Name: isolation of stem-like cells from 3-dimensional spheroid cultures
Uploaded: 2019-12-13T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures at JoVE.com

这项研究得到了国家癌症研究所R01-CA172220（普惠制，WYH），R01-ES02207（普惠制，WYH）的资助。我们感谢芝加哥伊利诺伊大学的流式细胞学核心在细胞分拣方面给予的帮助。

所有细胞处理和介质制剂均应在 II 类生物安全柜 （BSC） 中采用无菌技术进行。
1. 二维HPrEC细胞的培养与维护
<ol><li>在室温 （RT） 下，用 2 mL 的 2.5 μg/cm2纤维内丁溶液涂抹 100 mm 培养皿。吸出溶液，让培养皿在BSC中晾干45分钟。 Fibroinin涂层培养皿可在4°C下储存2~4周。</li>
	<li>将9 mL的前列腺上皮细胞生长培养基（例如PrEGM）加入纤维素涂层100毫米培养皿中，并在37°C的...

<ol>
	<li>Leong, K. G., Wang, B. E., Johnson, L., Gao, W. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+prostate+from+a+single+cell.">Generation of a prostate from a single cell.</a> Nature. 456, 804-808 (2008).</li><li>Xin, L., Lukacs, R. U., Lawson, D. A., Cheng, D., Witte, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+and+multilineage+differentiation+in+vitro+from+murine+prostate+stem+cells.">Self-renewal and multilineage differentiation in vitro from murine prostate stem cells.</a> Stem Cells. 25, 2760-2769 (2007).</li><li>Hu, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen-initiated+transformation+of+prostate+epithelium+derived+from+normal+human+prostate+stem-progenitor+cells.">Estrogen-initiated transformation of prostate epithelium derived from normal human prostate stem-progenitor cells.</a> Endocrinology. 152, 2150-2163 (2011).</li><li>Hu, W. Y., Shi, G. B., Hu, D. P., Nelles, J. L., Prins, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actions+of+endocrine+disrupting+chemicals+on+human+prostate+stem/progenitor+cells+and+prostate+cancer+risk.">Actions of endocrine disrupting chemicals on human prostate stem/progenitor cells and prostate cancer risk.</a> Molecular and Cellular Endocrinology. 354, 63-73 (2012).</li><li>Collins, A. T., Berry, P. A., Hyde, C., Stower, M. J., Maitland, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+tumorigenic+prostate+cancer+stem+cells.">Prospective identification of tumorigenic prostate cancer stem cells.</a> Cancer Research. 65, 10946-10951 (2005).</li><li>Vander Griend, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+CD133+in+normal+human+prostate+stem+cells+and+malignant+cancer-initiating+cells.">The role of CD133 in normal human prostate stem cells and malignant cancer-initiating cells.</a> Cancer Research. 68, 9703-9711 (2008).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetrical+and+asymmetrical+division+analysis+provides+evidence+for+a+hierarchy+of+prostate+epithelial+cell+lineages.">Symmetrical and asymmetrical division analysis provides evidence for a hierarchy of prostate epithelial cell lineages.</a> Nature Communications. 5, 4758 (2014).</li><li>Neumuller, R. A., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dividing+cellular+asymmetry:+asymmetric+cell+division+and+its+implications+for+stem+cells+and+cancer.">Dividing cellular asymmetry: asymmetric cell division and its implications for stem cells and cancer.</a> Genes and Development. 23, 2675-2699 (2009).</li><li>Cicalese, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tumor+suppressor+p53+regulates+polarity+of+self-renewing+divisions+in+mammary+stem+cells.">The tumor suppressor p53 regulates polarity of self-renewing divisions in mammary stem cells.</a> Cell. 138, 1083-1095 (2009).</li><li>Klein, A. M., Simons, B. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+multipotent+luminal+progenitor+cells+in+human+prostate+organoid+cultures.">Identification of multipotent luminal progenitor cells in human prostate organoid cultures.</a> Cell. 159, 163-175 (2014).</li><li>Hu, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+functional+interrogation+of+adult+human+prostate+epithelial+cells+at+single+cell+resolution.">Isolation and functional interrogation of adult human prostate epithelial cells at single cell resolution.</a> Stem Cell Research. 23, 1-12 (2017).</li><li>Bryant, D. M., Stow, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ins+and+outs+of+E-cadherin+trafficking.">The ins and outs of E-cadherin trafficking.</a> Trends in Cell Biology. 14, 427-434 (2004).</li><li>Clevers, H., Loh, K. M., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+signaling.+An+integral+program+for+tissue+renewal+and+regeneration:+Wnt+signaling+and+stem+cell+control.">Stem cell signaling. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control.</a> Science. 346, 1248012 (2014).</li><li>Madueke, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+WNT10B+in+normal+prostate+gland+development+and+prostate+cancer.">The role of WNT10B in normal prostate gland development and prostate cancer.</a> The Prostate. 79 (14), 1692-1704 (2019).</li><li>Guan, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+stem+cells.">Autophagy in stem cells.</a> Autophagy. 9, 830-849 (2013).</li></ol>

