Name: establishment and characterization of small bowel neuroendocrine tumor spheroids
Uploaded: 2019-10-14T13:00:00
Description: Watch this Scientific Journal Video about Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids at JoVE.com

这项工作得到了NIH授予P50 CA174521（J.R.豪和A.M.贝利齐）的支持。P.H. Ear 是 P50 CA174521 职业提升计划奖的获得者。

所有使用人类神经内分泌肿瘤样本的实验都已获得爱荷华大学医院和诊所IRB委员会的批准（协议号199911057）。所有材料和设备的列表在材料表中描述。增长介质和关键解决方案的列表见表1。
1. 小肠神经内分泌肿瘤（SBNET）采集和细胞分离
<ol><li>从外科病理学核心确认肿瘤组织后，获取切除的患者SBNET样本。</li>
	<li>将 SBNET 切成 5 mm 立方体，并储存在...

<ol>
	<li>Maxwell, J. E., Sherman, S. K., Howe, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+Diagnostics+and+Therapeutics+in+Pancreatic+Neuroendocrine+Tumors.">Translational Diagnostics and Therapeutics in Pancreatic Neuroendocrine Tumors.</a> Clinical Cancer Research. 22, 5022-5029 (2016).</li><li>Pfragner, 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=Establishment+of+a+continuous+cell+line+from+a+human+carcinoid+of+the+small+intestine+(KRJ-I).">Establishment of a continuous cell line from a human carcinoid of the small intestine (KRJ-I).</a> International Journal of Oncology. 8, 513-520 (1996).</li><li>Kolby, 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=A+transplantable+human+carcinoid+as+model+for+somatostatin+receptor-mediated+and+amine+transporter-mediated+radionuclide+uptake.">A transplantable human carcinoid as model for somatostatin receptor-mediated and amine transporter-mediated radionuclide uptake.</a> American Journal of Pathology. 158, 745-755 (2001).</li><li>Van Buren, G., 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+development+and+characterization+of+a+human+midgut+carcinoid+cell+line.">The development and characterization of a human midgut carcinoid cell line.</a> Clinical Cancer Research. 13, 4704-4712 (2007).</li><li>Pfragner, 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=Establishment+and+characterization+of+three+novel+cell+lines+-+P-STS,+L-STS,+H-STS+-+derived+from+a+human+metastatic+midgut+carcinoid.">Establishment and characterization of three novel cell lines - P-STS, L-STS, H-STS - derived from a human metastatic midgut carcinoid.</a> Anticancer Research. 29, 1951-1961 (2009).</li><li>Ellis, L. M., Samuel, S., Sceusi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Varying+opinions+on+the+authenticity+of+a+human+midgut+carcinoid+cell+line--letter.">Varying opinions on the authenticity of a human midgut carcinoid cell line--letter.</a> Clinical Cancer Research. 16, 5365-5366 (2010).</li><li>Hofving, T., 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+neuroendocrine+phenotype,+genomic+profile+and+therapeutic+sensitivity+of+GEPNET+cell+lines.">The neuroendocrine phenotype, genomic profile and therapeutic sensitivity of GEPNET cell lines.</a> Endocrine Related Cancer. 25, 367-380 (2018).</li><li>Moreno, 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=Antitumor+activity+of+rapamycin+and+octreotide+as+single+agents+or+in+combination+in+neuroendocrine+tumors.">Antitumor activity of rapamycin and octreotide as single agents or in combination in neuroendocrine tumors.</a> Endocrine Related Cancer. 15, 257-266 (2008).</li><li>Broutier, 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=Culture+and+establishment+of+self-renewing+human+and+mouse+adult+liver+and+pancreas+3D+organoids+and+their+genetic+manipulation.">Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation.</a> Nature Protocols. 11, 1724-1743 (2016).</li><li>Saito, 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=Establishment+of+Patient-Derived+Organoids+and+Drug+Screening+for+Biliary+Tract+Carcinoma.">Establishment of Patient-Derived Organoids and Drug Screening for Biliary Tract Carcinoma.</a> Cell Reports. 27, 1265-1276 (2019).</li><li>Park, S. 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=Detection+of+bone+marrow+metastases+of+neuroblastoma+with+immunohistochemical+staining+of+CD56,+chromogranin+A,+and+synaptophysin.">Detection of bone marrow metastases of neuroblastoma with immunohistochemical staining of CD56, chromogranin A, and synaptophysin.</a> Applied Immunohistochemisty and Molecular Morphology. 18, 348-352 (2010).</li><li>Clifton-Bligh, R. 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=Improving+diagnosis+of+tumor-induced+osteomalacia+with+Gallium-68+DOTATATE+PET/CT.">Improving diagnosis of tumor-induced osteomalacia with Gallium-68 DOTATATE PET/CT.</a> The Journal of Clinical Endocrinology and Metabolism. 98, 687-694 (2013).</li><li>Clinton, J., McWilliams-Koeppen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation,+Expansion,+and+Cryopreservation+of+Human+Primary+Tissue-Derived+Normal+and+Diseased+Organoids+in+Embedded+Three-Dimensional+Culture.">Initiation, Expansion, and Cryopreservation of Human Primary Tissue-Derived Normal and Diseased Organoids in Embedded Three-Dimensional Culture.</a> Current Protocols in Cell Biology. 82, 66 (2019).</li><li>Markovits, J., Roques, B. P., Le Pecq, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethidium+dimer:+a+new+reagent+for+the+fluorimetric+determination+of+nucleic+acids.">Ethidium dimer: a new reagent for the fluorimetric determination of nucleic acids.</a> Analytical Biochemistry. 94, 259-264 (1979).</li><li>Weeber, F., Ooft, S. N., Dijkstra, K. K., Voest, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Organoids+as+a+Pre-clinical+Cancer+Model+for+Drug+Discovery.">Tumor Organoids as a Pre-clinical Cancer Model for Drug Discovery.</a> Cell Chemical Biology. 24, 1092-1100 (2017).</li><li>Sachs, N., 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=A+Living+Biobank+of+Breast+Cancer+Organoids+Captures+Disease+Heterogeneity.">A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity.</a> Cell. 172, 373-386 (2018).</li><li>Puca, 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=Patient+derived+organoids+to+model+rare+prostate+cancer+phenotypes.">Patient derived organoids to model rare prostate cancer phenotypes.</a> Nature Communication. 9, 2404 (2018).</li><li>Singh, S. P., 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=SSTR2-based+reporters+for+assessing+gene+transfer+into+non-small+cell+lung+cancer:+evaluation+using+an+intrathoracic+mouse+model.">SSTR2-based reporters for assessing gene transfer into non-small cell lung cancer: evaluation using an intrathoracic mouse model.</a> Human Gene Therapy. 22, 55-64 (2011).</li></ol>

肿瘤3D培养物已成为临床前药物检测的宝贵资源。各种肿瘤有机体生物库最近已建立从乳腺癌和前列腺癌肿瘤16，17。在这项研究中，我们为培养SBNET作为球体提供了详细的方案，以及一种简单快捷的方法，通过免疫荧光和测试药物敏感性验证NET标记物的球体培养。根据我们的经验，SBNET球体可以在各种文化媒介中成长。它们在干细胞培?...

小肠神经内分泌肿瘤（SBNETs）起源于小肠的肠色素细胞。虽然SBNET通常已知生长缓慢，但它们通常转移至肝脏1。虽然手术切除或肿瘤消融可以考虑在许多情况下，复发几乎是普遍的，因此，药物治疗在管理中起着重要的作用。已经投入了巨大的努力来产生新的SBNET细胞系用于药物检测。然而，收效甚微。只报告了6个SBNET细胞系（KRJ-I、CND2、GOT1、P-STS、L-STS、H-STS）。不幸的是，一个细胞系不再表示NET标记6和三个其他SBNET细胞系（KRJ-I，L-STS，H-STS）被确定为从转化淋巴细胞，而不是NET7派生。为了加速识别针对SBNET的药物，需要采用替代方法进行体外药物检测。
在这里，我们利用被切除的SBNET的可用性，并建立了一种方法，培养这些患者衍生的SBNET作为在ECM中生长的球体。本手稿的总体目标是将一种将 SBNET 培养为三维 （3D） 培养方法描述为一种三维 （3D） 培养方法，并概述通过免疫荧光染色和免疫组织化学来描述这些球体以保留 SBNET 标记的过程。
此外，我们演示了这些SBNET球体如何可用于测试拉帕霉素的效果，一种抗癌药物NET8。该协议背后的原理是开发一种在体外培养SBNET细胞的新方法，并将其用于药物测试。与建立SBNET细胞系的传统方法相比，该技术的优点是可以快速获得SBNET的3D培养物，并在3周内进行药物测试。SBNET球体可能被用作执行体外药物屏幕的模型，以识别SBNET患者的新药。由于SBNET细胞系并不广泛可用，SBNET球体的3D培养物可以作为研究SBNET的新体外模型，并可在该领域的科学家之间共享。

