This project was funded by the National Science Foundation ECCS grant 10-02165, and the Interdisciplinary Innovation Initiative Program, University of Illinois grant 12035. M.Y. A. was funded at UIUC from NIH National Cancer Institute Alliance for Nanotechnology in Cancer &#8216;Midwest Cancer Nanotechnology Training Center&#8217; Grant R25 CA154015A. Immnunostaining and confocal microscopy imaging were carried out at the Institute for Genomic Biology (IGB), UIUC. M.Y.A. acknowledges the discussions with B. J. Williams of UIUC regarding the isolation experiments. M.Y.A. acknowledges C. Nemeh and Abdul Bhuiya of UIUC for assistance in schematic and materials list preparation.

下面所描述的协议如下UIUC人类研究伦理委员会的指导方针。 1.收集和外科组织消解<ol><li>收集后右结肠切除术（ 图1A和1B）的肿瘤组织样品。收集来自邻近部位的健康组织为好。 </li><li>马上转移的组织，以15毫升小瓶含12毫升HBSS解决方案。继续冰瓶内的绝缘泡沫箱。 </li><li>传送后45分钟的含有组织小瓶组织培养罩作进一步的处理。保持罩内的冰块上的小瓶。 </li><li>倒入供给组织成含有6-7毫升的HBSS溶液用移液管6孔板。 注意：每孔组织的量不在这个漂洗步骤的关键。 </li><li>保持一个冰块在6孔板。在冲洗液HBSS组织两次。 </li><li>干净切割组织与无菌分离SCIS节SOR。百果组织成更小的部分不超过1-2毫米3大小用无菌手术刀片更大。 </li><li>称重经标记的0.1％胰蛋​​白酶小瓶（含有1毫升0.1％的胰蛋白酶溶液）转移所述组织之前。转移〜20 mg组织到每个所述的0.1％胰蛋​​白酶的小瓶用吸管的。 </li><li>尽量减少的HBSS转移到胰蛋白酶小瓶，以避免进一步的稀释胰蛋白酶。 </li><li>权衡组织转移后的胰蛋白酶小瓶;文件的差异作为组织肿块。 </li><li>倾〜20毫克组织和1ml胰蛋白酶于24孔板的每个孔中。 </li><li>把24孔平板在摇床上于冰箱，在4℃下进行16-20小时。在此等待期间或更早的时候，准备聚丙烯酰胺凝胶基质和如在第3节描述的ECM的蛋白质功能化他们。 </li></ol>组织样本单细胞悬液2酶解和ECM功能化的弹性Substrat培养分离细胞ES和刚性聚苯乙烯餐具<ol><li>后在摇床胰酶以及在4℃下16-20小时温育的，从4℃的胰蛋白酶转移组织至4℃完全生长培养基小瓶（含有2-3ml的完全生长培养基的）用移液管。保证胰蛋白酶最小转移到生长介质。 注意：完全生长培养基配方如下：补充有马血清为10％和青霉素 - 链霉素至总溶液的1％的最终浓度的RPMI 1640基础培养基中。 </li><li>将包含媒体的小瓶中的温水浴，在37℃下进行12-15分钟的组织。 </li><li>转移组织到15ml小瓶中含有用吸管的HBSS溶液，轻轻摇动。 </li><li>重复2.3。 </li><li>从HBSS溶液，并转移到0.1％的胶原酶溶液（在HBSS中）含有1ml溶液小瓶去除组织。孵育45分钟，在37℃和5％CO 2的环境中的潮湿的细胞培养箱中。在等待期间，温growt在37℃] C H媒体在15ml小瓶在水浴中。 </li><li>所有的组织碎片拔出吸管将胶原酶最小化交叉污染。喷射刚组织碎片进入预热培养基瓶中。 </li><li>放置一个40μm的良好的插入滤波器入6孔板的每个孔中。滋润过滤用1毫升细胞培养基。 </li><li>磨碎组织在媒体用10毫升玻璃吸管数次，直至组织被完全离解。 </li><li>保持枪头尽可能接近到小瓶的底部。 </li><li>存款的解决方案到过滤，清除杂物及未解离部分。 </li><li>离心机过滤的细胞悬浮液中的媒体，在150×g离心5分钟，在RT。 </li><li>事后去除上清，重悬细胞沉淀用2毫升的新鲜培养基。 </li><li>计数使用血球（如果需要）的细胞。种子被隔绝在细胞外基质（ECM）官能化的凝胶和聚苯乙烯菜肴所需浓度的原代细胞。</li></ol> 3.准备和变刚度聚丙烯酰胺（PA）凝胶和聚苯乙烯基质功能化注意：对于视觉第3节的示范，观众/读者可以参考最近的杂志可视化实验（朱庇特）第17条。 <ol><li>采用协议公布的18，19，激活12毫米2玻璃盖玻片化学确保共价水凝胶结合。 <ol><li>首先，请客玻璃盖玻片3- Aminopropyltrymethoxysilane（ATS）在室温下7分钟。 </li><li>用DI水漂洗完全除去ATS和治疗盖玻片，用0.5％戊二醛（从70％戊二醛原液在PBS中稀释）处理30分钟溶液。 </li></ol></li><li>通过将5％w / v的丙烯酰胺溶液和0.05％的N，在10mM的HEPES缓冲盐水20 -N&#39;甲基双丙烯酰胺（BIS）溶液得到2千帕凝胶溶液。