Name: preparation of giant vesicles encapsulating microspheres by centrifugation of a water-in-oil emulsion
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Description: Watch this Scientific Journal Video about Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion at JoVE.com

我们感谢山口智子在图1绘制了示意图像。这项研究是由冈崎研究所自然科学theNational学院生物科学结合的冈崎ORION项目（NINS）的支持;由天体生物学中心项目NINS（无AB281010）;由格兰特 - 在援助来自日本学术振兴科学研究的创新领域（用于创建集成功能生物分子系统的动态排序）（JSPS）（无25102008）;由格兰特 - 在资助青年科学家（B）（以KK，没有15K17850）由日本学术振兴会; andby从JSPS格兰特 - 在援助研究活动启动（以YN，没有。15H06653）。额外的支持是由野口研究所，花王基金会艺术和科学的资助，并从栗田与环境基金会的资助拨款提供。

1.准备GVS由W / O乳化离心法<ol><li>制备1,2- dioleoyl- SN -glycero -3-磷酸胆碱的原液（DOPC，25毫摩尔）和德克萨斯红1,2- dihexadecanoyl- SN -glycero -3-磷酸的原液，三乙铵盐（德克萨斯红DHPE，0.20毫摩尔）在氯仿中并存储储备溶液在-20℃。 </li><li> 制备油溶液。 <ol><li>通过蒸发DOPC原液（51.0微升和流动的氮气下的德克萨斯红DHPE原液（19.2微升）的混合物形成5毫升玻璃小瓶的内表面上的脂质膜。 </li><li>孵育减压膜过夜，然后都加上1.0毫升的液体石蜡（0.86-0.89克/厘米3）的小瓶中。包裹在铝箔的小瓶中，并在80℃CO / N孵育它静止。 DOPC和德克萨斯红DHPE的最终浓度是1.3和3.8×10 - 3毫分别和DOPC:德克萨斯红DHPE摩尔比为100:0.3。 </li></ol></li><li> 制备内部水分散体。 <ol><li>在1.5ml带盖微型管，混合237.5微升的1.0微米的非荧光微球（2.5体积％）和12.5微升的1.0微米的荧光微球（2.5体积％）的分散体的分散体;这对应于95:非荧光的荧光微球5（V / V）的比率。 </li><li>添加64毫克蔗糖随后Tris缓冲溶液的125微升（TBS，1M）和875微升的去离子水。微球的最终体积分数为0.5体积％，和的Tris-HCl（pH为7.5）和蔗糖的最终浓度分别为0.1和0.15微米。 </li><li>涡流的微管30秒，然后超声处理它为10分钟。 </li></ol></li><li> 制备的外水溶液。 <ol><li>制备的10毫升Tris-缓冲溶液后（0.1M的Tris-HCl [pH为7.5]，0.15M GLUCOSE）中相同的操作作为内部水介质，放置1ml溶液中有盖微型管1.5毫升涡流的微管30秒，然后超声处理它为10分钟。 </li></ol></li><li> 制备含微球AW / O乳液。 <ol><li>混合1毫升油溶液（含有DOPC液体石蜡和德克萨斯红DHPE）用300μl的内水溶液的在1.5ml微管中。 </li><li>通过使用机械均化器（与该高速旋转叶片的搅拌器）乳化在微管中的两个组成部分在10,000rpm下操作在室温2分钟。 </li></ol></li><li> 沉淀的GVS。 <ol><li>轻轻在4℃在带盖微型管1.5ml的层300微升上1毫升的外水溶液的上表面上的w / o乳液中。冷却微管在4℃下10分钟。 </li></ol></li><li> 收集GVS。 <ol><li>立即冷却微管后，在30分钟18000×g的离心它。通过用图钉刺入微管的底部，并在无菌1.5毫升微型管收集一个液滴获得沉淀的GVS。 </li><li>稀释沉淀GV液滴10-100倍（体积）与外水溶液如果数量足够大，以使观测难以得到所获得的GVS。 </li></ol></li></ol> 2. GVS的显微镜观察<ol><li> 准备显微镜标本。 <ol><li>放置粘合剂孵育室用于在显微镜盖玻璃的顶部原位聚合酶链反应和杂交（室尺寸9毫米×9毫米×0.3mm厚）。 </li><li>用微量，存款25微升稀释沉淀的标本地区GVS并立即放置在孵化室的顶部的另一个玻璃盖（0.15mm左右厚）。 </li></ol></li><li> 记录微分干涉反政府GVS该效应的T型显微镜图像。 <ol><li>囊泡的记录显微镜图像用装有12V100WHAL-L卤素灯显微镜（10X，20X，和40X目标）。 </li></ol></li><li> 进行荧光显微镜观察。 <ol><li>插入U-FBNA和U-FMCHE荧光镜单元置于显微镜。适合的单位以470-495纳米和565-585纳米的激发滤光片，分别和与分别发送510-550纳米和600-690纳米的光，发射滤光片。 </li></ol></li></ol>