使用多种干细胞表面标记的流式细胞测定是干细胞研究常用的方法，尽管缺乏特异性和选择性1，5，6。虽然3D培养系统中的球形形成是从原发上皮细胞（包括HPrEC）中丰富稀有干细胞群的另一种有用方法，但产生的球体仍然是茎和祖细胞2、3、4的异质混合物。</su...

人类前列腺含有具有分泌功能的明上皮，其基础细胞以及不寻常的神经内分泌细胞成分。上皮细胞，在这种情况下，产生于罕见的前列腺干细胞群，在体内相对静止，并作为修复系统，以维持腺平衡在整个生命1。尽管取得了许多进展，前列腺干细胞的鉴定和功能分离仍然是该领域的一大挑战。干细胞生物标志物，包括基于细胞表面标记的方法，结合流式细胞测定，通常用于干细胞研究2、3、4。然而，由于标记组合和抗体特异性5，6的函数，富集和分离的结果差异很大，对分离细胞的身份提出了疑问。另一种广泛使用的干细胞富集方法是三维（3D）球体培养2，3，4。虽然常驻干细胞在体内相对静止，具有利基约束，但它们在3D基质培养（对称和非对称）中经历细胞分裂，产生干细胞和祖细胞，迅速繁殖，向血统承诺7，8。形成球体是一种异质混合物，含有干细胞和祖细胞，处于不同谱系承诺阶段，包括早期和晚期祖细胞。因此，使用整个球体的测定不是干细胞独有的，使得独特的干细胞特性的鉴定没有定论。因此，创建检测以识别和分离其女儿后代的前列腺干细胞至关重要。为此，目前协议的目标是建立一个检测系统，以便有效地识别和分离人类前列腺组织的干细胞，然后对其生物功能进行强有力的下游分析。
长期5-溴-2'-脱氧尿碱（BrdU）标签保留被广泛用于体内和体外血统追踪干细胞基于其长期倍增时间9，10。本文描述的前列腺干细胞鉴定和分离的目前方法基于其相对静止特性和在混合上皮种群中的标签保留特性。此外，根据不朽的链DNA假说，只有干细胞可以进行不对称细胞分裂。干细胞代表含有较老父母DNA的子细胞，而祖细胞（即一个承诺子细胞）则接收新合成的DNA。上述独特的干细胞特性被利用在原发培养物的亲子干细胞中执行BrdU标记，然后在转移到3D无锚球体培养物后跟踪其标签。虽然大多数原发性前列腺上皮细胞在二维培养中保留基底和中转扩增表型，但也有罕见的多能干细胞补充和维持上皮平衡，这在转移到3、12时形成球形或完全分化的有机体，具有相应的培养基。在我们目前的协议中，通过使用HPrEC前列腺球体或基于前球的BrdU、CFSE或远红保留性检测，然后进行荧光活化细胞分拣（FACS），我们在单细胞13级识别球体中保留标签的干细胞。
重要的是，我们进一步确认了在早期球体中贴片细胞的干细胞特性，与具有血统承诺的祖细胞相比。这些包括干细胞不对称分裂、体外球体形成能力和体内组织再生能力、自噬活性升高、核糖体生物发生增强和代谢活性下降。随后，进行了RNAeq分析。观察到在标签保留球体细胞中表达的不同基因，这些基因可作为人类前列腺干细胞的新型生物标志物。这种基于球体的标签保留方法可应用于癌症标本，以类似地识别少量癌症干细胞，从而提供转化机会来管理治疗耐药人群13。下面介绍以人类原发性前列腺上皮细胞（HPrEC）为例，采用基于前层的标签保留测定。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL tuberculin syringes</td>
			<td>Bectin Dickinson</td>
			<td>BD 309625</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes, sterile</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm culture dishes</td>
			<td>Corning/Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well culture plate</td>
			<td>Corning/Falcon</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Corning/Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>22 x 22 mm coverslips, sq</td>
			<td>Corning</td>
			<td>284522</td>
			<td>For MatTek 35 mm culture dish</td>
		</tr>
		<tr>
			<td>24 x 50 mm coverslips</td>
			<td>Corning</td>
			<td>2975245</td>
			<td></td>
		</tr>
		<tr>
			<td>26G x 1.5 inch hypodermic needle</td>
			<td>Monoject</td>
			<td>1188826112</td>
			<td></td>
		</tr>
		<tr>
			<td>2N HCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm culture dish with cover glass bottom</td>
			<td>MatTek Corp</td>
			<td>P35G-0-10-C</td>
			<td>Glass bottom No. 0, uncoated, <img src="files/ftp_upload/60357/60357img1.jpg"/> irradiated</td>
		</tr>
		<tr>
			<td>40 &#181;m pore nylon cell strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>5% CO2 culture incubator, 37 &#176;C</td>
			<td>Forma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td>Corning/Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL Polystyrene Round-Bottom Tube with strainer snap cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td>35 &#181;m nylon mesh</td>
		</tr>
		<tr>
			<td>6-well culture plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well chamber slides</td>
			<td>Millipore Sigma</td>
			<td>PEZGS0816</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqueous mounting medium containing DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>A nuclear fluorescent dye</td>
		</tr>
		<tr>
			<td>Biological safety cabinet, Level 2 certified</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BrdU (5-bromo-2'-deoxyuridine)</td>
			<td>Sigma-Aldrich</td>
			<td>B5002</td>
			<td>1 mM stock solution in DMSO</td>
		</tr>
		<tr>
			<td>Centrifuge for 1.5 mL microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge for 15 mL tubes</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Allegra 6</td>
		</tr>
		<tr>
			<td>CFSE (carboxyfluorescein succinimidyl ester)</td>
			<td>Thermo Fisher Scientific</td>
			<td>C34554</td>
			<td>5 mM stock solution in DMSO</td>
		</tr>
		<tr>
			<td>cytochalasin D</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHZ1063</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase 1U/mL</td>
			<td>StemCell Technologies</td>
			<td>07923</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS CellSorter MoFlo XDP</td>
			<td>Beckman Coulter</td>
			<td>s</td>
			<td></td>
		</tr>
		<tr>
			<td>Far-Red pro-dye</td>
			<td>Thermo Fisher</td>
			<td>C34564</td>
			<td>5 mM stock solution in DMSO</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>F0895</td>
			<td>For coating 100 mm culture dishes</td>
		</tr>
		<tr>
			<td>Fluorescent microscope with color digital camera</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Axioskop 20 fluorescent microscope; color digital Axiocamera</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher</td>
			<td>A-11029</td>
			<td></td>
		</tr>
		<tr>
			<td>HPrEC (Primary normal human prostate epithelial cells)</td>
			<td>Lifeline Cell Technology</td>
			<td>FC-0038</td>
			<td>Pooled from 3 young (19-21yr od) disease-free organ donors; 1 x 105 cells/mL; stored in liquid nitrogen</td>
		</tr>
		<tr>
			<td>ice bucket and ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microsope with digital camera</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel, low growth factor, phenol-red free</td>
			<td>Corning</td>
			<td>356239</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Corning</td>
			<td>A452-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-BrdU antibody</td>
			<td>Cell Signaling</td>
			<td>5292S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG antibody (negative control)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2025</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Vector Laboratories</td>
			<td>S-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS), pH 7.4</td>
			<td>Sigma-Aldrich</td>
			<td>P5368-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettors and tips, various sizes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PrEGM (ProstaLife Epithelial Cell Growth Medium)</td>
			<td>Lifeline Cell Technology</td>
			<td>LL-0041</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide (PI)</td>
			<td>R &#38; D Systems</td>
			<td>5135/10</td>
			<td>10 &#956;g/mL PI in PBS stored at 4 &#176;C in the dark</td>
		</tr>
		<tr>
			<td>Serological pipets, various sizes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software for sphere counting and size measurements</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software: 3D images using Imaris an imageing software with freeform drawing capabilities</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Millipore Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath, 37 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>z-stack images using a transmitted light inverted fluorescent confocal microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of stem-like cells from 3-dimensional spheroid cultures

