个实验清楚地不能涵盖所有发生在相互作用体内。但是，这也促使人们更好地了解PEI / MNP修饰细胞的靶向的潜力。其结果是，甚至当培养基中的活性摇动没有朝向磁铁54与细胞的运动干扰高效细胞靶向证明。为了这个目的，的miR-改性将HUVEC装载有至少〜0.16皮克铁/细胞。此外，磁响应细胞进行排序。应用于当前研究基于磁铁细胞分离过程已不损害所述miR / PEI / MNP-细胞的活力。重要的是，静磁场的应用（ 即，针对所涉及的磁场的应用为12个实验- 24小时），并没有对靶细胞产生破坏性的影响。因此，证明了生产磁化遗传修饰的细胞的进一步提供了在解决由磁力确保细胞保留的效率的体内研究的基础。值得注意的是，日È最成功的在采用磁化细胞的靶向51，静脉注射后54后，用过的55细胞保留局部给药而不是细胞指导体内研究。因此，在注射部位的基于磁体的保留用于进一步合乎需要出现在的miR / PEI / MNP-修饰的细胞的体内应用。

基因和细胞治疗是必须要解决的CVD处理当前挑战的潜在的强大工具。尽管这两种方法，目前正在临床试验，他们还没有准备好广泛的临床应用1。值得注意的是，以解决基因和细胞治疗的挑战的共同方法是开发适合于临床应用的多官能基因递送载体。缺乏安全，高效的基因传递系统是基因治疗的主要关注点。同时，细胞产物的基因工程移植前可以克服细胞治疗的严重挑战，如低效率（ 例如，在心脏字段，只有约5％的功能改善，实现后干细胞移植1 ）和差保留/植入在损伤部位（ 即，细胞保留低于5 - 10％几分钟到几小时内口服ST-应用，无论给药途径2,3，4）。 迄今为止，病毒载体大大超过在效率，这导致了在临床试验中其广泛应用方面非病毒系统（〜67％）5。然而，病毒载体携带在携带的遗传物质6的尺寸严重的风险，如免疫原性（和随后的炎症反应，有严重的并发症），致癌性，和限制。由于这些安全问题和病毒载体的生产成本高，使用非病毒系统的是在某些情况下7,8优选的。它特别适合用于需要瞬时遗传校正病症，诸如的调控血管生成（ 例如，用于CVD处理）的生长因子的表达或国内配送疫苗的RY。 本组的递送系统通过结合支链25-kDa的聚乙烯亚胺（PEI），并通过生物素-链霉亲和相互作用9结合在一起的超顺磁性氧化铁纳米颗粒（MNP）而设计的。该载体是用于细胞基因工程，从而允许他们的移植之前同时磁化的潜在工具。后者提供了一种用于磁性引导/保持，这是特别有前途的今天，先进的磁性靶向技术正在研制成功10的基础。此外，所得到的磁响应的细胞必须是潜在的非侵入性通过磁共振成像（MRI）或磁性粒子成像11,12监测。 在PEI / MNP载体的情况下，聚胺保证了从低分子化因子核酸缩合，从而保护<html> s时，细胞载体内化和内体逃逸5。所述的MNP补充PEI的特性，不仅在磁性引导的方面，还通过降低已知的PEI毒性7，13，14。此前，PEI / MNP载体性质在输送效率（ 即，质粒DNA和miRNA）和安全性方面，通过使用成纤维细胞和人类间质干细胞15,16调整。 在该手稿上PEI /的MNP的对于miRNA修饰细胞的生成应用程序的详细方案描述17。为了这个目的，将HUVEC被使用并且表示用于在体外血管生成一个建立的模型。他们是具有挑战性的转染，并很容易受到有毒影响18，19，屁股= "外部参照"&gt; 20。另外，我们提供一种算法来评估这些细胞在体外 ，包括它们的定位，细胞间通讯，和MRI检测。