使用8％w / v的丙烯酰胺和0.13％的N，在10mM的HEPES缓冲盐水-N&#39;甲基双丙烯酰胺（BIS）溶液为10千帕的凝胶20。 <ol><li>在这两种情况下，使用1：200的过硫酸铵（10％重量/体积）和1：2000 N，N，N&#39;，N&#39;四甲基乙二胺（TEMED），为中，引发剂和催化剂用于聚合过程分别。 </li><li>此外，添加100μl的荧光珠，以2千帕凝胶溶液作为受托标记21,22。 </li></ol></li><li>存款下降了20微升的12 平方毫米激活盖玻片预聚合物PA凝胶溶液。广场上的下降，另有12个平方毫米普通玻璃盖玻片。确保降盖玻片之间的利差，由于毛细作用。倒置三明治为2千帕的凝胶，以确保大多数的珠来对细胞培养物表面附近。 </li><li>治愈PA凝胶在室温45分钟。 </li><li>剥离使用单刃剃须刀顶盖滑。 注：在剥离，剥离从一个边缘进行的三明治。凝胶仍然附着在活化载玻片。 </li><li>使用之前储存在PBS液凝胶。 </li><li>官能PA凝胶和玻璃使用ECM分子，人纤连蛋白在50微克/毫升以下公开的方法23的浓度。 <ol><li>简单地说，孵化用2毫升纯水合肼O / N基板。 </li><li>除去水合肼和去离子水彻底冲洗衬底。 </li><li>洗，用5％乙酸在基板30分钟。 </li><li>除去乙酸和去离子水彻底冲洗衬底。 </li><li>保持浸渍于去离子水中30分钟的衬底。 </li><li>孵育氧化纤维连接蛋白35分钟的基板在50微克/毫升的浓度。 </li><li>冲洗在摇床上用PBS基板在低转速10分钟。 </li><li>电镀细胞之前孵育2-3毫升培养基所有基板在37℃下30分钟。 注：板细胞在人口稀少填充ð方式（1,000-3,000个/厘米2）。每个凝胶覆盖的玻璃滑需要被包含在35毫米的培养皿。使细胞的任何显微镜（至少O / N）之前完全遵守。 </li></ol></li></ol> 4.牵引力显微镜和免疫荧光显微测定<ol><li>牵引力显微镜分析<ol><li>暖0.25％胰蛋白酶-EDTA / 10％SDS在37℃溶液在水浴10分钟前牵引实验开始。 </li><li>除去从细胞培养培养箱一种凝胶在时间和地点上的荧光倒置显微镜阶段（32X放大倍率）。 </li><li>发现在视场的单个细胞。取出培养皿的盖子。 </li><li>取细胞的相衬图像（ 例如 ， 图3）。 </li><li>切换成像模式荧光和选择适当的过滤器。在此期间不要移动显微镜阶段或样品。 </li><li>取的荧光珠displac的图像由蜂窝牵引（ 图4C）编</li><li>加入1毫升胰蛋白酶/ SDS溶液到培养皿分离从凝胶中的细胞。在此期间不要移动显微镜阶段或样品。此外，取单元的控制图像，以确保完全除去从凝胶。 </li><li>取的珠的参考（零力）图像的单元除去后。 </li><li>对于所有其他凝胶重复步骤4.1.2-4.1.8。 </li><li>请使用ImageJ从步骤4.1.6和4.1.8每一种情况下获得的两个图像图像堆栈。要生成图像堆栈，用命令的ImageJ的下列顺序打开图片后：图像→堆叠→图像堆栈。 </li><li>为了获得位移场和牵引，使用ImageJ的插件下面公布的方法21，22获得这些插件使用下面的链接代码和详细的教程。 <a href="https://sites.google.com/site/qingzongtseng/imagejplugins">https://sites.google.com/site/qingzongtseng/ima</a>gejplugins。 <ol><li>对齐用的“模板匹配”插件堆栈中的图像。使用以下工作步骤4.1.10生成的图像堆栈后序列中的ImageJ命令：在堆栈插件→模板匹配→对齐→切片确定。保存图像堆栈因此。在下面的步骤使用这个新的图像堆栈。 </li><li>获得使用的PIV（粒子图像测速）插件（ 图4D）的位移场。使用下面的打开保存在步骤4.1.11.1图像堆栈后命令序列：插件→PIV→迭代PIV（基本）→OK→接受这个PIV和输出→确定。在同一目录下的PIV输出保存为原始图像堆栈。以此为输入下一个步骤。 </li><li>最后使用FTTC（傅立叶变换牵引术）的插件，以获得牵引力地图（ 图4E）。命令使用以下顺序：→插件→FTTC插件ERT的材料特性， 即 ，杨氏模量和PA的凝胶→确定→泊松比选择保存在步骤4.1.11.2→OK了PIV输出文件。下次FTTC在同一目录中的结果如在图像堆栈和PIV输出。 </li></ol></li></ol></li><li>免疫荧光显微镜测定<ol><li>就拿基板被免疫染色的层流罩，取出培养基。 </li><li>冲洗用PBS基板和固定细胞用PBS中的4％多聚甲醛20分钟，室温。 </li><li>为3次（每次5分钟）洗涤，用PBS基板。因此，孵育用500μl信号增强基板30分钟并漂洗用PBS。 </li><li>孵育细胞与单克隆抗粘着斑蛋白抗体以1：250稀释在PBS中在RT下45分钟。为3次（每次5分钟）洗涤，用PBS基板。 </li><li>孵育二级抗体的Alexa Fluor 488山羊抗 - 小鼠IgG的样品以1：200稀释在PBS中在RT下30分钟。为3次（每次5分钟）洗涤，用PBS基板。 </li><li>为了显现的F-肌动蛋白的结构，孵育细胞TRITC鬼笔环肽缀合物的浓度50微克/毫升，在室温45分钟。为3次（每次5分钟）洗涤，用PBS基板。 </li><li>图像（ 图5A - 5D）使用共焦扫描激光显微镜的样品。 </li></ol></li></ol>

<ol>
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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+rigidity+regulates+cancer+cell+growth+and+cellular+phenotype.">Matrix rigidity regulates cancer cell growth and cellular phenotype.</a> PLoS ONE. 5 (9), e12905 (2010).</li><li>Ulrich, T. A., de Juan Pardo, E. M., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+rigidity+of+the+extracellular+matrix+regulates+the+structure,+motility,+and+proliferation+of+glioma+cells.">The mechanical rigidity of the extracellular matrix regulates the structure, motility, and proliferation of glioma cells.</a> Cancer Res. 69, 4167-4174 (2009).</li><li>Williams, B. J., Anand, S. V., Rajagopalan, J., Saif, M. T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+self-propelled+biohybrid+swimmer+at+low+Reynolds+number.">A self-propelled biohybrid swimmer at low Reynolds number.</a> Nat. Commun. 5, (2014).</li></ol>

The authors have nothing to disclose.

蜂窝牵引强调最近成为转移状态13一个潜在的生物物理指标。然而，随着原发肿瘤细胞没有实验数据的牵引文献中存在的日期。此外，还未见报道的是不同的刚度聚丙烯酰胺凝胶直接分离培养原发性结肠癌细胞。因此，我们建立对凝胶和聚苯乙烯（ 图2）优化的原发性结肠癌细胞的培养条件。在软凝胶制剂邻近细胞培养表面的荧光微珠封装允许位移场和由人原代细胞（ ?...

