这项研究得到了授予R01NS062842国家神经紊乱和中风研究所，国立卫生研究院（VVD）和国立神经疾病和中风，国家卫生研究院通过复苏法案（VVD）和R21的R21 NS064403补助EB006301国家生物医学成像和生物工程研究所，国立卫生（VVD）研究院。

分子机器的检测部分，首先是因为他们的准备需要更多的时间比分子器件的装配，应准备。兴建工程，以及5 -6μm厚的部分切多聚甲醛固定，石蜡包埋组织块。使用幻灯片的品牌如ProbeOn另外收取和precleaned幻灯片“（Fisher Scientific这）或类似的保留部分，。我们建议在第一次使用与知名的DNA损伤的模式，其中包含两个DNA酶I -和DNase II型断裂，如地塞米松治疗的细胞凋亡大鼠胸腺13,14 ，一个组织。 1。节的制备<ol><li>放置在幻灯片机架和二甲苯脱蜡15分钟的部分，转移到了一个额外的5分钟的新鲜二甲苯浴。 </li><li>经过梯度乙醇浓度，补充水分：96％乙醇2x5min; 80％的乙醇 - 5分钟;水 - 2X5分钟。 </li><li>精华部分与Proteinas发送光使用100μL，每节50μg/mL的解决方案。孵育15&#39;在湿盒室温（23℃）。取决于组织类型，时间可能需要调整。硬组织可能需要更长的消化。 15-25分钟的时间通常使用。消化不足可能会导致信号较弱。另一方面过量信号消失和部分中断。 </li><li>在蒸馏水冲洗2X10分钟。 </li><li>应用100μL2％BSA preblocking每节。孵育15分钟在室温（23℃）。在这段时间内的分子机器组装。 </li></ol> 2。分子机器装配所有试剂比例为25μL总体积，这是足以在一个平均大小的组织切片（10x10mm）的单一检测。根据需要可以扩展该卷。 <ol><li>在一个小塑料管相结合的顺序如下： </li></ol><table border="1"><tbody> &lt;TR&gt; <td><ol><li> Bidistilled水; </li><li> 15％聚乙二醇8000; </li><li> 66毫米的解决方案三盐酸，pH值7.5，5毫米氯化镁 2，0.1毫米dithioerythritol，1毫米的ATP（即定期T4 DNA连接酶缓冲） ; </li><li> 70寡核苷酸1 pmoles， </li><li> VACC TOPO 215 pmoles（1.76 - 7.1微克） </li><li> 10个单位的T4 DNA连接酶（500 U /毫升） </li></ol></td></tr></tbody></table><ol start="2"><li>轻轻吹打混合。几乎立刻自组装的分子构造在此解决方案，可用于立即在室温（23℃）。提高温度至37 ° C块的T4 DNA连接酶为基础的组件标签。 </li></ol> 3。在组织切片双标签5&#39;OH和5&#39;PO4 DNA断裂的分子机器<ol><li>吸preblocking解决方案，并申请25μL混合充分反应，含有70 pmoles寡核苷酸1，53 - 215 pmoles（1.76 - 7.1微克）VACCTOPO和10个单位的T4 DNA连接酶（500 U / mL）的66毫米的Tris盐酸，pH值7.5，2 5毫米氯化镁，0.1毫米dithioerythritol，1毫米的ATP和15％聚乙二醇8000解决方案。相同的序列寡携带一个单一的FITC标记的标签，寡核苷酸2，可以用来代替单一类型的DNA断裂的检测。在这种情况下，省略连接酶标记反应。 50MM的Tris - HCl，pH值7.4，解决方案，同时在这种情况下15％PEG - 8000可以用来代替T4 DNA连接酶缓冲。 </li><li>在室温（23℃），在一个用塑料盖玻片的湿盒孵育1小时。防止光线照射。温度降低到16 ° C降低连接酶为基础的信号，温度升高至37 ° C完全消除了连接酶为基础的信号。部分抑制，有时会出现一个连接酶在反应混合物，可能是由于与拓扑异构酶制剂引入的污染物。因此，潜伏期较长（2-4小时），可能需要，尤其是当S连接酶标记符的ignificant号码都存在。连接酶信号连接酶和Topo信号虽然可以观察到在这个阶段，在许多情况下，可以重新申请反应组合进一步加强，没有VACC TOPO和双标记的寡核苷酸，但包含一个发夹形的寡核苷酸（核苷酸3） （35毫克/毫升）和T4 DNA连接酶（250 U /毫升）。为了提高信号进行第3步，见没有反应增强转到步骤5。 </li><li>取出盖玻片，轻轻地沉浸在Coplin在室温下的水罐子的幻灯片垂直。吸干多余的水分。 </li><li>加强连接酶信号申请没有VACC TOPO和寡核苷酸1 25μL反应混合物，但含有寡核苷酸3：66毫米三盐酸，pH值7.5，MgCl 2的 5毫米，0.1毫米dithioerythritol，1毫米ATP，15％的聚乙二醇8000，5个单位的T4 DNA连接酶，35毫克/毫升寡核苷酸3（钝结束发夹）。总体积的标签解决方案CAn是扩大规模，以适应更大的部分。在室温（23℃），在一个用塑料盖玻片的湿盒孵育18小时（过夜）。 </li><li>取出盖玻片，轻轻地沉浸在Coplin在室温下的水罐子的幻灯片垂直。然后洗节3x10分钟蒸馏水。 </li><li>与碳酸氢钠液冲洗。碱性溶液冲洗增强FITC荧光，这是pH敏感，并显著降低pH值低于7。 </li><li>抗衰落的解决方案（用DAPI Vectashield），盖玻片覆盖的部分，并用荧光显微镜分析信号。与5&#39;OH双链DNA断裂会发出荧光绿，5&#39;PO 4游-会发出荧光红色（图2）。 </li></ol> 4。代表性的成果<img alt="图2。" src="/files/ftp_upload/3628/3628fig2.jpg" /> 图1半人工分子机器检测两类。DNA损伤原位 。荧光机自组装时VACC TOPO的结合双发夹的32 - mer。机器开始分为两个检测器的操作通过分裂本身拓扑异构酶切识别序列3&#39;端。这会导致一个循环过程，FITC标记的单位不断分离罗丹明标记的单位和religates。这种情况持续存在，直到遇到了可检测的DNA断裂。当这种替代受体（5&#39;OH钝端DNA断裂）是在组织切片中，FITC标记的一部分，将结扎。其余诺丹明部分将到5&#39;PO 4 DNA T4 DNA连接酶的帮助下钝断头的重视。因此，同时检测DNA断裂两种类型。 <img alt="图1。" src="/files/ftp_upload/3628/3628fig1.jpg" /> 图2。分子机器两种类型的DNA断裂的双重标签在地塞米松治疗的胸腺组织部分。我和DNase II型平末端的DNA断裂的DNA酶在胸腺皮质区凋亡检测​​。细胞质绿色荧光（5&#39;OH DNA断裂），标志着消化皮质巨噬细胞凋亡的胸腺细胞的核材料。这个信号是由VACC TOPO中，定位与DNA含有5&#39;OH双链断裂11,12 phagolysosomes。红色荧光（5&#39;PO 4休息时间）标 ​​签不是由巨噬细胞吞噬的凋亡胸腺细胞的细胞核。胸腺细胞大规模同时进行伴随着一代5&#39;PO 4双链断裂的细胞凋亡，通过连接酶为基础的标签的可视化。这些休息是位于核周边，形成环形图案。所有细胞的细胞核用DAPI（蓝色荧光）counterstaining可视化。

