这项工作是由卫生研究第1阶段临床医生，科学家奖（AG），一个加拿大学院和操作格兰特从癌症研究协会（AG）的支持。作者感谢约翰·迪克博士对他的支持这个项目。

该协议的示意图示于图1。 1。人肝癌样本的处理取得初级人类肝癌标本，并以书面征得患者同意，并与机构研究伦理委员会的批准。这些协议已经进行了在我们的机构与大学健康网络研究伦理委员会批准，符合人类福祉的所有机构，国家和国际准则。 尽快收集新鲜肝癌标本下一次合适的样本已经用于临床目的的外科手术。理想情况下这应该从患者体内取出组织后需要在30分钟内到位。如示于图2中，至少1cm 3的肿瘤的周围得到的样品是最优的，因为肿瘤的中心部分可以是坏死。还没有接收到任何治疗之前，切除诸如放疗，化疗，或消融肿瘤是优选的，为了最大限度地提高的可能性，肿瘤细胞是可行的。处理的主要人体组织按照与生物危险品标准的个人防护协议。进行肿瘤组织和细胞制剂的所有实验室操作使用无菌技术II类生物安全柜。 <ol><li>将新鲜的HCC样本中10-25毫升的无血清的Dulbecco改良Eagle培养基/火腿的F12，在4℃并在冰转移到实验室进行立即处理和制备的组织碎片和/或细胞对异种移植到小鼠中。 </li><li>用无菌镊子，将肿瘤样品在一个100毫米×20毫米的培养皿或其它合适的无菌的工作​​表面。肿瘤样本分成约2-3毫米3用10号手术刀刀片碎片。此时，考虑管理单元冻结或福尔马林固定S而其他实验青梅肿瘤碎片或分析的要求。 </li><li>对肿瘤片段的异种移植中，将一些肝癌片段到含有足够的Matrigel以允许碎片保持淹没一个或多个微量离心管中。置于冰上这些管子。 </li><li>用于制备肿瘤细胞悬浮液，可使用外科手术刀刀片剁碎其余肝癌组织尽可能并用5-10毫升的DMEM-F12的混合在50ml锥形管这取决于组织糜的体积。 </li><li>加胶原酶IV型和分散酶II的最终浓度为200单位/ ml和0.8单位/毫升分别。吸取混合物上下以及用25毫升吸管。 </li><li>密封该管并从步骤1.6孵育混合物在37℃下在5％二氧化碳培养箱中培养30-60分钟取决于肿瘤组织的柔软度。移液器的混合物上下几次每10分钟，以评估酶消化的进展。 </li><li>二后拥塞完成后，通过100微米的细胞过滤通过肿瘤的解决方案。轻轻捣碎用25毫升移液管尖，以使肿瘤细胞的最大数量通过在细胞过滤器的剩余组织。收集应变细胞悬浮在15毫升锥形管中。 </li><li>离心肿瘤细胞悬浮液在1,200 rpm离心5分钟，在4℃下</li><li>轻轻倒出上清液。取决于颗粒的大小，加2-5毫升冰冷的1X红血细胞（RBC）的裂解缓冲液，轻轻地吹吸，以重悬沉淀。置于冰上5分钟。 </li><li>添加的DMEM-F12的15毫升离心的总体积为1,000 rpm离心5分钟，在4℃下洗出的RBC裂解缓冲液。 </li><li>轻轻倒出上清液，重悬在DMEM-F12的红细胞游离肿瘤细胞沉淀。 </li><li>无论是手动或自动细胞计数器采用台盼蓝染色计数活细胞。 </li><li>将含有所需NUMBE离心机肿瘤细胞等分细胞为如上所述的注塑R，重悬所得的细胞沉淀中加入30μl冰冷的基底膜，并在冰上储存。 </li></ol> 选择性：1.11步骤之后，我们经常从大块肿瘤细胞悬浮液耗尽人类CD45 +细胞（白细胞）和/或使用流式细胞术或免疫磁珠纯化的肿瘤细胞亚群。对这些技术的详细方案由相关抗体，珠子，和流式细胞仪的制造商有很好的描述。 注意：该协议如上所述，也可用于处理来自小鼠，以执行连续移植收获的人类肿瘤异种移植物，取代的异种移植物组织为在步骤1.1的初级人HCC组织中。在此情况下，步骤1.11后，我们经常消耗浸润的细胞悬浮液鼠细胞利用抗体对小鼠组织相容性抗原H2K。 2。异种移植开展符合所有动物的程序与批准的机构动物护理委员会的协议。本文所述的程序已经在按照和遵守所有相关法规和体制机构，规定和准则批准大学健康网络动物保护委员会的特定动物使用协议完成。 设备交付吸入挥发性麻醉剂的小动物应根据动​​物设施和研究机构的标准作业程序予以确认。开展利用无菌技术和无菌器械的II类生物安全柜的所有外科手术。利用非肥胖糖尿病重症联合免疫缺陷（NOD / SCID）或NOD / SCID /白细胞介素2受体γ链空（NSG）的菌株任一性别的小鼠6-8周龄（杰克逊实验室，缅因州巴港） 9,10。这些老鼠必须被容纳并保持在能够提供适合于免疫缺陷动物无病原体条件的设施。 在腔室中于1L /氧的分供给5％（体积/体积）异氟醚吸入制备小鼠进行手术。维持麻醉，直到有角膜和脚趾反射损耗在动物（S）。对于皮下异种移植，剃一个或多个小区域上的动物（S）的背部和清洗用70％乙醇的皮肤。