这项工作是由台湾国科会grants- NSC 99-2320-B-007-005-MY2（CC）和NSC 101-2628-E-007-011-MY3（CMC），以及荣总部分支持医院和台湾联合研究计划大学系统（CC）。

操作步骤的总体框图如图1中提供。使用道德实践，我们收集了来自健康受试者的血液样本，并通过台中荣民总医院（TCVGH），台中，台湾在IRB批准协议获得房水样本的患者（ IRB TCVGH号CF11213-1）。 1.制造纸张的设备<ol><li>切色谱纸放入5mm的圆的直径，以提供相同的布局为一个96孔微量滴定板。夹着这些纸片具有两个聚苯乙烯片材与注册的通孔，和层压体。 </li><li>修改使用在下面的步骤28中所述的化学缀合方法纸装置。 <ol><li>处理纸的设备用氧等离子体（100毫瓦，1％的氧气，30秒）在等离子体室中。 注意：用3-巯丙基三甲氧基硅烷和 N-γ-马来酰亚胺琥珀酰亚胺工作时特别小心，必须采取酯（GMBS），因为它们都是湿气敏感和毒性。戴防护手套，避免吸入或与皮肤和眼睛接触。保持股市瓶子盖紧并允许它打开，以避免冷凝水之前平衡至RT。或者，打开股票瓶内充氮的手套袋中或框。分装成小药瓶，以避免频繁开股票瓶。 </li><li>立即处理的孵育在4％（体积/体积）的3-巯基丙基三甲氧基硅烷的乙醇（200标准强度）为30分钟，溶液纸装置。 </li><li>冲洗纸装置，用乙醇和孵化它们与0.01微摩尔/毫升GMBS在乙醇中15分钟。 </li><li>冲洗，用乙醇（200标准强度），并培育纸装置用10μg/ ml的抗生物素蛋白在磷酸盐缓冲盐水（PBS），在4℃的溶液1小时。如果需要储存在4℃下，并在4周内使用。 </li></ol></li><li>湿纸的每个测试区与10微升的PBS含有1％（重量/体积）牛血清白蛋白（BSA）为察觉10微升生物素化的捕获分子3×10分钟前，3×10分钟。使用抗CD63抗体（20微克/毫升的PBS含有1％（重量/体积）BSA和0.09％（重量/体积）叠氮化钠）或膜联蛋白V（1:20，体积/体积在膜联蛋白V结合缓冲液），为捕获分子。 </li><li>冲洗掉，用10微升相应的PBS含有1％（重量/体积）BSA或膜联蛋白V结合缓冲液进行3×1分钟，分别未结合的抗CD63抗体或膜联蛋白V的分子。 </li></ol> 2.血清和房水样品采集与处理<ol><li>血清集合。 <ol><li>在血清分离管中收集10毫升外周血通过静脉穿刺，轻轻颠倒试管5次。设置该管处于垂直位置，并等待30分钟。 </li><li>离心机在1200×g下15分钟。再次传输从顶层血清到一个干净的试管中，离心以3000×g的30分钟。 </li><li>通过0.8微米FIL通过上清器。保持样品在-80℃下直到使用。 </li></ol></li><li>房水的收集：收集房水样本的患者可直接通过侵入性操作和储存样品在-80°C，直到使用诊断眼科医生。 </li></ol> 3.隔离外囊泡<ol><li>点样到每个纸测试区以5微升/分钟的速率。 </li><li>冲洗掉未结合的电动汽车用10微升相应的PBS含有1％（重量/体积）BSA或膜联蛋白V结合缓冲液进行3×1分钟，用于分别官能用抗CD63抗体或膜联蛋白V的分子，造纸设备。执行以下下游试验。 </li></ol> 4.下游测定实施例1：扫描Electromicrographs <ol><li>修复捕获使用10微升0.5×Karnovsky固定液10分钟官能试纸测试区的电动车。 </li><li>冲洗了充足的PBS样品2×5分钟。 </li><li>脱水样品随后35％乙醇10分钟，50％乙醇为2×10分钟，70％乙醇中2×10分钟，95％乙醇为2×10分钟，100％乙醇为4×10分钟。 </li><li>临界干燥和溅射涂层与钯/金的样品中，并且检查使用在低电子加速电压操作的扫描型电子显微镜（〜5千伏）。 </li></ol> 5.下游分析实施例2：基于纸张的ELISA <ol><li>在PBS 1000稀释含电动车捕捉每一个测试区：添加含有抗CD9一抗5微升溶液中1。等待1分钟。 </li><li>冲洗样品用30ml PBS中振摇在100rpm下30秒。 </li><li>添加5微升含有溶液辣根过氧化物酶（HRP）为1 - 连接的二级抗体：1,000稀释在PBS中，并等待1分钟。 </li><li>冲洗样品用30ml PBS中振摇在100rpm下30秒。 </li><li>添加含有1 5微升比色底物：过氧化氢的1体积比ð3,3&#39;，5,5&#39;-四甲基联苯胺（TMB）和扫描使用台式扫描器。 </li></ol> 6.下游试验例3：RNA分离<ol><li>浸泡样品中35微升的聚乙烯吡咯烷酮为基础的RNA分离援助和265微升裂解/结合缓冲液裂解捕获电动车。涡大力。 </li><li>加在分离试剂盒的裂解物提供30微升匀浆。涡大力和雪藏了10分钟。 </li><li>使用在下面的步骤中描述的酸苯酚 - 氯仿分离提取的RNA。 <ol><li>取330微升酚 - 氯仿从瓶底层并添加到匀浆/溶胞产物。涡大力。 </li><li>离心以10,000×g离心5分钟。水性（上部）相转移到一个干净的试管，并注意除去的体积。 </li></ol></li><li>沉淀的总RNA使用本身是在前面的步骤和共除去水相的1.25倍的体积的体积的100％乙醇llect在预热至95℃的洗提溶液的核糖核酸。 </li><li>浓缩和进一步纯化根据生产商的方案使用RNA净化试剂盒的RNA。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

为胞外囊泡的子组的成功分离的最重要的步骤是：1）一个良好的纸张选择; 2）稳定和高的纸纤维的表面上的捕获分子的覆盖范围; 3）样品的妥善处理; 4）一般实验室卫生习惯。 多孔材料已经被用于许多廉价且设备无测定。它们可以具有可调孔径，多用途功能，成本低和高的表面与体积之比，允许液体的被动的芯吸。我们选择色谱纤维素纸1级主要针对其适当的孔径和低蛋白吸...

Extracellular vesicles (EVs) are heterogeneous membranous particles that range in size from 40 nm to 5,000 nm and are released actively by many cell types via different biogenesis routes1-9. They contain unique and selected subsets of DNA, RNA, proteins, and surface markers from parental cells. Their involvement in a variety of cellular processes, such as intercellular communication10, immunity modulation11, angiogenesis12, metastasis12, chemoresistance13, and the development of eye diseases9, is increasingly recognized and has spurred a great interest in their utility in diagnostic, prognostic, therapeutic, and basic biology applications.
EVs can be classically categorized as exosomes, microvesicles, apoptotic bodies, oncosomes, ectosomes, microparticles, telerosomes, prostatosomes, cardiosomes, and vexosomes, etc., based on their biogenesis or cellular origin. For example, exosomes are formed in multivesicular bodies, whereas microvesicles are generated by budding directly from plasma membrane and apoptotic vesicles are from apoptotic or necrotic cells. However, the nomenclature is still under refined, partly due to a lack of thorough understanding and characterization of EVs. Several methods have been developed to purify EVs, including ultracentrifugation14, ultrafiltration15, magnetic beads16, polymeric precipitation17-19, and microfluidic techniques20-22. The most common procedure to purify EVs involves a series of centrifugations and/or filtration to remove large debris and other cellular contaminants, followed by a final high-speed ultracentrifugation, a process that is expensive, tedious, and nonspecific14,23,24. Unfortunately, technological need for rapid and reliable isolation of EVs with satisfactory purity and efficiency is not yet met. 
We have developed a paper-based immunoaffinity device that provides a simple, time- and cost-saving, yet efficient way to isolate and characterize subgroups of EVs22. Cellulose paper cut into a defined shape can be arranged and laminated using two plastic sheets with registered through-holes. In contrast to the general strategy to define the fluid boundary in paper-based devices by printing hydrophobic wax or polymers25-27, these laminated paper patterns are resistant to many organic liquids, including ethanol. Paper test zones are chemically modified to provide stable and dense coverage of capture molecules (e.g., target-specific antibodies) that have high affinity to specific surface markers on EV subgroups. Biological samples can be pipetted directly onto the paper test zones, and purified EVs are retained after rinse steps. Characterization of isolated EVs can be performed by SEM, ELISA, and transcriptomic analysis.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Chromatography Paper</td>
			<td>GE Healthcare Life Sciences</td>
			<td>3001-861</td>
			<td>&#160;Whatman&#174; Grade 1 cellulose paper</td>
		</tr>
		<tr>
			<td>(3-Mercaptopropyl) trimethoxysilane</td>
			<td>Sigma Aldrich</td>
			<td>175617</td>
			<td>This chemical reacts with water and moisture and should be applied inside a nitrogen-filled glove bag. Avoid eye and skin contact. Do not breathe fumes or inhale vapors.</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818</td>
