这项工作得到了霍华德·休斯医学研究所（HHMI）协同创新奖。林支持信令和细胞调节研究所的T32 GM008759卫生培训资助和NIH的露丝L. Kirschstein前博士后研究员（CA165349-01）。我们要感谢他们的宝贵意见教授迈克尔·斯托厄尔（铜博尔德），教授道格拉斯里斯和罗布·菲利普斯教授（加州理工学院）。

1。脂质体的制备<ol><li>检索与聚四氟乙烯内衬盖20 mL的玻璃小瓶。 </li><li>清理所有与氯仿的玻璃器皿和注射器使用前，以防止污染。 </li><li>转移100μL试剂级氯仿的玻璃小瓶，用250μL气密玻璃注射器。 </li><li>加入30μL试剂级甲醇相同的玻璃小瓶，用100μL气密玻璃注射器。 </li><li>准备一个2毫米7:1.5:1.5卵磷脂（POPC）：磷脂（教皇）：胆固醇脂解，转移216μL10毫克/ 10毫克/毫升教皇和20μL11毫克/毫升，42毫升POPC微升在氯仿的胆固醇解决方案，以20 mL的玻璃小瓶。 </li><li>蒸发缓慢流动的脂质薄膜，直到被观察瓶底部氩气或氮气使用的有机溶剂。 </li><li>在真空干燥器不封顶的玻璃小瓶放置至少30分钟，以除去残留的溶剂。 </li><li>风帆ansfer 2缓冲液，通过0.2微米的过滤器，玻璃小瓶水合物血脂。 </li><li>孵育的混合物在4°C过夜，并在48小时内使用。 </li></ol> 2。仅冻结和解冻：脂质体大小为30 - 100纳米<ol><li>冻结15秒的脂质体悬浮在液氮。 </li><li>解冻加热块的脂质体悬浮液在42°C的温度〜3分钟。 </li><li>重复步骤2.1和2.2共5个周期。 </li></ol> 3。挤压按照Avestin说明正确组装的Liposofast的LF-50挤出机，使用仪器纲要“在他们的指导书。 <ol><li>将大洞，支持屏幕支持过滤基地（圆孔），其次是循环烧结盘（无孔），一个排水盘（直径25毫米），和一个聚碳酸酯膜（直径25毫米）。 </li><li>将黑色小膜顶部的O型圈，以确保它的支持过滤基地。 </li><li>附加4中的4个对应的孔的螺丝拧紧顶端的过滤挤出机。 </li><li>组装过滤器下方的大型挤出机机筒挤出机组。添加到缸筒的脂质体的解决方案。为了挤压筒的顶部放置以下内容：大型的圆形O型圈，窄帽，大型的圆形O型圈，圆帽。 </li><li>将气体调节挤出机机筒顶部，并关闭所有阀门，以防止空气泄漏，包括泄压阀。将玻璃小瓶20毫升或50 mL锥形瓶中挤出过滤装置下。安全阀连接到稳压器，并会释放，如果压力超过600磅。打开氮气，并打开煤气阀连接到挤出机。 </li><li>增加氮气压力为400纳米脂质体为100纳米脂质体，125磅，30纳米脂质体的400-500磅到25磅。 </li><li>观看脂质体悬液ension弹出，而氮的容器推入。保持氮的流动，直到你不再遵守任何通过挤出过滤器的玻璃小瓶中的液体通过。 </li></ol> 4。动态光散射（DLS）分析<ol><li>准备50μL20μM脂质体解决方案。 </li><li>打开电源和灯源。 </li><li>打开DynaPro软件。 </li><li>设置软件的MS / X算法模型检测30纳米和100纳米和MS800算法模型检测&gt; 100纳米脂质体的脂质体。 </li><li>连接硬件。 </li><li>吸取14μL脂质体样品的石英比色皿，并插入到细胞支架。 </li><li>按下启动。 </li><li>按停止后约20 - 30收购。 </li><li>分析直方图记录峰脂质体的平均直径。 </li></ol> 5。纳米粒子跟踪分析（NTA）的<ol><li>准备了500微升，0.1微米的脂质体解决方案。 </li><li>冲洗水和乙醇的样品室。 </li><li>与无棉绒纸巾擦干样品室。 </li><li>打开激光电源和电脑。 </li><li>提供300μL0.1微米脂质体解决方案的样品室。 </li><li>打开温度控制和纳米粒子跟踪分析软件。 </li><li>按捕捉按钮，打开激光。 </li><li>使用水平和垂直调整动议阶段，调整显微镜的焦点。 </li><li>设置所需的温度（20℃）和录音时间（至少30秒）。 </li><li>按下“录制”按钮，采取多种相框指定的时间量的脂质体颗粒。分析相应的直方图，它跟踪每个粒子的运动脂质体直径大小山峰。 </li></ol> 6。代表结果计划概述了挤压方法是p在图1愤慨。为了获得最佳的结果，直径为30纳米脂质体的制备要求的高压〜500 psi和100纳米的直径，需要一个125磅的压力，实现了快速的过滤率。对于直径为400纳米，低了25磅压力的建议，以达到过滤速率较慢，这使得囊泡拉长，形成较大的，均匀的脂质体。我们进行了一系列实验，以确定最佳生产一致的亚微米级囊泡大小压力。我们不同的压力，以及挤压通过孔径30，100和400 nm的聚碳酸酯过滤器传递的数量，为每个所需的大小，并发现了适当的压力。