<table border="0" cellpadding="0" cellspacing="0" style="width:1009px;" width="1009">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Anti-rabbit FITC</td>
			<td style="width:196px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">11-095-152</td>
			<td style="width:437px;">Secondary antibody couple to a green fluorophore</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antigen Retrieval Solution</td>
			<td>Agilent Dako</td>
			<td style="width:119px;">S2367</td>
			<td>Solution at pH 9 for preparing slides for IHC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Autostainer Link 48</td>
			<td>Agilent Dako</td>
			<td style="width:119px;">Not Available</td>
			<td>Automated system for antibody staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell freezing container</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td>5100-0001</td>
			<td>Container to for freezing cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">CellSence</td>
			<td style="width:196px;">Olympus</td>
			<td style="width:119px;">Version 1.18</td>
			<td>Computer software for using fluorescent microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromogranin A antibody</td>
			<td>Abcam-45179</td>
			<td style="width:119px;">RB-9003-PO</td>
			<td>Antibodies for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromogranin A antibody (clone LK2H10)</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td>MA5-13096</td>
			<td>Antibodies for IHC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase</td>
			<td style="width:196px;">Sigma</td>
			<td style="width:119px;">C0130</td>
			<td>Enzyme for digesting tumor tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:196px;">Gibco</td>
			<td style="width:119px;">11965-092</td>
			<td>Medium for tissue preparation</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">DMEM/F12</td>
			<td style="width:196px;">Gibco</td>
			<td style="width:119px;">11320-033</td>
			<td>Medium for organoid cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td style="width:196px;">Sigma</td>
			<td>D8418</td>
			<td>Solvent for dissolving drug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNAse</td>
			<td style="width:196px;">Sigma</td>
			<td style="width:119px;">DN25</td>
			<td>Enzyme for digesting tumor tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethidium Homodimer</td>
			<td style="width:196px;">Chemodex</td>
			<td style="width:119px;">CDX-E0012-T1E</td>
			<td>DNA and RNA binding dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td style="width:196px;">Gibco</td>
			<td style="width:119px;">16000044</td>
			<td>Reagent for culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Fluorescent microscope</td>
			<td style="width:196px;">Olympus</td>
			<td style="width:119px;">CKX35</td>
			<td>Microscope for taking pictures of SBENT spheroids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamine</td>
			<td style="width:196px;">Gibco</td>
			<td style="width:119px;">A2916801</td>
			<td>Reagent for culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">ImageJ</td>
			<td style="width:196px;">National Institutes of Health</td>
			<td style="width:119px;">Version 1.51</td>
			<td>Computer software for image analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin</td>
			<td style="width:196px;">Sigma</td>
			<td style="width:119px;">I0516</td>
			<td>Reagent for culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel</td>
			<td style="width:196px;">Corning</td>
			<td>356235</td>
			<td>Matrix to embed and anchore organoids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting medium (VECTASHIELD)</td>
			<td style="width:196px;">Vector Laboratories</td>
			<td>H-1200</td>
			<td>Fixative for labelled-cells with a nuclear stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nicotinamide</td>
			<td style="width:196px;">Sigma</td>
			<td>72340</td>
			<td>Reagent for culture media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Paraformaldehyde</td>
			<td style="width:196px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td>Reagent to fix cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PEN/STREP</td>
			<td style="width:196px;">Gibco</td>
			<td style="width:119px;">15140-122</td>
			<td>Reagent for culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PT Link</td>
			<td>Agilent Dako</td>
			<td style="width:119px;">Not Available</td>
			<td>Automated system to prepare slides for IHC staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rapamycin</td>
			<td style="width:196px;">Alfa Aesar</td>
			<td style="width:119px;">J62473</td>
			<td>Drug that can inhibit NET growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Secondary antibodies for IHC</td>
			<td style="width:196px;">Agilent Dako</td>
			<td style="width:119px;">K8000</td>
			<td>Secondary antibodies for IHC using Polymer-based EnVision FLEX system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SSTR2 antibody</td>
			<td style="width:196px;">GeneScritp</td>
			<td style="width:119px;">A01591</td>
			<td>Antibodies for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SSTR2 antibody (clone UMB1)</td>
			<td style="width:196px;">Abcam</td>
			<td>ab134152</td>
			<td>Antibodies for IHC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synaptophysin antibody</td>
			<td style="width:196px;">Abcam</td>
			<td>32127</td>
			<td>Antibodies for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synaptophysin antibody (clone DAK-SYNAP)</td>
			<td>Agilent Dako</td>
			<td style="width:119px;">M7315</td>
			<td>Antibodies for IHC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TritonX</td>
			<td style="width:196px;">Mallinckrodt</td>
			<td style="width:119px;">3555 KBGE</td>
			<td>Reagent to permeablize cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Y-2763 ROCK inhibitor</td>
			<td style="width:196px;">Adipogen</td>
			<td style="width:119px;">AG-CR1-3564-M005</td>
			<td>To improve SBNET spheroid viability after freeze thaw</td>
		</tr>
	</tbody>
</table>

