作者要感谢一月汉斯曼（弗劳恩霍夫IGB，斯图加特）对他的技术支持，以发展生物反应器和生物反应器的孵化器。

1。该BioVaSc脱细胞<ol><li>冲洗猪空肠段的血管系统通过插管动脉进入和肠腔用PBS - 。重复，直到它完全干净。 </li><li>准备一个直径200毫米的玻璃容器4的适配器，并通过硅管将它们连接到蠕动泵（ISMATEC）。压力控制单元可以通过一个连接到一个无菌的一次性圆顶（参见图1）的压力传感器来监测。 </li><li>填写水库瓶脱细胞（DZ）解决方案，并检查管道系统可能的气泡。 </li><li>连接肠腔用电缆扎带将玻璃连接器管腔流。泵500毫升DZ溶液到血管系统中的红色动脉进入（ 图1B）。中断泵送过程每隔15分钟很快手动按出整个肠腔。 </li><li>在decellularizati上的过程中，缓冲溶液的压力应在80 - 100毫米汞柱，自然血压后建模。 </li><li>洗BioVaSc用PBS -直到它是免费的细胞残余的（“全白”）。 </li><li>完全与DZ的解决方案填补了BioVaSc和孵化它在DZ的解决方案，淹没了晚上，在4℃的摇摆振动筛。 </li><li>重复步骤1.6。 </li><li>将BioVaSc在DNA酶溶液孵育过夜，4℃的摇摆振动筛。 </li><li>去除DNA酶液和冲洗用洗涤缓冲液。 </li><li> γ-灭菌用25千戈瑞</li></ol> 2。在不同的细胞类型 2.1分离的原代人皮肤微血管内皮细胞（mvECs）和成纤维细胞的<ol><li>切开皮肤活检（最好是阳茎外鞘）为2条- 3毫米宽用手术刀和冲洗一下，用PBS 3倍-解决方案。 </li><li>盖上分散酶溶液中的组织和培养了16 - 18小时，在4℃下</li><li>从真皮层有2个独立的镊子表皮和两个分别转移到装有PBS +培养皿。 </li><li>清洗真皮剥离1X与维尔烯。 </li><li>加入10 ml胰蛋白酶/ EDTA溶液到真皮片孵育它在孵化40分钟。 </li><li>立即停止用1％FCS的酶反应。 </li><li>皮肤转移到带培养皿充满VascuLife和挠出每个条带用手术刀8X每边，增添了几分压力。 </li><li>通过细胞过滤器传送所生成的细胞悬浮液到一个离心管中，冲洗细胞3次过滤器与VascuLife。 </li><li>离心管中以1200×g离心5分钟，重悬细胞沉淀用VascuLife。 </li><li>为了分离成纤维细胞，真皮印章剥离到使用手术刀的小件。 </li><li>加入10 mL的胶原酶溶液至真皮件。 </li><li>孵育它在孵化器45分钟，然后离心它，小心地取出苏pernatant。 </li><li>用DMEM +10％FCS +％PenStrep，离心洗沉淀1x和小心取出上清液。 </li><li>重悬沉淀在培养基中，并将其传输到一个T75培养瓶中，以使细胞生长出的组织。 </li></ol> 2.2肿瘤细胞系S462 肿瘤细胞系S462（由尼古拉HOLTKAMP博士，查理特大学医学柏林惠赠）是从一个女性患者的恶性神经鞘瘤产生与遗传性肿瘤易感综合征型神经纤维瘤病1 9。 S462是培养于DMEM补充有10％FCS。介质已被改变，每2 - 3天。每周一次的细胞必须被分割。 3。肿瘤测试系统：与动态文化在生物反应器系统静态培养条件相比<ol><li>切管SIS-MUC打开一侧并固定两个金属环（细胞冠，直径10毫米，自我℃之间onstructed）。覆盖SIS-MUC在细胞培养液中过夜。 </li><li>种子分离的细胞中所定义的细胞数目（参见下文）上的SIS（单 - 或共培养建立）的一侧或两侧。 <ol><li>种子8000细胞初级mvECs在100微升的总体积到SIS（原浆膜）的基底外侧表面/厘米2。 3小时后填入良好，中等，确保淹没的文化。 </li><li>允许内皮细胞粘附3天。 180°翻转静态培养系统，并将其转移到一个12孔板中。 </li><li> 500微升的SIS（原管腔的一侧）的顶表面的总体积内的种子初级真皮成纤维细胞（8000个细胞/ cm 2）和肿瘤细胞（15,000细胞/ cm 2）的混合物。 </li><li>让细胞附着3小时，并填充以及与介质（浸没培养，培养基：50％Vasculife +50％DMEM补充有10％FCS）。 </li><li>在静态条件下的肿瘤测试系统培养T 37℃，5％CO2培养箱额外的14天。更换培养液，每2 - 3天。 </li></ol></li><li>对于动态的文化，修复SIS-MUC两个金属环之间和3.2.1中描述的种子主要mvECs。 3天之后，从金属环中删除SIS-MUC并插入细胞膜进入流反应器（参见图2C和2D）。申请的首要真皮成纤维细胞和肿瘤细胞，用注射器和插管在基质中的生物反应器，以及使细胞与培养液填充所述生物反应器系统之前附着3小时。 </li><li>在翌日动态培养条件与常数的介质流量（3.8毫升/分钟，37℃，5％CO 2）可以启动。动态培养保持14天，培养基后7天内改变。 </li><li>动态测试装置是在培养的自建孵化器，通过泵和必要的温度和CO 2提供媒体流</sub&gt;内容。 </li></ol> 4。表征方法分析 4.1固定和石蜡包埋的种子胶原基质的<ol><li>对于（免疫）组织学表征除去培养基并修复组织用4％多聚甲醛2小时。 </li><li>拆下4％多聚甲醛和金属嵌件传送SIS的一个组织包埋盒。水组织以除去残留的固定液和脱水石蜡浸润。 </li><li>之前在石蜡块包埋，切SIS在2 - 3片，将它们放置在一个石蜡填充金属底座模具使得切割面朝下。增加对模具为依托的顶级组织录像带。 </li><li>切5μm的切片，它们漂浮在40℃水浴拉直，然后装入滑动到合适的载玻片上。未涂布的载玻片用于H＆E染色，聚赖氨酸包被的载玻片进行免疫组织化学染色，以改善附件。让切片彻底干燥。 </li><li>融化的石蜡，用二甲苯将其删除，并补充水分切片染色后。 </li></ol> 4.2染色<ol><li>再水合片可以用苏木精/伊红染色作为一种标准化的概述染色。 </li><li>为immunhistological染色，固定和石蜡渗透组织切片必须经过一个抗原修复，以允许抗体识别和结合到其特异性抗原决定簇。因此，脱石蜡和再水化的载玻片放置在蒸汽烹调器以20分钟加热的柠檬酸盐缓冲液（pH 6.0）。 </li><li>转移到载玻片洗涤缓冲液（0.5M TBS缓冲液+0.5％吐温）和圆切片用PAP笔，以尽量减少染色溶液至所需体积。 </li><li>将滑动在湿气室内。以取得的抗原 - 抗体结合特定的辣根过氧化物酶介导的可视化，将内源性过氧化物酶必须是饱和的，用3％的过氧化氢。 </li><li>主要antibODY稀释液施加到片，温育1小时，在室温，小心地用洗涤缓冲液洗掉。 </li><li>用于检测特定的抗原 - 抗体绑定的超视界双币种检测系统HRP DAB（Thermo Scientific）进行，根据所建议的协议中使用。 </li><li>细胞核用苏木精复，持续1分钟。 </li><li>滑动安装与含水介质，干燥，并用倒置显微镜成像。 </li></ol>

