Name: isolation of exosome-enriched extracellular vesicles carrying granulocyte-macrophage colony-stimulating factor from embryonic stem cells
Uploaded: 2021-11-11T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Exosome-Enriched Extracellular Vesicles Carrying Granulocyte-Macrophage Colony-Stimulating Factor from Embryonic Stem Cells at JoVE.com

我们感谢阿尔卡迪乌斯·斯卢萨尔奇克先生和肯塔基生物医学研究基础设施网络（KBRIN，P20GM103436）获得传输电子显微镜图像。这项工作得到了NIH AA018016-01（J.W.E.）、肯塔基联邦研究挑战信托基金（J.W.E.）、NIH CA106599和CA175003（C.L.）、NIH CA198249（K.Y.）和自由呼吸研究补助金（K.Y.）的资助。

1. 培养ES-D3细胞
<ol><li>要生成无外显子的母牛血清 （FBS），在 4 °C 下以 100，000 x g 的速度将 FBS 装载到超中心和离心机中，持续 16 小时。 离心后，收集血清超高纳特作为无外奥体的FBS，用于培育粘膜ESC细胞系ES-D3和获得富含外体的EV。</li>
	<li>在电镀ES-D3细胞之前，在室温下使用明胶（0.1%）涂抹15厘米的组织培养皿30分钟。</li>
	<li>按照先前描述的协议16，培养ES-D3细胞没?...

<ol>
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本研究展示了一种高效的方法，可以产生携带免疫刺激蛋白GM-CSF的富含外体的EV，可用于研究富含外体的电动汽车的免疫调节作用。一些研究表明，外显体表现出免疫调节和抗肿瘤功能22。因此，来自表达转基因-CSF的ESC的外显子可能也具有调节免疫反应的生物活动。在本协议中，外源性粘液GM-CSF通过转染在木质ES-D3细胞中稳定过度表达（图1）。重要的是，?...

