This study was supported by National Research Foundation of South Korea: Mid-career scientist grant (2014002855), and Ministry of Industry, Trade, and Natural Resources: World Premier Material Development Program. This work is also supported by in part by Center for Nature-inspired Technology (CNiT) in KAIST Institute for NanoCentury (KINC).

所有的动物护理和实验是按照由KAIST（科学与韩国高等科技学院）提供的道德协议IRB（伦理审查委员会）执行。 1. TAPE形成<ol><li>制备的TA溶液，放置在磁力搅拌器上4ml的大小的玻璃小瓶中，并加入1毫升蒸馏水有搅拌棒。 1克鞣酸加入到小瓶中，并通过温和搅拌以200rpm以上1小时溶解在水中。当TA完全溶解，该混合物变成透明的褐色。 </li><li>通过由涡旋几秒到使白色浆液将它们混合加入1g的PEG粉末（4-臂，10 kDa的，以及直链的，4.6 kDa的）至1毫升蒸馏水，随后制备的PEG溶液。保持在孵化该浆料在60℃下10分钟。直到白某变得完全清楚。 注:PEG的10 kDa的分子量的熔点为约55- 60℃，并在4 kDa的一个是53 - 58℃。熔化的PEG变成水混溶性，使得PEG的水高达1克高浓度/毫升，可以实现为透明的溶液。一旦明确PEG溶液在高温下形成的，该溶液仍然是冷却后在室温下是稳定的。 </li><li>添加PEG的329微升（4-臂，10 kDa）的溶液中制备1.2至671微升在步骤1.1中制备的TA溶液（在具有4.6 kDa的线性PEG的情况下，添加的PEG溶液的311微升至689微升在微型离心管中的TA溶液）。轻轻地融合与一个狭窄的铲两粘性和蜂蜜般的解决方案，它们均匀混合。 注意:这两种解决方案都相当粘稠，所以科学家必须缓慢而充分地拉起来 ​​，用微量的传输解决方案。 </li><li>旋在12300 xg离心在装有固定角转子的离心分离机，在步骤1.3制备3分钟的混合物。 </li><li>仔细REM奥雅纳尽可能多使用微量上清液尽可能的，并收集已定下来的产品是:这是完全形成胶带。后胶带的形成，它存放在冰箱（4 - 8℃）长达数周。注 :TAPE能够用γ射线或在外科应用在使用前的电子束处理来灭菌。 </li></ol> 2.带粘合强度的测量<ol><li>制备猪皮肤组织的两片由在皮肤上的组织去除所有脂肪后，用活检穿孔切割直径为6毫米。 注:从健康猪侧翼的皮肤得到的猪皮肤组织，并从位于大田的韩国当地肉类市场购买。 </li><li>适用的商业氰基丙烯酸酯胶的每个组织的外侧，并且组织附着到金属杆。 注:金属杆被用作补充手柄，使织物a重不直接由机器夹住。因此，它可以用下面的拉伸机的结构的其他材料来代替。 </li><li>应用带的压降（TAPE一滴大约为3 - 6毫克）于组织的一侧。然后，散布在胶带均匀地使用在其内两侧的两个组织之间的另一个组织，以便它们所连接， 如图2A所示 。 </li><li>然后，手动附加和分离组织双方几次混匀并最大限度地提高各组织和磁带之间的接口。 </li><li>用UTM，小心地夹住杆的每一侧。的粘接强度将通过分离附着有胶带两种组织所需的力来确定。首先，应用的20 N的力1分钟。接着，用机，以1mm /分的速度拉在相反方向上各棒。直至组织完全分离。 注意:数据将给出的力-距离由运动检测（FD）曲线的每个杆。 </li><li>除以在步骤2.5通过样品表面区域取得的FD曲线所示的最大力（kN）计算带的粘合强度，也就是3.14×（0.003米）2。 </li><li>对于在水的存在下监测的粘接强度，加入20微升的水在分离的区域两种组织之间，并立即附加它们。随着机，再次进行测试支队。 </li></ol> 3. 体外降解测试<ol><li>切微型离心管的盖子（D = 8 MM），并权衡帽将其定义为W C。 </li><li>装满150毫克磁带的帽子，再权衡所有一起将其设置为一个总的初始重量W 0。 注意:不要在帽超载TAPE。带的高度应该比盖的顶部低，因为它可以是一个物理屏障以在步骤3.4中孵育期间由搅拌过程中产生的PBS缓冲液的流。 </li><li&gt;把含有TAPE到细胞培养瓶的帽（75 平方厘米），并加入50毫升的PBS缓冲液（1×，pH 7.4）中，以细胞培养烧瓶，以便在盖的磁带完全浸入PBS缓冲液，如在图3A所示（N = 5）。 </li><li>孵育在步骤3.3中，在37℃，类似于生理条件的轨道摇动孵化器中制备的细胞培养瓶中，在温和搅拌下（50rpm）进行。 注意:保持搅拌条件以50rpm。更高的转速可能导致TAPE的崩溃。 </li><li>在每个时间点，采取与从细胞培养烧瓶TAPE帽，然后通过吹氮气干燥。称量装有剩余磁带上限。在每个时间点为W t将重量。再次更换新鲜的PBS，并在每个时间点测量W¯¯ 吨后再次摇晃。 </li><li>计算相对其余重量（％）在下面的等式。 相对剩余重量（％）=（W 吨 </suB&gt; - W C）/（W 0 - W C）×100％ </li></ol> 4.带止血能力 注:所有的动物实验应按照卫生和福利部韩国提供的准则和道德协议来完成。 <ol><li>为了评估在体内的止血功能，如参考文献24中描述审查出血鼠肝模型。 </li><li>麻醉15只小鼠（正常ICR小鼠，6周，30 - 35克，雄性）以替来他明 - 唑拉西泮（33.333毫克/千克）和赛拉嗪（7.773毫克/千克）的腹膜内注射（N =每组5只）。要确认正确的麻醉，轻轻一捏动物的爪子和观察运动，如抽出爪子等 ，没有动作表明，该动物是充分麻醉做手术。 </li><li>为防止动物的眼睛干涩，下充分而申请的兽医药膏眼睛感知。暴露经由中线腹部切口肝脏，并用18G的针以诱导出血刺肝脏。 </li><li>除去流动的血液用无菌纱布，并把100微升TAPE或纤维蛋白胶（阳性对照）的立即在出血部位。 注:在应用胶带后，需要因对伤口组织中的高血耐粘接性能没有进一步的缝合。为阴性对照，无处理发生在出血的部位。 </li><li>在每一种情况下，把一个滤纸与已知质量的肝脏下方，以收集来自损伤部位的血液。用新的，每30秒，4次（ 即 ，2分钟）更换纸张。 </li><li>测量所收集的每30秒每滤纸吸收血液的质量。动物实验后，通过牺牲二氧化碳窒息安乐死的老鼠。 </li></ol>

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The authors have nothing to disclose.

我们开发了一个全新的由植物衍生的多酚化合物，TA的抗水分子相互作用的启发止血胶粘剂命名的胶带。 TA是一个代表可水解单宁已显著受到关注，因为它的抗氧化，抗菌，抗诱变，和抗致癌特性。 使磁带的过程是非常简单的，可扩展和环境友好的，因为它仅仅是两种水溶液随后不经任何进一步的化学合成方法离心的一步法混合。 双组分混合协议是形成?...

在过去的十年中，已经进行了努力，以取代目前的手术缝合线和缝合钉关闭与生物可降解/生物可吸收的粘合剂伤口由于在外科手术处理它们在使用和低组织侵袭性的便利。市售的组织粘合剂分为四种类型:（1）氰基丙烯酸酯衍生物1，（2）通过酶转化从纤维蛋白原形成由凝血酶2,3至纤维蛋白聚合物的血纤维蛋白胶，（3）基于蛋白质的材料，如化学或物理交联的白蛋白和/或明胶4,5，和（4）为基础的聚合物合成的6。尽管它们已经在许多临床应用中使用，所有的粘合剂具有其自己固有的不足和缺陷，可以是它们的广泛使用的障碍。氰基丙烯酸酯系胶粘剂显示出高的粘合强度的组织，但它们的毒性副产物如氰基和甲醛降解过程中形成的，常会导致符号量占用度的炎症反应7。血纤维蛋白胶水和白蛋白或明胶基材料具有关于传染性成分，如动物来源的病毒的传输安全性的问题:人血浆纤维蛋白胶水和动物包括牛，鸡，猪和鱼明胶基胶水8。虽然一些基于合成聚合物的粘合剂已通过美国联邦药物管理局（FDA），由合成聚合物的大多数粘合剂继续在减少制造过程中的步骤和实现生物相容性9困难。最重要的是，所有的胶水贫困机械和粘接强度遭受湿纸巾10。近日，由贻贝11-13，14壁虎，壁虎与贻贝15和内寄生蠕虫启发16仿生组织粘合剂已经逐渐成为有前途的替代品，以目前的医疗胶由于其可调的机械和粘接性能与生物相容性。然而，到了今天，仍有待解决的问题，他们成为商业化产品17日之前。 在这里，我们报告一个称为胶带全新型医疗胶是由植物来源的粘附分子，单宁酸（TA）和一个生物惰性聚合物聚（乙二醇）（PEG）之间的分子间氢键制备正如其名称所示。 TA是植物的次生代谢过程中发现普遍存在的代表水解单宁。它已引起人们的关注，因为它的抗氧化剂，抗诱变，和抗致癌特性，并已被证明参与超分子相互作用与许多聚合物，如聚（N- -isopropylacrylamide）（PNIPAM）和聚（N -乙烯基吡咯烷酮）（PVPON），以形成层-层（LBL）膜18-20和药物释放的微胶囊21-23。在这项研究中，我们发现，TA可以作为一个有效的行动耐水粘接性官能基团，以形成医用粘合剂，胶带。通过用TA简单混合，非结垢聚合物PEG变得与超分子胶2.5倍与商业纤维蛋白胶相比增加粘合强度，并且此粘附保持在整个装卸的多达20个循环，甚至在水存在。其止血能力上的肝出血模型的体内测试，并显示出良好的止血能力以停止在几秒钟内出血。录像带的相关领域作为第一植物性粘合剂可以揭示新的洞察解决与生物启发方法目前存在的问题的弊端其显著的意义。我们也期望在各种医疗和药物应用如粘膜粘合剂，药物释放的补丁，伤口护理敷料以及其他的广泛使用磁带，由于其制备方法简单，可扩展性，可调谐的生物降解速率，以及高湿耐adhes离子的特性。

<table>
	<tbody>
		<tr>
			<td>Tannic acid</td>
			<td>Sigma-aldrich</td>
			<td>403040</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene oxide), 4-arm, hydroxy terminated</td>
			<td>Aldrich</td>
			<td>565709</td>
			<td>Averge Mn ~10,000</td>
		</tr>
		<tr>
			<td>Poly(ethylene glycol)</td>
			<td>Aldrich</td>
			<td>373001</td>
			<td>Average Mn 4,600</td>
		</tr>
		<tr>
			<td>Biopsy punch</td>
			<td>Miltex</td>
			<td>33-36</td>
			<td>Diameter = 6 mm</td>
		</tr>
		<tr>
			<td>Aron Alpha&#174;</td>
			<td>Toagosei Co., Ltd.</td>
			<td></td>
			<td>Instant glue</td>
		</tr>
		<tr>
			<td>Universal testing machine (UTM)</td>
			<td>Instron</td>
			<td>5583</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>SPL life science</td>
			<td>60015</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>SPL life science</td>
			<td>10090</td>
			<td>90 x 15 mm</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Sigma</td>
			<td>S5011</td>
			<td>1x PBS ingredient</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma</td>
			<td>S5136</td>
			<td>1x PBS ingredient</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Duchefa biochemie</td>
			<td>S0520.5000</td>
			<td>1x PBS ingredient</td>
		</tr>
		<tr>
			<td>Incubating shaker</td>
			<td>Lab companion</td>
			<td>SIF6000R</td>
			<td></td>
		</tr>
		<tr>
			<td>ICR mice</td>
			<td>Orient bio</td>
			<td>Normal ICR mouse</td>
			<td>6 weeks, 30-35 g, male</td>
		</tr>
		<tr>
			<td>Tiletamine-zolazepam (Zoletil 50)</td>
			<td>Virbac</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zylazine (Rompun)</td>
			<td>Bayer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlideTM needle (18 G)</td>
			<td>BD</td>
			<td>302032</td>
			<td>18 G</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td>1001 125</td>
			<td>Diameter = 125 mm</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis Flexible Pakaging</td>
			<td>PM996</td>
			<td></td>
		</tr>
	</tbody>
</table>

tape: a biodegradable hemostatic glue inspired by a ubiquitous compound in plants for surgical application

此视频描述了制备可生物降解的手术胶水具有比商业组织粘合剂的有效的止血能力和更大的耐水粘合强度的最简单的协议。医用粘合剂已经由于其在使用方便与微创备受关注的潜在的替代工具，缝线和U形钉。虽然有几种协议制定组织粘合剂包括那些市售如纤维蛋白胶水和氰基丙烯酸酯基材料，主要是它们需要一系列的有机分子，或复杂的蛋白质纯化方法的化学合成的，在生物驱动材料的情况下（ 即纤维蛋白胶）。此外，手术胶水的发展显示出高的粘合性，同时维持生物降解性仍然是一个挑战，因为在实现本体的潮湿环境良好的性能的困难。我们说明了一种新的方法来准备医用胶，称为胶带，由植物来源的，湿耐粘合剂分子的物理混合后形成的水不混溶性的超分子聚集体的重量为基础的分离，T annic 的 CID（TA），以及公知的生物聚合物，聚（乙烯）乙二醇（PEG）。随着我们的做法，录像显示粘接强度高，比在有水存在的商业纤维蛋白胶的2.5倍。此外，磁带是在生理条件下可生物降解的，并且可以用作抗组织出血一种有效的止血胶水。我们预期在各种医疗环境和药物递送的应用中，广泛使用磁带的诸如用于粘膜粘附更为聚合物，药物贮藏和。

带是超分子聚集体离心含有TA（1微克/毫升蒸馏水中）和PEG（1微克/毫升的蒸馏水中）与2两种水溶液的混合物后稳定下来:1的体积比（ 图1A）。混合比是在实现高粘附强度的关键因素;当带通过一个2形成的:1混合比，20个单位在25个单位的TA的羟基（-OH）的与PEG各醚基（-O-）相互作用，导致最高的分子间氢键形成具有最大粘附性能。 -OH的其余五个单位似乎通过与TA?...

Watch this Scientific Journal Video about TAPE: A Biodegradable Hemostatic Glue Inspired by a Ubiquitous Compound in Plants for Surgical Application at JoVE.com

TAPE: A Biodegradable Hemostatic Glue Inspired by a Ubiquitous Compound in Plants for Surgical Application

我们描述制备生物可降解医用胶，有一个有效的止血能力的最简单的协议。带通过单宁酸的混合制备的水不混溶的超分子聚集体，一个无处不化合物在植物中发现，和聚（乙烯）乙二醇，与商业纤维蛋白胶相比，得到一个2.5倍的耐水附着性。

TAPE:一种生物可降解止血胶由无处不在的复方植物外科应用的启发

This video describes the simplest protocol for preparing biodegradable surgical glue that has an effective hemostatic ability and greater water-resistant ...

tape-biodegradable-hemostatic-glue-inspired-ubiquitous-compound

Research

JoVE Journal

Bioengineering

14.0K 次观看.  Korea Advanced Institute of Science and Technology (KAIST). 我们描述制备生物可降解医用胶，有一个有效的止血能力的最简单的协议。带通过单宁酸的混合制备的水不混溶的超分子聚集体，一个无处不化合物在植物中发现，和聚（乙烯）乙二醇，与商业纤维蛋白胶相比，得到一个2.5倍的耐水附着性。 

视频: TAPE: A Biodegradable Hemostatic Glue Inspired by a Ubiquitous Compound in Plants for Surgical Application

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

科研

生物工程

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

构建交付的干细胞壳聚糖微球的胶原凝胶

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

活微生物组共培养与人的微工程肠绒毛在肠道上一个芯片微流体装置

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

基于自动对准的大面积扫描探针光刻及其在细胞培养研究中的应用

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

大鼠体内部分肝工程的外科技术基础

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

可伸缩、双通道、微流控芯片制造

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

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

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

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

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

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A general protocol is described for the preparation of V. natriegens crude cell extracts using common laboratory equipment. This high yielding protocol has been specifically optimized for user accessibility and reduced cost. Cell-free protein synthesis (CFPS) can be carried out in small scale 10 &#956;L batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of &#62; 260 &#956;g/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1&#8722;2 full days by a single user. This protocol can be easily integrated into existing protein synthesis pipelines to facilitate advances in bio-production and synthetic biology applications.

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template.

快速生长的纳氏弧菌的无细胞蛋白表达

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A ...