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作者宣称，他们没有竞争的经济利益。

<html>内部水分散介质和外侧水溶液的比重必须仔细选择。为w / o乳液，以沉淀成在进行离心处理期间外水溶液，内部水分散介质的比重必须比外水溶液大。我们试图制备使用内和外解决方案，而糖GVS，但我们得到的这些条件下没有GVS，因为内水溶液没有足够的质量，以跨越两相之间的干扰。如果有上述两种溶液，GVS该沉淀入外水溶液可以收缩或破裂之间的大的渗透压差。因此，内，外GVS渗透压必须相等。要做到这一点，我们使用蔗糖作为在内部水分散体和葡萄糖作为在外层水溶液溶质溶质;两个糖是在相同的浓度。这两种盐SS ="外部参照"&gt; 22和糖23，35，38已被用于这种目的，但通常使用的糖，因为它是毒性更小，比盐更可溶。然而，如果加入过多的糖，则GVS会与玻璃盖和崩溃的底部接触。有避免这几款策略。一种策略是降低内，外的水溶液，使得GVS温和条件下沉淀之间的比重差;具体地，沉淀的GV分散液的上清液可以用一个解决方案是相同的内水溶液通过粘接，以减少对盖玻璃作为浮力的结果囊泡破裂的可能性进行交换。另一种策略是用脂质薄膜29预涂两个盖玻片。 适当的准备和孵化乳液都重要。我们使用的液体石蜡来制备油溶液。矿物油26，31，十二烷21，33，和角鲨烷33，39也经常用作溶剂油。因为这些材料的比重，粘度和表面张力而变化，囊泡不同数量时相同的离心条件使用33甚至形成。以获得具有目标性能的囊泡，它是必不可少的，以优化比重和内外水溶液粘度以及油相的离心acceleration.For制备，孵育必须发生在高温下，在干燥环境如培养箱或脱水。在这项研究中，我们加热液体石蜡至80℃以完全溶解脂质molecules.In此外，乳剂应根据需要，应立即进行离心，因为它是不稳定的是制备后立即和仅制备的W / O型微滴容易熔合到彼此。该乳液可大量通过超声处理，涡旋，或攻丝来制备。然而，使用均化器允许大量乳液的快速制备和更容易乳化油具有高的粘度。同样重要的是，该乳液的外水溶液轻轻和迅速，然后在4℃冷却分层。缩短乳化和离心之间的时间，油外水溶液系统可以将乳液在其上层叠之前预冷，并immediately.If白浊出现比平常更快或更慢，机械整个系统然后可以离心均质必须进行彻底漂洗，以除去清洁剂。此外，乳化之前，油溶液，内水溶液和乳化剂必须为returned至室温。 适当的离心加速度也很重要。通过18000×g下离心，我们能够以高浓度包封单个内部材料制备GVS。当需要多种材料的封装，最好是减小离心加速度。例如，肌动蛋白组装系统包封个化合物，用离心低于350×g的35来实现的。在其离心是不希望的情况下，GVS可以通过调节糖浓度或通过其自身质量38，40，41的作用下沉淀该乳液得到。 本文所报导的方法有两个主要的限制。其中之一是，油分子（石蜡，在这种情况下）可以在GV膜被溶解，如已经指出的WEITZ基团21。当膜蛋白进入GV膜的插入是期望的，在蛋白质上共存的油分子的影响必须考虑。另一限制是体积分数的变化。在这项研究中，我们估计，微球在GVS的体积分数从4-30体积％范围;的体积分数不是完全相同用于GV制备的内水溶液的体积分数。虽然，我们能够在一个体积分数为显微镜观察高到足以封装在GVS微球，这种方法是不适合于制备GVS的具有均匀体积分数分布。已经报道了微球的体积分数的分布离心34期间变化。 在w / o乳液离心法通常用于含有包封材料GVS的形成。然而，很少有报道描述为PGVS微型封装材料34，42的赔偿。近日，包含封装器件的DNA或分子器件的分子机器人已建成43，44。与车厢GVS是这类应用的首选;因此，技术，如我们的，这可用于包封的磁性微球和微球与不同的表面官能可以预期是有用的44。

<html>建设性，或合成的，生物学的方法是探索生活之间的边界和非生命物质的一个迷人的途径。因为该细胞是生命所需要的最小单位，因为我们目前所知，各种研究人员试图构造简单，易于理解的化学物质的人造细胞，使这种人工细胞中发生的现象，可以研究，以阐明的最终目标生命的起源和研究活细胞1，2，3的基本功能。特别是，小泡4，它是制成两性分子球形微区和可以封装生物分子如蛋白质5,6和DNA 7，8，9，小姑娘="外部参照"&gt; 10，经常被用来作为生物膜的模型。 囊泡可以被分类为小（定义为具有直径&lt;100nm的），大（直径&lt;1微米），或巨（直径&gt; 1微米）。巨囊泡（GVS），因为它们类似于活细胞的大小，形状和结构已被广泛研究。由于GVS的大小，在GV膜的形态学变化可以很容易地进行实时的光学显微镜下观察。 制备GVS几种方法已经报道了11，包括水合方法12，13中 ，冻融法14，electroformation方法15，16和流体装置的方法17,18。然而，包封蛋白质和其它MACROM通过这些方法的装置在GVS olecules在高浓度是困难的。尤其是，它是非常具有挑战性的封装在足够量（20-30体积％）的生物材料，以模仿细胞内部19，20中的拥挤的环境。为形成GVS瞬间，韦茨和他的同事建立了一个水包油（W / O）乳液离心法21，22。这种方法有五个重要的特性。首先，因为用这种方法制备GVS具有低层数23，24，它们的膜是如此之薄，他们可以很容易地变形。由的FtsZ（细菌细胞分裂的蛋白质），微管蛋白，和其它大分子诱导GV膜变形已经研究25，26，27，28，和我们观察polyhe<html>通过微球29，30的封装诱导GV膜德龙状变形。第二，膜蛋白可以插入通过这种方法，小泡膜尽管有困难31。例如，YOMO组使用这种方法来研究膜蛋白α溶血素32的体外合成和孔形成活性。第三，有可能产生非对称GVS，其中内和外小叶的脂质组分是不同的22。例如，Whittenton 等。与内小叶来封装带负电荷的多核苷酸的阳离子脂质，并与外部小叶中性脂质产生不对称GVS降低毒性和非特异性细胞摄取33。第四，物质的GVS内的浓度和体积分数可以是相对高的S ="外部参照"&gt; 28，34。第五，多种类型的材料可封装35。例如，Nishimura 等人的封装体外转录-翻译系统进入GVS和所使用的系统的GVS 36内表达绿色荧光蛋白（GFP）。这五个特点使W / O乳化离心产生细胞模仿GVS不可缺少的方法。 在先前的工作中，通过离心分离生成的GVS通过装有用含一些最终水溶液22的长为16G的不锈钢针注射器装置收集。在经验的技术人员手中，这种收集方法很容易导致一些油的GV的污染。在这项研究中，我们使用由YOMO组23，37开发的w / o乳液离心协议其中，通过在离心管中，他们制备的底部上的孔打开沉淀GVS被收集。我们制备GVS包封1.0微米的微球，其在尺寸上与细胞内细胞器相似。使用的微球的允许我们通过计算它们的体积分数来估计其浓度。准备GVS，其中材料被密密麻麻方法的建立是创造人造细胞的重要一步。为了证实我们的各类内的材料的协议的效用，我们还证明了GFP和一个小的水溶性荧光分子（荧光素钠）可在GVS进行封装。

<table><tbody><tr><td>&lt;strong&gt;REAGENTS:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1,2-Dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine (DOPC)</td><td>Avanti Polar Lipids</td><td>850375C</td><td>Vesicular membrane molecule</td></tr><tr><td>Texas Red 1,2-dihexadecanoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red DHPE)</td><td>Thermo Fisher Scientific</td><td>T1395MP</td><td></td></tr><tr><td>1 M Tris-HCl (pH 7.5)</td><td>Nippon Gene Co.</td><td>318-90225</td><td>Buffer solution</td></tr><tr><td>D(+)-Glucose</td><td>Wako Pure Chemical Industries</td><td>049-31165</td><td>For outer water solution</td></tr><tr><td>Sucrose</td><td>Wako Pure Chemical Industries</td><td>196-00015</td><td>For inner water solution</td></tr><tr><td>Polybead carboxylate microspheres 1.0 &amp;micro;m</td><td>Polysciences</td><td>08226-15</td><td>Nonfluorescent, 2R = 1.0 &amp;micro;m</td></tr><tr><td>Fluoresbrite YG carboxylate microspheres 1.0 &amp;micro;m</td><td>Polysciences</td><td>15702-10</td><td>Fluorescent, 2R = 1.0 &amp;micro;m</td></tr><tr><td>Fluoresbrite YG carboxylate microspheres 0.10 &amp;micro;m</td><td>Polysciences</td><td>16662-10</td><td>Fluorescent, 2R = 0.10 &amp;micro;m</td></tr><tr><td>Fluorescein sodium salt (uranine)</td><td>Sigma Aldrich Japan</td><td>F6377-100G</td><td></td></tr><tr><td>GFP standard (recombinant)</td><td>Vector Laboratories</td><td>MB-0752</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;EQUIPMENT:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge 5427R</td><td>Eppendorf&amp;nbsp;</td><td>5409000233</td><td>Centrifuge rotor: FA-45-48-11</td></tr><tr><td>Physcotron</td><td>Microtec Co.</td><td>NS-310EIII</td><td>Handy microhomogenizer</td></tr><tr><td>Generator shaft</td><td>Microtec Co.</td><td>NS-4</td><td>Homogenizer attachment</td></tr><tr><td>Frame-Seal incubation chambers for &lt;em&gt;in situ &lt;/em&gt;PCR and hybridization</td><td>Bio-Rad Laboratories</td><td>SLF0201</td><td>Hybridization chamber volume: 25 &amp;micro;L&lt;br/&gt; Chamber size: 9 &amp;times; 9 &amp;times; 0.3 mm</td></tr><tr><td>Frame-Seal incubation chambers for &lt;em&gt;in situ&lt;/em&gt; PCR and hybridization</td><td>Bio-Rad Laboratories</td><td>SLF0601</td><td>Hybridization chamber volume: 65 &amp;micro;L&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&lt;br/&gt; Chamber size: 15 &amp;times; 15 &amp;times; 0.3 mm</td></tr><tr><td>Microman E</td><td>Gilson</td><td>FD10006</td><td>Pipette for viscous liquid. We measured liquid paraffin by using this.</td></tr><tr><td>Inverted microscope</td><td>Olympus Co.</td><td>IX73</td><td></td></tr><tr><td>Mirror unit</td><td>Olympus Co.</td><td>U-FBNA</td><td>Excitation filter: 470 - 495 nm&lt;br/&gt; Emission filter: 510 - 550 nm</td></tr><tr><td>Mirror unit</td><td>Olympus Co.</td><td>U-FMCHE</td><td>Excitation filter: 565 - 585 nm&lt;br/&gt; Emission filter: 600 - 690 nm</td></tr></tbody></table>

preparation of giant vesicles encapsulating microspheres by centrifugation of a water-in-oil emulsion

建设性生物学和合成生物学的方法来创建人工生命涉及生物或非生物材料的自下而上装配。这种方法已经得到极大的重视科研的生活和非生命物质的之间的边界上，并已使用在过去的二十年里建造人造细胞。特别是，巨人囊泡（GVS）经常被用作人造细胞膜。在本文中，我们将介绍GVS封装高度封装的微球含有高度浓缩生物分子的细胞模型的准备。在GVS通过一个简单的水包油乳剂离心法的方法制备。具体地，均化器，使用乳化含有待包封的材料的水溶液与含有溶解的磷脂的油，将得到的乳液的另一种水溶液的表面上小心分层。然后分层系统离心ŤØ生成GVS。采用这种强大的方法来封装不等的小分子微球材料。

在w / o乳液离心法是照相并示意性示出在图1中。 图1中的概略图像表明，这种方法的成功的最重要的决定因素在于，所述内水溶液的比重必须比外水溶液的直径，从而使GVS将离心分离期间沉淀。此外，在W / O型接口的脂质单层的形成要求系统被冷却10分钟，将乳液在外水溶液分层后。因为由乳液液滴横跨的w / o接口传输的GVS形式，在内部和外部的水层的渗透压必须相同。作为对照实验，在图1示出和步骤1.3，所不同的是内水溶液我们由该方法的装置不含微球体还制备GVS无球准备。 离心后沉淀的GVS的集合示于图2。除了在微管的最底部的半透明相，我们也观察到在外层水溶液（ 图2a）的白色混浊的中间阶段。这中间阶段是丰富的微球和油的聚合，而底部阶段包含的GVS。因此，用图钉（ 图2b）刺入微管的底部后，我们只收集的首次下降（ 图2c），其中载GVS的大量。以确保不超过两个滴被收集是非常重要的;任何额外的液滴可能含有微球和脂质聚集，这将导致在GVS的较低的密度。得到的泡分散液通常载有GVS外封装材料，因为ofte的GVS离心在n个破裂。以获得仅GVS，排序应该选择一个分选方法，例如透析，凝胶过滤，或荧光激活细胞，由于包封材料的大小。如有必要，为宗旨的量，沉淀的GVS是被使用，它们可以与外水溶液稀释。在一些情况下，中间相延伸到管的底部，这表明所形成的任何囊泡是由油保持在一起。 我们得到的GVS的微分干涉显微镜和荧光显微镜图像，而不微球（ 图3A，3B）中，用微球（ 图3c -3 F）。与以前的报告23，37相一致，具有低层数GVS通过我们的协议形成。与得克萨斯红DHPE，它会发出红色fluores结合脂质萤光，使用使囊泡形成可以由薄膜的可视化可以直接证实。我们得到的160 GVS的，55包封的微球和105是空的，给予34％的包封的比率。 我们通过以下的方法来确定在GVS微球的体积分数（φ，体积％）。因为每个GV含有几十到几百微球，在光学显微镜下计数所有的微球是具有挑战性的。因此，我们用少量荧光微球，其中手动荧光显微镜下计数混合的非荧光1.0微米的微球。 （基于原始95:非荧光的5 [V / V]比荧光微球）包封的微球的总数（N）的通过用20手工计数荧光微球的数量（n）相乘来计算。的价值66;然后估计为NV 100 / V，其中v是微球的体积，V是各个GV的体积。注氮从n表示估计引起计数错误，并且这些错误必须计算φ时加以考虑。 φ值由N，将其直接计算为20 N估计，而这又导致了该φ概率 准确地反映真正的价值为&lt;50％。事实上，N波动围绕20 N一定程度上，所以我们需要把它作为20（N + i），其中i为n中的错误。我们为了使n的概率± 我可以是泊松分布的基础上，获得了超过50％的估计的I。我们估计的I，这反过来又允许我们计算20（ 正 ±i）和值66;其中包括计数误差的GVS直径的10和15微米（ 表1）。根据该过程，在图3c中所示的GV微球的体积分数估计为11±3％（体积）。所计算的体积分数的精度为10〜30％。 我们的研究结果表明，我们成功地封装在GVS 1微米的微球在高体积分数。我们使用相同的外水溶液和相同的协议（ 图4）还能够封装的其它材料为100％摩尔DOPC GVS。具体地讲，从含有0.1M的Tris-HCl（pH值7.5），0.15M蔗糖和0.5体积％的荧光微球由含有1.0微米的微球GVS描述的协议的装置（Tris-缓冲溶液制备含有0.1微米的微球GVS 图4a）。继协议中所述ABOV即，DOPC GVS含有GFP（0.1M的Tris-HCl [pH为7.5]，0.15M蔗糖，和100微克/毫升的GFP; 图4b）和含有荧光素钠GVS（0.1M的Tris-HCl [pH为7.5]，0.15M蔗糖，和30微米荧光素;还制备图4c）。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> ñ </td><td> ñ </td><td> φ（10微米直径） </td><td> φ（15微米直径） </td></tr><tr><td> 3 </td><td> 60±20 </td><td> 6.0±2.0 </td><td> 1.8±0.6 </td></tr><tr><td> 4 </td><td> 80±20 </td><td> 8.0±2.0 </td><td> 2.4±0.6 </td></tr><tr><td>五</td><td> 100±20 </td><td> 10±2 </td><td> 3.0±0.6 </td></tr><tr><td> 6 </td><td> 120±30 </td><td> 12±4 </td><td> 3.6±1.2 </td></tr><tr><td> 10 </td><td> 200±32 </td><td> 20±3 </td><td> 5。9±0.9 </td></tr><tr><td> 20 </td><td> 400±45 </td><td> 40±5 </td><td> 12±2 </td></tr><tr><td>三十</td><td> 600±55 </td><td> - </td><td> 18±2 </td></tr><tr><td colspan="4">如文中所述进行确定的误差; N =手工计数微球的数目; N =总数量封装的微球。 </td></tr></tbody></table> 表1: 微球上 数字和体积分数（φ， 体积％）。如文中所述进行确定的误差; N =手工计数微球的数目; N =总数量封装的微球。 <img alt="图1" src="/files/ftp_upload/55282/55282fig1.jpg" /> 图1: 流动通道<html>艺术和W / O乳化离心法示意图。 （a）由DOPC和德克萨斯红DHPE的油溶液（100:0.3摩尔比）在液体石蜡中。 （b）由蔗糖和微球体在Tris-HCl缓冲液的内水分散液。 （c）由葡萄糖在Tris-HCl缓冲液外水溶液。 （ 四 ）1毫升的油溶液和300μl的内水分散体的混合物。 （ 五 ）乳化用均化（10,000rpm下，2分钟）。 （f）在W / O乳液。 （G）于1毫升外水溶液以300μl乳液分层。 （H）沉淀GVS刚过离心。 （i）的w / o乳液离心方法的原理的示意图。黑色箭头指示的离心加速度的方向。.JPG"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/55282/55282fig2.jpg" /> 图2: 离心管穿孔。 （ 一 ）沉淀的GVS只是离心（也描述于图1小时 ）之后。红色椭圆表示沉淀的GV分散的液滴。用图钉微管（ 二 ）穿刺。通过用图钉刺入底部附近的微管，并从孔收集液滴得到含有GVS沉淀。 （c）该沉淀的GVS与外水溶液稀释分散体（由黄色箭头指示）的液滴。 <a href="http://ecsource.jove.com/files/ftp_upload/55282/55282fig2.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3:GVS 的显微图像。 （ 一 ）GV的微分干涉对比显微镜图像而没有微球。 GV的直径约为10微米。比例尺= 10微米。一个GV（b）的荧光显微镜图像而没有微球。红色荧光由德克萨斯红DHPE射出。含有微球GV（ 三 ）微分干涉对比显微镜图像。 （ 四 ）荧光显微镜的1.0微米的微球（YG羧酸微球体）一个GV内部图像。计数荧光微球（N）的数量为6，我们预计基于泊松分布错误（I = 2）。然后，我们估计内球总数（N = 20（N + I））为120±40。有人计算过，GV包含1.0微米的微球在大约11±3％（体积），其中v是，微球的体积，V是GVS的体积的体积分数（φ= NV 100 / V）。 （ 五 ）荧光显微镜一GV膜的形象。红色荧光由德克萨斯红DHPE射出。 （ 六 ）合并面板中的D和E的图像的图像。 <a href="http://ecsource.jove.com/files/ftp_upload/55282/55282fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55282/55282fig4.jpg" /> 图4:GVS 的微分干涉对比和荧光显微镜图像不同封装材料。微分干涉相衬显微镜（顶部）和荧光显微镜（下图）的图像含有（A）0.1微米的微球，（B）绿色荧光蛋白，和（c）荧光素钠GVS。比例尺= 10微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55282/55282fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion at JoVE.com