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没有利益冲突的声明。

在这段视频中，我们将演示如何组装和使用双标签的DNA损伤传感器。传感器是一个由生物分子发动机，病毒编码的蛋白质VACC TOPO的人工组件联系在一起驱动的分子机器。所提出的发展充分体现了一个生物功能的做法，主张适应的无毒分子尺度器件12,15设计的生物结构，架构和实际零件和电池组件。这种方法可以解决两个问题的内在体内的纳米传感器的领域： 1。使用传统的纳米材料?...

<ol><li>二甲苯</li><li> 80％和96％的乙醇。 </li><li> 2％的蒸馏水中牛血清白蛋白（BSA）的解决方案。 </li><li>寡核苷酸1：双簪，双荧光素和四甲标记： </li></ol> 5&#39; - AAG GGA CCT的气相色谱F GCA的GGT的CCC认证TTA ACG的CAT R在GCG TT - 3&#39;，F - FITC - DT，R -四甲- DT。其他红移荧光团，如肾上腺素TR，罗丹明或TAMRA，可以用来代替四甲为R。 另外，您可以使用寡核苷酸2 - 双发夹单（单一类型的DNA断裂（DNA酶II型只）检测用荧光素标记的单荧光携带探针相当便宜，方便，每当单一DNA断裂的类型必须标明：5&#39; - AAG GGA CCT的GC F GCA的GGT的CCC认证TTA ACG的CAT ATG总工会的T - 3&#39;，F - FITC - DT </p&gt; <ol start="5"><li>寡核苷酸3。钝端罗丹明标记的发夹，在T4 DNA连接酶的原位结扎信号增强：5&#39; - GCG GAC中注册税务师彗星 R G GTC豪CGC - 3&#39;，R -四甲- DT </li><li>痘苗病毒DNA拓扑异构酶（TOPO VACC）升 - 3000 U /μL（生动的技术）。在最初的实验中，我们使用215％每25μL的反应组合VACC TOPO pmoles（7.1微克）。然而，拓扑异构酶浓度可显着降低，而不损失灵敏度。后来我们用了类似的结果四次少量的VACC TOPO（1.76微克）在25μL的反应混合物中每节每节。减少880纳克（25μL的反应组合）的酶量在信号较弱VACC TOPO和8.8纳克，导致生产没有检测到信号。 </li><li> T4 DNA连接酶5 U /μL（罗氏公司）。这种高度集中的连接酶制剂提供了在本实验条件下的最佳信号。 </l我&gt; <li> 10 × T4 DNA连接酶反应缓冲液：660毫米，50毫米的Tris - HCl MgCl 2的 10毫米（ATP，pH值7.5，10毫米dithioerythritol，20℃）（罗氏公司）。 ATP的反应缓冲液很容易被破坏，在解冻冷冻的重复周期。分装在15-20微升小部分，并存储在缓冲区 - 20 ° C使用一次。 </li><li> bidistilled水PEG - 8000（Sigma公司）的30％（W / V）的解决方案。 15％PEG - 8000在反应混合物强烈刺激的检测，其成员的有效浓度增加量排斥。 </li><li>蛋白酶K（罗氏）20 mg / ml的蒸馏水原液。储存在 - 20 ° C反应使用50mg/mL的解决方案，在PBS准备从股市。不要重复使用。 </li><li>用DAPI（媒介实验室）Vectashield。 </li><li>碳酸氢钠缓冲液：50米中号碳酸氢钠3，15M M氯化钠，pH值8.2。 </li><li> 22x22mm或22x40mm的玻璃或塑料盖玻片。塑料盖玻片是可取的，在反应过程中，他们一个重新容易从一节中删除。 </li></ol>

in vitro assembly of semi-artificial molecular machine and its use for detection of dna damage

自然发生的生物分子机器工作在每一个活细胞，并显示各种设计1-6。然而，人工分子机器设备上，即分子马达定向运动的能力，以及按比例缩小的机械部件（车轮，axels，吊坠等）7-9的中心发展。这模仿的宏观机，这些设备的基本的物理性质，化学性质，如惯性和动量守恒，即使不是在10纳米世界环境使用。替代设计，不按机械macromachines计划和使用生物分子的进化发展的机制，可以利用纳米世界的具体条件优势。此外，适应实际的生物分子纳米设计的目的，降低了纳米技术的产品可能会造成的潜在危险。在这里，我们展示了这样一个启用生物构造的组装和应用，EMI人工分子器件相结合的人工组件的一个自然发生​​的分子机器。从酶学点来看，我们的结构是一个设计师荧光酶 - 底物复合物放在一起执行特定的有用的功能。这个大会是由定义的分子机器，因为它包含一个 12 。然而，其一体化工程的一部分-荧光双发夹-重新定向到一个新的任务标签DNA损伤12。 我们的构造，组装出32 MER DNA和一种酶的牛痘拓扑异构酶I（VACC TOPO）。本机，然后使用自己的材料来制造两个荧光标记的检测器（图1）。单位之一（绿色荧光）进行VACC TOPO的共价连接到其3&#39;end和另一个单位（红色荧光），是同一个终端3&#39;OH发夹。的单位是短命的，并迅速重新组合成原来的构造，随后recleaves。在DNA的情况下，打破了这两个循环的方式不断独立和religate单位。在与DNA损伤，探测器单元的拓扑异构酶选择性重视5&#39;OH（DNA酶II型断裂）11,12平末端的DNA断裂，荧光标签的组织切片。第二，酶寡核苷酸裂解后形成的发夹，将结扎一个5&#39;PO 4钝端突破（DNA酶I型断裂）11,12，T4 DNA连接酶是在解决方案 13,14中。当T4 DNA连接酶将被添加到一个组织部分或溶液中的DNA与5&#39;PO 4休息钝端，连接酶与5&#39;PO 4 DNA末端反应，形成半稳定酶- DNA复合物。钝端发夹将与这些复合物释放的DNA连接酶和共价连接的发夹，从而标签5&#39;PO 4平末端的DNA断裂。 这方面的发展充分体现了新的切实可行的办法，以日E的分子机器的设计，并提供有用的一个固定的细胞和组织的细胞凋亡和DNA损伤检测传感器。

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In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

我们证明由拓扑异构酶蛋白驱动的分子尺度器件的组装和应用。构造是一个标签由组织切片中的DNA断裂的两个主要类型，其两端连接两个不同的荧光团的生物分子传感器。

在检测DNA损伤的体外半人工分子机器，其使用的大会

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

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Bioengineering

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

11.5K 次观看.  Baylor College of Medicine. 我们证明由拓扑异构酶蛋白驱动的分子尺度器件的组装和应用。构造是一个标签由组织切片中的DNA断裂的两个主要类型，其两端连接两个不同的荧光团的生物分子传感器。

视频: In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

科研

生物工程

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

CometChip：一种高通量的96孔平台的微阵列人类细胞测量DNA损伤

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

模块化 DNA 设备的自动机器人液体处理组件

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

对细胞的超快磁化阳离子化Magnetoferritin的合成

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation.

电刺激室的构建与应用, 促进间充质茎间质细胞体外成骨分化

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

长期纳米材料暴露后体外基因毒性测试的高级 3D 肝脏模型

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.