肝内异种移植，从腋窝下至腹股沟区域剃除动物（S）的腹侧胸部和腹部和清洗，用70％乙醇在皮肤上。 肿瘤片段的2.1皮下植入<ol><li>将麻醉的小鼠易发生，保持吸入异氟醚麻醉组（2％（V / 1升V）/分钟O 2）与喉舌。适用撕裂凝胶，保护动物的眼睛免受外伤。 </li><li>用优碘消毒背部剃光面积（S）手术擦洗后70％的乙醇，最后用聚维酮碘溶液。 </li><li>使用无菌锋利的剪刀做5毫米的皮肤切口。 </li><li>插入一个封闭钝剪刀轻轻插入皮下空间和蔓延轻轻地开发一个口袋大到足以容纳一个肿瘤片段。 </li><li>插入使用无菌细镊子准备步骤1.3到皮下口袋肿瘤片段。 </li><li>关闭使用缝线或夹子皮肤切口。 </li><li>提供术后护理，以如下所述的鼠标。 </li></ol> 2.2皮下注射肿瘤细胞<ol><li>准备按照步骤2.1.1和2.1.2中所述的动物。 </li><li>加载的肿瘤细胞在基质胶制备步骤1.13到胰岛素注射器用29 G 1/2针头的悬浮液。 </li><li>将针头插入皮下空间和排出注射器的内容物。推进沿皮下平面针几毫米远神父OM穿刺部位皮肤可以防止肿瘤细胞悬液泄漏出来的穿刺部位后撤出针的。 </li><li>提供术后护理，以如下所述的鼠标。 </li></ol>肿瘤片段的2.3肝内植入<ol><li>在1毫升注射器针头在使用27 G 1/2，管理350μl，以麻醉动物的脖子背部无菌生理盐水皮下注射，以弥补术中流体的损失。镇痛，管理350微升无菌正常生理盐水含丁丙诺啡（0.1毫克/千克）皮下注射对动物的侧面，采用了27G的1/2-英寸的针在1毫升注射器。 </li><li>将鼠标仰卧在预加热垫与位于所述嘴件内以提供维护吸入异氟醚麻醉（2％（体积/ 1升五）/分钟O 2）的鼻子和嘴。 </li><li>延长四肢及它们固定用胶带给作业面优化曝光腹腹部和胸部。 </li><li>下一个放大镜灯进行程序优化的可视化。 </li><li>消毒的腹侧的腹部和胸部的皮肤剃光用聚维酮碘外科擦洗后70％的乙醇，最后用聚维酮碘溶液。 </li><li>使用无菌锋利的剪刀，作一横双边肋缘下皮肤切口并划分肌肉层进入腹膜腔，使整个肝脏足够的曝光。 </li><li>放置一个缝在上面剑突皮肤，并固定用胶带为了喉舌，以便更好地暴露肝脏及周围结构。 </li><li>使用相邻的两个棉签涂药，后到肝脏以使其稳定。 </li><li>做一个切口3毫米的长度和深度用无菌10号解剖刀刀片肝脏的表面上。 </li><li>立即申请的Surgicel和温和的压力，切口部位，以达到止血;后60-90秒删除并继续只有彻底止血已经实现。 </li><li>将肿瘤片段的步骤1.3进入肝脏切口用无菌细镊子或用18号针头。 </li><li>取一小片的Surgicel超过肝脏切开，以防止肿瘤片段的位移，保证持续止血。 </li><li>关闭切口缝合或剪辑。 </li><li>提供如下所述的术后护理</li></ol>肿瘤细胞通过直接注射到肝脏2.4肝内植入<ol><li>准备在2.3.1的步骤描述2.3.7鼠标在上面。 </li><li>加载的肿瘤细胞悬浮液（在步骤1.13的方法制备）为使用一个29 G 1/2在针的胰岛素用注射器。 </li><li>用棉尖涂药与肝企稳，将胰岛素注射器针头进入肝脏，推进嘴伸入穿刺部位沿包膜下飞机几毫米。 </li><li>轻轻地排出注射器和t中的内容母鸡从肝脏取出针。 </li><li>这里的Surgicel在穿刺部位和应用用棉签轻轻按压，以防止肿瘤细胞悬浮液的泄漏，并实现完全止血。 </li><li>关闭切口缝合或剪辑，并提供如下所述的术后护理。 </li></ol>肿瘤细胞通过注射入脾胃2.5异种移植肝内<ol><li>作为准备步骤2.3.1描述2.3.5鼠标在上面。 </li><li>使用无菌剪刀尖锐，使1厘米左肋下切口进入腹腔。 </li><li>用棉签对颅和动物的右侧反映胃，以揭露脾。 </li><li>通过处理周围的脂肪组织，用无菌细无创伤钳，交付脾插入切口，然后将棉签涂药脾后面以使其稳定。 </li><li>用5-0丝线缝合，地点围绕上述下极脾宽松预绑结。 </li><li>加载的肿瘤细胞悬浮液（在步骤1.13的方法制备）为使用一个29 G 1/2在针的胰岛素用注射器。 </li><li>将胰岛素注射器针头插入脾脏下极，推进它过去的宽松预先系结的水平。 </li><li>慢慢地排出注射器的内容物，从脾脏取出针，并拧紧结，以防止注射肿瘤细胞悬浮液的任何泄漏。 </li><li>更换脾进入腹腔。 </li><li>关闭切口缝合或剪辑，并提供如下所述的术后护理。 </li></ol> 2.6部分肝切除术，以促进人类肿瘤组织肝内移植植入<ol><li>作为准备步骤2.3.1描述2.3.7鼠标。 </li><li>用无菌剪刀尖锐，分附着在肝脏的中叶镰状韧带。 </li><li>用棉签，调动绕左叶5-0丝线缝合肝和位置的松脱预先系在一起的结的左叶。推进松散的结尽可能接近朝着左叶胆血管蒂，然后拧紧结。 </li><li>用无菌剪刀，留下一个小树桩，防止结，随后出血打滑切除左叶远端结扎。 </li><li>使用类似的技术，结扎并切除大部分肝脏中叶，避免了胆囊。 </li><li>使用的Surgicel和温和的压力沿肝实质的切割表面棉签涂药，达到完全止血。 </li><li>对于肿瘤片段或肿瘤细胞的异种移植肝内，继续进行步骤2.3.8至2.3.11或2.4.2至2.4.5如上所述。 </li><li>对于肿瘤细胞经脾注射肝内异种移植，使用棉签涂药轻轻地反映胃颅和动物的右侧，exposi纳克脾。继续进行步骤2.5.4至2.5.9如上所述。 </li><li>关闭切口缝合或剪辑，并提供如下所述的术后护理。 </li></ol> 2.7术后护理<ol><li>从吸入麻醉喉舌移开鼠标。 </li><li>放置在笼子里的老鼠在大约20分钟的加热灯，直到从麻醉中恢复，并动员全。 </li><li>在第一次手术后2-3天重复上述丁丙诺啡剂量为每8-12小时。 </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+and+liver+drug+uptake+following+hepatic+artery+and+portal+vein+infusion.">Tumor and liver drug uptake following hepatic artery and portal vein infusion.</a> J. Clin. Oncol. 5, 1836-1840 (1987).</li><li>Mitchell, C., Willenbring, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reproducible+and+well-tolerated+method+for+2/3+partial+hepatectomy+in+mice.">A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice.</a> Nat. Protoc. 3, 1167-1170 (2008).</li><li>Michalopoulos, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration+after+partial+hepatectomy:+critical+analysis+of+mechanistic+dilemmas.">Liver regeneration after partial hepatectomy: critical analysis of mechanistic dilemmas.</a> Am. J. Pathol. 176, 2-13 (2010).</li><li>Zhang, D. Y., Friedman, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrosis-dependent+mechanisms+of+hepatocarcinogenesis.">Fibrosis-dependent mechanisms of hepatocarcinogenesis.</a> Hepatology. 56, 769-775 (1002).</li><li>Chen, K., Ahmed, S., Adeyi, O., Dick, J. E., Ghanekar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+solid+tumor+xenografts+in+immunodeficient+mice+are+vulnerable+to+lymphomagenesis+associated+with+Epstein-Barr+virus.">Human solid tumor xenografts in immunodeficient mice are vulnerable to lymphomagenesis associated with Epstein-Barr virus.</a> PLoS One. 7, e39294 (2012).</li></ol>