			<td>Absolute, 200 Proof, molecular biology grade</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>BioShop Canada Inc.</td>
			<td>ALB001</td>
			<td>Often referred to as Cohn fraction V.</td>
		</tr>
		<tr>
			<td>N-g-maleimidobutyryloxy succinimide ester (GMBS)</td>
			<td>Pierce Biotechnology</td>
			<td>22309</td>
			<td>GMBS is an amine-to-sulfhydryl crosslinker. GMBS is moisture-sensitive.</td>
		</tr>
		<tr>
			<td>Avidin</td>
			<td>Pierce Biotechnology</td>
			<td>31000</td>
			<td>NeutrAvidin has 4 binding sites for biotin and its pI value is 6.3, which is more neutral than native avidin</td>
		</tr>
		<tr>
			<td>Biotinylated mouse anti-human anti-CD63</td>
			<td>Ancell</td>
			<td>215-030</td>
			<td>clone AHN16.1/46-4-5</td>
		</tr>
		<tr>
			<td>biotinylated annexin V</td>
			<td>BD Biosciences</td>
			<td>556418</td>
			<td>Annxin V has a high affinity for phosphotidylserine (PS)</td>
		</tr>
		<tr>
			<td>Primary anti-CD9 and secondary antibody</td>
			<td>System Biosciences</td>
			<td>EXOAB-CD9A-1</td>
			<td>The secondary antibody is horseradish peroxidise-conjugated</td>
		</tr>
		<tr>
			<td>Serum separation tubes</td>
			<td>BD Biosciences</td>
			<td>367991</td>
			<td>Clot activator and gel for serum separation</td>
		</tr>
		<tr>
			<td>Annexin V binding buffer</td>
			<td>BD Biosciences</td>
			<td>556454</td>
			<td>10X; dilute to 1X prior to use.</td>
		</tr>
		<tr>
			<td>TMB substrate reagent set</td>
			<td>BD Biosciences</td>
			<td>555214</td>
			<td>The set contains hydrogen peroxide and 3,3&#8217;,5,5&#8217;-tetramethylbenzidine (TMB)</td>
		</tr>
		<tr>
			<td>RNA isolation kit</td>
			<td>Life Technologies</td>
			<td>AM1560</td>
			<td>MirVana RNA isolation kit</td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone-based RNA isolation aid</td>
			<td>Life Technologies</td>
			<td>AM9690</td>
			<td>Plant RNA isolation aid contains polyvinylpyrrolidone (PVP) that binds to polysaccharides.</td>
		</tr>
		<tr>
			<td>RNA cleanup kit</td>
			<td>Qiagen Inc.</td>
			<td>74004</td>
			<td>MinElute RNA cleanup kit is designed for purification of up to 45 &#956;g RNA.</td>
		</tr>
		<tr>
			<td>Plasma chamber</td>
			<td>March Instruments</td>
			<td>PX-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>Hitachi Ltd.</td>
			<td>S-4300</td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop scanner</td>
			<td>Hewlett-Packard Company</td>
			<td>Photosmart B110</td>
			<td>8-bit color images were captured. Cameras and smart phones may be also used.</td>
		</tr>
		<tr>
			<td>Image-record system</td>
			<td>J&#38;H Technology Co</td>
			<td>GeneSys G:BOX Chemi-XX8</td>
			<td>16-bit fluroscence images were captured. Fluroscence microscopes may be also used.</td>
		</tr>
	</tbody>
</table>

paper-based devices for isolation and characterization of extracellular vesicles

胞外囊泡（电动汽车），从不同类型的细胞释放的膜的颗粒，持有用于临床应用的巨大潜力。它们含有核酸和蛋白质的货物和正日益被视为间通讯双方真核生物和原核生物细胞利用的手段。然而，由于它们的尺寸小，对电动汽车的隔离电流协议往往费时，繁琐，并且需要较大的样品体积和昂贵的设备，如超速离心。为了解决这些限制，我们开发了一种基于纸张免疫平台，用于分离电动汽车的亚组是容易的，高效的，并且需要的样品体积低至10微升。生物样品可以直接吸移到已经被化学修饰以捕获分子具有高亲和力的特定的EV表面标记纸测试区。我们验证该测定通过使用扫描电子显微镜（SEM），纸基酶联immunosorben吨测定（P-ELISA），和转录组分析。这些文件为基础的设备将使临床和研究设置电动车的研究，以帮助促进我们的健康和疾病EV功能的理解。

造纸设备的隔离电动汽车的亚组的能力有效地依靠其敏感性和特异性识别的EV表面标志。纸纤维与捕获分子的稳定修饰是通过使用抗生物素蛋白-生物素化学如别处28-30所描述实现的。化学缀合的物理吸附方法的有效性，并且在使用基于荧光的读数评估。纸张测试区被以下置换为20微克/毫升的荧光团R-藻红蛋白缀合的生物素分子（PE-生物素），在步骤1.3协议步骤1），所不同的捕获分子制备。...

Watch this Scientific Journal Video about Paper-based Devices for Isolation and Characterization of Extracellular Vesicles at JoVE.com

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

该协议细节的方法分离细胞外囊泡（电动汽车），从细胞中释放的小的膜的颗粒，从少至10微升血清样品。这种方法绕过需要超速离心，需要测定时间仅几分钟，并且使电动车从有限体积的样品的分离。

基于纸张的设备进行隔离外囊泡与表征

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

paper-based-devices-for-isolation-characterization-extracellular

Research

JoVE Journal

Bioengineering

11.3K 次观看.  National Tsing Hua University. 该协议细节的方法分离细胞外囊泡（电动汽车），从细胞中释放的小的膜的颗粒，从少至10微升血清样品。这种方法绕过需要超速离心，需要测定时间仅几分钟，并且使电动车从有限体积的样品的分离。 

视频: Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

定量和大小分析外囊泡的使用可调电阻脉冲检测

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

科研

生物工程

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

三维纸基微流体装置的制造用于免疫分析

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

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 ...

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 ...

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

Here we present a protocol to design and fabricate custom microfluidic devices with minimal financial and time investment. The aim is to facilitate the adoption of microfluidic technologies in biomedical research laboratories and educational settings.

用于研究和教育应用的定制微流体器件的快速制造

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

用于刺激生物组织的电动和磁场设备

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

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.

标记细胞外囊泡以监测软骨外植体的迁移和摄取

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many consider that NTA instruments and their software packages can be easily utilized following minimal training and that size calibration is feasible in-house. As both NTA acquisition and software analysis constitute EV characterization, they are addressed in Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018). In addition, they have been monitored by Transparent Reporting and Centralizing Knowledge in Extracellular Vesicle Research (EV-TRACK) to improve the robustness of EV experiments (e.g., minimize experimental variation due to uncontrolled factors).
Despite efforts to encourage the reporting of methods and controls, many published research papers fail to report critical settings needed to reproduce the original NTA observations. Few papers report the NTA characterization of negative controls or diluents, evidently assuming that commercially available products, such as phosphate-buffered saline or ultrapure distilled water, are particulate-free. Similarly, positive controls or size standards are seldom reported by researchers to verify particle sizing. The Stokes-Einstein equation incorporates sample viscosity and temperature variables to determine particle displacement. Reporting the stable laser chamber temperature during the entire sample video collection is, therefore, an essential control measure for accurate replication. The filtration of samples or diluents is also not routinely reported, and if so, the specifics of the filter (manufacturer, membrane material, pore size) and storage conditions are seldom included. The International Society for Extracellular Vesicle (ISEV)&#39;s minimal standards of acceptable experimental detail should include a well-documented NTA protocol for the characterization of EVs. The following experiment provides evidence that an NTA analysis protocol needs to be established by the individual researcher and included in the methods of publications that use NTA characterization as one of the options to fulfill MISEV2018 requirements for single vesicle characterization.

Nanoparticle tracking analysis (NTA) is a widely used method to characterize extracellular vesicles. This paper highlights NTA experimental parameters and controls plus a uniform method of analysis and characterization of samples and diluents necessary to supplement the guidelines proposed by MISEV2018 and EV-TRACK for reproducibility between laboratories.

提高可重复性以满足细胞外囊泡研究的最少信息 2018纳米颗粒跟踪分析指南

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many ...

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider implications for the well-being of societies and economic stability. Recent years have seen several new isothermal diagnostic modalities emerge to meet the need for rapid, low-cost molecular diagnostics. We have contributed to this effort through the development and patient validation of toehold switch-based diagnostics, including diagnostics for the mosquito-borne Zika and chikungunya viruses, which provided performance comparable to gold-standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR) based assays. These diagnostics are inexpensive to develop and manufacture, and they have the potential to provide diagnostic capacity to low-resource environments. Here the protocol provides all the steps necessary for the development of a switch-based assay for Zika virus detection. The article takes readers through the stepwise diagnostic development process. First, genomic sequences of Zika virus serve as inputs for the computational design of candidate switches using open-source software. Next, the assembly of the sensors for empirical screening with synthetic RNA sequences and optimization of diagnostic sensitivity is shown. Once complete, validation is performed with patient samples in parallel with RT-qPCR, and a purpose-built optical reader, PLUM. This work provides a technical roadmap to researchers for the development of low-cost toehold switch-based sensors for applications in human health, agriculture, and environmental monitoring.

Access to decentralized, low-cost, and high-capacity diagnostics that can be deployed into the community for decentralized testing is critical for combating global health crises. This manuscript describes how to build paper-based diagnostics for viral RNA sequences that can be detected with a portable optical reader.

纸基脚趾开关诊断开发和患者验证的设计到实施研究

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

2D人干细胞来源心肌细胞中心脏收缩力调节疗法的评估

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...