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作者什么都没有透露。

<html>当比较的二维和三维培养系统在肿瘤研究，3D系统，尽管是更昂贵的方法，已被证明在模拟生物微环境的条件较好。它可以表明，某些肿瘤细胞的生长慢得多的三维培养比在普通2D培养12，这是根据在一个真正的肿瘤的情况。比斯尔和同事显示在他们的工作中致癌乳房细胞的行为反映了体内的情况，包括细胞形态和信令，更准确地，当一个基体中的三维培养提供了细胞-细胞外基质相互作用。此外，他们强调通过证明改变环境的相互作用导致了恶性细胞的复归到正常表型的细胞外环境中的三维的重要性。此外，最重要的是，这些结果也可以在体内动物模型10,11证实。 ontent“&gt; 在体内动物实验和体外组织模型的直接比较显示出的优点和缺点，在这两个系统。 体外模型的一个优点是一个更好的实时或固定成像显微镜的权限。一个限制是它们模仿静态或短期的条件，而在体内系统往往进步，目前缺乏血管和小分子的正常的运输，宿主的免疫应答，和其它细胞-细胞相互作用的体外模型12的进一步的缺点，因此，三维体外系统在本研究中提出提供了一个有前途的除了动物实验，他们提供了一个更好的可比性，以人的机体，因此尽量减少实验误解。仿生体内模型系统将会因此变得更加相关研究癌症和转移扩散是如何依赖该规范tumorig微环境条件香根草11。 我们的研究表明，由SIS-MUC提供的三维环境导致更瘤样组织形成的细胞，这是不常见的二维细胞培养物观察到（参见图3A）。此外，使用从肿瘤活检源性原代细胞是实现个性化医学，一门学科，旨在确定依赖于患者的个性化需求的最佳治疗非常重要的一步。结合主要针对具体患者的肿瘤细胞从活检材料中分离将允许在治疗策略体外测试。这样的测试系统将使得有可能在时间和节省成本的高通量筛选研究不同的药物和它们的组合。此外，肿瘤相关的基质细胞，如本研究中的整合是为个人化的方法很重要，因为肿瘤的微环境影响肿瘤进展和13可能被证明为suitab乐治疗靶点。 可替换地，以个人化的方式，我们的肿瘤模型可以被修改以作为广义的肿瘤的测试系统所确立的致瘤细胞系的结合。这是一种很有前途的适应基础研究的目的。对于这两种药物测试接近的血管结构的存在是必需的，以测试的治疗性物质的分布和吸收。在SIS-MUC矩阵使得基底播种原发性MVEC阻隔吸收的研究中，BioVaSc的保留血管结构的补种将进一步提高给药的研究。 为了创建组织模型中，三维生物可降解基质可作为框架对不同类型的细胞14的共培养。使用这样的三维基质通常是由不存在的功能性血管的限制。这个问题可以通过使用BioVaSc，它提供了保藏血管海峡的解决uctures，它可以被重新接种内皮细胞。此外，BioVaSc提供外成分，这确保了细胞的粘附和促进组织分化。它还使之生物人工肝三维组织7,8,15长时间的组织特定功能。的前提，功能性血管代用品的工程是人的生理和生物力学条件的模仿。因此，生物反应器系统，它可以在体外实现这些需求，对于创建生物肿瘤模型中极大的兴趣。 该BioVaSc的组合，所述生物反应器技术和共培养不同的细胞类型是一个非常有前途的方法来生成血管化的肿瘤组织，这将允许相关的癌症的进展，如血管生成和转移的机制的研究。我们通过提供同等看到这样的肿瘤模型作为一种很有前途的方法来补充动物研究人类肿瘤生理学。

新的药品必须考虑到他们的质量，安全和市场认可之前疗效进行验证。迄今为止，动物实验是药物测试和验证的标准方法。然而，由于物种特异性差异，动物实验往往不全面地评价在人1的化合物的效果。出于这个原因，它以产生可用于新的药物和物质的体外试验人体组织模型是重要的。 我们的一个小组的重点是建立在体外试验模型与我们的生物血管支架（BioVaSc）2,3。该BioVaSc可以用作静态或动态三维矩阵系统。对于静态的文化，脱细胞猪空肠段（小肠黏膜下层SIS-MUC）被放置在一个金属插入细胞重新植入。各种细胞，如肿瘤细胞和内皮细胞可以培养在支架上。 <p级=“jove_content”&gt;对于动态文化，BioVaSc附加到应用流在整个脉管系统或穿过支架的表面的生物反应器系统。当前的生物反应器实现生物，机械或电刺激后的细胞4的分化或增殖，其作用。对于在组织工程领域的生物反应器，其基本概念是为了模拟在人体内的条件。其特征在于，将细胞提供了一种自然的环境，使它们可以彼此和它们周围的细胞外基质相互作用。用于生产的体外试验系统或移植，以模仿细胞的天然环境与适当的载体结构和生物反应器系统的能力是至关重要的5。因此，更复杂和技术要求的设备必须开发为了完成这些任务6。 而且，可以使用我们的脚手架为滹一个血管模型的由于保留的管状结构，其包括供血动脉，静脉和连接毛细血管床ishment。所有猪细胞需要通过化学，机械和酶脱细胞被除去，并且该支架γ-灭菌的。还原的管状血管结构可以随后被重新接种使用的再循环灌注生物反应器7，它模仿生物力学和/或生物化学参数如pH，温度，压力，营养供应和废物清除6人微血管内皮细胞。的再内皮化管状结构的创建人血管等效的胶原支架3,7内。在下面的步骤中，前腔（黏膜）的表面可以与人原代细胞被接种到建立共培养3,7,8。 在这项研究中一个三维肿瘤测试系统设置为通过共培养原发性ST肿瘤细胞系romal细胞在SIS-MUC静态和动态条件下。

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Collagenase solution</td>
			<td>SERVA</td>
			<td>17454</td>
			<td>(500 U/ml)</td>
		</tr>
		<tr>
			<td>Dispase solution</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>(2.0 U/ml)</td>
		</tr>
		<tr>
			<td>DMEM, high-glucose</td>
			<td>PAA</td>
			<td>G0001,3010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>ROCHE</td>
			<td>10104159001</td>
			<td>200 mg solved in 500 ml PBS+ + 1% PenStrep</td>
		</tr>
		<tr>
			<td>DZ solution</td>
			<td>Roth</td>
			<td>3484.2</td>
			<td>34 g Sodium Desoxychelate, in 1 L Ultra-pure water</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>LONZA</td>
			<td>DE14-801F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IHC-Kit DCS SuperVision 2 HRP</td>
			<td>DCS</td>
			<td>PD000KIT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>medical pressure transducer</td>
			<td>MEMSCAP</td>
			<td>SP844</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>monoclonal mouse anti-human Von Willebrand Factor</td>
			<td>DAKO Cytomation</td>
			<td>M0616</td>
			<td>Clone F8/86 0.12 &mu;g/ml</td>
		</tr>
		<tr>
			<td>mouse monoclonal anti-human p53</td>
			<td>DAKO Cytomation</td>
			<td>IS616</td>
			<td>Clone DO-7 ready-to-use</td>
		</tr>
		<tr>
			<td>peristaltic pump</td>
			<td>Ismatec</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>sterile disposable dome</td>
			<td>MEMSCAP</td>
			<td>844-28</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin / EDTA solution</td>
			<td>PAA</td>
			<td>L11-003</td>
			<td>0,05%</td>
		</tr>
		<tr>
			<td>VascuLife (VEGF-Mv)</td>
			<td>Lifeline</td>
			<td>LL-0003</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Versene</td>
			<td>Gibco</td>
			<td>15040-033</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

tissue engineering of a human 3d in vitro tumor test system

癌症是人类死亡的主要原因之一。目前的治疗策略主要开发的二维培养系统，这充分体现在体内生理条件。生物3D矩阵提供的细胞中，细胞能够自我组织的环境，使组织的组织和细胞分化的研究。这样的支架可以被接种了不同的细胞类型，以研究直接三维细胞 - 细胞相互作用的混合物。要模仿癌症肿瘤的三维复杂，本集团已开发出一种三维体外肿瘤测试系统。 我们的3D组织的测试系统模型的恶性外周神经鞘瘤（MPNSTs），这是我们与我们的脱细胞猪空肠段取得的生物血管支架（BioVaSc）建立了在体内的情况。在我们的模型中，我们重新植入一个修改BioVaSc矩阵初级成纤维细胞，微血​​管内皮细胞（mvECs）和S462肿瘤细胞系。对于静态文化，BioVaSc的血管结构被删除，剩下的脚手架被切开一侧（小肠黏膜下层SIS-MUC）。由此产生的矩阵，然后两个金属环（细胞冠）之间固定。 另一种方法是培养的细胞接种SIS-MUC在流动生物反应器系统，它公开了细胞的剪切应力。这里，所述生物反应器被连接到在自建培养箱的蠕动泵。一种计算机通过调节参数例如血压，温度和流速的动脉氧气和营养供应。这种设置允许动态文化，无论是压力调节脉动或流量恒定。 在这项研究中，我们可以成功地同时建立一个静态的和动态的三维培养系统MPNSTs。建模癌症肿瘤中更自然的三维环境的能力将使发现，测试和验证未来医药在人类类似的模型。

如图1B所示 ，我们脱细胞猪空肠段（长度为约2米，直径20毫米）与毛细管网络的保藏的管状结构。经过化学，酶促和机械脱细胞中，我们得到的胶原Ⅰ/Ⅲ支架，它可以被用于三维细胞培养物。甲富尔根试验以证明矩阵（数据未示出）的纯度（无DNA的残余）。 图2A和2B显示SIS-MUC的静态培养的细胞牙冠固定。我们固定的SIS-MUC在内部设计的生物反应器（ 图2C）进行动态文化。 图2D说明模拟动态流过生物 ​​反应器的腔。所述生物反应器被放置在一个自建培养箱系统和用蠕动泵连接。此设置允许动态的文化，无论是压力调节脉动流或c<html>恒定流量。 图3给出了在二维单作静态培养的S462肿瘤细胞系的概述（ 图3A）和在三维共培养（ 图3B-3D）。图3B示出的肿瘤细胞S462和SIS的根尖侧初级成纤维细胞的三重培养-MUC（原内腔侧）和MVEC的基底侧（原浆膜侧）。不同类型的细胞的识别可以通过染色细胞类型特异性标志物，如血管性血友病因子标记MVEC（ 图3C）。在p53阳性S462细胞可以从p53阴性初级成纤维细胞（ 图3D）和细胞的三维分布，可以分析区分开来。 图4显示了染色相当于图3中的动态培养的三重培养物。 <html> 0460/50460fig1.jpg“/&gt; 图1。脱细胞设置。 （一）生物反应器和泵的设置，用来在脱细胞BioVaSc，由PC监控。（B）脱细胞BioVaSc在玻璃缸。管腔和动脉入口被连接到适配器。 。 <img alt="图2" src="/files/ftp_upload/50460/50460fig2.jpg" /> 图2。以上观点不同的文化调校的。 （一）CAD节静态文化体系中微量滴定孔板图，（B）金属嵌件的静态文化，（C）介质流动模拟（速度场[米/秒]）为流动生物反应器的上盖动态文化（D）流生物反应器连接到蠕动泵动态培养。 <a href="https://www.jove.com/files/ftp_upload/50460/50460fig2large.jpg" target="_blank">点击这里查看大图</a> ENT“FO：保持together.within页=”总是“&gt; <img alt="图3" fo:content-src="/files/ftp_upload/50460fig3highres.jpg" fo:content-width="5.5in" src="/files/ftp_upload/50460/50460fig3.jpg" /> 图3。静态三维肿瘤模型的免疫组织学特征的概述。 （一）苏木S462细胞上和Permanox幻灯片的静态培养2D单文化的污点，静态培养的3D三重文化（二）H＆E染色，箭头标记血管内皮细胞，（三）免疫组化染色血管性血友病因子（D ）免疫组化染色p53基因。 <a href="https://www.jove.com/files/ftp_upload/50460/50460fig3large.jpg" target="_blank">点击这里查看大图</a> <img alt="图4" fo:content-src="/files/ftp_upload/50460fig4highres.jpg" fo:content-width="6in" src="/files/ftp_upload/50460/50460fig4.jpg" /> <st荣&gt;图4。动态三维肿瘤模型的免疫组织学特征的概述。 （一）培养动态3D三重文化的H＆E染色，箭头标记血管内皮细胞，（B）免疫组化染色血管性血友病因子（C）免疫组化染色p53基因。 <a href="https://www.jove.com/files/ftp_upload/50460/50460fig4large.jpg" target="_blank">点击这里查看大图</a>

Watch this Scientific Journal Video about Tissue Engineering of a Human 3D in vitro Tumor Test System at JoVE.com

Tissue Engineering of a Human 3D in vitro Tumor Test System

方法来创建三维人体肿瘤组织的测试系统进行了描述。这些技术是基于脱细胞生物血管支架（BioVaSc），原代人细胞和肿瘤细胞系，可在静态中的流动生物反应器中的动态条件下培养，以及。

一个人的3D组织工程<em&gt;在体外</em&gt;肿瘤测试系统

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

tissue-engineering-of-a-human-3d-in-vitro-tumor-test-system

Research

JoVE Journal

Bioengineering

Tissue Engineering of a Human 3D in vitro Tumor Test System

21.2K 次观看.  University Hospital Würzburg. 方法来创建三维人体肿瘤组织的测试系统进行了描述。这些技术是基于脱细胞生物血管支架（BioVaSc），原代人细胞和肿瘤细胞系，可在静态中的流动生物反应器中的动态条件下培养，以及。 

视频: Tissue Engineering of a Human 3D in vitro Tumor Test System

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

组织工程：建设多细胞三维支架的层状细胞片交货

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

科研

生物工程

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

肿瘤微环境基质与应用癌细胞侵袭组织工程

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

一种混合的3D组织工程<em&gt;体外</em&gt; /<em&gt;在硅片</em在具体的突变背景预测药物的有效性&gt;肺肿瘤模型

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Force System with Vertical V-Bends: A 3D In Vitro Assessment of Elastic and Rigid Rectangular Archwires

A proper understanding of the force system created by various orthodontic appliances can make treatment of patients efficient and predictable. Reducing the complicated multi-bracket appliances to a simple two-bracket system for the purpose of force system evaluation will be the first step in this direction. However, much of the orthodontic biomechanics in this regard is confined to 2D experimental studies, computer modeling/analysis or theoretical extrapolation of existing models. The objective of this protocol is to design, construct and validate an in vitro 3D model capable of measuring the forces and moments generated by an archwire with a V-bend placed between two brackets. Additional objectives are to compare the force system generated by different types of archwires among themselves and to previous models. For this purpose, a 2 x 4 appliance representing a molar and an incisor has been simulated. An orthodontic wire tester (OWT) is constructed consisting of two multi-axis force transducers or load cells (nanosensors) to which the orthodontic brackets are attached. The load cells are capable of measuring the force system in all the three planes of space. Two types of archwires, stainless-steel and beta-titanium of three different sizes (0.016 x 0.022 inch, 0.017 x 0.025 inch and 0.019 x 0.025 inch), are tested. Each wire receives a single vertical V-bend systematically placed at a specific position with a predefined angle. Similar V-bends are replicated on different archwires at 11 different locations between the molar and incisor attachments. This is the first time an attempt has been made in vitro to simulate an orthodontic appliance utilizing V-bends on different archwires.

Force System with Vertical V-Bends: A 3D In Vitro Assessment of Elastic and Rigid Rectangular Archwires

The method presented here is designed to construct and validate an in vitro 3D model capable of measuring the force system generated by different archwires with V-bends placed between two brackets. Additional objectives are to compare this force system with different types of archwires and to previous models.

垂直 V 形弯曲力系统: 3D 弹性和刚性矩形矫正线的体外评价

A proper understanding of the force system created by various orthodontic appliances can make treatment of patients efficient and predictable. Reducing ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

在微流体平台内开发 3D 组织人类心脏组织

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...