作者宣称，他们有没有竞争的财务权益。

我们已经描述了多种技术来建立皮下和肝内人类肝癌异种移植物中，可以应用到各种各样的实验问题，并测定免疫缺陷小鼠。而皮下异种移植物已被广泛用于研究肝癌生物学的各个方面，肝内异种移植物在文献中很少描述。此外，大多数研究描述了使用异种移植物的从良好建立的细胞系产生的这些。给定的肿瘤细胞系中模拟人类肿瘤生物学12的限制，我们一直激励，以验证上述利用原代人...

肝细胞癌（HCC）是全球第五大最常见的癌症和癌症死亡的北美最迅速增加的原因。肝细胞癌的最普遍的危险因素是肝硬化，最经常是由于慢性病毒性肝炎，酒精滥用，自身免疫性疾病，或遗传性代谢紊乱1发生。 尽管在全球范围内的人群所施加肝癌沉重的疾病负担，肝癌的病理生理了解，相较于其他常见的癌症如大肠癌，乳腺癌或前列腺癌相对较差。例如，特定的分子和细胞事件驱动肿瘤的发生仍有待明确界定2。像大多数其他固体上皮癌，基因组学方法揭示异质性与肝癌3相关的像差。许多研究已经揭示了多种参与细胞增殖，外加信号传导途径的紊乱的活性存活力，分化和血管生成4。此外，癌症干细胞在HCC病理生物学中的作用仍有待阐明5。 与肝癌病理生理的了解有限，为有效的治疗肝癌的医疗设备也仍然相对有限。早期患者肿瘤局限于肝脏是适合采用肿瘤切除或手术切除根治性治疗，但复发是常见的。为病人提供更先进的疾病，化疗和放疗是有限的功效，并主要用于与姑息意图6疾病控制。 高品质的人类肝癌的体内实验模型从而提供了一个宝贵的平台，为急需的基础研究到人类肝癌的病理生理，以及对新的治疗方法的评价。由于与使用的细胞系或高度定义的小鼠模型，PRI的移植比较玛丽人类肿瘤的免疫缺陷小鼠已经成为这类研究的重要工具，因为它们能够扼要人类疾病具有高保真度，同时也捕获的异质性是内和不同的患者之间7,8存在的。为此，我们开发了多种方法来建立人肝癌裸鼠移植免疫缺陷小鼠。虽然大多数已发表的研究涉及肝癌移植瘤的描述使用的行之有效的人肝癌细胞系用于此目的，我们把重点放在优化我们的分析产生手术切除的患者后，立即获得原发性肝癌标本移植。 可能需要为不同的研究应用不同异种移植技术。例如，正在迅速产生肿瘤碎片产生的皮下移植瘤，很容易被监控，并且可能更适合于新型疗法方便本地管理监测肿瘤反应。肝内移植可能是更相关的有关肝微环境在肝癌生物学中的作用研究。从肿瘤细胞悬浮液产生的异种移植物中是必需的肿瘤起始细胞亚群的鉴定和表征，或适用于需要在肿瘤细胞之前，异种移植的体外实验操作。我们就此开发并验证以下协议，建立皮下或肝内移植从初级人类肝癌标本来源的细胞悬液或肿瘤碎片。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Dulbecco's Mod. Eagle Medium/Ham's F12 50/50 Mix x1(DMEM-F12)</td>
			<td>WISENT Bioproducts</td>
			<td>319-075-CL</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase TypeIV</td>
			<td>Sigma-Aldrich</td>
			<td>C5138</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Stemcell Technologies</td>
			<td>7923</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Matrigel Matrix</td>
			<td>Becton-Dickinson Biosciences</td>
			<td>354234</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 % Buffered Formalin solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.9 % Saline Solution (NaCl), sterile</td>
			<td>House Brand</td>
			<td>1011-L8001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Betadine surgical scrub</td>
			<td>Purdue Pharma</td>
			<td>NPN 00158313</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LORIS 10% PVP-I Solution</td>
			<td>LERNA Pharma Inc.</td>
			<td>109-09</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Buprenorphine (Temegesic) NR 0.3 mg/ml</td>
			<td>Reckitt Benckiser</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isoflurane USP, 99.9 %, inhalation anesthetic</td>
			<td>Pharmaceutical Partners of Canada Inc.</td>
			<td>M60302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tear-Gel</td>
			<td>Novartis Pharmaceuticals</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Frozen section compound</td>
			<td>VWR</td>
			<td>95057-838</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryomold, Tissue -Tek</td>
			<td>Sakura Finetek</td>
			<td>4566</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Precision Glide Needle 18G 1 &frac12;</td>
			<td>Becton-Dickinson Biosciences</td>
			<td>305196</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Precision Glide Needle 27G &frac12;</td>
			<td>Becton-Dickinson Biosciences</td>
			<td>305109</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Insulin syringe, 3/10 cc U-100, 29G&frac12;</td>
			<td>Becton-Dickinson Biosciences</td>
			<td>309301</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical blade No.10</td>
			<td>Feather Safety Razor Co.</td>
			<td>08-916-5A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>#5-0 Soft silk surgical suture, 3/8" taper point needle</td>
			<td>Syneture</td>
			<td>VS-880</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Transpore surgical tape</td>
			<td>3M Health care</td>
			<td>1577-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cotton applicator</td>
			<td>Medpro</td>
			<td>018-425</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgicel, oxidized regenerated cellulose</td>
			<td>Ethicon</td>
			<td>1951</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell strainer 100 &mu;m nylon</td>
			<td>Becton-Dickinson Biosciences</td>
			<td>352360</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Magnification lighting with mobile base</td>
			<td>Benson medical Industries Inc.</td>
			<td>model: RLM-CLT-120V</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Petridish sterile 100x20 mm</td>
			<td>Sarstedt</td>
			<td>821474</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tissue forcep, 1x2 teeth, 4-1/2"</td>
			<td>Almedic</td>
			<td>A10-302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Adson dressing forcep 4-3/4"</td>
			<td>Almedic</td>
			<td>A10-220</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Eye dressing forcep, serrated, straight, 4"</td>
			<td>Almedic</td>
			<td>A19-560</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hartman Hemostatic Forceps, curved, 3-1/2"</td>
			<td>Almedic</td>
			<td>A12-142</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Iris scissor, curved, 4-1/4"</td>
			<td>Almedic</td>
			<td>A8-690</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Iris scissor, straight, 4-1/2"</td>
			<td>Almedic</td>
			<td>A8-684</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olsen-Hegan needle driver, 5-1/2"</td>
			<td>Almedic</td>
			<td>A17-228</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

generation of subcutaneous and intrahepatic human hepatocellular carcinoma xenografts in immunodeficient mice

在体内肝细胞癌（HCC）的实验模型，概括了人类疾病为研究疾病的病理生理学和新疗法的临床前评价一个宝贵的平台。我们提出了多种方法来产生皮下或原位人类肝癌异种移植物中，可以在各种研究应用中使用的免疫缺陷小鼠。重点是使用从接受手术切除为起点的患者原发肿瘤组织中，我们描述了制备细胞悬液或肿瘤片段的异种移植。我们描述了特定的技术来异种移植这些组织ⅰ）皮下或ii）肝内，无论是由肿瘤细胞或片段的直接植入到肝脏，或间接通过细胞注射入小鼠脾。我们还描述了使用原生小鼠肝脏部分切除术在异种移植时的一种策略诱导的活性肝再生的状态下在受体小鼠可便于原代人肿瘤细胞的肝内移植。这些技术的预期结果如图。所述的协议主要使用人肝癌样本和移植，通常表现强劲少比得到广泛使用，经常引用的文献行之有效的人肝癌细胞系已经被验证。与细胞系的比较中，我们讨论的因素，可能有助于原发性肝癌移植相对较低的机会，在异种移植模型，对可能影响移植瘤生长的动力学技术问题发表意见。我们还建议，应适用，以确保获得准确的移植类似于父肝癌组织方法。

图3展示了一个皮下人肝癌异种移植物以及肿瘤的组织病理学对应的外观的典型外观。皮下移植瘤的发展和增长可以通过受体小鼠的日常检查来随时监测。异种移植肿瘤和显影之间的时间间隔可能会变化很大取决于组织的类型（肿瘤片段与细胞悬液），源组织（主病人样本，传代的异种移植物，或细胞系），和组织的数量植入（细胞的数目或肿瘤片段的大小）。例如，相当大的异种移...

Watch this Scientific Journal Video about Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice at JoVE.com

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

在免疫缺陷小鼠人类肿瘤异种移植是有价值的工具来研究癌症生物学。特定的协议来生成从人肝癌细胞或肿瘤碎片皮下和肝内移植瘤进行了描述。肝再生诱导的受体小鼠肝部分切除术是作为一项战略，以促进肝内移植。

皮下及肝内人肝癌裸鼠移植瘤的免疫缺陷小鼠的产生

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

generation-subcutaneous-intrahepatic-human-hepatocellular-carcinoma

Research

JoVE Journal

Medicine

35.7K 次观看.  University Health Network. 在免疫缺陷小鼠人类肿瘤异种移植是有价值的工具来研究癌症生物学。特定的协议来生成从人肝癌细胞或肿瘤碎片皮下和肝内移植瘤进行了描述。肝再生诱导的受体小鼠肝部分切除术是作为一项战略，以促进肝内移植。 

视频: Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

C57BL/6J小鼠乳腺间质细胞和癌细胞的移植

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

科研

医学

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

浸润性膀胱移行细胞癌中的完整的人MUC1转基因小鼠免疫诱导免疫治疗发展模型

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

创建精确的解剖学和人脑肿瘤的重现性颅内移植瘤

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

该<em&gt;卵内</em&gt;鸡胚尿囊膜（CAM）含量为肝癌的有效异种移植模型

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of infection-induced immunopathology. Although Th17 cells are implicated in antifungal defense, their role in immunopathology is unclear. This study presents a method for establishing oral Th17 immunopathology associated with oral candidal infection in immunodeficient mice. The method is based on reconstituting lymphopenic mice with in vitro cultured Th17 cells, followed by oral infection with Candida albicans (C. albicans). Results show that unrestrained Th17 cells result in inflammation and pathology, and is associated with several measurable read-outs including weight loss, pro-inflammatory cytokine production, tongue histopathology and mortality, showing that this model may be valuable in studying OPC immunopathology. Adoptive transfer of regulatory cells (Tregs) controls and reduces the inflammatory response, showing that this model can be used to test new strategies to counteract oral inflammation. This model may also be applicable in studying oral Th17 immunopathology in general in the context of other oral diseases.

Although Candida infection models are available to study host immune resistance, a model to study T cell mediated immunopathology in the context of Candida infection is absent. Here we describe a method to establish Th17 immunopathology associated with oral Candida infection in immunodeficient mice.

口咽念珠菌病的免疫缺陷小鼠Th17细胞炎症模型

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

“以病人为像”肝癌原位同源小鼠模型的癌转移

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

Osmotic pumps continuously deliver compounds at a constant rate into small animals. This article introduces a standard protocol used to induce aortic aneurysms via subcutaneous infusion of angiotensin II (AngII) from implanted osmotic pumps. This protocol includes calculation of AngII amount and dissolution, osmotic pump filling, implantation of osmotic pumps subcutaneously, observation after pump implantation, and harvest of aortas to visualize aortic aneurysms in mice. Subcutaneous infusion of AngII through osmotic pumps following this protocol is a reliable and reproducible technique to induce both abdominal and thoracic aortic aneurysms in mice. Infusion durations range from a few days to several months based on the purpose of the study. AngII 1,000 ng/kg/min is sufficient to provide maximal effects on abdominal aortic aneurysmal formation in male hypercholesterolemic mouse models such as apolipoprotein E deficient or low-density lipoprotein receptor deficient mice. Incidence of abdominal aortic aneurysms induced by AngII infusion via osmotic pumps is 5 - 10 times lower in female hypercholesterolemic mice and also lower in both genders of normocholesterolemic mice. In contrast, AngII-induced thoracic aortic aneurysms in mice are not hypercholesterolemia or gender-dependent. Importantly, multiple features of this mouse model recapitulate those of human aortic aneurysms.

Subcutaneous implantation of osmotic pumps provides a convenient approach for prolonged and consistent delivery of compounds. This approach has been used extensively to study both abdominal and thoracic aortic aneurysms in mice.

使用渗透皮下血管紧张素Ⅱ输液泵诱导主动脉瘤小鼠

Osmotic pumps continuously deliver compounds at a constant rate into small animals.  This article introduces a standard protocol used to induce aortic ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying the mechanisms of normal and malignant hematopoiesis and are the gold standard for identifying effective chemotherapies for many malignancies. PDX models are possible because many of the mouse cytokines also act on human cells. However, this is not the case for all cytokines, including many that are critical for studying normal and malignant hematopoiesis in human cells. Techniques that engineer mice to produce human cytokines (transgenic and knock-in models) require significant expense before the usefulness of the model has been demonstrated. Other techniques are labor intensive (injection of recombinant cytokine or lentivirus) and in some cases require high levels of technical expertise (hydrodynamic injection of DNA). This report describes a simple method for generating PDX mice that have exogenous human cytokine (TSLP, thymic stromal lymphopoietin) via weekly intraperitoneal injection of stroma that have been transduced to overexpress this cytokine. Use of this method provides an in vivo source of continuous cytokine production that achieves physiological levels of circulating human cytokine in the mouse. Plasma levels of human cytokine can be varied based on the number of stromal cells injected, and cytokine production can be initiated at any point in the experiment. This method also includes cytokine-negative control mice that are similarly produced, but through intraperitoneal injection of stroma transduced with a control vector. We have previously demonstrated that leukemia cells harvested from TSLP-expressing PDX, as compared to control PDX, exhibit a gene expression pattern more like the original patient sample. Together the cytokine-producing and cytokine-negative PDX mice produced by this method provide a model system that we have used successfully to study the role of TSLP in normal and malignant hematopoiesis.

Described here is a method for producing exogenous cytokine in patient-derived xenograft (PDX) mice via weekly intraperitoneal injection of a cytokine-transduced stromal cell line. This method broadens the utility of PDX and provides the option for transient or sustained exogenous cytokine delivery in a multitude of PDX models.

通过细胞因子转导的基质细胞系注射外源性细胞因子在患者来源的异种移植物中的表达

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying ...