作者承认Armenise哈佛基金会，玛丽 - 居里特伦蒂诺COFUND（ACS）自治省特伦托（ECOMM），和CIBIO资金。

1。准备DNA模板<ol><li>从一个标准的实验室菌株大肠杆菌中纯化质粒大肠杆菌 ，如大肠杆菌大肠杆菌 DH5a中或新商业套件蓝。可替换地，直链的PCR产物，可以类似地用市售的试剂盒纯化。洗脱DNA与H 2 O只。 </li><li>苯酚-氯仿提取的DNA溶液26。 </li><li>确定DNA浓度和纯度， 例如，通过紫外线吸光率或其他合适的方法。高效转录翻译，重要的是要使用高纯度的DNA。 </li></ol> 2。准备薄的脂质膜<ol><li>称量干脂质粉末溶解在溶剂中。对于1 -棕榈酰-2 -油酰-SN-甘油-3 -磷酸胆碱（POPC），储备溶液的制备在氯仿中，在浓度为40毫克/毫升。 注:有机溶剂必须始终处理的玻璃吸管和瓶子。换句话说，决不能使用塑料。商店s滴答的解决方案中的气密性，抗溶剂的琥珀色玻璃瓶在-20℃下，优选在氩气下。 </li><li>等分试样12微摩尔到5毫升的圆底烧瓶中加入220微升40毫克/毫升原液（POPC）。 </li><li>减压蒸除溶剂，用旋转蒸发器（ 图1），以产生薄的脂质膜。 <ol><li>牢固地连接的圆底烧瓶中，蒸馏管，有一个圆形的剪辑，并开始在烧瓶的旋转。 </li><li>启动的循环水通过冷凝器盘管。使用的热浴中，是没有必要的，因为氯仿61.2°C在正常大气压下具有低的沸点。 </li><li>确保检查活塞打开，该系统是开放的气氛。打开真空泵慢慢地关闭活栓到系统应用真空。 </li><li>的薄的脂质膜沉积在圆底烧瓶中的壁形成一个不透明的膜在旋转蒸发这是肉眼可见的。让旋转蒸发持续0.5-2小时。要停止的过程中，慢慢释放真空压力，停止了转动，取下圆底烧瓶中。 </li></ol></li></ol> 3。脂质再悬浮和囊泡同质化<ol><li>薄的脂质膜，直接在圆底烧瓶中加入1毫升18.2MΩH 2 O。溶液剧烈振荡以最大的速度，约每分钟3200转，从玻璃上分离，用肉眼可观察到脂质膜。 </li><li>脂质分散转移到2毫升离心管中。 </li><li>建立了一个环站举行匀浆5毫米的小费。将下面的均质牢牢固定脂质分散离心立场。冲洗的均化分散体浸渍18.2MΩH 2 O中的前端和运行几秒钟。 </li><li>将分散体直接将脂质分散体，以确保次的尖端处的微量离心管的底部不接触。均质功率4级或14000转1分钟。 </li></ol> 4。囊泡挤压和冻干<ol><li>的组装部件的小型挤出机（ 图2），它由一个外壳和容纳两个聚四氟乙烯内部膜支持，两个O形环，和一个特富龙轴承的保持器螺母。 <ol><li>将两个O形圈进入膜内部支持的凹槽。预湿两个过滤器和一个400nm的膜。该过滤器将被放置对聚四氟乙烯O型圈的内侧，与它们之间的膜。 </li><li>将膜放置在支持到挤出机中的外壳与膜包围的两个过滤器之间的O形环。将特富龙轴承外壳内，并附加固定螺母并用手拧紧。 </li></ol></li><li>冲洗两个注射器，并填补一个18.2MΩ水的。将注射器针头插入到小的holes聚四氟乙烯的挤出机组件的任一端上。针应该很容易滑动，不要强行针。固定注射器进入挤出机挤出住房和紧固。 </li><li>通过挤出机出来的水缓慢地推入一个注射器和成其他的通水。这代表一个通道。重复一共有三个通道，确保不存在任何泄漏。取下注射器水和处置的水。 </li><li>与样品填充一个注射器，连接到挤出机中，并如上所述在步骤4.3中，通过膜囊泡溶液缓慢通过。重复10倍，总的11通道。由于挤压过程的进行，样品会变得混浊，更容易推动穿过膜。突然减少阻力，但是，通常表示一个破裂的膜。 </li><li>挤出囊泡溶液转移到微量离心管中，将40μl等分的囊泡。闪光冻结各等份，在任一干冰或液氮。 </li><li>用离心蒸发器在30℃下过夜冻干各等份存储冻干空的囊泡在-20°C </li></ol> 5。封装转录翻译机械<ol><li>转录 - 翻​​译反应中混合各成分，并加入20个单位的RNA酶抑制剂。在冰上孵育。 </li><li>添加DNA模板。控制反应，250毫微克质粒编码mVenus的或类似的荧光蛋白后面的T7转录启动子和一个强大的E.大肠杆菌核糖体结合位点的建议。 </li><li>用无RNA酶的水使终体积为25微升。 </li><li>水合物冻干囊泡的等分试样（来自步骤4.6）与10μl的反应组装在步骤5.3中。简言之涡的混合物，直至囊泡悬浮。这应小于30秒。 </li><li> 3在冰上孵育反应0分钟，以允许膨胀的小泡。 </li><li>稀释20倍，至终体积为30μl加入到27.0微升的50mM Tris-盐酸，50 mM氯化钠，pH值7.4和20.2毫克/毫升的蛋白酶K DNA酶和RNA酶1.5微升1.5微升囊泡的囊泡混合物也可以被添加在这一点上，作为一种替代蛋白酶K降解extravesicular材料。 </li><li>孵育至少2.5小时，在37℃下</li></ol> 6。显微镜<ol><li>检查在不同时间点的囊泡和荧光蛋白的生产进度。囊泡将有较大的直径大于400nm的孔隙的膜用于囊泡挤出的大小。 <ol><li>准备样品室放置一个20×5毫米的硅垫片到一个标准的显微镜幻灯片。吸取10微升囊泡进入样品室。在室放置一个硅化玻璃盖玻片。 </li><li>观察囊泡与63X油分散Ø类似ŗ的明场和荧光显微镜使用适当的过滤器设置为利用荧光蛋白的目的。 </li></ol></li></ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Large+Monodisperse+Vesicles.">Preparation of Large Monodisperse Vesicles.</a> PLoS ONE. 4, 1-4 (2009).</li></ol>

什么都没有透露。

虽然无细胞合成生物学是仍处于起步阶段，进展奠定了基础，日益复杂的细胞样系统可以。完全定义的组件内囊泡28的重组转录翻译机器尤为显着，在促进以后努力建设环境敏感人工cells17 18。同样地，人工细胞的研究已被用来探测进化过程4,29,30，RNA和蛋白质的合成22,31，影响代谢load32 33，和组装病毒颗粒34的机理的细节。足够的知识，更重要的是，现在存在里面泡在实验室后，这些先前的报告和本文所描述的协议，可以重组，基本的细胞功能。 除了容易，所描述的封装程序ocedure有几个好处。例如，许多空的，冻干的囊泡等分试样可以预先制造，并储存在-20℃下以供以后使用。该协议并不受生物分子的有机溶剂，温度急剧变化或长时间的透析。我们预计，温柔的程序，会促进将因为需要额外的元件。我们还没有观察到改变的脂质组成的膜封装或转录翻译效率的不利影响。因此，脂质膜蛋白，具体的形态，或可视化纳入更适合可以想见，被人利用。 上述方法的主要限制是，由此产生的囊泡大小和层数（lamellarity）不是同质的。对于许多应用，这些困难不会干扰对数据的解释。然而，如果需要的话，额外的步骤可以并入窄次E尺寸分布和减少层膜，如进一步挤压轮封装后，冻结解冻，或透析35。无疑是更好的方法规避这些和其他问题将得到发展。直到这时，我们发现这里描述的协议非常适合蜂窝模仿建设。

无细胞， 在体外转录翻译反应和合成脂质囊泡代是什么新鲜事。然而，成蜂窝模拟相结合的两个显着更多challenging1-6 E。带或不带T7 RNA聚合酶的大肠杆菌细胞提取物可用于作为源转录-翻译机器7,8。细胞提取物受益于额外的细胞成分的存在下，可以方便蛋白的表达和折叠。或者，单独纯化的RNA和蛋白质分子的混 ​​合， 即在PURE系统9，可以用于介导合成intravesicular蛋白4,10-14。纯系统可以完全定义蜂窝模仿，建设并不会受到细胞提取物中发现核酸酶活性。实际上，这意味着，需要少得多的DNA模板，从而促进低包封11的流程与</sup&gt;。虽然不经常使用，可内置模拟蜂窝，细胞提取物是来自于真核cells15。到目前为止，基因编码封装的瀑布和细胞感知周围环境的模仿已经报道16-18。 监测转录 - 翻​​译反应最简单的方法是测量基因编码的元素的荧光或发光。通常情况下，萤火虫荧光素酶19或GFP的使用，虽然在体外的反应，经常测定放射性标记。荧光检测还可以让囊泡20,21人群的监测，通过基于流式细胞仪的方法，从而提供一些洞察类似生物过程的随机性。这些监测方法已被用来定义一个小的设计规则和部件，从而建立从图书馆，包括荧光蛋白的集合， 与 i体外转录-翻译22，22表达的基因的组织的影响，西格玛因子16的活性，和转录终止子效率23。尽管如此，仍然有许多增加的能力， 在体外建立可预测，基因编码的设备，需要做的事情。 有许多方法可以使囊泡。最常用的方法取决于在玻璃表面上，然后再悬浮在水溶液中solution24薄的脂质膜的生成。如果该水溶液含有转录 - 翻​​译机器，例如，则形成囊泡的一小部分将包含蛋白质的生产所需的元件。然而，这样的方法包封为低，这意味着，只有一小部分的囊泡是活跃的。许多替代的方法，其特征在于高得多的封装EFF的iciency利用囊泡的油包水型乳状液的液滴转换。虽然这是可能的，这种方法将在未来是司空见惯，目前这些方法存在需要专门的设备和给囊泡有改变膜组合物的25。的油包水型囊泡的方法的一个显着优点是控制膜的层数（lamellarity）的电位。本文所述的方法是根据在薄的脂质膜YOMO实验室11所述的协议，有轻微的修改，包括一个额外的均质化步骤。这个方法很简单，价格便宜，而且提供了强大的囊泡转录翻译机器的封装非常适合。

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the encapsulation of cell-free transcription and translation machinery in vesicles for the construction of cellular mimics

从单个分子的分子系统随着利率的变化，越来越多的实验室已设法建立从下往上蜂窝模仿细胞生命的复杂性，更好地代表。迄今为止，也有一些可以采取，建立条块蜂窝模仿的路径，包括开采的油包水型乳状液，微流体装置，和囊泡。可用的选项中的每一个具有特定的优点和缺点。例如，油包水型乳剂给予较高的包封率，但没有模仿活细胞的通透性屏障。本文所述的方法的主要优点是，它们都容易和便宜的实施。转录 - 翻​​译机械是封装内的磷脂小泡通过一个过程，它利用常见的仪表，如离心式蒸​​发器和挤出机。荧光光谱反应监测。该协议s可以适于重组蛋白的表达，细胞模仿建设，探索细胞生命的最低要求，或遗传的电路组件的。

荧光显微镜揭示了该荧光观察囊泡内，因为extravesicular材料酶降解（ 图3）。 ，的intravesicular荧光如果在mVenus表达开始1.5小时后，观察到在37℃下，6小时内达到最大荧光强度。最佳的温度和温育时间可以有所不同，这取决于所使用的特定构造。例如，不同的荧光蛋白的成熟的完全不同的温度依赖性。换句话说，观察蛋白质生产并不仅仅取决于蛋白质的合成和折叠，但对发色基团的形成。总体蛋白质的合成，可以增加通过整合膜蛋白毛孔允许耗尽后，组件27的转录和翻译所必需的大量涌入。 建议开展一个类似的转录的翻译在囊泡的情况下，以确保利用的遗传构建功能重刑反应。该控制反应更容易监测荧光光谱法，而不是显微镜图4示出了在体外转录-翻译一个构建体编码mVenus的反应。未封装的反应给远高于总荧光强度比同类intravesicular反应。这是由于包封率，因为总的切除此体积比extravesicular体积（ 即稀释的效果）要少得多。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51304/51304fig1.jpg" width="600px" /> 图1。旋转蒸发器和真空泵等 。 <a href="https://www.jove.com/files/ftp_upload/51304/51304fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 图2所示的挤出机的外壳和部件分开 。从左至右黑色O型圈面对彼此，挤出机的外壳，和第二注射器，注射器，固定螺母，聚四氟乙烯轴承，膜内部支持， <a href="https://www.jove.com/files/ftp_upload/51304/51304fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51304/51304fig3.jpg" width="600px" /> 图3。荧光图像，在脂质体中的蛋白质生产mVenus A和C:明场图像多层囊泡1.5小时和2.5小时后，B＆＃160，D:的生产mVenus是荧光（颜色为绿色）后分别为1.5和2.5小时，可视化。比例尺为20微米。 <a href="https://www.jove.com/files/ftp_upload/51304/51304fig3highres.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51304/51304fig4.jpg" width="600px" /> 图4。 在体外控制反应的未封装的体外转录和翻译的mVenus中 。测定荧光强度超过4小时，每5分钟。被收购的数据在实时PCR仪。 <a href="https://www.jove.com/files/ftp_upload/51304/51304fig4highres.jpg" target="_blank">点击此处查看大图</a> 。

Watch this Scientific Journal Video about The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics at JoVE.com

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

在这里，我们介绍简单的方法编制泡，封装的转录和翻译机制，监测蛋白质的生产。将得到的无细胞系统可以用来作为一个起点，从而建立越来越复杂的细胞模仿。

无细胞的转录和翻译的机械囊泡的封装建设模拟蜂窝

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

the-encapsulation-cell-free-transcription-translation-machinery

Research

JoVE Journal

Bioengineering

15.0K 次观看.  University of Trento. 在这里，我们介绍简单的方法编制泡，封装的转录和翻译机制，监测蛋白质的生产。将得到的无细胞系统可以用来作为一个起点，从而建立越来越复杂的细胞模仿。 

视频: The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

卵泡在蛋白质降解的3维系统的封装和文化的一种方法

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

科研

生物工程

Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome

While the transcription regulation of protein coding genes was extensively studied, little is known on how transcription factors are involved in transcription of non-coding RNAs, specifically of microRNAs. Here, we propose a strategy to study the potential role of transcription factor in regulating transcription of microRNAs using publically available data, computational resources and high throughput data. We use the H3K4me3 epigenetic signature to identify microRNA promoters and chromatin immunoprecipitation (ChIP)-sequencing data from the ENCODE project to identify microRNA promoters that are enriched with transcription factor binding sites. By transfecting cells of interest with shRNA targeting a transcription factor of interest and subjecting the cells to microRNA array, we study the effect of this transcription factor on the microRNA transcriptome. As an illustrative example we use our study on the effect of STAT3 on the microRNA transcriptome of chronic lymphocytic leukemia (CLL) cells.

Herein we propose a strategy to study the effect of a transcription factor of interest on the microRNA transcriptome using publically available data, computational resources and high throughput data from microRNA arrays after transfecting cells with small hairpin (sh)RNA targeting a transcription factor of interest.

描述了微RNA转录的转录因子依赖调节

While the transcription regulation of protein coding genes was extensively studied, little is known on how transcription factors are involved in ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

构建模块化水凝胶片的微模式化宏观缩放3D细胞结构

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

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 ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

闪速纳米沉淀法包聚物纳米粒子中疏水亲水化合物的作用

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Evaluation of the Storage Stability of Extracellular Vesicles

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

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

细胞外囊储存稳定性的评价

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

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Finite Element Modelling of a Cellular Electric Microenvironment

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).

This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation.

细胞电微环境的有限元素建模

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the ...

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.