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

巨囊泡封装微球通过一个水包油乳液的离心制备

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

preparation-giant-vesicles-encapsulating-microspheres-centrifugation

Research

JoVE Journal

Biochemistry

14.4K 次观看.  Japan Women's University. Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials. 

视频: Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

高吞吐量的单细胞和多细胞微胶囊

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

科研

生物工程

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

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

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 ...

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

无洗涤剂超快重组膜蛋白成脂双层使用融合补充充电 Proteoliposomes。

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

生物化学

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping.

We present a technique for contactless micromanipulation of vesicles, using localized calcium ion gradients. Microinjection of a calcium ion solution, in the vicinity of a giant lipid vesicle, is utilized to remodel the lipid membrane, resulting in the production of membrane tubular protrusions.

局部钙离子梯度对大泡囊膜重塑的响应

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local ...

化学

Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular recognition within biosensors for diagnosis, functional membranes for artificial cells, and development of bioinspired micro/nano-reactors. In all of these applications, the characterization of their membrane properties is of fundamental importance. Among existing characterization techniques, micropipette aspiration, pioneered by E. Evans, allows the measurement of mechanical properties of the membrane such as area compressibility modulus, bending modulus and lysis stress and strain. Here, we present all the methodologies and detailed procedures to obtain giant vesicles from the thin film of a lipid or copolymer (or both), the manufacturing and surface treatment of micropipettes, and the aspiration procedure leading to the measurement of all the parameters previously mentioned.

The goal of the protocol is to reliably measure membrane mechanical properties of giant vesicles by micropipette aspiration.

通过电形成和微移液器测量其机械性能，对巨型Unilamellar混合囊泡的吸收

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

含脂肪酸脂质体的制备、纯化及应用

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

用于复杂蛋白质网络的脂质囊泡包封方案

Construction of Out&#45;of&#45;Equilibrium Metabolic Networks in Nano&#45; and Micrometer&#45;Sized Vesicles

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove non-incorporated components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameters by fluorescence readout. Our membrane protein-containing vesicles of 100-400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs are&#160;trapped in microfluidic devices for analysis by optical microscopy.

作者聚焦:应对合成细胞工程中的挑战

We present a protocol for reconstituting membrane proteins and encapsulating enzymes and other water-soluble components in lipid vesicles of sub-micrometer and micrometer size.

在纳米和微米大小的囊泡中构建失衡代谢网络

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, ...

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

生物活性亲水球状蛋白的纳米脂质体制备与表征

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...

巨囊泡封装微球通过一个水包油乳液的离心制备

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在纳米和微米大小的囊泡中构建失衡代谢网络

在纳米和微米大小的囊泡中构建失衡代谢网络

在纳米和微米大小的囊泡中构建失衡代谢网络

生物活性亲水球状蛋白的纳米脂质体制备与表征