ESC来自植入前胚胎1的胚泡阶段。作为多能干细胞，ESC有能力自我更新并分化成任何类型的胚胎细胞。由于其显著的发展潜力和长期增殖能力，ESC对生物医学研究具有极其宝贵的价值。目前的研究工作主要集中在ESC治疗各种重大病理疾病的潜力，包括糖尿病，心脏病和神经退行性疾病2，3，4。
众所周知，哺乳动物细胞，包括ESC，会向细胞外环境释放大小不一的囊泡，这些EV由于在细胞间通信中的作用而具有许多生理和病理功能。在不同的子类型EV中，外显子是小膜囊从不同细胞类型释放到细胞外空间后融合的中间内分泌室，多血管体（MVB），与等离子膜6。据报道，外显体可以调解细胞间的交流，并严重参与许多（病理）生理过程7，8。外显子从它们自己的母细胞中继承了一些生物功能，因为外体含有从细胞溶胶中获取的生物材料，包括蛋白质和核酸。因此，相关的抗原或刺激特定疾病免疫反应的因素被封装在特定类型的细胞9的外源中。这为探索肿瘤衍生外显体作为抗癌疫苗10的临床试验铺平了道路。
GM-CSF是由不同类型的免疫细胞11分泌的细胞因子。新出现的证据表明，GM-CSF激活和调节免疫系统，并在抗原呈现过程中发挥着至关重要的作用。例如，一份临床报告表明，GM-CSF刺激免疫反应肿瘤作为疫苗辅助剂13。在14日的临床试验中，研究了几种基于GM-CSF的癌症免疫治疗策略，以利用GM-CSF强大的免疫刺激活性。其中，由辐照GM-CSF分泌肿瘤细胞组成的癌症疫苗通过诱导细胞和幽默抗肿瘤反应以及随后转移肿瘤15中的坏死，在晚期黑色素瘤患者中显示出一定的希望。
由于来自ESC的外显子具有与原始ESC相似的生物活动，因此可能来自ESC的转基因-CSF携带的外体可以起到无细胞囊泡的作用，以调节免疫反应。本文介绍了从ESC中生产出高质量外体浓缩电动汽车的详细方法，该方法表达了GM-CSF。这些富含外体的电动汽车有可能作为免疫调节囊泡来调节免疫反应。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alkaline phosphate, Calf Intestinal</td>
			<td>New England Biolabs</td>
			<td>M0290S</td>
			<td>Dephosphorylating DNA plasmid</td>
		</tr>
		<tr>
			<td>anti-Annexin V mAb</td>
			<td>Santa Cruz Biotechnology</td>
			<td>clone H-3, sc-74438</td>
			<td>Western blot, RRID:AB_1118989</td>
		</tr>
		<tr>
			<td>anti-CD81 mAb</td>
			<td>Santa Cruz Biotechnology</td>
			<td>clone B-11, sc-166029</td>
			<td>Western blot, RRID:AB_2275892</td>
		</tr>
		<tr>
			<td>anti-cytochrome c mAb</td>
			<td>Santa Cruz Biotechnology</td>
			<td>clone A-8, sc-13156</td>
			<td>Western blot, RRID:AB_627385</td>
		</tr>
		<tr>
			<td>anti-Flotillin-1 mAb</td>
			<td>Santa Cruz Biotechnology</td>
			<td>clone C-2; sc-74566</td>
			<td>Western blot, RRID:AB_2106563</td>
		</tr>
		<tr>
			<td>anti-GAPDH pAb</td>
			<td>Rockland</td>
			<td>600-401-A33S</td>
			<td>Western blot, RRID:AB_11182910</td>
		</tr>
		<tr>
			<td>anti-mouse IgG, goat, peroxidase-conjugated</td>
			<td>Thermo Fisher</td>
			<td>31430</td>
			<td>Western blot, RRID:AB_228307</td>
		</tr>
		<tr>
			<td>anti-Oxphos COX IV-subunit IV mAb</td>
			<td>Thermo Fisher</td>
			<td>clone 20E8C12 A21348</td>
			<td>Western blot, RRID:AB_221509</td>
		</tr>
		<tr>
			<td>anti-protein disulfide isomerase (PDI) pAb</td>
			<td>Enzo</td>
			<td>ADI-SPA-890</td>
			<td>Western blot, RRID:AB_10616242</td>
		</tr>
		<tr>
			<td>anti-rabbit IgG, goat, peroxidase-conjugated</td>
			<td>Thermo Fisher</td>
			<td>31460</td>
			<td>Western blot, RRID:AB_228341</td>
		</tr>
		<tr>
			<td>BCA (bicinchoninic acid) assay</td>
			<td>Thermo Fisher</td>
			<td>23223</td>
			<td>Determining protein concentrations</td>
		</tr>
		<tr>
			<td>Bis-Tris PAGE Gel, ExpressPlus, 4-20%</td>
			<td>Genscript</td>
			<td>M42015</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Carbenicillin, Disodium Salt</td>
			<td>Thermo Fisher</td>
			<td>10177012</td>
			<td>Selecting E. coli colonies</td>
		</tr>
		<tr>
			<td>Centrifuge, Avanti J-26 XPI</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Low speed centrifugation</td>
		</tr>
		<tr>
			<td>Centrifuge rotor, JA-10</td>
			<td>Beckman Coulter</td>
			<td>09U1597</td>
			<td>Low speed centrifugation</td>
		</tr>
		<tr>
			<td>Centrifuge bottle, Nalgene PPCO</td>
			<td>Thermo Fisher</td>
			<td>3120-0500PK</td>
			<td>Low speed centrifugation</td>
		</tr>
		<tr>
			<td>Cu grids with carbon support film</td>
			<td>Electron Microscopy Sciences</td>
			<td>FF200-Cu</td>
			<td>Acquiring electron microscopy images</td>
		</tr>
		<tr>
			<td>EcoRI</td>
			<td>New England Biolabs</td>
			<td>R0101</td>
			<td>Digesting DNA plasmid</td>
		</tr>
		<tr>
			<td>Enhanced chemiluminescence detection system</td>
			<td>Thermo Fisher</td>
			<td>32106</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>FACScalibur flow cytometer</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td>Examining GFP levels of ES-D3 cells</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>ATCC</td>
			<td>SCRR-30-2020</td>
			<td>Medium for ES-D3 cells</td>
		</tr>
		<tr>
			<td>Fisherbrand Sterile Cell Strainers; Mesh Size: 40&#956;m</td>
			<td>Thermo Fisher</td>
			<td>22-363-547</td>
			<td>Filtering ES-D3 cells for FACS sorting</td>
		</tr>
		<tr>
			<td>Gelatin (0.1%)</td>
			<td>Thermo Fisher</td>
			<td>ES006B</td>
			<td>Culturing ES-D3 cells</td>
		</tr>
		<tr>
			<td>GM-CSF ELISA kit</td>
			<td>Thermo Fisher</td>
			<td>88733422</td>
			<td>Determining GM-CSF concentrations</td>
		</tr>
		<tr>
			<td>KnockOut Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td>Thermo Fisher</td>
			<td>10-829-018</td>
			<td>Medium for ES-D3 cells</td>
		</tr>
		<tr>
			<td>Leukemia Inhibitory Factor</td>
			<td>Thermo Fisher</td>
			<td>ESG1106</td>
			<td>Medium for ES-D3 cells</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>VWR</td>
			<td>VWRL0131-0100</td>
			<td>Medium for ES-D3 cells</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 transfection reagent</td>
			<td>Thermo Fisher</td>
			<td>11668019</td>
			<td>Transfecting ES-D3 cells</td>
		</tr>
		<tr>
			<td>Microplate reader, PowerWave XS</td>
			<td>BioTek</td>
			<td></td>
			<td>Determining GM-CSF concentrations</td>
		</tr>
		<tr>
			<td>MoFlo XDP high-speed cell sorter</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Isolating single ES-D3 cell clones</td>
		</tr>
		<tr>
			<td>NEB 5-alpha Competent E. coli</td>
			<td>New England Biolabs</td>
			<td>C2988J</td>
			<td>Generating GM-CSF expression plasmid</td>
		</tr>
		<tr>
			<td>Neomycin</td>
			<td>Thermo Fisher</td>
			<td>10-131-035</td>
			<td>Selecting ES-D3 clones</td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Thermo Fisher</td>
			<td>SH3023801</td>
			<td>Medium for ES-D3 cells</td>
		</tr>
		<tr>
			<td>Non-fat dry milk</td>
			<td>Thermo Fisher</td>
			<td>NC9022655</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher</td>
			<td>31985062</td>
			<td>Transfecting ES-D3 cells</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Acquiring electron microscopy images</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>VWR</td>
			<td>sc45000-652</td>
			<td>Medium for ES-D3 cells</td>
		</tr>
		<tr>
			<td>Plasmid pEF1a-FD3ER-IRES-hrGFP</td>
			<td>Addgene</td>
			<td>37270</td>
			<td>Generating GM-CSF expression plasmid</td>
		</tr>
		<tr>
			<td>PVDF membranes</td>
			<td>Millipore EMD</td>
			<td>IPVH00010</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (250)</td>
			<td>QIAGEN</td>
			<td>27106</td>
			<td>Generating GM-CSF expression plasmid</td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit (50)</td>
			<td>QIAGEN</td>
			<td>28704</td>
			<td>Generating GM-CSF expression plasmid</td>
		</tr>
		<tr>
			<td>Quick Ligation Kit</td>
			<td>New England Biolabs</td>
			<td>M2200S</td>
			<td>Generating GM-CSF expression plasmid</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>Hitachi</td>
			<td>HT7700</td>
			<td>Acquiring electron microscopy images</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>VWR</td>
			<td>45000-660</td>
			<td>Culturing ES-D3 cells</td>
		</tr>
		<tr>
			<td>Ultracentrifuge, OptimaTM L-100 XP</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>High speed centrifugation</td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotor, 45Ti</td>
			<td>Beckman Coulter</td>
			<td>09U4454</td>
			<td>High speed centrifugation</td>
		</tr>
		<tr>
			<td>Ultracentrifuge polycarbonate bottle</td>
			<td>Beckman Coulter</td>
			<td>355622</td>
			<td>High speed centrifugation</td>
		</tr>
		<tr>
			<td>UranyLess staining solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>22409</td>
			<td>Acquiring electron microscopy images</td>
		</tr>
	</tbody>
</table>

isolation of exosome-enriched extracellular vesicles carrying granulocyte-macrophage colony-stimulating factor from embryonic stem cells

胚胎干细胞（ESCs）是多能干细胞，能够自我更新和分化到所有类型的胚胎细胞。像许多其他细胞类型一样，ESC 将小膜囊泡（如外显子）释放到细胞外环境中。外显子是细胞间交流的重要中介，在许多（病理）生理过程中发挥着基本作用。粒细胞-巨噬细胞群刺激因子（GM-CSF）作为细胞因子调节免疫反应。GM-CSF在外显子中的存在有可能增强其免疫调节功能。在这里，GM-CSF在穆林ESC细胞系ES-D3中被稳定地过度表达。开发了一个协议，将高质量的外体富集细胞外囊泡（EV）与ES-D3细胞分离出来，从而过度表达GM-CSF。孤立的外体丰富的电动汽车的特点是各种实验方法。重要的是，在富含外显子的电动汽车中发现了大量GM-CSF。总的来说，来自ESC的具有转基因-CSF的外生丰富型电动汽车可以作为无细胞囊泡来发挥其免疫调节作用。

GM-CSF 在穆林 ESC 中表达过度。 为了在ES-D3细胞中稳定地过度表达GM-CSF，将MM-CSF cDNA克隆成一个转染载体，以生成表达载体pEF1+-mGM-CSF-IRES-hrGFP（图1A）。通过转染，GM-CSF在ES-D3细胞中表达过度，约20%的瞬态瞬态瞬态ES-D3细胞为GFP阳性。细胞克隆稳定过度表达GM-CSF或空矢量控制被FACS收购。如图1 B</stron...

Watch this Scientific Journal Video about Isolation of Exosome-Enriched Extracellular Vesicles Carrying Granulocyte-Macrophage Colony-Stimulating Factor from Embryonic Stem Cells at JoVE.com

Isolation of Exosome-Enriched Extracellular Vesicles Carrying Granulocyte-Macrophage Colony-Stimulating Factor from Embryonic Stem Cells

这项研究描述了一种将携带免疫刺激性粒细胞巨噬细胞刺激因子的外细胞富集细胞外囊泡从胚胎干细胞中分离的方法。

分离外体富集的细胞外囊泡携带颗粒细胞-巨噬细胞-胚胎干细胞的细胞外刺激因子

Embryonic stem cells (ESCs) are pluripotent stem cells capable of self-renewal and differentiation into all types of embryonic cells. Like many other cell ...

我们的协议可用于从胚胎干细胞中产生高质量的外体浓缩细胞外囊泡，表达免疫刺激因子，GM-CSF。外体富集的细胞外囊携带GM-CSF，有可能提供无细胞免疫调节囊泡，可以调节免疫反应。接受过基本分子和细胞生物学训练的研究人员应该能够像这个协议一样轻松，但是任何第一次执行这个协议的人都应该密切关注这个方向。由于我们的协议是复杂的，可视化每一步的复杂细节将有助于其他研究人员产生外体免费FBS，超中心融合FBS所需的体积，并收集外体免费超高纳特。在电镀ESD三细胞之前，在室温下用0.1%的明胶涂抹15厘米的组织培养皿30分钟。在 37 摄氏度的 5% 二氧化碳加湿培养器中，通过吸入和培养 ESD 三个没有支线层细胞的 ESD 三细胞去除明胶，直到细胞达到 90% 的汇流。用每道菜0.05%的5毫升洗涤几乎汇流的培养物，然后在37摄氏度的新鲜试丁香中孵育5分钟。在孵化结束时，将分离的细胞拉入离心机管中，用五毫升的新鲜培养基灭活试剂。通过离心沉积细胞，并在新鲜介质中重新悬挂托盘，用于计数以进行传递， 板五倍10至第六ESD三细胞在15毫升的细胞培养介质到新的明胶涂层板三天的培养之前，分栽培细胞收集细胞培养超高分子分离外体富集细胞外囊板1倍10至第七ESD三细胞在15每块新的明胶涂层板的细胞培养介质的毫升在收集细胞培养超分子之前三天，为了外体富集额外的细胞囊泡分离，首先通过离心从72小时培养ESD三个细胞的超分子中沉积出大细胞片段。收集超高分子后，超中心将样品放样并丢弃超纳特。根据制造商的说明，每次洗涤用一毫升PBS轻轻冲洗每片片，去除任何残留培养物超高分子，并用Bicinchonin酸检测量化外体富集的细胞外囊素蛋白质含量。然后以每微米浓度六微克的浓度重新悬挂 PBS 中富含体的细胞外囊，以零下 80 摄氏度储存。通过传输电子显微镜可视化外体富集的细胞外囊泡，每毫升细胞外囊泡修复三到五微克，最终浓度为 2%电子显微镜网格双酚醛，在室温下为两小时。在孵化结束时，用碳支持膜将固定样品的 10 微升加载到铜网格上一分钟，然后用滤纸排出网格，根据制造商的协议，使用适当的染色溶液留在网格中，并使用钳子将网格转移到一块滤纸上， 然后根据标准协议，使用放大5万倍的传输电子显微镜获取电子显微镜图像。要准备整个细胞提取物，从培养物中收集ESD三个细胞，并重新暂停PBS中收获的细胞进行计数。第二次离心后，将每个 SDS 页面加载缓冲器的 5 倍 10 到第三个细胞重新悬挂到包含 0.5% SDS 浓度的微升波器中，并将样品在 10% 振幅的声波器上进行 10 秒的声波处理。然后在100摄氏度下加热样品5分钟。为准备外体富集的细胞外囊泡的裂解剂，在 SDS 页面加载缓冲中重新暂停外体浓缩的细胞外囊泡，该缓冲器以每微升浓度 1.2 微克的速度包含 0.5% SDS，并将样品进行 10 秒的声波测量，然后在 100 摄氏度下加热 5 分钟。对于西 Blot 分析，将 10 微升批发提取物和外体富集的细胞外囊裂解剂样品装载到最佳应力页凝胶的单独井中。在运行结束时，将蛋白质转移到PVDF膜上，并用适当的一级和二级抗体孵育膜，然后根据制造商的说明使用增强的化学发光检测套件检测蛋白质表达。评估外体富集细胞外囊中的GM-CSF量， 使用套件 Murine GM-CSF 涂上 ELISA 板与抗 GM-CSF 捕获抗体，仅用 100 微升 PBS 或 PBS 加 0.05%tween 20 在室温下处理 0.6 微克外体富集细胞外囊泡样品 30 分钟。在孵化结束时，将经过处理的样品添加到准备好的 ELISA 板的单独井中，在室温下孵育一小时，然后单独用 PBS 或 PBS 加 0.05%tween 20 清洗相应的井。洗涤后，在样品中加入检测抗体，在室温下孵育一小时，然后用PBS或PBS加0.05%tween 20进行洗涤，然后在样品中加入Avidin马萝卜过氧化酶，在室温下孵育30分钟，然后在450纳米的微板读卡器上清洗并测量每口井的吸水量。GM-CSF 表达 ESD 三细胞线或 ESD 三细胞线表示空矢量的 GFP 荧光强度远远高于其父母的荧光强度。ELISA显示，ESD三个表达GM-CSF的细胞在其细胞培养中产生的GMCSF水平明显高于空载体控制细胞。此外，GM-CSF表达ESD三个细胞产生的GM-CSF量与表达GM-CSF的STO成纤维细胞产生的量相似。从移位载体和GM-CSF转导细胞培养物中分离出的细胞外囊的传输电子显微镜揭示了不同尺寸的囊泡，这些囊泡的直径在预期的30至100纳米范围内。与西方斑点分析的相应批发提取物相比，细胞外囊泡中外切除标记（包括CD81、附素V和浮石-1）的表达明显增强。重要的是，在ESD中未检测到其他亚细胞间标记物的三个衍生细胞外囊泡，包括内质质标记、蛋白质脱硫异酶、线粒体标记、细胞色素C和Oxphos复合IV-子单元IV和细胞溶性标记GAPDH。洗涤0.05%tween 20后，在控制细胞外囊泡中检测到的背景GM-CSF水平显著降低。相比之下，GM-CSF表达细胞的细胞外囊中的GM-CSF水平显著提高了20。获得携带 GMCSF 的高品质外体浓缩细胞外囊泡是本协议成功最重要的步骤。研究人员可以进行额外的实验，如研究转基因-CSF依赖细胞系和动物，以确定外体丰富的细胞外囊囊护理GM-CSF是否可以作为无细胞免疫调节囊泡。然后，这些外体丰富的细胞外囊泡可用于探索免疫反应的调节如何影响不同的疾病条件。

Research

JoVE Journal

Cancer Research

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

在斑马鱼胚胎人乳腺癌细胞的侵袭行为

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

科研

癌症研究

Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples

利用DNA染色体引发的侧群体表型，从生物样品中检测和纯化癌细胞

Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells ...

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

骨肉瘤前小鼠模型对肿瘤与间充质干细胞细胞外囊泡介导通信的定义

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

来自沙柯玛患者衍生异种移植物的肿瘤启动细胞的分离和表征

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

正常结肠和结直肠癌患者内皮细胞的分离--一种改进的协议

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

骨髓间充质干细胞在浆组织中的分离及与癌细胞联合培养研究其相互作用

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Extraction of Extracellular Vesicles from Whole Tissue

全组织细胞外囊的提取

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or ...

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

突变黑色素细胞干细胞引发小鼠黑色素瘤的时空控制

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

从3维球体培养中分离出类似干细胞的细胞

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...