为30纳米的孔隙，低于500 psi的压力将降低流速，引起伸长，从而较大的囊泡大小。一个稳定的流量为100纳米的孔隙，达到125磅。低压（25 psi）的为400纳米的毛孔，使囊泡拉长成较大的囊泡小号izes。一个缓慢下降明智的过滤流量是最佳，以创造更大的亚微米泡13。 我们完成了当值律师服务，以确定通过三个不同直径的大小，即30，100和400 nm的挤压脂质体大小。 DLS是一个既定的方法，收集散射光，以确定粒子的直径。 2毫米，通过在500 psi的聚碳酸酯30纳米膜5张通行证通过过滤孔; 100 nm的聚碳酸酯薄膜在125磅与5科及格通过过滤孔和400 nm的聚碳酸酯在25 psi的膜2我们挤压水合脂质体通过过滤器的孔隙。脂质体和用DLS测量为30，100和400 nm孔径的直径分别为66±28，138±18和360±25纳米（ 图2）。暂停50纳米聚苯乙烯珠被用来作为校准标准，在图2所示，它的直径为47±16纳米。 ％polydispersity显示，脂质体的尺寸范围内有没有重叠。这是典型的观察直径小于30纳米时，使用动态光散射分析，由于已知的偏见，该仪器对有较大的颗粒12。同时从一个检测方法收集从大，小颗粒的散射光强度，从而更难解决脂质体在暂停12。尽管这种仪器的限制，校准曲线的描述附近的线性关系。 NTA是一种新技术，测量每个粒子在液体介质中，独立粒子的折射率和密度扩散的直接观测的大小。这种高分辨率的技术可以用来补充脂质体与DLS测量。国家旅游局记录了直径为95±48和356±51纳米，两个100纳米和400纳米聚苯乙烯解决方案被用于校准。观察到的30纳米和100纳米的脂质解决方案有更多的排版<html>耕地直径相对如图3所示，生产平均粒径为29±14 95±17和359±73纳米的DLS。国家旅游局，可能是一个更一般的表征技术来量化，因为它的灵敏度允许它来衡量50 - 1000 nm的粒子微观粒子。校准曲线显示了一个与记录国税局直径的聚碳酸酯膜孔之间的线性相关。 <table border="1"><tbody><tr><td> 脂质体大小 </td><td> 检测阈值 </td><td> 预计最小颗粒尺寸（nm） </td></tr><tr><td> 30纳米</td><td> 11 </td><td> 30 </td></tr><tr><td> 100纳米</td><td> 11 </td><td> 100 </td></tr><tr><td> 400毫微米</td><td> 21 </td><td> 400 </td></tr></tbody></table> 表1。Nanosight参数。 ad/4151/4151fig1.jpg“/&gt; 图1。挤压法，流程图描述如何的压力和氮气流量控制不同的脂质体直径的均匀性。脂质体水化后，不同尺寸的单层囊泡是通过不同的聚碳酸酯膜过滤器在不同压力下毛孔挤压。 <img alt="图2" src="/files/ftp_upload/4151/4151fig2.jpg" /> 图2。动态光散射（DLS）数据描述挤压后定量脂质体的大小。 （一）棒图显示，描述三种脂质体样品的直径。校准标准的50纳米聚苯乙烯（PS）珠被用来作为参考。脂质体的平均直径是上述每个样品的酒吧。 x轴描述的解决方案，其中挤压通过孔径。介绍用DLS记录y轴的直径。虽然30纳米和100纳米大小的记录编辑值高于其孔径较大的400纳米大小录得略低的大小，校准曲线（b）显示附近的线性关系，其中x轴表示聚碳酸酯膜孔大小，y轴的描述用DLS录得的脂质体直径。 <img alt="图3" src="/files/ftp_upload/4151/4151fig3.jpg" /> 图3纳米粒子跟踪分析（NTA）的数据描述挤压脂质体大小。 （一）条形图代表各脂质体样品的直径。 x轴描述的解决方案，其中挤压通过孔径。 y轴描述了由国家旅游局录得的直径大小。脂质体的平均直径是上述每个样品的标记。 （二）NTA校准曲线显示比之间的记录的直径与孔径的过滤膜的动态光散射校正曲线的线性相关。 x轴描述聚碳酸酯膜孔大小。 y轴的描述由国家旅游局的记录脂质体直径。 <img alt="图4" src="/files/ftp_upload/4151/4151fig4.jpg" /> 图4。负染色透射电子显微镜（TEM）图像显示每个脂质体的大小（500微米），其次受到挤压。呈负出院前碳福尔瓦网格来样染色，其中1％醋酸铀水用于染色的样品前干燥和34000×放大倍率成像。大小30，100和400 nm明显有别于对方。放大倍率为25,000×。比例尺表示0.5微米。 <img alt="图5" src="/files/ftp_upload/4151/4151fig5.jpg" /> 图5。一个时间课程实验进行了三种脂质体的尺寸。动态光散射（DLS）测量每个脂质体的解决方案后立即挤压。脂质体解决方案储存在4℃C过夜。 DLS的过夜培养后，其直径均录得。几乎没有变化，观察后16小时的潜伏期。

<ol>
	<li>Connor, J., Bucana, C., Fidler, I. J., Schroit, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation-dependent+expression+of+phosphatidylserine+in+mammalian+plasma+membranes:+quantitative+assessment+of+outer-leaflet+lipid+by+prothrombinase+complex+formation.">Differentiation-dependent expression of phosphatidylserine in mammalian plasma membranes: quantitative assessment of outer-leaflet lipid by prothrombinase complex formation.</a> Proc. Natl. Acad. Sci. U.S.A. 86, 3184-3188 (1989).</li><li>Smith, S. A., Morrissey, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+incorporation+of+tissue+factor+into+liposomes.">Rapid and efficient incorporation of tissue factor into liposomes.</a> J. Thromb. Haemost. 2, 1155-1162 (2004).</li><li>Hui, E., Johnson, C. P., Yao, J., Dunning, F. M., Chapman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptotagmin-mediated+bending+of+the+target+membrane+is+a+critical+step+in+Ca(2+)-regulated+fusion.">Synaptotagmin-mediated bending of the target membrane is a critical step in Ca(2+)-regulated fusion.</a> Cell. 138, 709-721 (2009).</li><li>Loughrey, H. C., Choi, L. S., Cullis, P. R., Bally, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+procedures+for+the+coupling+of+proteins+to+liposomes.">Optimized procedures for the coupling of proteins to liposomes.</a> J. Immunol. Methods. 132, 25-35 (1990).</li><li>Mui, B., Chow, L., Hope, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrusion+technique+to+generate+liposomes+of+defined+size.">Extrusion technique to generate liposomes of defined size.</a> Methods Enzymol. 367, 3-14 (2003).</li><li>Gruner, S. M., Cullis, P. R., Hope, M. J., Tilcock, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+polymorphism:+the+molecular+basis+of+nonbilayer+phases.">Lipid polymorphism: the molecular basis of nonbilayer phases.</a> Annu. Rev. Biophys. Biophys. Chem. 14, 211-238 (1985).</li><li>Guven, A., Ortiz, M., Constanti, M., O'Sullivan, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+method+for+the+size+separation+of+homogeneous+fluorescein-encapsulating+liposomes.">Rapid and efficient method for the size separation of homogeneous fluorescein-encapsulating liposomes.</a> J. Liposome. Res. 19, 148-154 (2009).</li><li>Zimmerberg, J., Kozlov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+proteins+produce+cellular+membrane+curvature.">How proteins produce cellular membrane curvature.</a> Nature Reviews Molecular Cell Biology. 7, 9-19 (2006).</li><li>Chonn, A., Cullis, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+liposomal+drug-delivery+systems.">Recent advances in liposomal drug-delivery systems.</a> Curr. Opin. Biotechnol. 6, 698-708 (1995).</li><li>Mayer, L. D., Hope, M. J., Cullis, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicles+of+variable+sizes+produced+by+a+rapid+extrusion+procedure.">Vesicles of variable sizes produced by a rapid extrusion procedure.</a> Biochim. Biophys. Acta. 858, 161-168 (1986).</li><li>Matsuoka, K., Schekman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+liposomes+to+study+COPII-+and+COPI-coated+vesicle+formation+and+membrane+protein+sorting.">The use of liposomes to study COPII- and COPI-coated vesicle formation and membrane protein sorting.</a> Methods. 20, 417-428 (2001).</li><li>Dragovic, R. A., Gardiner, C., Brooks, A. S., Tannetta, D. S., Ferguson, D., Hole, P., Carr, B., Redman, C., Harris, A. L., Dobson, P. J., Harrison, P., Sargent, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sizing+and+phenotyping+of+cellular+vesicles+using+Nanoparticle+Tracking+Analysis.">Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis.</a> Nanomedicine. , (2011).</li><li>Hunter, D. G., Frisken, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+extrusion+pressure+and+lipid+properties+on+the+size+and+polydispersity+of+lipid+vesicles.">Effect of extrusion pressure and lipid properties on the size and polydispersity of lipid vesicles.</a> Biophys J. 74, 2996-3002 (1998).</li></ol>

没有利益冲突的声明。

我们使用的Avestin Liposofast LF-50挤出机，并演示了如何小型，合成脂质体通过压力控制系统的准备。重要的是要注意多室囊泡自发形成脂质体水化后，这可能会导致更小的纳米粒子的生产。这些小的多室囊泡，将不可避免地流过较大的聚碳酸酯膜孔径，导致异质性的解决方案由一个大型的过滤孔的单层囊泡。因此，建议，以减少氮流量压力，当使用大孔径直径为400纳米以上所述毛孔（即25磅）为了?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>氯仿</td><td> Sigma-Aldrich公司</td><td> 02432-25ML </td><td> 95％的稳定剂</td></tr><tr><td>高品位的甲醇</td><td> Sigma-Aldrich公司</td><td> 179337-4L </td><td></td></tr><tr><td> liposofast LF-50挤出机</td><td> avestin，公司</td><td></td><td></td></tr><tr><td>磷脂</td><td>阿凡提极性脂</td><td></td><td></td></tr><tr><td>聚碳酸酯毛孔</td><td> avestin，公司</td><td></td><td>直径25毫米</td></tr><tr><td>排水光盘体育</td><td> avestin，公司</td><td> 230600 </td><td>直径25毫米</td></tr></tbody></table>

constant pressure-controlled extrusion method for the preparation of nano-sized lipid vesicles

脂质体是人为编制的模拟平台，研究蛋白质和脂质蛋白相互作用3，监测给药4,5，和4封装膜作为细胞被广泛使用的天然和合成磷脂组成的囊泡。磷脂自然弯曲的脂质双层，本身区别于胶束。6脂质体是双层，即大（LUVs）的单层囊泡，小囊泡（越野车）单层和多室囊泡（MLV更具）的大小和数量由传统分类。特别是，编制各种大小均匀脂质体是重要的研究膜的曲率，在细胞信号传递中扮演了至关重要的作用，内和胞吐作用，膜融合，蛋白质贩运8。几组分析蛋白质如何被用来调节过程，涉及膜的曲率，从而准备脂质体直径&lt;100 - 400纳米研究其对细胞功能的行为3。其他专注于脂质体药物包封，学习车辆进行并交付利息9药物的脂质体。可以实现药物包封在脂质体形成9报告。我们的挤压步骤应该没有影响，原因有两个封装的药物，即（1）药物包封应事先取得到了这一步，（2）脂质体应保留其天然生物物理稳定性，安全携带药物在水溶液中的核心。这些研究的目标，进一步表明需要优化的方法来设计稳定的亚微米级的脂质囊泡。 尽管如此，目前的脂质体的制备技术（10超声，冷冻和解冻10，沉淀）不容许高度的一致性和高效率10,5，这限制了生物物理曲面（即直径小于100纳米）的脂质体的制备研究1 emergi会吴膜曲率传感领域。在此，我们提出了各种生物相关的脂质体健壮的制备方法。 使用气密注射器和聚碳酸酯薄膜10,5手动挤压是一种常见的做法，但经常被观察到的异质性时，使用孔径小于100纳米，由于由于手动施加压力的变异。我们采用恒压控制挤出设备，准备合成脂质体，其直径范围在30和400 nm之间。被用来量化脂质体的大小，在我们的协议与商业聚苯乙烯（PS）作为校准标准用于珠，动态光散射（DLS）10 11，电子显微镜和纳米粒子跟踪分析（NTA）的12。一个附近的线性相关就业孔径和实验测定脂质体之间，说明我们的压力控制脂质体的制备高保真会见HOD。此外，我们已经表明，这种脂质囊泡的制备方法是普遍适用的，独立于各种脂质体的大小。最后，我们在一个时间过程的研究还表明，这些准备脂质体是稳定的，长达16小时。下面演示了一个有代表性的纳米脂质体的制备协议。