近年来它已成为越来越明显的是，机械微环境中，除了生物化学因素，在调节细胞功能中起重要作用。细胞可以感知并就它们所附着到（如在2D培养）或（在3D培养如）包围3-7基板刚度作出响应。通过这样做，细胞可以调节它们的分化3，形态4，迁移/蠕动5，生物物理性质6，生长7，等工序。 癌细胞也使用粘附受体的协调，分层机械化学组合和相关的信号转导的膜蛋白，细胞骨架结构，以及分子马达1,2的二维和三维矩阵刚度作出响应。例如，乳腺上皮细胞（的MECs）上为150Pa衬底培养时形成正常腺泡实质是类似于刚度健康的乳腺组织。有趣的是，它们表现出的显影肿瘤的标志，其结构和转录，当较硬基板（&gt; 5000帕），模仿一个肿瘤基质8的刚度进行培养。此外，另一实验表明，乳房肿瘤发生是伴随着胶原交联和ECM加强9。最近的实验表明，人结肠癌（HCT-8）细胞显示样表型（MLP）转移时，他们就具有生理学相关刚度（20-47千帕）2D基板培养，但不能在非常僵硬（3.6 GPA）衬底-10- 12。这些细胞的第一种形式的肿瘤样细胞团，然后相互分离，从外围开始。作为这种上皮到圆形形态（E至R转变）发生变化时，它们增殖，减少细胞 - 细胞和细胞-ECM粘附，并成为迁移的。 HCT-8细胞培养上很辛苦聚苯乙烯基板没有表现出这些恶性特征。因此，已经假定HCT-8细胞成为转移性由于其暴露于适当的微环境。值得注意的是，这些实验进行了永生化癌细胞株或鼠衍生的原代细胞，不与原代人癌细胞。 最近的一项研究提出，扩充蜂窝牵引应力可被用作一个潜在的生物物理签名为转移细胞13。研究涉及测量牵引力为在聚丙烯酰胺凝胶不同的人类肿瘤细胞株。据发现，转移性癌细胞可以发挥显著较高牵引应力相比，非转移性的细胞在所有情况下13。然而，这些结果直接矛盾对鼠衍生的乳腺癌细胞系14中的早些时候发表的研究结果。此外，最近的一项研究强调了他们的细胞骨架重构亲永生和初级人体细胞之间的显着差异蛋白分析和细胞存活蛋白表达15。因此，重新许多生物物理测定法包括牵引原发性人类癌细胞的是重要的。这将解决的问题的原代细胞是否概括永生化癌细胞系牵引趋势。 这里所描述的协议被优化用于原代人结肠细胞（包括健康的和癌性的），并为它们对软底物（聚丙烯酰胺凝胶），以及在培养皿中培养的隔离。该协议基于消化外科组织样品成单细胞悬浮液16的结果酶解。据我们所知，这是直接在软水剂基质与牵引嵌入荧光微球培养流式细胞仪分离原发性结肠癌肿瘤和正常细胞的第一个示范。透明凝胶基质也让免疫。这个实验表明在F-肌动蛋白组织和差异粘着斑在原代人结肠癌细胞作为基材硬度的变化。该细胞培养平台开辟了探索原代人细胞的各种生物物理性质，如细胞刚度和牵引作为癌症预后参数的可能性。

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td ></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr >
			<td >HBSS</td>
			<td>Life technologies</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr >
			<td >PBS</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin</td>
			<td>Worthington</td>
			<td>LS003736</td>
			<td></td>
		</tr>
		<tr >
			<td >Collagenese</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td></td>
		</tr>
		<tr >
			<td >3- Aminopropyltrymethoxysilane (ATS)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td >281778</td>
			<td></td>
		</tr>
		<tr >
			<td >Glutaraldehyde</td>
			<td>Polysciences, Inc.</td>
			<td>01201-5</td>
			<td></td>
		</tr>
		<tr >
			<td >Acrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>A4058</td>
			<td></td>
		</tr>
		<tr >
			<td >N- methylenebisacrylamide (bis)</td>
			<td>Sigma-Aldrich</td>
			<td>M1533</td>
			<td></td>
		</tr>
		<tr >
			<td >HEPES buffer solution</td>
			<td>Sigma-Aldrich</td>
			<td >83264</td>
			<td></td>
		</tr>
		<tr >
			<td >Ammonium persulfate</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td></td>
		</tr>
		<tr >
			<td >N&#714;-tetramethylethylenediamine (TEMED)</td>
			<td>Bio-Rad</td>
			<td>161-0801</td>
			<td></td>
		</tr>
		<tr >
			<td >Human fibronectin</td>
			<td>BD biosciences</td>
			<td >354008</td>
			<td></td>
		</tr>
		<tr >
			<td >Hydrazine hydrate</td>
			<td>Sigma-Aldrich</td>
			<td >18412</td>
			<td>Hazardous</td>
		</tr>
		<tr >
			<td >Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr >
			<td >Paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>RT15710</td>
			<td></td>
		</tr>
		<tr >
			<td >Signal enhancer</td>
			<td>Life technologies</td>
			<td>I36933</td>
			<td></td>
		</tr>
		<tr >
			<td >Monoclonal anti vinculin antibody&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>V9131</td>
			<td></td>
		</tr>
		<tr >
			<td >Alexa fluor 488 goat anti-mouse IgG</td>
			<td>Life technologies</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr >
			<td >TRITC phalloidin conjugates&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1951</td>
			<td></td>
		</tr>
		<tr >
			<td >0.1 &#181;m fluroscent beads</td>
			<td>Life technologies</td>
			<td>F8801</td>
			<td></td>
		</tr>
		<tr >
			<td >0.25% Trypsin-EDTA</td>
			<td>Life technologies</td>
			<td>25200-056</td>
			<td></td>
		</tr>
	
		<tr >
			<td ></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		</tr>
		<tr >
			<td >12 mm2 glass cover slips</td>
			<td>Corning</td>
			<td>2865-12</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of primary human colon tumor cells from surgical tissues and culturing them directly on soft elastic substrates for traction cytometry

癌细胞响应不同癌细胞使用粘附受体组成的协调，分层机械化学系统和相关的信号转导的膜蛋白，细胞骨架结构，以及分子马达1,2与基质的机械刚度以复杂的方式。Mechanosensitivity 体外是与永生化细胞系或鼠衍生的原代细胞，不与原代人肿瘤细胞研究主要。因此，知之甚少初级人结肠癌细胞的体外 mechanosensitivity。这里，一个优化的协议被开发，描述从健康和癌外科人类组织样品的原代人结肠细胞的分离。孤立的结肠细胞，然后软（2千帕刚度）和僵硬（10千帕刚度）聚丙烯酰胺水凝胶和刚性聚苯乙烯（〜3.6 GPA刚度）由细胞外基质官能化基底（纤连蛋白培养成功在这种情况下）。荧光微珠嵌入在细胞培养物表面附近软凝胶，和牵引试验被执行以评估使用免费开放存取软件蜂窝收缩应力。此外，在不同的刚度衬底免疫荧光显微镜提供有关主细胞形态，细胞骨架组织及纽蛋白含有粘着斑作为底刚度的函数的有用信息。

上述协议被成功地用于对来自四个不同患者的多个组织样品（n = 12）下的机构审查委员会的指导方针。 图1A示出了从该组织切片的细胞培养物获得的手术后右一代表结肠直肠肿瘤。在转移到层流罩，以便进一步处理后的HBSS溶液的典型组织截面示于图1B。消化外科组织样品成单细胞悬浮液酶解的示意图示于图2。 成功的组织分离后，分离原?...

Watch this Scientific Journal Video about Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry at JoVE.com

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

从手术组织小学的人结肠癌细胞的分离和培养直接它们柔软的弹性基底牵引术

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

isolation-primary-human-colon-tumor-cells-from-surgical-tissues

Research

JoVE Journal

Bioengineering

15.4K 次观看.  University of Illinois at Urbana-Champaign. A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses. 

视频: Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

从病人的血液样本可行的循环肿瘤细胞的快速分离

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

科研

生物工程

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

工程骨骼肌组织中的小鼠成肌细胞的内皮祖细胞与应用电刺激

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

质量流式细胞术：协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

在光刻技术定义的聚对二甲苯-C细胞图案：二氧化硅<sub&gt; 2</sub&gt;基板

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

手术取出，分离和<em&gt;在体外</em&gt;扩展人类前十字韧带衍生细胞组织工程的应用

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

人体软组织的生物力学特性采用压痕和拉伸试验

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

人肺泡骨膜间充质干细胞的分离及维生素 D 对骨膜衍生细胞成骨活性的影响

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

用 Widefield 荧光显微镜研究软硅基美国匹茨堡的刚度测量

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.