这项研究是由抵消计划，美国国立卫生研究院（NIH），办公室主任，以及环境卫生科学研究所（NIEHS）授权号码U54 ES015678（CWW）的支持。 SA也由儿童医院的矿业合作试点奖＃G0100394和儿童医院科罗拉多研究所研究试点奖＃G0100471的科罗拉多州/科罗拉多学院的支持。

1。大鼠心肌细胞培养<ol><li>根据批准的机构动物护理和使用委员会，IACUC协议被执行的所有实验。 </li><li>从雄性大鼠使用前面4描述的方法的心（心室）（240-260克）获得大鼠心肌细胞。简单地说，用腹腔注射戊巴比妥麻醉动物（100毫克/千克;用脚趾捏法确认麻醉），然后取出心，10.0毫升，1毫米的Ca 2 +含克雷布斯Ringer缓冲液，pH值7.4。 </li><li>漂洗心中5-6倍，除去血液，然后切换到Ca 2 +的免费林格克雷布斯含有0.02％蛋白酶和0.06％胶原酶（5.0毫升/心脏）的缓冲区。孵育的心在此溶液中10-15分钟，在37℃，偶尔摇动。 </li><li>经过10-15分钟，洗出的酶溶液与Ca 2 +的免费的Krebs Ringer缓冲额外5分钟。从弛缓性组织我们释放细胞吹打悬浮上下几次ING 25毫升吸管。 </li><li>通过70微米尼龙网过滤从组织分离细胞，并允许他们定居在含有0.1毫米的Ca 2 +的Krebs Ringer缓冲。暂停的细胞沉淀在1.0ml含0.2毫米的Ca 2 +的Krebs Ringer缓冲液。 </li><li>仔细层以上5.0毫升60微克/毫升牛血清白蛋白，牛血清白蛋白，在15ml离心管中，向心肌细胞从nonmyocytes分开，并允许他们沉降30分钟。心肌较重，移动到管的底部。小心取出上清液细胞和介质。重复此步骤一次，以进一步纯化细胞。 </li><li>洗（使用0.25毫米，0.5毫米，0.75毫米和1.0毫米的Ca 2 +的缓冲液的步骤）的心室肌细胞/心肌细胞，以1.0毫米的Ca 2 +的缓冲液和重悬浮在由Dulbecco改良的Eagle培养基ACCT介质逐渐过渡，DMEM containi纳克2毫克/毫升牛血清白蛋白，2mM的L-肉碱，5mM的肌酸，5mM的牛磺酸，100国际单位/毫升青霉素和100微克/毫升链霉素。 </li><li>板ACCT介质的心肌细胞在100〜150 个 / mm 2 100毫米或35毫米层粘连蛋白的浓度包被预包被层粘连蛋白（1微克/厘米2）的塑料培养皿或40×22毫米的玻璃盖玻片上。 1小时后，洗碗用2.0ml ACCT，除去未贴壁细胞。加入10.0毫升新鲜培养基孵育细胞。 </li></ol>人呼吸道上皮的基底细胞2。分化气 - 液界面（ALI）培养<ol><li>根据机构审查委员会促使人体气管，支气管组织从国家疾病研究交换批准的协议。细胞收获和使用已发布的程序5,6进行文化。本程序采用细胞培养，因为他们是人类吸入风险，以信托投资一个精确的模型，但是，如果需要的话可以隔离和增长ALI月使用和/或大鼠呼吸道上皮细胞7,8。 </li><li>简单地说，删除所有结缔组织和淋巴结后，使组织的小碎片（〜6.3英寸）。在乳酸林格氏液冲洗组织几次。加组织到一个50ml锥形管中，并加入蛋白酶溶液（1％Protease/0.01％DNA酶在最小必需培养基，MEM，组织，以液比（1:10））。 </li><li>岩石的组织在4℃过夜。通过添加10％胎牛血清的最终组织的离解。刮除上皮表面用外科手术刀并通过离心（2000 rpm离心10分钟）收集细胞。 </li><li>板在细胞上通过Fulcher的等 9中详细描述的方法制备在特殊ALI介质胶原包被的snapwells通道之一。培养，用于通过除去根尖媒体改变到空气 - 液体界面（ALI）前5天。 </li><li>饲料采用ALI媒体每隔一天细胞，并允许细胞分化为另外的2-3周（进行曝光前，观察无数跳动的纤毛和黏液分泌）。用温热的PBS随着媒体的变化洗净顶面。 </li></ol> 3，氯曝光<ol><li>含氯曝光系统（CES）上一个合格的化学罩内有100英尺，可提供必要的二次密封，以防止意外氯气泄漏事件的人员暴露的操作表面风速。 </li><li>经营体制下轻微正压（0.5英寸水柱）。干燥的空气被送入在15升/分钟和氯被馈送到达到所期望的最终的氯浓度适当的水平。该室有一个锁盖与4迷你BCU锁和低硬度硅胶垫片，以提供压力密封。 </li><li>通过质量流量控制器提供了2氯混合。在CES使用含有1.0％的C18：2在干燥的氮气压缩气体钢瓶。 </li><li>通过调节稀释气流定制设计的控制面板，同样调节氯离子浓度输送至曝光室。低流量采样泵就会从室成氯分析仪排气监测的浓度，然后记录在连接到分析仪的数据采集器。 </li><li>在曝光之前使用流量计来保证平等送货和排气率测量中的腔室的流速。 </li><li>通过除去上清液介质并加入新鲜培养基（在ALI培养基底外侧培养基）制备的细胞培养物进行曝光。与代理商的任何预处理可以在这个时候进行。 </li><li>在两个密封的聚砜生物控制腔室暴露的气道上皮细胞的培养ALI到Cl 2中的气体（50，100，或300 ppm的30分钟）。心肌细胞（浸没或汇合在膜上培养物）暴露于50或100ppm Cl 2中15分钟。 </li><li>曝光后冲洗室与空气，直至C升2水平低于1ppm的，可以安全地打开，取出细胞（5分钟内）。 </li></ol> 4，跨上皮电阻（TER）测量<ol><li>测量气 - 液界面的TER，ALI，使用上皮voltohmmeter有一对氯化银“筷子”电极文化。 </li><li>平衡筷子电极在使用前，ALI媒体15分钟。 </li><li>添加温暖的媒体心尖（1.0毫升）和基底（2.0毫升）表面，并使用voltohmmeter的筷子电极测量TER。 </li><li>浸电极在心尖媒体短臂和基底介质的长臂。点击voltohmmeter的“测量”按钮来评价的电阻。 </li><li>该voltohmmeter具有测量欧姆或k欧姆的选项。减去电阻两端的电阻的无细胞培养支持在每个细胞层测量，得到反上皮电阻（TER）。 </li></ol> 5，胱天蛋白酶测量<ol><li>加入新鲜ALI媒体（2.0毫升）的基底表面暴露后孵育的细胞在室温下。收集上清液媒体在4至24小时。 </li><li>通过使用商用的caspase 3/7检测试剂盒测量在媒体上清的caspase 3/7活性。 </li></ol> 6，Western blot和免疫细胞化学<ol><li>用细胞裂解液如前文所述10进行Western印迹。暂停蛋白裂解液（20微克）的5倍减少上样缓冲液，煮沸5分钟。若使蛋白质裂解物进行SDS-PAGE（4-15％）和电泳印迹法所分离的蛋白质转移到硝酸纤维素膜上。 </li><li>方框孵育膜用5％脱脂乳的洗涤缓冲液（PBS +0.1％洗涤剂）的非特异性结合，并与反SERCA2或肌节肌动蛋白一抗探测膜在1:1000稀释，过夜，在4℃下。接下来，清洗和膜孵育与各自的过氧化物酶标记的二抗和使用商业过氧化物酶检测试剂盒10之前描述的发展进行检测。 </li><li>对于免疫细胞化学治疗生长在插入或盖玻片的6孔​​板中以0.4％的10mM柠檬酸钠缓冲液的Triton-X-100的PBS漂洗20分钟后的活细胞。 </li><li>块由非特异性治疗的细胞，用5％驴血清20分钟，然后于室温孵育与非特异性IgG或个人的初级抗体特异性，以肌动蛋白或Ki-67的约束力。 </li><li>用PBS洗涤细胞，并用荧光标记的二抗孵育，以检测一抗和核染色剂（1μg/ ml的DAPI）。 </li><li>用PBS洗涤，并使用商业安装介质安装盖玻片。 </li><li>用荧光显微镜可视化染色。 </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

当一个人呼吸有毒化学进入肺部急性毒性暴露的最常见的类型出现。这些化学物质也可以迅速吸收到血液中，并可能影响其他器官，如大脑和心脏。利用动物模型各种药剂吸入毒性进行了研究和广泛报道，但是机制了解较少。这是开发有效的治疗的一个主要障碍。 体外照射系统的缺席的背后是缺乏机械的见解的一个主要原因。在这里，我们描述了心脏肌肉和呼吸道，研究吸入有毒的化学物...

接触有毒化学物质吸入（国际信托）/气体如氯（Cl 2）保持在意外风险，以及在其作为化学威胁代理可能使用一个持续的健康问题。虽然肺是主要的目标，器官，如心脏和大脑也受到影响1-3。 体内模型通常用于从国际信托测试毒性，但在体外实验进行毒性评估更简单，更快速，更具成本效益。 在体外模型还允许对可能难以在体内评估代理-细胞相互作用广泛的调查。这种体外曝光系统是罕见的，而且，在有毒物质被添加到其中的细胞被淹没培养基某些常规型号，代理商的属性可以改变由于相互作用和结合的培养基成分。在这样的情况下的细胞培养系统，例如气 - 液界面（ALI人原代气道上皮细胞，在这里提出，可直接暴露于气态试剂的）培养物可能是有希望的。 上皮细胞衬里的气道是抵御吸入有毒化学品的第一线。人类气道上皮细胞形成在肺中的腔管和相关的细胞之间的物理屏障，并参与肺的响应。它产生的许多细胞因子和其他亲和抗炎剂，以及分泌粘液/气道表面液体（ASL）覆盖的上皮。一个在常规浸没在体外培养系统中的限制也是覆盖上皮表面ASL和粘液除去或稀释。这并不反映被暴露在空气中的肺上皮细胞的生理条件。因此， 在体外系统的理想旅游业议会毒性试验应重复这种架构。目前在开发快速筛选米的极大兴趣ethods的预测体内毒性。在ALI生长上皮细胞分化并具有分化良好的结构和功能相比，细胞生长淹没和服务于呼吸道的卓越典范。 在这项研究中，我们描述了使用人的气道（支气管）上皮细胞，用于测试有毒气体吸入毒性气 - 液界面培养物，并将其与心肌细胞的浸没式细胞培养进行比较，从而研究毒性的另一个重要目标。

<table border="0" cellpadding="0" cellspacing="0" width="1128">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Name</td>
			<td style="width:329px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Rats</td>
			<td style="width:329px;">Harlan Laboratories</td>
			<td>Sprague-Dawley&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Pentobarbital</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P3761</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Chlorine</td>
			<td style="width:329px;">AirGas, Inc</td>
			<td style="width:147px;">X02NI99CP163LS1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Caspase 3/7 kit&#160;</td>
			<td style="width:329px;">Promega</td>
			<td style="width:147px;">G8091</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Epithelial voltohmmeter and chopstick electrode</td>
			<td style="width:329px;">World Precision Instruments</td>
			<td style="width:147px;">EVOM and STX2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Snapwell inserts</td>
			<td style="width:329px;">Corning</td>
			<td style="width:147px;">07-200-708</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">70 micron nylon cell strainer</td>
			<td style="width:329px;">Corning</td>
			<td style="width:147px;">#352360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polysulfone biocontainment chambers&#160;</td>
			<td style="width:329px;">BCU, Allentown Cage Equipment</td>
			<td style="width:147px;">BCU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:329px;">Life technologies</td>
			<td style="width:147px;">12491-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sarcomeric actin antibody</td>
			<td style="width:329px;">Abcam Cambridge, MA</td>
			<td style="width:147px;">ab28052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SERCA2 antibody</td>
			<td style="width:329px;">&#160;Affinity Bioreagents, Golden, CO</td>
			<td style="width:147px;">MA3-9191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ki-67 antibody</td>
			<td style="width:329px;">&#160;Dako, Carpinteria, CA</td>
			<td style="width:147px;">M7248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa-488-conjugated secondary antibody</td>
			<td style="width:329px;">&#160;Invitrogen, Grand Island, NY</td>
			<td style="width:147px;">A11029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">BSA</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Carnitine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C0283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Taurine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">T8691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Creatinine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C6257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Krebs Ringer Buffer</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">K4002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Protease</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P5147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagenase</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C6885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; DNAase</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">DN-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Lactated Ringer solution</td>
			<td style="width:329px;">Abott Laboratories</td>
			<td style="width:147px;">7953</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Donkey serum</td>
			<td style="width:329px;">Fisher Scientific</td>
			<td style="width:147px;">017-000-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">PBS, phosphate buffered saline</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">D1408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">4-15% SDS-PAGE gels</td>
			<td style="width:329px;">Bio-Rad</td>
			<td style="width:147px;">456-1083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Nitrocellulose membrane</td>
			<td style="width:329px;">Bio-Rad</td>
			<td style="width:147px;">162-0115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Dergent, Tween&#160;</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Peroxidase detection kit</td>
			<td style="width:329px;">Pierce</td>
			<td align="right" style="width:147px;">3402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">DAPI</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Mounting media, Fluormount G</td>
			<td style="width:329px;">eBiosciences</td>
			<td style="width:147px;">00-4958-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium citrate</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">71497</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagen</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C7521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">MEM</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">M8028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Laminin</td>
			<td style="width:329px;">BD biosciences</td>
			<td style="width:147px;">354259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Penicillin/Streptomycin</td>
			<td style="width:329px;">Life Technologies</td>
			<td style="width:147px;">15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">FBS</td>
			<td style="width:329px;">Gibco</td>
			<td style="width:147px;">200-6140AJ</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro cell culture model for toxic inhaled chemical testing