Watch this Scientific Journal Video about Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles at JoVE.com

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

本协议描述了一个准备一个高度同质化的亚微米大小的脂质体的挤压方法。此方法使用脂质体的制备与控制氮流量压力控制系统。脂质准备<sup&gt; 1,2</sup&gt;，脂质体挤压，大小特征将在此呈现。

恒压控制的挤压方法制备纳米脂质体

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

constant-pressure-controlled-extrusion-method-for-preparation-nano

Research

JoVE Journal

Bioengineering

23.8K 次观看.  University of Colorado Boulder. 本协议描述了一个准备一个高度同质化的亚微米大小的脂质体的挤压方法。此方法使用脂质体的制备与控制氮流量压力控制系统。脂质准备 1,2，脂质体挤压，大小特征将在此呈现。

视频: Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

科研

生物工程

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

对于I型胶原蛋白的制备皮肤方法的改进

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

脂质双层囊泡代使用微流控喷射

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

支持的脂双层云母制备的高分辨率光学显微成像

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

生物膜制作的溶剂辅助脂质双分子层（SALB）方法

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a high-protein crambe meal that has limited value, as it is not fit for food or feed use. However, it contains proteins that could potentially make it a suitable raw material for higher-value products. The purpose of this study was to find methods of making this side stream into extruded films, showing that products with a higher value can be produced. The study mainly considered the development of material compositions and methods of preparing and extruding the material. Wheat gluten was added as a supportive protein matrix material, together with glycerol as a plasticizer and urea as a denaturant. The extrudate was evaluated with respect to mechanical (tensile testing) and oxygen barrier properties, and the extrudate structure was revealed visually and by scanning electron microscopy. A denser, more homogeneous material had a lower oxygen transmission rate, higher strength, and higher extensibility. The most homogeneous films were made at an extruder die temperature of 125-130 &#176;C. It is shown here that a film can be extruded with promising mechanical and oxygen barrier properties, the latter especially after a final compression molding step.

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

The side stream from plant oil production of Crambe abyssinica has limited value. The purpose of this study was to find methods for extruding materials based on this side stream, demonstrating that products with a higher value can be produced. The extrudates were found to have promising properties.

薄膜挤出<em&gt;海甘蓝abyssinica</em&gt; /谷朊粉共混物

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a ...

Evaluation of the Storage Stability of Extracellular Vesicles

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical development, not only their pharmaceutical activity is important but also their production needs to be evaluated. In this context, research focuses on the isolation of EVs, their characterization, and their storage. The present manuscript aims at providing a facile procedure for the assessment of the effect of different storage conditions on EVs, without genetic manipulation or specific functional assays. This makes it possible to quickly get a first impression of the stability of EVs under a given storage condition, and EVs derived from different cell sources can be compared easily. The stability measurement is based on the physicochemical parameters of the EVs (size, particle concentration, and morphology) and the preservation of the activity of their cargo. The latter is assessed by the saponin-mediated encapsulation of the enzyme beta-glucuronidase into the EVs. Glucuronidase acts as a surrogate and allows for an easy quantification via the cleavage of a fluorescent reporter molecule. The present protocol could be a tool for researchers in the search for storage conditions that optimally retain EV properties to advance EV research toward clinical application.

Here we present a readily applicable protocol to assess the storage stability of extracellular vesicles, a group of naturally occurring nanoparticles produced by cells. The vesicles are loaded with glucuronidase as a model enzyme and stored under different conditions. After storage, their physicochemical parameters and the activity of the encapsulated enzyme are evaluated.

细胞外囊储存稳定性的评价

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure single-molecular binding kinetics, assess mechanical properties of ligand-receptor interactions, visualize protein dynamic conformational changes and more excitingly elucidate receptor mediated cell mechanosensing mechanisms. More recently, BFP has been used to measure the spring constant of molecular bonds. This protocol describes the step-by-step procedure to perform molecular spring constant DFS analysis. Specifically, two BFP operation modes are discussed, namely the Bead-Cell and Bead-Bead modes. This protocol focuses on deriving spring constants of the molecular bond and cell from DFS raw data.

A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP.

分子弹簧常量分析由生物元原力探针光谱

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure ...