Name: extraction of plant-based capsules for microencapsulation applications
Uploaded: 2016-11-09T14:00:00.000+00:00
Duration: 10 min 54 s
Description: Watch this Scientific Journal Video about Extraction of Plant-based Capsules for Microencapsulation Applications at JoVE.com

This work was supported by the National Research Foundation (NRF-NRFF2011-01) and the National Medical Research Council (NMRC/CBRG/0005/2012).

1.孢粉外壁胶囊的提取（秒）从L. clavatum孢子注意:美国证券交易委员会的提取过程涉及的易燃粉（L. clavatum），热腐蚀性酸和易燃溶剂，因此，适当的个人防护装备（护目镜，面罩，手套，实验室外套），使用情况获得批准的风险评估和处置由授权的实验室工作人员的化学品是必不可少的。 <ol><li> 孢子脱脂 <ol><li>通过使用玻璃冷凝器，循环水系统，和水浴（ 图1）准备在通风橱中回流设置。 </li><li>称取百克L. clavatum孢子不要产生灰尘和任何火源。 注意:这里使用的是商业上获得孢子（见材料清单）。 </li><li>转印孢子慢慢到1L聚四氟乙烯（PTFE）的圆底烧瓶中有磁力搅拌棒。 </li><li> 500ml丙酮添加到在孢子聚四氟乙烯烧瓶中并轻轻摇动烧瓶中以形成均匀悬浮液。 </li><li>放置在水浴集聚四氟乙烯烧瓶在50℃下，并连接到回流冷凝器。 </li><li>在丙酮进行回流6小时，以200rpm搅拌，然后允许在室温冷却。 </li><li>使用过滤器真空过滤纸暂停并收集孢子脱脂大（直径15厘米）的玻璃培养皿中。 </li><li>通过用铝箔与溶剂蒸发的孔覆盖干燥，在室温（通风柜）的孢子。 </li></ol></li><li> 酸解 <ol><li>制备85％（V / V）磷酸，用去离子（DI）水和脱脂干孢子转移到一个1升的PTFE烧瓶中。 </li><li> 500毫升的磷酸（85％V / V）添加到脱脂孢子。 </li><li>放置在水浴集聚四氟乙烯烧瓶在70℃，并连接到回流冷凝器。 </li><li>在磷酸执行温和回流（70℃）30小时，然后允许凉爽室温。 </li><li>通过用500毫升温去离子水稀释悬挂和使用滤纸真空过滤收集SEC的。 </li><li>转移SEC的一个3升的玻璃烧杯中进行一系列洗涤的。 </li></ol></li><li> SEC清洗 <ol><li>放置装有肝窦在通风橱中的3升玻璃烧杯中。 </li><li> 800毫升热（50℃）去离子水添加到SEC的温和搅拌10分钟。 </li><li>通过使用滤纸和真空过滤设置进行过滤悬浮液中。 </li><li>收集SEC的在干净的3升的玻璃烧杯中。 </li><li>重复步骤1.3.1。 1.3.4。五次。 </li><li>添加600毫升热（45℃）丙酮SEC的温和搅拌10分钟。 </li><li>通过过滤收集SEC的真空下和SEC的转移到一个干净3升玻璃烧杯中。 </li><li>重复步骤1.3.6。 1.3.7和。用600毫升热（50℃）的2M盐酸。 </li><li>重复步骤1.3.6。 1.3.7和。使用600毫升浩T（50°C）2M的氢氧化钠。 </li><li>重复步骤1.3.1和1.3.4八次。 </li><li>重复步骤1.3.6。 1.3.7和。用600毫升热（45℃）丙酮。 </li><li>重复步骤1.3.6。 1.3.7和。用600毫升热（45℃）乙醇。 </li><li>重复步骤1.3.6。 1.3.7和。使用800毫升热（50℃）去离子水。 </li><li>转移清洁SEC的六个大的玻璃培养皿中，并干燥在通风橱中在室温下放置24小时以除去所有的水含量。 </li><li>干燥在真空烘箱SEC的在60℃和1毫巴下真空10小时或直至恒重。 </li><li>收集干燥的SEC的并转移到聚丙烯瓶中。 </li><li>在干燥的柜子存放SEC的。 </li></ol></li></ol> 2. SEC的表征<ol><li>使用在不同阶段产生的孢子和肝窦进行扫描电子显微镜分析41。 </li><li>使用在不同阶段产生的孢子和SEC的执行元素分析41。 ðRY在60℃元素分析之前至少1小时的所有样品，并计算使用具有6.25乘法系数氮百分比的蛋白质含量。11获取使用用于每个样品一式三份测量的结果。 </li><li>使用动态图像颗粒分析仪进行粉体学性质分析。41 </li><li>使用共聚焦激光扫描显微镜扫描未处理，处理，和FITC-BSA的加载肝窦41 </li></ol> 3.生物大分子封装真空载荷技术<ol><li>准备使用去离子水，并转移到50毫升聚丙烯试管的1.2ml 125毫克/毫升牛血清白蛋白（BSA）溶液。 </li><li>重150毫克肝窦的并传送到BSA溶液。 </li><li>涡管10分钟，以形成均匀的溶液。 </li><li>封面用滤纸筒。 </li><li>在1毫巴管转移到冻干机瓶和干燥4小时真空。 </li><li>执行步骤3.1到3.5。三个独立的批次。 </li><li>收集的BSA加载肝窦并使用研钵和杵除去团块。 </li><li>传送BSA的加载肝窦至50ml管中并加2ml DI水以去除残留的BSA。 </li><li>收集离心SEC的在4704×g离心5分钟并弃上清。 </li><li>重复用DI水洗涤一次。 </li><li>覆盖含有用滤纸BSA加载肝窦管。 </li><li>转移SEC的一个冰箱（-20℃），并冷冻1小时。 </li><li>冷冻干燥SEC的在冷冻24小时，直到店进一步鉴定。 </li><li>准备用同样的步骤不含BSA安慰剂SEC的如在步骤3.1。 3.13。 </li><li>使用相同的程序，在3.1的步骤制备FITC-BSA加载肝窦。 3.13。 </li></ol> 4.测定包封率<ol><li>称量5毫克BSA-加载肝窦在2ml聚丙烯管中。 </li><li>加入1.4毫升磷德缓冲盐水pH为7.4（PBS）中，并涡旋5分钟。 </li><li>探针超声处理在40％振幅的悬浮液10秒的3个循环。 </li><li>过滤用0.45微米的聚醚砜（PES）过滤器解决方案。 </li><li>执行相同的步骤，在步骤4.1。 4.4。使用安慰剂SEC的。 </li><li>测量在使用安慰剂作为空白280nm的吸光度。 </li><li>量化使用在PBS中BSA标准曲线（200至1000微克/毫升）包封的BSA的量。42 </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+better+understanding+of+pharmacological+activities+and+uses+of+phytochemicals+of+Lycopodium+clavatum:+A+review.">A better understanding of pharmacological activities and uses of phytochemicals of Lycopodium clavatum: A review.</a> J. Pharmacogn. Phytochem. 3 (1), 207-210 (2014).</li><li>Mundargi, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+sporopollenin+exine+capsules+from+sunflower+pollen+grains.">Extraction of sporopollenin exine capsules from sunflower pollen grains.</a> RSC Adv. 6 (20), 16533-16539 (2016).</li><li>Mathias, P. M., Chen, C. C., Walters, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Polyethylene+Fractionation+Using+the+Statistical+Associating+Fluid+Theory.">Modeling Polyethylene Fractionation Using the Statistical Associating Fluid Theory.</a> , (2000).</li><li>El-Bayaa, A., Badawy, N., Gamal, A., Zidan, I., Mowafy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+wet+process+phosphoric+acid+by+decreasing+iron+and+uranium+using+white+silica+sand.">Purification of wet process phosphoric acid by decreasing iron and uranium using white silica sand.</a> J. Hazar. Mater. 190 (1), 324-329 (2011).</li><li>Carr, J., Zhang, L., Davis, M., Ravishankar, S., Flieg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale+Controlling+Chemical+Additives+for+Phosphoric+Acid+Production+Plants.">Scale Controlling Chemical Additives for Phosphoric Acid Production Plants.</a> Procedia Engineering. 83, 233-242 (2014).</li><li>Mundargi, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eco-friendly+streamlined+process+for+sporopollenin+exine+capsule+extraction.">Eco-friendly streamlined process for sporopollenin exine capsule extraction.</a> Sci. Rep. 6, 1-14 (2016).</li><li>Smith, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamentals of Fourier transform infrared spectroscopy. , 182 (2011).</li></ol>

作者什么都没有透露。

在这项工作中，从L. SEC提取的系统分析clavatum孢子提出并此报告表明，它能够生产更高质量的胶囊剂，同时实现11的预先存在的常用协议。一个显著精简与此相反的现有协议需要高的处理温度（180℃）和一个较长的过程持续时间（7天）， 第11的当前SEC提取处理优化主要是集中于降低酸解步骤的温度和持续时间。 脱脂，碱处理提供了从最小的孢子?...

有在微胶囊化应用从植物孢子和花粉获得使用天然植物为基础的胶囊显著的兴趣。1-15在自然界，孢子和花粉反对恶劣的环境条件敏感的遗传物质提供保护。植物孢子和花粉的基本结构通常包括外壳层（外壁）中，内壳层（内壁），和内部细胞质材料。外壁是由称为孢粉化学鲁棒生物聚合物1,9,10,13,16和内壁是主要由纤维素材料16-18空胶囊可通过各种方法7,9中分离除去细胞质材料，蛋白质，和内壁层。2,12,16这些孢粉外壁胶囊（秒），由于其窄的尺寸分布和均匀的形态提供令人信服的替代合成密封剂。7,9,13,19,20的标准化的过程，以获得来自不同植物物种，如伸筋草肝窦的发展，打开了一个广泛的药物递送，食品和化妆品等领域的微胶囊的应用的潜力。6,10-13,21 为了获得肝窦，研究人员首先处理孢子和花粉与有机溶剂和碱性溶液回流以除去细胞质内容22-25然而，剩下的胶囊结构被确定为仍然含有纤维素内壁层。为了除去此，研究人员使用盐酸，热硫酸或热磷酸在若干天，22-25用磷酸成为SEC内壁去除的优选方法探讨了使用延长的酸性水解处理2然而，正在进行研究多年来已经表明，各种孢子和花粉具有不同程度的弹性恶劣处理我thods常用。26,27一些孢子和花粉是完全降解并失去在强碱性溶液中的所有结构完整性，或变得严重损坏在强酸性溶液16在SEC对治疗的反应条件的变异性是由于在化学细微变化结构和物种之间的孢粉外壁材料的外壁形态28由于孢粉外壁胶囊（秒）的鲁棒性的可变性，有必要以优化孢子和花粉的各种处理的条件。 从种属植物孢子clavatum已成为肝窦的最广泛研究的单一来源。因此建议，这主要是由于其广泛的可用性，成本低，单分散性，和化学坚固性9,29孢子可以在1分组的形式来方便地收获并含有sporoplasmic内容- 2微米细胞器和biomolecules 11 L. clavatum孢子已被用来作为一种天然粉末润滑剂，30,31一个化妆品的基础上，30和草药32-36为广泛的治疗应用。从L.获得SEC的clavatum已被证明是更有弹性，以来自其它物种的孢子和花粉。2处理之后的处理比肝窦，所得SEC的已显示出保留其错综复杂microridge结构和同时提供用于包囊的大的内部空腔高形态的均匀性。7研究表明，L。 clavatum SEC的可用于药物，10,13疫苗，11种蛋白质，7,14细胞，8油，5-7,9和食品补充剂的封装。5,15观察SEC负载效率相对较高比常规封装材料。7也有一些好处报道SEC的封装诸如掩盖味道，6,10，并提供一定程度的天然保护的抗氧化作用。12在现有的研究的能力，为L.最常用的SEC提取方法clavatum基于四个主要步骤。第一步是在50℃下在丙酮溶剂回流达12小时，脱脂孢子11第二步是在120℃到12小时以除去细胞质和蛋白质材料在6％的氢氧化钾的碱性回流11步骤三是在180℃下在85％的磷酸酸回流至7天以除去纤维素内壁材料11第四步是一个全面的洗涤过程中使用的水，溶剂，酸和碱以除去所有剩余的非外壁材料和化学残留物。 相对于封装应用SEC提取的主要目标是产生胶囊它们是空的细胞质材料制成的，自由往复中号潜在变应原性蛋白质和形态完好。2.37然而，从工业制造的角度来看，它也是重要的考虑附加的经济和环境因素，比如，能源效率，生产持续时间，安全性，和产生的废物。至于能量效率，兼具高的温度和长的处理时间会影响生产成本以及环境的影响。生产时间和周转时间是影响加工盈利的关键因素。特别值得关注的是，高温磷酸处理提高了安全性问题，并已知会导致腐蚀性缩放这导致在基础设施维护显著增加和延迟在批量周转时间。38-40在可能的情况，尽量减少可能会导致所需的步数到显著减少所产生的废物。然而，常用的L的四个步骤clavatum SEC提取具有简单的EV从几十年的研究和olved已很少实际过程的优化。近日，穆恩达尔吉等人 ，41通过系统地评估和优化的最常见的SEC提取技术之一提出在这一领域正在进行的工作显著的贡献。 在这项研究中的第一部分:孢子脱脂论证利用丙酮处理在50℃下6小时; sporoplasm和内壁去除过程证明利用85％的磷酸处理，在70℃，30小时;与水，溶剂，酸和碱充分洗涤用于演示的去除残留sporoplasmic内容;和SEC干燥证明利用对流干燥和真空烘箱干燥。在第三部分中，SEC真空装载证明利用模型蛋白的真空加载，牛血清白蛋白（BSA），其次是BSA-加载-SEC洗涤和冷冻干燥。在第四部分中，determin的BSA包封率通货膨胀论证利用离心，超声探头和紫外/可见光光谱。

<table><tbody><tr><td>&lt;em&gt;Lycopodium clavatum&lt;/em&gt; spores (s-type)</td><td>Sigma</td><td>19108-500G-F</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A2153-50G</td><td></td></tr><tr><td>FITC-conjugated BSA</td><td>Sigma</td><td>A9771-250MG</td><td></td></tr><tr><td>Phosphoric acid (85% w/v)</td><td>Sigma</td><td>438081-2.5L</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma</td><td>V800202&amp;nbsp;</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma</td><td>S5881-1KG&amp;nbsp;</td><td></td></tr><tr><td>Acetone</td><td>Sigma</td><td>V800022</td><td></td></tr><tr><td>Ethanol</td><td>AcME&amp;nbsp;</td><td>C000356</td><td></td></tr><tr><td>Deionized water</td><td>Millipore purified water&amp;nbsp;</td><td></td><td></td></tr><tr><td>Qualitative filter paper (grade No. 1, cotton cellulose)</td><td></td><td></td><td></td></tr><tr><td>Polystyrene microspheres (50 &amp;plusmn; 1 &amp;micro;m)&amp;nbsp;</td><td>Thermoscientific (CA, USA)</td><td>4250A</td><td></td></tr><tr><td>Vectashield&amp;nbsp;</td><td>Vector labs (CA, USA)</td><td>H-1000</td><td></td></tr><tr><td>Sticky-slides, D 263 M Schott glass, No.1.5H (170 &amp;mu;m, 25 mm x 75 mm) unsterile glass slide</td><td>Ibidi GmbH (Munich, Germany)</td><td>10812</td><td></td></tr><tr><td>Commercial Lycopodium SECs (L-type)</td><td>Polysciences, Inc. (PA, USA)</td><td>16867-1</td><td></td></tr><tr><td>Heating plates</td><td>IKA, Germany</td><td></td><td></td></tr><tr><td>Scanning electron microscope</td><td>Jeol, Japan</td><td>&amp;nbsp;JFC-1600</td><td></td></tr><tr><td>Elemental analyzer&amp;nbsp;</td><td>Elementar, Germany</td><td>VarioEL III</td><td></td></tr><tr><td>FlowCam: The benchtop system</td><td>Fluid Imaging Technologies, USA</td><td>FlowCamVS</td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Carl Zeiss, Germany</td><td>LSM710</td><td></td></tr><tr><td>Freeze dryer</td><td>Labconco, USA&amp;nbsp;</td><td></td><td></td></tr><tr><td>UV Spectrometer</td><td>Boeco, Germany</td><td>S220</td><td></td></tr></tbody></table>

extraction of plant-based capsules for microencapsulation applications

从植物为基础的孢子或衍生的微胶囊提供一个多元化的微胶囊化应用提供了强大的平台。当孢子或花粉进行处理，以除去内部sporoplasmic内容孢粉外壁胶囊（秒）获得。得到的中空微胶囊表现出高度的粉体学均匀性并保留相关的特定植物物种错综复杂显微结构特征。在此，我们展示了从伸筋草孢子生产SEC的和为亲水性化合物进入这些SEC的加载一个简化的流程。当前SEC分离过程最近已经优化以显著减少其常规SEC中单独使用的处理的要求，并确保生产完整微囊。自然L. clavatum孢子脱脂用丙酮，用磷酸处理过的，并充分洗涤以除去sporoplasmiC含量。丙酮脱脂后，用85％的磷酸一单个处理步骤已被证明以除去所有sporoplasmic内容。通过限制酸处理时间为30小时，这是可以分离干净肝窦和避免SEC压裂，这已被证明与延长处理时间发生。用清水充分冲洗，稀酸，稀碱和溶剂确保所有sporoplasmic物质和化学残留物被充分去除。真空加载技术被利用来加载一个模型蛋白（牛血清白蛋白），为代表的亲水性化合物。真空装载提供了一种简单的技术来装载的各种化合物，而不需要这往往需要在其他微囊协议苛刻的溶剂或不希望的化学品。根据这些隔离和装载协议，SEC的提供一种在各种不同的微胶囊的应用，如，治疗剂，食品，化妆品使用一个有前途的材料，和个人护理刺UCTS。

对于孢粉外壁胶囊简化提取工艺 该L. clavatum SEC提取由三个主要步骤实现:（1）用丙酮脱脂; （2）酸解使用磷酸85％（体积/体积）;并使用（3）广泛SEC洗涤溶剂。流线型SEC提取处理的流程在图1中提示的-我简言之，该方法涉及在50℃下用丙酮回流一个脱脂步骤6小时，然后将脱脂孢子在?...

Watch this Scientific Journal Video about Extraction of Plant-based Capsules for Microencapsulation Applications at JoVE.com

Extraction of Plant-based Capsules for Microencapsulation Applications

这个协议/手稿描述了生产由伸筋草芽孢孢粉外壁胶囊（秒）的和为亲水性化合物为这些肝窦的装载一个简化的流程。

基于植物的提取胶囊微胶囊化应用

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

extraction-of-plant-based-capsules-for-microencapsulation-applications

Research

JoVE Journal

Bioengineering

11.9K 次观看.  Nanyang Technological University. 这个协议/手稿描述了生产由伸筋草芽孢孢粉外壁胶囊（秒）的和为亲水性化合物为这些肝窦的装载一个简化的流程。 

视频: Extraction of Plant-based Capsules for Microencapsulation Applications

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

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

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 ...

科研

生物化学

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

发育生物学

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 ...

生物工程

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

一种可定制的酶化生产与纯化天然产物的方法

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

Bacterial Cellulose Spheres that Encapsulate Solid Materials

Bacterial cellulose (BC) spheres have been increasingly researched since the popularization of BC as a novel material. This protocol presents an affordable and simple method for BC sphere production. In addition to producing these spheres, an encapsulation method for solid particles has also been identified. To produce BC spheres, water, black tea, sugar, vinegar, and bacterial culture are combined in a baffled flask and the contents are agitated. After determining the proper culture conditions for BC sphere formation, their ability to encapsulate solid particles was tested using biochar, polymer beads, and mine waste. Spheres were characterized using ImageJ software and thermal gravimetric analysis (TGA). Results indicate that spheres with 7.5 mm diameters can be made in 7 days. Adding various particles increases the average size range of the BC capsules. The spheres encapsulated 10 - 20% of their dry mass. This method shows low-cost sphere production and encapsulation that is possible with easily obtainable materials. BC spheres may be used in the future as a contaminant removal aid, controlled release fertilizer coating, or soil amendment.

This protocol presents an easy, inexpensive method of forming bacterial cellulose (BC) spheres. This biomaterial can function as an encapsulation medium for solid materials, including biochar, polymer spheres, and mine waste.

封装固体材料的细菌纤维素球体

Bacterial cellulose (BC) spheres have been increasingly researched since the popularization of BC as a novel material. This protocol presents an ...

工程学

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications.

使用简单搅拌容器在藻酸盐珠中的哺乳动物细胞包封

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

封装产热前脂肪细胞移植到脂肪组织仓库

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

医学

Synthesis of Black Seed Oil-loaded-, Alginate-based, pH-sensitive Beads Using Electrospraying Technique

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in the context of colon cancer. Its active compound, thymoquinone, may help inhibit cancer cell growth and induce apoptosis in colon cancer cells. Additionally, black seed oil's anti-inflammatory and antioxidant effects could contribute to a healthier gut environment, potentially reducing cancer risk. Therefore, this study synthesized pH-sensitive alginate beads to deliver BSO into the colon in a controlled-release manner without releasing the drug at pH 1.2 (stomach), thus providing a well-defined release pattern at pH 6.8. The use of electrospray technology improves process performance by making it easier to formulate small, homogeneous beads with a higher rate of swelling and diffusion in the gastrointestinal medium. 
The formulated beads were characterized by an ex-vivo mucoadhesive strength test, bead size, sphericity factor (SF), encapsulation efficiency (EE), scanning electron microscope (SEM), in vitro swelling behavior (SB), and in vitro drug release in acidic and buffer media. All these manufactured beads demonstrated modest sizes of 0.58 &plusmn; 0.01 mm and spherical shape of 0.03 &plusmn; 0.00 mm in this testing. The formulation showed promising floating and releasing properties in vitro. With a very low cumulative percentage of beads, the oil EE of 90.13% &plusmn; 0.93% was high, and the release study demonstrated more than 90% in pH 6.8 with good floating nature in the stomach. Additionally, the beads were evenly spaced throughout the intestine. The electrospraying approach used in this protocol can be reproducible, yielding consistent outcomes. Therefore, this protocol can be used for large-scale production for commercialization purposes.

A technique employing high electric voltage and a targeted, active ingredient-loaded emulsion to fabricate pH-responsive, uniform microbeads is presented.

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in ...

化学

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 ...

Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and scalability. In this paper, we describe a strategy for the robust and reproducible formation of hPSC spheroids where a co-axial flow focusing device is utilized to entrap hPSCs inside core-shell microcapsules. The core solution contained single cell suspension of hPSCs and was made viscous by the incorporation of high molecular weight poly(ethylene glycol) (PEG) and density gradient media. The shell stream comprised of PEG-4 arm-maleimide or PEG-4-Mal and flowed alongside the core stream toward two consecutive oil junctions. Droplet formation occurred at the first oil junction with shell solution wrapping itself around the core. Chemical crosslinking of the shell occurred at the second oil junction by introducing a di-thiol crosslinker (1,4-dithiothreitol or DTT) to these droplets. The crosslinker reacts with maleimide functional groups via click chemistry, resulting in the formation of a hydrogel shell around the microcapsules. Our encapsulation technology produced 400 &#181;m diameter capsules at a rate of 10 capsules per second. The resultant capsules had a hydrogel shell and an aqueous core that allowed single cells to rapidly assemble into aggregates and form spheroids. The process of encapsulation did not adversely affect the viability of hPSCs, with &gt;95% viability observed 3 days post-encapsulation. For comparison, hPSCs encapsulated in solid gel microparticles (without an aqueous core) did not form spheroids and had &lt;50% viability 3 days after encapsulation. Spheroid formation of hPSCs inside core-shell microcapsules occurred within 48 h after encapsulation, with the spheroid diameter being a function of cell inoculation density. Overall, the microfluidic encapsulation technology described in this protocol was well-suited for hPSCs encapsulation and spheroid formation.

This article describes encapsulation of human pluripotent stem cells (hPSCs) using a co-axial flow focusing device. We demonstrate that this microfluidic encapsulation technology enables efficient formation of hPSC spheroids.