<table border="0" cellpadding="0" cellspacing="0" width="653">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">PEI 25 kDa</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:119px;">408727</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">EZ-Link Sulfo-NHS-LC-Biotin</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">21335</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:197px;">PD-10 Desalting Columns</td>
			<td style="width:159px;">GE Healthcare</td>
			<td style="width:119px;">17085101</td>
			<td style="width:179px;">Containing Sephadex G-25 Medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:197px;">Ninhydrin Reagent solution 2%</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:119px;">7285</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">Glycine</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:119px;">410225</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:197px;">Pierce Biotin Quantitation Kit</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">28005</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:197px;">&#160;Microplate reader Model 680</td>
			<td style="width:159px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:197px;">Streptavidin MagneSphere Paramagnetic Particles</td>
			<td style="width:159px;">Promega</td>
			<td style="width:119px;">Z5481</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">Millex-HV PVDF Filter</td>
			<td style="width:159px;">Merck</td>
			<td style="width:119px;">SLHV013SL</td>
			<td style="width:179px;">0.45&#181;m</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:197px;">Libra 120 transmission electron microscope&#160;</td>
			<td style="width:159px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:179px;">Acceleration Voltage 120KV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">Sapphire X-ray detector</td>
			<td style="width:159px;">EDAX-Amatek</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">Cell culture plastic</td>
			<td style="width:159px;">TPP</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">NHS-Esther Atto 565</td>
			<td style="width:159px;">ATTO-TEC GmbH</td>
			<td style="width:119px;">AD 565-31</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">NHS-Esther Atto 488&#160;</td>
			<td style="width:159px;">ATTO-TEC GmbH</td>
			<td style="width:119px;">AD 488-31</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">Cy5 miRNA Label IT kit</td>
			<td style="width:159px;">Mirus Bio</td>
			<td style="width:119px;">MIR 9650</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">Biotin Atto 565</td>
			<td style="width:159px;">ATTO-TEC GmbH</td>
			<td style="width:119px;">AD 565-71</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">Collagense Type IV Gibco</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">17104019</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:197px;">Endothelial growth medium, EGM-2</td>
			<td style="width:159px;">Lonza</td>
			<td style="width:119px;">CC-3156 &#38; CC-4176</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">Penicillin/Streptomycin</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">15140122</td>
			<td style="width:179px;">100 U/ml, 100&#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">Matrigel</td>
			<td style="width:159px;">BD Biosciences</td>
			<td style="width:119px;">356234</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">anti-PECAM-1 antibody</td>
			<td style="width:159px;">Santa Cruz</td>
			<td style="width:119px;">sc-1506</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">MS MACS columns</td>
			<td style="width:159px;">Miltenyi Biotec&#160;</td>
			<td style="width:119px;">130-042-201</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:197px;">Near-IR Live/Dead Cell Stain Kit</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">L10119</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:197px;">Cy3 Dye-Labeled Pre-miR Negative Control</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">AM17120</td>
			<td style="width:179px;">&#34;Cy3-miR&#34; or &#34;Cyanine-miR3&#34; in the manuscript</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:197px;">Pre-miR miRNA Precursor Molecules - Negative Control&#160;</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">AM17110</td>
			<td style="width:179px;">&#34;scr-miR&#34; in the manuscript</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:197px;">Anti-hsa-miR92a-3p synthetic Inhibitor&#160;</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">AM10916</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">LSM 780 ELYRA PS.1 system</td>
			<td style="width:159px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">Paraformaldehyde</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:119px;">158127</td>
			<td style="width:179px;">4% solution in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">DAPI nuclear stain</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">D1306</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">NucleoSpin RNA isolation Kit</td>
			<td style="width:159px;">Machery-Nagel</td>
			<td style="width:119px;">740955</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">mirVana miRNA Isolation Kit</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">AM1560</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:197px;">TaqMan MicroRNA Reverse Transcription Kit</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">4366596</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:197px;">StepOnePlus Real-Time PCR System</td>
			<td style="width:159px;">Applied Biosystems</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:197px;">High-Capacity cDNA Reverse Transcription Kit</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">4368814</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:197px;">hsa-miR-92a TaqMan assay</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">000431</td>
			<td style="width:179px;">Mature miRNA Sequence: UAUUGCACUUGUCCCGGCCUGU</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastGene Taq Ready Mix</td>
			<td style="width:159px;">Nippon Genetics</td>
			<td style="width:119px;">LS27</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">ITGA5 TaqMan assay</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">Hs01547673_m1</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">RNU6B TaqMan assay</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">001093</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:197px;">18S rRNA Endogenous Control</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">4333760F</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">Gelatin</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:119px;">G7041</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:197px;">CellTrace Calcein Red-Orange</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">C34851</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">PBS</td>
			<td style="width:159px;">Pan Biotech</td>
			<td style="width:119px;">P04-53500</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">BSA</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">MACS buffer</td>
			<td style="width:159px;">Miltenyi Biotec&#160;</td>
			<td style="width:119px;">130-091-221</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">Agarose</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:119px;">A9539</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:197px;">7.1 Tesla animal MRI system</td>
			<td style="width:159px;">Bruker Corporation</td>
			<td style="width:119px;">A7906</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:197px;">ImageJ software</td>
			<td style="width:159px;">National Institutes of Health</td>
			<td style="width:119px;"></td>
			<td style="width:179px;">upgraded with an AngiogenesisAnalyzer (NIH)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">MPS device</td>
			<td style="width:159px;">Bruker Biospin</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">Matlab software</td>
			<td style="width:159px;">Mathworks</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:197px;">Ring Neodym Magnet&#160;</td>
			<td style="width:159px;">magnets4you GmbH</td>
			<td style="width:119px;">RM-10x04x05-G</td>
			<td style="width:179px;">&#248; 10 mm; remanescence is ~1.3T, coercivity &#8805; 955 kA/m</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:197px;">Click-iT EdU Alexa Fluor 647 Imaging Kit</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">C10340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">FluorSave Reagent</td>
			<td style="width:159px;">Merck</td>
			<td style="width:119px;">345789</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">Ultrasonic bath</td>
			<td style="width:159px;">Bandelin electronic</td>
			<td style="width:119px;">Type: RK 100 SH</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation and in vitro characterization of magnetized mir-modified endothelial cells

迄今为止，用于治疗心血管疾病的可用外科和药物治疗（CVD）是有限的，经常姑息。与此同时，基因和细胞的疗法可用于CVD处理极有希望的替代方法。然而，基因治疗的广泛临床应用极大地受到缺乏合适的基因递送系统的限制。的合适的基因递送载体的发展可以提供解决方案，以在细胞疗法目前的挑战。特别是，现有的缺点，例如，在受伤器官限定的效率和低的细胞保留，可以通过适当的细胞工程来克服移植（ 即遗传）之前进行。所提出的协议描述了使用聚乙烯亚胺的超顺磁性纳米颗粒（PEI / MNP）系递送载体的内皮细胞的有效且安全的瞬时变形。此外，算法和细胞表征方法定义。成功intracellu微RNA（MIR）到人脐静脉内皮细胞（HUVECs）的拉尔递送已经在不影响细胞生存力，功能，或者细胞间的通信被实现。此外，这种方法被证明会导致导入的外源的miR强大的功能效果。重要的是，这种基于MNP-矢量的应用确保细胞磁化，伴随磁靶向和非侵入性的MRI跟踪的可能性。这可以提供对于可以非侵入性地监测与MRI磁性引导，遗传工程细胞疗法的基础。

所提出的协议的主要目的是产生磁响应的miR-修饰的细胞，并进行其精确的表征（ 图1）。其结果是，有效地转染的细胞中，响应于磁选择和指导和可检测的与MRI，应当获得。 首先，分离的内皮细胞的身份，通过与内皮细胞标记物CD31染色的典型（PECAM）（ 图2A）和通过以在适当的基底膜基质（ 图2B）的管的能力证实。将所得到的细胞拿起所述miR非常有效地引入由PEI / MNP;显微镜表明，所有的细胞均为阳性标记的miR（Cy3标记）24小时后转染（ 图3A）的信号。重要的是，没有细胞死亡被记录相比，未经处理的细胞（ 图3B），并且normal外观保持（ 图3A）。如共聚焦激光扫描显微镜观察，的Cy3的miR的信号被主要位于细胞内。此外，转染载体和miR的所有部分的细胞内定位的更详细的调查，使用3色标记的复合物更高的分辨率下进行。 SIM，的miR，PEI，和MNP信号在核周区域（ 图3C）的细胞质中检测到的所有。 此外，转染载体的安全的详细调查已经证明了miR / PEI / MNP-的HUVEC能够在适当的基底膜基质和形成管的所产生的网络比得上用作阳性对照未处理的细胞（ 图4A ）。此外，转染的细胞保持GJIC，如3D-FRAP实验揭示。特别是，当细胞加载荧光GJ-柏美亚BLE染料和它们中的一个被漂白，其荧光恢复由于与相邻小区通信，将所得染料转移（ 图4B）。 重要的是，由于超顺磁性化合物40（ 图5A）的存在下，PEI / MNP应用不仅允许细胞转染，而且还同时磁化。使用MPS（ 图5B）为最佳的范围内的所有施加MNP浓度将得到的细胞内铁的量进行测定（2.5皮摩尔/ cm 2的的miR; NP 25补充了5-25微克/毫升MNP）:0.37±0.079至0.7± 0.150 PG铁/细胞17。由于使用磁分离柱的证明的，对于所有这些MNP浓度，磁响应细胞在转染细胞的总体积的量为〜70％（ 图5C）。此外，转^ hUVECs 在体外琼脂糖幻影的内部与MRI（ 图5D）可见，模仿小鼠组织易感性。此外，在体外模拟动态条件下转染的HUVEC的磁性靶向被证明是有效的（ 图6）。在这个实验装置，甚至培养基的不断剧烈运动并没有阻止在靠近磁体的应用领域盛行细胞生长。 <img alt="图1" src="/files/ftp_upload/55567/55567fig1.jpg" /> 图1.磁化的miR-修改内皮细胞和他们的分析生产中的示意图。的PEI / MNP（聚乙烯亚胺/基于超顺磁性纳米颗粒载体）被施加到传送微RNA（MIR）到人脐静脉内皮细胞（HUVEC）。将得到的细胞产物的特征在于如下参数:安全（包括细胞活力，功能两者均，以及用于细胞间通信容量）;转染效率（ 即，的miR吸收效率和之后的功能）;磁中的静态和动态模拟条件体外靶向;磁共振成像（MRI）检测。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55567/55567fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55567/55567fig2.jpg" /> 图2.表征分离的HUVEC的。与内皮CD31标记染色分离培养的HUVEC A.代表性图像。细胞核复用DAPI（蓝色）。比例尺为20μm。对分离的内皮细胞进行的管形成测定的B.代表性结果;使用激光扫描共聚焦显微镜图像被记录（帧间差ference对比度）上基底膜基质细胞接种后18小时。比例尺为100μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55567/55567fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55567/55567fig3.jpg" /> 图3. 交货的miR人脐静脉内皮采用PEI / MNP。 A.标记的miR的摄取（Cy3的红色）24小时后转染细胞和正常细胞的外观的维护（的Cy3标记的miR，NP 25和MNP 25微克/毫升的2.5皮摩尔/ cm 2）的被示出。图像使用一个514-nm激发激光（Cy3标记，红色）和微分干涉对比采取共聚焦激光扫描显微镜。比例尺为50μm。 B.细胞生存力24小时转染后，通过流式细胞术评估。该图表示HUVEC得到的结果S采用以下复杂组合物处理:的Cy3的miR的2.5皮摩尔/厘米2; NP比25补充有5至25微克/毫升的MNP的。杆描绘的平均数量的活细胞和整个细胞群体之间的比率（N = 6;误差条:SEM）。 3色的C.结构照明显微镜（SIM）成像标记的由血管内皮细胞吸收的miR / PEI / MNP络合物。如上所述，温育转染所述细胞，和固定24小时后处理;细胞核用DAPI复染。原始SIM数据记录在Z-栈和处理;代表图像是最大强度投影的结果。下面激发激光器施加:405纳米（DAPI，蓝色），488纳米（Atto488-PEI，橙），565纳米（Atto565-MNP，青色）和633纳米（Cy5的-MIR，红色）。比例尺为5μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55567/55567fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55567/55567fig4.jpg" /> 图4的HUVEC变形例的安全与的miR / PEI / MNP。 48小时后处理; A.甲管形成测定用转染的细胞（NP 25和MNP 25微克/毫升SCR-的miR的2.5皮摩尔/ cm 2）的执行。图像用激光扫描共聚焦显微镜获取（ 即，微分干涉对比）上基底膜基质细胞接种后18个小时。用的miR / PEI转染的HUVEC被用作一个控制毒性和未处理的细胞作为阳性对照。比例尺为100μm。该图反映了转染的HUVEC和使用若干参数未处理的细胞之间的比例:管长度，分枝数目，和结（n = 3时，误差条:SEM）。 B.的miR / PEI / MNP改性的HUVEC的间隙连接介导的细胞间通信，评价24小时后transfec灰。为了这个目的，将细胞装载有GJ可渗透染料钙黄绿素（橙色）;光漂白（FRAP）后荧光恢复是通过在Z-堆叠扫描测试细胞在三维检查。钙黄绿素装载的细胞的代表性图像漂白之前，直接漂白后，并在15分钟后的脱色（用回收的荧光）被描绘。比例尺为20μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55567/55567fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55567/55567fig5.jpg" /> 图5.细胞磁化从与PEI / MNP及其应用转染得到的。过滤的MNP的A.形象代表，与TEM拍摄，说明他们的集群形态。一个小尺寸 - 单氧化铁颗粒（导致超顺磁性）的（〜5 15纳米）由此证实。的比例尺为100nm。 B.转染的细胞的细胞内装载物通过磁性粒子光谱（MPS）24小时后转染进行评估。代表面板描绘检查样品的MPS光谱。特别是，纯MNP悬浮液（50μL）作为参照（蓝色正方形）;圆圈是两个转染的细胞样品（红色圆圈:MNP 25微克/毫升;绿色圆圈:MNP 5微克/毫升）的MPS光谱，而灰色三角表示没有任何样品通过测量获得的检测到的背景光谱。注意的幅度（A）的对数标度。 C.磁性修饰的细胞的量通过转染的HUVEC的应用定量（2.5皮摩尔/ cm 2的的miR; NP 25补充了5-25微克/毫升的MNP），通过胰蛋白酶消化24小时后处理收集到的磁细胞分离柱。将得到的磁性阳性分数（ 即，残留在柱）进行计数，其百分比出转染细胞的整个量的被反映在曲线图对于每个MNP浓度（红色三角形）。细胞活力随后通过台盼蓝排除测定（灰色菱形）检查。数据表示为平均值±SEM（N = 3）。转染的HUVEC的D.说明性MRI图像（300,000个细胞; 2.5皮摩尔/ cm 2的的miR; NP 25 25微克/毫升的MNP）嵌入到琼脂糖幻像记录有7.1Ť动物MRI系统。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55567/55567fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/55567/55567fig6.jpg" /> 图6. 在模拟动态条件的miR / PEI / MNP-HUVEC的 体外 磁性靶向。血管内皮细胞收集24小时后转染离子（2.5皮摩尔/厘米的Cy3的miR的2，NP 25 MNP 25微克/毫升），并重新接种在孔中的新鲜培养基，与本地连接到所述侧壁上的小磁铁。反过来，这培养板固定到摇床上以150rpm旋转。细胞附着和生长进行12个小时后评估通过用PFA固定细胞，用DAPI染色它们的原子核，并进行激光扫描共聚焦显微镜（激发激光器:405纳米，DAPI，蓝色; 514纳米，CY3，橙色）。代表性图像描绘与磁铁申请区域的细胞生长;无需磁铁应用;并在培养物的中心孔，其中，液体流被浓缩细胞。该比例尺是50微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55567/55567fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells at JoVE.com

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

这个手稿描述的miR的有效的，非病毒递送通过PEI / MNP矢量和它们的磁化与内皮细胞。因此，除了遗传修饰，该方法允许用于磁性细胞的指导和MRI可检测性。该技术可以被用来改善的治疗性细胞产品的特性。

制备和<em&gt;体外</em&gt;磁化的表征的miR-改性内皮细胞

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

preparation-vitro-characterization-magnetized-mir-modified

Nanyang Technological University

Research

JoVE Journal

Medicine

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

7.6K 次观看.  University of Rostock. 这个手稿描述的miR的有效的，非病毒递送通过PEI / MNP矢量和它们的磁化与内皮细胞。因此，除了遗传修饰，该方法允许用于磁性细胞的指导和MRI可检测性。该技术可以被用来改善的治疗性细胞产品的特性。 

视频: Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection3-6.
Initially, cornea donor posterior lamellar dissection for DSEK was performed manually1 resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing.
Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in terms of the quality of the tissue7 or patient outcomes8,9 using eye bank precut tissue versus surgeon-prepared tissue for DSAEK surgery. For most corneal surgeons, the availability of precut DSAEK corneal tissue saves time and money10, and reduces the stress of performing the donor corneal dissection in the operating room. In part because of the ability of the eye banks to provide high quality posterior lamellar corneal in a timely manner, DSAEK has become the standard of care for surgical management of corneal endothelial disease.
The procedure that we are describing is the preparation of the posterior lamellar cornea at the eye bank for transplantation in DSAEK surgery (Figure 1).

Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.

供体角膜内皮角膜移植术的组织准备

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full ...

科研

医学

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 ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

作为脑微血管内皮细胞（CEND）的伸展<em&gt;体外</em创伤性脑损伤模型血脑屏障

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche.&#160;The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines.&#160;Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions.&#160;Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs.&#160;Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions.&#160;However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly.&#160;CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs.&#160;Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques.&#160;In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system.&#160;This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions.

在体外的方法来观察派生病人和人内皮细胞前列腺循环肿瘤细胞之间的E-选择素介导的相互作用

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to ...

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, along with its basement membrane (Descemet&#39;s membrane) is introduced onto the recipient&#39;s posterior stroma3. Unlike Descemet&#8217;s Stripping Automated Endothelial Keratoplasty (DSAEK), where additional donor stroma is introduced, no unnatural stroma-to-stroma interface is created. As a result, the natural anatomy of the cornea is preserved as much as possible allowing for improved recovery time and visual acuity4. Endothelial Keratoplasty (EK) is the procedure of choice for treatment of endothelial dysfunction. The advantages of EK include rapid recovery of vision, preservation of ocular integrity and minimal refractive change due to use of a small, peripheral incision1. DSAEK utilizes donor tissue prepared with partial thickness stroma and endothelium. The rapid success and utilization of this procedure can be attributed to availability of eye-bank prepared precut tissue. The benefits of eye-bank preparation of donor tissue include elimination of need for specialized equipment in the operating room and availability of back up donor tissue in case of tissue perforation during preparation. In addition, high volume preparation of donor tissue by eye-bank technicians may provide improved quality of donor tissue. DSAEK may have limited best corrected visual acuity due to creation of a stromal interface between the donor and recipient cornea. Elimination of this interface with transplantation of only donor Descemet&#39;s membrane and endothelium in DMEK may improve visual outcomes and reduce complications after EK5. Similar to DSAEK, long term success and acceptance of DMEK is dependent on ease of availability of precut, eye-bank prepared donor tissue. Here we present a stepwise approach to donor tissue preparation which may reduce some barriers eye-banks face in providing DMEK grafts.

Endothelial keratoplasty is a surgical technique continuously evolving as advancements result in transplantation of thinner grafts with each succession1. Here we present a technique for controlled manual tissue dissection to allow safe and repeatable separation of endothelium and Descemet&#39;s membranes from donor corneoscleral buttons for transplantation2.

角膜供体组织准备后弹力层内皮角膜移植术

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, ...

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

在小鼠大肠传入结尾的体外功能研究

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

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 ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

分离培养犬从动脉和静脉主血管内皮细胞

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

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 ...