细胞培养是必不可少的开发和研究治疗药物，其在动物模型中使用前药效。我们的模型以及分化的人类气道上皮细胞和心脏肌肉细胞的独特能力。这可能是非常有用的工具来研究的毒性吸入的化学物质，如氯，可以通常与细胞表面相互作用，并且在与水反应，并限制了它们在液态培养的影响形成的各种副产物的有害影响。采用高分化人支气管上皮细胞培养在空中liqiuid接口我们的模型绕过这个限制，以及提供了一个机会，以评估潜在的有毒化学物质吸入毒性的关键机制。我们描述了增强的损失细胞膜的完整性，一旦吸入有毒化学蛋白酶的释放和死亡，如氯曝光。在这篇文章中，我们提出的方法氯气暴露在哺乳动物心脏和呼吸道上皮C型号英语学习者的文化和简单的测试，以评估其对这些细胞类型的影响。

主要杆状心肌细胞上附着层粘连蛋白基质和蔓延，并分化成融合培养（ 图1A和它的插图）。这些细胞的特征还在于肌节肌动蛋白和SERCA2表达（ 图1B和1C）的基础上。大鼠心肌细胞极易受到氯气的毒性为15分钟暴露于100ppm的氯引起广泛的细胞变圆，死在水下文化和融合层破坏的生长层粘连蛋白涂层膜（ 图1D）细胞。以前也有增强细胞凋亡通过半胱天?...

Watch this Scientific Journal Video about In vitro Cell Culture Model for Toxic Inhaled Chemical Testing at JoVE.com

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

此协议的目的是证明细胞培养物对吸入有毒化学品的曝光方法。曝光的区别空气 - 液体界面的气道上皮细胞（ALI）的培养物提供了呼吸道暴露于有毒气体，如氯气的独特模式。在这个手稿，我们描述对上皮细胞的气-液界面培养氯曝光和心肌细胞的液体培养的效果。 体外曝光系统允许重要机理研究，以评估，然 ​​后可以用于开发新的治疗剂的途径。

对于中吸入有毒化学测试体外细胞培养模型

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

in-vitro-cell-culture-model-for-toxic-inhaled-chemical-testing

Research

JoVE Journal

Bioengineering

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

10.6K 次观看.  University of Colorado. 此协议的目的是证明细胞培养物对吸入有毒化学品的曝光方法。曝光的区别空气 - 液体界面的气道上皮细胞（ALI）的培养物提供了呼吸道暴露于有毒气体，如氯气的独特模式。在这个手稿，我们描述对上皮细胞的气-液界面培养氯曝光和心肌细胞的液体培养的效果。 体外曝光系统允许重要机理研究，以评估，然 ​​后可以用于开发新的治疗剂的途径。 

视频: In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

微流控装置重新创建一个肿瘤微环境<em在体外</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

科研

生物工程

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

形状记忆聚合物主动细胞培养

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

自体内皮祖细胞播种技术和生物相容性在大动物模型的心血管设备的测试

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

旋转的人类细胞培养的细胞培养系统：作为一种模式的滋养细胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

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 ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

大鼠视网膜仿生化学调节的神经递质谷氨酸的体外实验方法

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

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 ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

利用大分子挤出的人类食性增大疤痕的体外模型

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...