尽管成人干细胞研究取得了进展，但从组织标本中鉴定和分离干细胞仍然是一个重大挑战。虽然常驻干细胞在成人组织中具有利基约束，但它们进入无锚三维（3D）培养的细胞周期，并经历对称和非对称细胞分裂，产生干细胞和祖细胞细胞。后者迅速增殖，是处于不同谱系承诺阶段的主要细胞群，形成异质球体。使用原发正常的人类前列腺上皮细胞（HPrEC），开发了一种基于球体的标记保留测定，允许在单个细胞分辨率下识别和功能分离球形启动干细胞。
HPrEC 或细胞系与 BrdU 一起培养 10 天，以允许将其纳入所有分裂细胞的 DNA，包括自更新干细胞。洗涤开始转移到3D培养体5天，在此期间，干细胞通过不对称分裂自我更新，并启动球形形成。虽然相对静止的女儿干细胞保留了带有BrdU标记的亲子DNA，但女儿后代迅速增殖，失去了BrdU标签。BrdU可以用CFSE或远红亲染料替代，允许FACS进行活干细胞分离。干细胞特性通过体外球形形成、体内组织再生测定和记录其对称/不对称细胞分裂来确认。下游分子和生物研究，包括RNA-seq、ChIP-seq、单细胞捕获、代谢活性、蛋白酶分析、免疫细胞化学、有机体形成和体内组织再生，可以严格探究分离的标记保留干细胞。重要的是，这种无标记的功能干细胞分离方法从新的癌症标本和来自多个器官的癌细胞系中识别干细胞样，表明其广泛适用性。它可用于识别癌症干细胞样生物标志物，筛选针对癌症干细胞的药物，并发现癌症中新的治疗靶点。

原发正常人前列腺上皮细胞被放入纤维素涂层培养皿中，细胞生长在二维培养中保持（图1a）。当转移到具有基底膜基质的3D培养中时，分化的上皮细胞慢慢消失。只有前列腺干细胞才能在无锚培养物中存活，并在5天内形成球形（图1b）。
在2D培养中前列腺上皮细胞的双重标记，随后在3D培养中形成球形，?...

Watch this Scientific Journal Video about Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures at JoVE.com

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

我们使用人类原发性前列腺上皮细胞，报告一种新的无生物标记方法，通过基于球体的标签保留测定，对干细胞进行功能表征。介绍了 BrdU、CFSE 或远红色 2D 细胞标记的分步协议;三维球形形成;通过免疫细胞化学进行标签保留干细胞鉴定;和隔离由FACS。

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 ...

干细胞分离仍然是一个主要的挑战，因为使用当前方法共同隔离祖先细胞。我们开发了一种新型无生物标志物的检测方法，从混合祖体中分离出静态干细胞。这种基于球形的标签保留分析的主要优点是，它可以利用CFSE或远红标签，通过FACS从其子祖先中扩展和分离活干细胞。虽然传统疗法可以根除大多数前列腺癌细胞，但癌症干细胞仍然由于治疗阻力而存在，从而推动疾病进展。干细胞样细胞分离将允许识别新的基因，这些基因可以在治疗上共同定位，以有效缓解疾病。我们的标签保留测定适用于从各种组织和细胞中分离的正常干细胞，以及癌症干细胞研究。重要的是，这种测定适用于其他上皮细胞癌，如乳腺癌和结肠直肠癌。首先用两毫升2.5微克纤维素溶液涂上100毫米培养盘，并在室温下孵育过夜。然后吸气溶液，让培养皿在生物安全柜中晾干45分钟。将前列腺上皮细胞生长介质的9毫升加入纤维素涂层的盘中，并在37摄氏度的二氧化碳培养箱中保持温暖。在37摄氏度的水浴中解冻一小瓶冷冻人类前列腺上皮细胞，并在10毫升温暖介质中重新给细胞。将电池悬浮液以500倍g离心5分钟。然后吸气并丢弃上一液。将细胞重新悬浮在一毫升温暖的培养基中，用预热培养基将悬浮液的一毫升直接转移到纤维素涂层的盘中。然后在37摄氏度的二氧化碳培养箱中孵育。如果共同标记细胞，则准备 10 天 2D 培养的 BrdU 标记细胞，如文本手稿中所述。然后加入5微摩尔CFSE或远红，孵育细胞30分钟。孵育后，用染料小心吸气介质，用五毫升热 PBS 清洗细胞两次。接下来，根据手稿指示，使用05%的三辛EDTA进行酶消化。将细胞以500倍g离心5分钟，然后丢弃上一代。细胞在冰冷的一对一地下室膜基质和培养基组合中重新被重新吸收，用于3D前球的形成。要在 3D 地下室基质系统中准备表层培养，请将地下室膜基质在四摄氏度下解冻，并在使用前将基底膜基质放在冰上。然后轻轻加入一毫升的冰冷基底膜基质，加入同等量的冰冷培养基中。上下移液混合，注意不冒气泡。在冰冷的一对一地下室膜基质和培养基质组合中，将5倍10至1/4的人类前列腺上皮细胞重新吸收，总体积为500微升。将溶液移入每井的底部边缘，并旋转板以均匀分布混合物。将板放在37摄氏度的二氧化碳培养箱中30分钟，使基质凝固。然后覆盖它每井一毫升温暖的培养介质，确保不打扰环。要收获 CFSE 标记的 prostasphere，请用两毫升的分散和移液器上下混合多次，以替代介质。在37摄氏度下孵育板30分钟，消化基质，然后将球体混合物收集到15毫升离心管中。将球体以 500 倍 g 离心五分钟，然后丢弃上一代。将颗粒重新放入 500 微升加热 05% trypsin EDTA 中，并将它们转移到 1.5 毫升离心管中。在37摄氏度下孵育球体5分钟，然后加入500微升热PBS，10%FBS。用26米针通过一毫升注射器，完全分离球体。将细胞以500倍g离心5分钟。然后吸气并丢弃上一液。将细胞重新用一毫升温暖的培养基中，用每毫升碘化钠一微克来染色死细胞，并在室温下孵育一分钟。重复离心，丢弃上经。然后用一毫升的温介质清洗细胞。重复离心，丢弃上经。将细胞重新用一毫升温暖的培养基重新暂停，并通过35微米孔径细胞滤株卡帽过滤，将细胞收集在五毫升聚苯乙烯圆形底部管中。对多普辛分散的CFSE标记的蛋白酶球细胞执行FACS分析。使用非标记细胞作为负对对，使用 CFSE 标记的细胞作为正对照，以设置门，运行样本以排序和收集分馏 CFSE 高和 CFSE 低细胞的子组。原发性正常人前列腺上皮细胞被放入纤维素涂层培养皿中，细胞生长在二D培养中保持。在通过基底膜基质转移到3D培养物时，分化的上皮细胞慢慢消失，只剩下前列腺干细胞。2D培养中前列腺上皮细胞的双重标记，3D培养中的球体形成，显示了BrdU、CFSE和远红在同一标签保留细胞中的结离。双免疫着色表明，标签保留细胞表现出较低的细胞角蛋白14水平，减少细胞结蛋白E-cadherin，增加干细胞早期标记蛋白Wnt10b和ALDH1A1，增加自噬蛋白LC3，增加米奥辛国际IB。此外，基于球形的标签保留检测成功检测前列腺癌标本中的癌症干细胞。CFSE标记保留干细胞和球体从人类前列腺癌标本中衍生出的球体显示的E-cadherin蛋白相对于非标记的祖细胞减少。在尝试此协议时，保持液体形式的矩阵对于培养介质制备非常重要。在四度下过夜，在配药矩阵之前将移液器提示冷藏在冰冷的PBS中。使用基于球类的标签保留测定分离干细胞后，可以进行单细胞RNA测序和蛋白质组分析，以确定干细胞中的特定基因和新型生物标志物。通过基于球体的标签保留检测分离活体癌症干细胞，使研究人员有可能在体外生长癌症干细胞衍生的肿瘤，并筛选出新型抗癌药物。

Research

JoVE Journal

Cancer Research

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

巨蟹座的建立源于人类传统的骨肉瘤细胞培养

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

科研

癌症研究

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

利用功能基因组筛选识别在肿瘤细胞球体培养潜在的新药物靶标

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

3维球形共培养胰腺癌细胞和成纤维细胞的制备及代谢试验

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

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 ...

A Spheroid Killing Assay by CAR T Cells

一种用汽车 t 细胞进行的切杀检测

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

三维细胞培养中胰腺肌囊内增生的模拟与操纵

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

评估癌细胞3D球形培养中的细胞活力和死亡

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres

大肠癌HT29衍生癌症干细胞类肿瘤球中的驱动基因发现

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

在结肠直肠癌细胞的三维球体培养物中使用无细胞益生菌超常剂评估细胞死亡

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...