1。总结筹备组织的表面张力的测量。 对于贴壁细胞，球体可以使用的悬滴法或产生一个连贯的表，然后将其削减到1毫米的碎片细胞形成。 总结形成悬滴法： <ol><li>近融合的贴壁细胞培养应增长到90％汇合，届时单层应该用PBS漂洗两次。排水良好后，加入2毫升，0.05％胰蛋白酶1毫米EDTA（100毫米板块），并在37 ° C，直到细胞分离。停止加入2 MLS的完全培养基，轻轻地用5毫升吸管磨碎的混合物，直至细胞悬浮液中的胰蛋白酶。细胞转移到15毫升锥形管。 </li><li>加入40μl和10毫克/毫升的DNA酶原液为5分钟，在室温下孵育。涡短暂离心5分钟，在200 XG。 </li><li>弃上清，用1毫升完整的组织培养基颗粒。重复，然后在2 MLS的完整的组织培养液中的悬浮细胞。 </li><li>计数细胞，使用hemacytometer，或自动细胞计数，调整浓度为2.5 × 10 6细胞/ ml 。 </li><li>删除从60毫米组织培养皿并将其放置在底部的菜5毫升的PBS的盖子。这将作为一个水化室。 </li><li>倒置的盖子，并用20μL移液器存款10μL滴上的盖子底部。确保，滴放置足够的距离，以不碰。这是可能的地方至少有20％菜滴。 </li><li>反转到的PBS填充的底部室和孵化在37 ° C / 5％的CO 2 / 95％湿度盖子，监察滴每天孵育直到细胞表或聚集已经形成。 </li><li>一旦表的形式，他们可以转移到圆底玻璃振动筛瓶含有3 MLS和完全培养基在摇晃水浴孵育37 ° C和5％，直到球体形成的CO 2。 </li></ol>总结形成的细胞表方法： <ol start="9"><li>如上所述，但浓度调整为1 × 10 6细胞/ ml单细胞悬液准备。 </li><li> 3毫升细胞悬液转移到10 ml圆底烧瓶（贝尔科，葡萄园，新泽西州）。 </li><li>孵育一个回旋水浴/摇床（新不伦瑞克省的科学，爱迪生，新泽西州）在37 ° C，5％CO2 4H 2 120转的烧瓶，直到从胰蛋白酶的细胞恢复。 </li><li>细胞转移到玻璃圆底离心管和200 X克成细颗粒的离心。另外24小时的孵育，直到一个连贯的表的形式。 </li><li>轻轻摇动培养管逐出表，并倒入一个小的，无菌的玻璃组织培养皿中培养管的内容。 </li><li>用微型手术刀，切成不同大小的片段的纸张。 </li><li>孵育37 ° C下在120转的回旋水浴摇床5％的CO 2 2至3天，或直至他们成为球形片段。 </li></ol> 2。骨料的表面张力的测量<ol><li> 组织表面张力 。该仪器是在图所示。 1和2。压缩单元（图3）两院组成。外室（OC），连接到37℃的循环水泵，是监管的内宅（IC）的温度。该商会是建造精聚甲醛和含有石英聚合的可视化窗口。使用较低的大会（LA）螺钉进入内宅的基础和为1）的位置在内宅的总和; 2）密封内腔的底部; 3）提高总启动压缩;（4 ）控制的平行，因此总的压缩之间的距离。大会（CC）的核心是可调的。核心提示（底座）组成的顺利聚四氟乙烯和较低的压缩板（LCP）的行为。上部压缩板（UCP）是聚四氟乙烯缸15毫米长，从平衡臂挂起（B）由火焰摆正镍铬丝（NCW）*在实验过程中，细胞聚合（一）是定位于下盘，并提出，直到它接触的上盘。上盘连接到平衡臂（二）。总的压缩会导致平衡臂的位移。的平衡是一个卡恩/ Ventron模型2000录音electrobalance，空平衡的原则运作。平衡臂的支点有一个永久磁场内的电枢。平衡运行时，它不断地调节通过电磁大会，这反过​​来又保持在水平位置平衡臂的电流通过。当一个对象被暂停的平衡臂，电压，适用于在horizo​​保持平衡手臂NTAL位置，是成正比对象的权衡的。此电压措施适用于总的外部压力。聚集的形状是通过25 x尼康SMZ10A配备图像采集卡的计算机的立体视觉观察监测。为了尽量减少细胞聚集粘附压缩板，无论是下限和上限的压缩板涂有聚（2 - hydroxyethylmethacrylate）{聚（HEMA）}，聚合物材料，细胞不坚持 16 。 *丝火焰理顺夹紧，挂着一个反驳的立场15英寸长的电线和一个小粘结剂剪辑到最后。本生灯然后运行线的长度，直到该导线呈红色。截弯取直的电线，然后可以切成适当的长度。小钩是弯曲的线约一英寸¼从使用两个刀片的最终形成。另一端，然后插入到每桶较低的压缩板。 </li><li> 总结压缩。 <ol><li>内宅是充满预热CO 2独立的组织培养基（GIBCO / BRL，NY） 。 </li><li>聚集在大小不等，从约200-300μ定位较低的压缩板（图4A）。聚合加载吸在组织培养基了巴斯德吸管尖端装有硅胶球中途中的聚合，吸管转移到内宅，定位上述LCP的吸管尖。骨料，然后轻轻地驱逐到LCP轻轻挤压灯泡。另外，总重力下降到LCP。 </li><li>上部压缩板（UCP）是定位高于总可以来台定居，建立预压明显UCP的重量基线。 </li><li>在LCP，然后提出，直到总压缩是对跟单信用证统一惯例（图4B）。调整的降低设备的内在核心的高度将控制不同程度的压缩。压缩是通过配备的CCD摄像头在解剖显微镜观察监测。 </li><li>总结图像被捕获，数字化，并使用ImageJ分析。视UCP体重变化是连续记录上的一个带状图记录，被卡恩平衡的电压输出练级小康表示形状均衡的成果。每个聚合受到不同程度的压缩。 </li></ol></li><li> 计算总的表面张力 。 在造型的平衡，可以得到的压缩细胞之间的平行板，它不坚持的聚集cohesivity，从青年Laplace方程（图5），其中σ是cohesivity，F是力压缩总，πr3 2施加力F总表面面积，R 2和R 3，分别压缩总的赤道半径和定义它的表面轮廓正常的弧形半径压缩板和扩大它们之间的（图6）。在力量和形状平衡的压缩力和几何测量和应用这些测量杨- Laplace方程，产生明显的组织表面张力的数值。聚集，达到平衡后，σ1计算，解压缩，并允许接近第二个平衡和 σ2的计算如上所述。 </li><li> 确认总的流动性 。 液体总量计算出的表面张力，当两个不同的按压将保持不变。的比例在这样的聚集σ2 /σ1等于1，将不到的力量在每一个连续的压缩（F 2 / F 1）的应用比例。相比之下，弹性聚合计算表面张力将服从虎克定律，并增加施加的力。的比例，对于弹性聚合σ2 /σ1不会等于1，而将方法F 2 / F 1的比例。表面张力的液体总量也将独立的总大小2,17。只有表面张力施加的力和规模的独立的测量是用来计算平均σ为每个细胞株。 </li></ol> 3。代表性的成果： 下面是一个典型的TST结果大鼠前列腺成纤维细胞（爱国阵线）和大鼠前列腺平滑肌细胞（RPSMC）的聚合表。在图中可以看出。 7卢旺达爱国阵线细胞聚集体的表面张力为22.8 ± 1.1达因/厘米。此外，平均压缩后1和2压缩后的表面张力测量值进行统计学IDENTICAL时由配对t检验比较。我们还比较σ2 /σ1和F 2 / F 1，以确保这些总量不服从胡克定律，因为他们，如果他们表现为弹性固体的比率。表1表明，σ2 /σ1确实接近1.0的比例。此外，F 2 / F 1的比例大大超过σ2 /σ1（配对t检验，P &lt;0.05），进一步证实了这些总量不服从虎克定律，实际上表现为液体。相比之下RPSMCs服从胡克定律。作为是显而易见的，在表1，σ2 /σ1的比例明显大于1，并且无统计学比F 2 / F 1的不同。为了进一步证明液体状的行为，我们探索的表面张力（σ）和总体积之间的关系。在图中可以看出。 8，体积是sigma的独立爱国阵线细胞（红色回归线，R 2 = 0.002），而出现一些sigma的RPSMCs（蓝色的回归线，R 2 = 0.146）卷上的依赖。这些数据进一步证实，卢旺达爱国阵线聚集的行为在液体状的方式，而RPSMCs出现更多的表现为弹性固体。只有那些表现为液体的聚合获得mesurements，将用于计算表面张力。 <img src="/files/ftp_upload/2739/2739fig1.jpg" alt="图1"/> 图1。组织表面张力的概述。 <img src="/files/ftp_upload/2739/2739fig2.jpg" alt="图2"/> 图2。张力室（右图）的一个更详细的视图。 <img src="/files/ftp_upload/2739/2739fig3.jpg" alt="图3"/> 图3。示意图张力室。 <img src="/files/ftp_upload/2739/2739fig4.jpg" alt="图4"/> 图4未压缩的（A）和压缩（二）聚集的图片。 <img src="/files/ftp_upload/2739/2739fig5.jpg" alt="图5"/> 图5。拉普拉斯方程。 <img src="/files/ftp_upload/2739/2739fig6.jpg" alt="图6"/> 图6。液滴之间的两个平行板，它坚持理想形状的平衡，压缩图。 R 1和R 2是两个小学曲率半径，在液滴的赤道平面通过其对称轴分别。 R 3的是液滴的接触要么压缩板的圆形区域的半径。 H是上部和下部的压缩板之间的距离。 X是一个直角三角形斜边ř一侧2延伸到液滴的表面，要么压缩板之间的接触点。 <table border="1"><tbody><tr><td></td><td> σ1（达因/厘米± SEM） </td><td> σ2（达因/厘米± SEM） </td><td> Pσ1 VSσ2 </td><td> σ1,2（达因/厘米± SEM） </td><td> σ2 /σ1 </td><td> F 2 / F 1 </td><td> Pσ2 /σ1和F 2楼1 </td></tr><tr><td>卢旺达爱国阵线</td><td> 22.6 ± 1.7 </td><td> 22.9 ± 1.4 </td><td> &gt; 0.05 * </td><td> 22.8 ± 1.1 </td><td> 1.04 ± 0.04 </td><td> 1.47 ± 0.06 </td><td> &lt;0.05 </td></tr><tr><td> RPSMC </td><td> 15.0 ± 2.8 </td><td> 23.0 ± 3.2 </td><td> 0.039 </td><td>不适用</td><td> 1.9 ± 0.3 </td><td> 1.6 ± 0.1 </td><td> 0.16 * </td></tr></tbody></table> 图7。尖沙咀测量和确认的总流动性大鼠前列腺成纤维细胞和平滑肌细胞的聚集。 <img src="/files/ftp_upload/2739/2739fig8.jpg" alt="图8"/> 图8。Sigma和卢旺达爱国阵线的聚集量（红线）和RPSMCs（蓝线）之间的关系。

<ol>
	<li>Foty, R. A., Forgacs, G., Pfleger, C. M., Steinberg, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+properties+of+embryonic+tissues:+Measurement+of+interfacial+tensions.">Liquid properties of embryonic tissues: Measurement of interfacial tensions.</a> Phys Rev Lett. 72, 2298-2301 (1994).</li><li>Foty, R. A., Pfleger, C. M., Forgacs, G., Steinberg, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+tensions+of+embryonic+tissues+predict+their+mutual+envelopment+behavior.">Surface tensions of embryonic tissues predict their mutual envelopment behavior.</a> Development. 122, 1611-1620 (1996).</li><li>Schotz, E. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+differences+in+tissue+surface+tension+influence+zebrafish+germ+layer+positioning.">Quantitative differences in tissue surface tension influence zebrafish germ layer positioning.</a> HFSP Journal. 2, 42-56 (2008).</li><li>Jia, D., Dajusta, D., Foty, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+surface+tensions+guide+in+vitro+self-assembly+of+rodent+pancreatic+islet+cells.">Tissue surface tensions guide in vitro self-assembly of rodent pancreatic islet cells.</a> Dev Dyn. 236, 2039-2049 (2007).</li><li>Schwarz, M. A., Zheng, H., Legan, S., Foty, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+Self-Assembly+is+Modulated+by+Tissue+Surface+Tensions.">Lung Self-Assembly is Modulated by Tissue Surface Tensions.</a> Am J Respir Cell Mol Biol. , (2010).</li><li>Ryan, P. L., Foty, R. A., Kohn, J., Steinberg, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+spreading+on+implantable+substrates+is+a+competitive+outcome+of+cell-cell+vs.+cell-substratum+adhesivity.">Tissue spreading on implantable substrates is a competitive outcome of cell-cell vs. cell-substratum adhesivity.</a> Proc Natl Acad Sci U S A. 98, 4323-4327 (2001).</li><li>Foty, R. A., Steinberg, M. 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+differential+adhesion+hypothesis:+a+direct+evaluation.">The differential adhesion hypothesis: a direct evaluation.</a> Dev Biol. 278, 255-263 (2005).</li><li>Robinson, E. E., Foty, R. A., Corbett, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+matrix+assembly+regulates+alpha5beta1-mediated+cell+cohesion.">Fibronectin matrix assembly regulates alpha5beta1-mediated cell cohesion.</a> Mol Biol Cell. 15, 973-981 (2004).</li><li>Robinson, E. E., Zazzali, K. M., Corbett, S. A., Foty, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha5beta1+integrin+mediates+strong+tissue+cohesion.">alpha5beta1 integrin mediates strong tissue cohesion.</a> J Cell Sci. 116, 377-386 (2003).</li><li>Winters, B. S., Raj, B. K., Robinson, E. E., Foty, R. A., Corbett, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+regulates+Raf-1+expression+to+modulate+fibronectin+matrix+assembly.">Three-dimensional culture regulates Raf-1 expression to modulate fibronectin matrix assembly.</a> Mol Biol Cell. 17, 3386-3396 (2006).</li><li>Caicedo-Carvajal, C. E., Shinbrot, T., Foty, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha5beta1+integrin-fibronectin+interactions+specify+liquid+to+solid+phase+transition+of+3D+cellular+aggregates.">Alpha5beta1 integrin-fibronectin interactions specify liquid to solid phase transition of 3D cellular aggregates.</a> PLoS One. 5, e11830-e11830 (2010).</li><li>Foty, R. A., Steinberg, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+tumor+cell+cohesion+and+suppression+of+invasion+by+E-+or+P-cadherin.">Measurement of tumor cell cohesion and suppression of invasion by E- or P-cadherin.</a> Cancer Res. 57, 5033-5036 (1997).</li><li>Foty, R. A., Corbett, S. A., Schwarzbauer, J. E., Steinberg, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dexamethasone+up-regulates+cadherin+expression+and+cohesion+of+HT-1080+human+fibrosarcoma+cells.">Dexamethasone up-regulates cadherin expression and cohesion of HT-1080 human fibrosarcoma cells.</a> Cancer Res. 58, 3586-3589 (1998).</li><li>Winters, B. S., Shepard, S. R., Foty, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+measurement+of+brain+tumor+cohesion.">Biophysical measurement of brain tumor cohesion.</a> Int J Cancer. 114, 371-379 (2005).</li><li>Foty, R. A., Cummings, K. B., Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+surface+tensiometry:+a+novel+technique+for+predicting+invasive+potential+of+prostate+tumors+based+on+tumor+cell+aggregate+cohesivity+in+vitro.">Tissue surface tensiometry: a novel technique for predicting invasive potential of prostate tumors based on tumor cell aggregate cohesivity in vitro.</a> Surgical Forum L. , 707-708 (1999).</li><li>Folkman, J., Moscona, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cell+shape+in+growth+control.">Role of cell shape in growth control.</a> Nature. 273, 345-349 (1978).</li><li>Foty, R. A., Forgacs, G., Pfleger, C. M., Steinberg, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+properties+of+embryonic+tissues:+Measurement+of+interfacial+tensions.">Liquid properties of embryonic tissues: Measurement of interfacial tensions.</a> Physical Review Letters. 72, 2298-2301 (1994).</li><li>Guevorkian, K., Colbert, M. J., Durth, M., Dufour, S., Brochard-Wyart, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspiration+of+biological+viscoelastic+drops.">Aspiration of biological viscoelastic drops.</a> Phys Rev Lett. 104, 218101-218101 (2010).</li></ol>

尖沙咀总的凝聚力测量相对比较简单。有，但是，为了生成可用的尖沙咀数据，必须要掌握的关键步骤; 1）总量必须是“健康”。这可以确保聚合形成与细胞的最佳汇合前支队开始控制。骨料粒径和文化也必须加以控制，以尽量减少坏死核心内总的发展; 2）另一个影响尖沙咀测量的参数，可以是总的附着力程度上部或下部的压缩板。因此，聚甲基丙烯酸羟乙酯的最佳浓度，用于涂层板必须凭经验?...

<ul><li>水浴/振动筛（新不伦瑞克省的科学，爱迪生，新泽西州） </li><li> 10 ml圆底烧瓶（贝尔科，葡萄园，新泽西州） </li></ul>

measurement of aggregate cohesion by tissue surface tensiometry

严格测量间的结合能只能使用接地系统中的热力学原理，在平衡的方法。我们已经开发出组织表面tensiometry（TST），专门用于测量表面的细胞之间相互作用的自由能。生物物理概念的基本尖沙咀此前已在详细的1,2描述。该方法是基于观察，细胞相互凝聚力，如果维持在振荡培养，会自发地聚集到集群。随着时间的推移，这些集群将全面形成球体。这四舍五入的行为模仿的液体系统的行为特征。细胞间的结合能测量压缩在定制设计的组织表面张力平行板之间的球形集合体。相同的数学方程，用来衡量一个液滴的表面张力是用来衡量组织的三维球形聚合表面张力。相当于液体的表面张力的细胞间的结合能，或更普遍的是，组织cohesivity。我们实验室以前的研究表明，组织的表面张力（1）预测两组胚胎干细胞如何将与另一1-5（2）可以强烈地影响着组织的能力与生物材料的 6相互作用，（3）可以改变，不仅通过直接操纵的钙粘蛋白为基础的 7间凝聚力，但也操纵关键的ECM分子如FN 8-11日和4）相关肺癌12 13，纤维肉瘤，脑肿瘤和前列腺肿瘤的侵袭潜力细胞系15。在这篇文章中，我们将描述仪器，细节产生球体所需的步骤，加载到张力腔的球体，启动总压缩，并组织产生的表面张力测量，分析和验证。

Watch this Scientific Journal Video about Measurement of Aggregate Cohesion by Tissue Surface Tensiometry at JoVE.com

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

我们描述的测量结合能源，作为组织的表面张力表达的方法，细胞内的三维组织状集合体之间。在组织表面张力的差异已经证明关联，肺，肌肉和脑肿瘤的侵袭，并建立不同类型的细胞之间的空间关系的基本决定因素。

总结凝聚力测量组织表面Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

measurement-of-aggregate-cohesion-by-tissue-surface-tensiometry

Research

JoVE Journal

Bioengineering

12.5K 次观看.  UMDNJ-Robert Wood Johnson Medical School. 我们描述的测量结合能源，作为组织的表面张力表达的方法，细胞内的三维组织状集合体之间。在组织表面张力的差异已经证明关联，肺，肌肉和脑肿瘤的侵袭，并建立不同类型的细胞之间的空间关系的基本决定因素。

视频: Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

科研

生物工程

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

通过结合表面接枝和修改后的过程光敏感膜的制备

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.

Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces.

细菌表面电位测量用开尔文探针力显微镜

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of exosomes to determine if they carry disease discriminatory biomarkers. For performing exosomal analysis, it is necessary to develop a method for extracting and analyzing exosomes from target biofluids without damaging the internal content.
Electric field-induced release and measurement (EFIRM) is a method for specifically extracting exosomes from biofluids, unloading their cargo, and testing their internal RNA/protein content. Using an anti-human CD63 specific antibody magnetic microparticle, exosomes are first precipitated from biofluids. Following extraction, low-voltage electric cyclic square waves (CSW) are applied to disrupt the vesicular membrane and cause cargo unloading. The content of the exosome is hybridized to DNA primers or antibodies immobilized on an electrode surface for quantification of molecular content.
The EFIRM method is advantageous for extraction of exosomes and unloading cargo for analysis without lysis buffer. This method is capable of performing specific detection of both RNA and protein biomarker targets in the exosome. EFIRM extracts exosomes specifically based on their surface markers as opposed to size-based techniques.
Transmission electron microscopy (TEM) and assay demonstrate the functionality of the method for exosome capture and analysis. The EFIRM method was applied to exosomal analysis of 9 mice injected with human lung cancer H640 cells (a cell line transfected to express the exosome marker human CD63-GFP) in order to test their exosome profile against 11 mice receiving saline controls. Elevated levels of exosomal biomarkers (reference gene GAPDH and protein surface marker human CD63-GFP) were found for the H640 injected mice in both serum and saliva samples. Furthermore, saliva and serum samples were demonstrated to have linearity (R = 0.79). These results are suggestive for the viability of salivary exosome biomarkers for detection of distal diseases.

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves.

通过电场诱导释放和测量（EFIRM）检测外体生物标志物

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

最大等长收缩力的测量通过透骨骼肌纤维生成

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

适应静电过程中提供三个独特的环境为一个三层次<em&gt;在体外</em&gt;的气道壁模型

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

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 ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

点化学法现场定向固定骨形态发生蛋白2到固体表面

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.