establishment and characterization of small bowel neuroendocrine tumor spheroids

小肠神经内分泌肿瘤（SBNETs）是肠道肠色素细胞的罕见癌症。这一领域的研究是有限的，因为很少患者衍生的SBNET细胞系被生成。分化良好的SBNET细胞生长缓慢，难以繁殖。已经建立的少数细胞系不是现成的，在培养中一段时间后可能无法继续表达NET细胞的特性。产生新的细胞系可能需要很多年，因为SBNET细胞有很长的倍增时间，并且需要许多浓缩步骤来消除快速分裂的癌症相关成纤维细胞。为了克服这些限制，我们开发了一种方案，从手术切除的肿瘤中培养SBNET细胞，将其作为细胞外基质（ECM）中的球形体。ECM 形成一个三维矩阵，封装 SBNET 细胞，并模仿肿瘤微环境，允许 SBNET 细胞生长。在这里，我们描述了SBNET球体的生长速率，并描述了使用免疫荧光显微镜和免疫组织化学识别SBNET标记物的方法，以确认球体是神经内分泌肿瘤细胞。此外，我们使用SBNET球体来测试拉帕霉素的细胞毒性。

目前只有2个SBNET细胞系建立和出版2，3，4，5，它们不是现成的许多研究人员。在这里，我们建议将 SBNET 培养为 ECM 中的球体，并将其用作研究 SBNET 药物敏感性的替代模型。从转移到肝脏的SBNET患者衍生的肿瘤被收集，消化以释放SBNET细胞，并与液体ECM混合，以建立SBNET球体培养物（图1A...

Watch this Scientific Journal Video about Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids at JoVE.com

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

神经内分泌肿瘤（NETs）来自神经峰的神经内分泌细胞。它们生长缓慢，对文化充满挑战。我们提出了一个替代策略，通过培养它们作为球体，从小肠中种植NET。这些球体有小肠NET标记，可用于药物测试。

小肠神经内分泌肿瘤类球体的建立与特征

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

分化良好的SBNET细胞生长缓慢，难以繁殖。少数已建立的细胞系对研究人员不易获得。为了克服这些限制，我们开发了此 3D 培养协议。与传统建立SBNET细胞系的方法相比，该技术的优点是可以获得3D培养，并在三周内完成药物测试。该方法可应用于其他神经内分泌肿瘤，如胰腺神经内分泌肿瘤。在获得经过切除的患者 SBNET 样本后，将其切成 5 毫米立方体，并将其存放在 25 毫升 DMEM F12 介质中，用于锥形管运输。根据手稿说明在洗涤介质中孵育肿瘤15分钟。然后将它们转移到一个新的菜，用无菌弯曲的剪刀把它们切碎到不到一毫米的碎片。将切碎的组织转移到含有 25 毫升洗涤介质的新的 50 毫升管中，然后在 4 摄氏度下以 500 倍 g 离心样品，15 分钟。丢弃上一杯，用胶原酶和DNase将颗粒重新用10毫升的洗涤介质中重新暂停。让切碎的肿瘤在37摄氏度下消化，缓慢摇晃90分钟。重要的是不要过度消化肿瘤样本。太多的酶或更长的孵育时间会杀死SBNET细胞。消化完成后，在4摄氏度下以500倍g离心管15分钟。丢弃上一杯，将颗粒重新在 15 毫升的洗涤介质中。将 70 微米细胞过滤器放在新的 50 毫升管上，并通过过滤器转移 10 毫升的洗涤缓冲液。旋转管内的洗涤缓冲液，防止细胞粘在两侧。然后通过过滤器过滤细胞悬浮液。在4摄氏度下以500倍g离心细胞15分钟。丢弃上一杯，将颗粒重新在200微升洗涤介质中。将管子放在冰上，使用五微升细胞进行细胞计数，使用血细胞计。计算细胞数量，然后重复离心，以去除上清液，然后以每毫升一百万个细胞的浓度重新暂停液体 ECM 中的细胞。接下来，将 ECM 中 5 到 20 微升的 SBNET 球体转移到 96 井板中，将板放在 37 摄氏度的培养箱中，使液体 ECM 凝固。如前文所述，将200微升的SBNET培养培养剂添加到每个含有球状物质或培养干细胞培养的井中。使用 P1000 移液器将管体转移到 1.5 毫升管中，并在 1，500 次 g 下将管离心一分钟，以去除上流液。用一毫升 PBS 清洗培养物，然后再次离心以去除上一代。然后通过加入500微升的半甲醛并孵育15分钟来修复器官。孵育后，用一毫升PBS洗涤培养。添加 500 微升 PBS 和 3%BSA 和 0.1% Triton X-100 以渗透培养。孵育管5分钟，然后用1毫升PBS和3%BSA洗三次管。接下来，用原抗体孵育培养组一小时，然后用PBS和BSA重复洗涤。与二次抗体孵育并重复洗涤，确保吸气并丢弃所有上流剂。要对球体进行成像，请添加五个包含 DAPI 的安装介质微升，并使用 P20 移液器将球体的五微升转移到玻璃幻灯片中。使用荧光显微镜使用 10、20 和 40 倍目标用盖玻片和图像密封幻灯片。使用 P1000 移液器机械地断开 ECM，然后将其转移到无菌的 1.5 毫升管中。在4摄氏度下将管子以1000倍g离心。然后取出所有上一日，将管子放在冰上。将新 ECM 的体积增加 2 到 4 倍到颗粒，通过上下移液 10 次混合管内装物，确保避免冒气泡。将 ECM 和 SBNET 混合物的 5 到 20 微升转移到新的 96 井板中，使 ECM 凝固。然后用新的 SBNET 介质盖住凝固的 ECM，将板放入培养箱中。如果将 SBNET 球体运送到另一个实验室，则将带新 ECM 的球体转移到 T25 烧瓶中，并允许 ECM 凝固。用 SBNET 球状介质填充烧瓶，拧紧盖子，并准备装运包装。接收球体区域性后，删除区域性介质并重复前面的步骤，将球体放回区域性中。通过显微成像监测SBNET球体生长速率。当区域性介质每周更改一次时，SBNET 球体的大小需要大约 14 天才能翻倍。在培养14天后，SBNET球体的大小不会增加。球体沾染了特异性对抗突触物理、色球蛋白 A 和 SSTR2 的抗体，并使用免疫荧光显微镜进行成像。数据表明，这些标记在细胞质和SBNET细胞膜中局部化。为了确保抗体的特异性，在胰腺肿瘤的器官线上进行了相同的染色程序，该根体不表达突触物理、色罗酮A和SSTR2，并且未检测到绿色信号。免疫荧光成像的主要优点是，它可以在四小时内完成，并给出与免疫组织化学类似的染色信息。将SBNET培养为球体是识别可抑制SBNET生长的药物的宝贵技术。球类用拉帕霉素和mTOR抑制剂治疗。五天后，拉帕霉素处理球体形成葡萄状结构，成为凋亡或坏死。尝试此程序时，必须用洗涤介质预湿细胞过滤器和收集管。这将防止SBNET细胞粘附在细胞过滤器和塑料管上。通过该协议，科学家和临床医生可以从被切除的肿瘤中建立SBNET球状培养物，与其他实验室共享，并使用它们来测试FDA批准的药物。

Research

JoVE Journal

Cancer Research

Operating Procedures of the Electrochemotherapy for Treatment of Tumor in Dogs and Cats

在电化学疗法的操作程序在肿瘤治疗中的狗和猫

Electrochemotherapy (ECT) is a local approach which is used for treating solid tumors of different histologies. Its mechanism is based on cell membrane ...

科研

癌症研究

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

单细胞下游特征的靶细胞预富集与全基因组扩增

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

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 ...

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

高通量三维肿瘤球体药物筛选的生成

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

三维肿瘤球体的纵向形态和生理监测

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

免疫荧光对非小细胞肺癌患者循环肿瘤细胞的半自动PD-L1特征化及枚举

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids

用于肿瘤球体半高通量分析的快速光学清除

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the ...

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

评估从3D多细胞肺肿瘤球体中分离的癌症相关成纤维细胞的线粒体健康

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

从患者来源的肿瘤样本中建立三维微球并评估其对药物的敏感性

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

生成用于药物评估研究的 3D 肿瘤微球

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...