部分支持这项研究是由美国航空航天局内布拉斯加空间格兰特和国家科学基金会资助号0926880。

1。乳胶容器的制备<ol><li>修复乳胶船只刺软管连接，这是一个坚固的工作台固定两端。 </li><li>测量面积乳胶船只，以确定视野的利息。感兴趣的支架试验区应集中之间的铁丝网软管接头，包括约一英寸支架两侧，以观察支架区以外的菌株。 </li><li>记录从一个倒钩软管接头的外边缘之间的连接器，该也是乳胶船只的近似中心的中心位置的距离。转换到导管测量了导管支架中心的距离。然后标记一个标记导管。 </li><li>取下乳胶船只从刺软管接头。 </li><li>准备通过与随机模式的白色和黑色喷漆喷涂感兴趣的领域或标记的乳胶船只使用永久性标记的随机点感兴趣的领域。对于较小的样品和更精细的随机模式是必需的。 </li></ol> （2） 在体外测试系统和校准阿拉米斯系统<ol><li>选择校准面板是稍大面积在第1步中测得的利息比。 </li><li>刺软管接头之间的校准面板放置在感兴趣的领域和确保光线充足，感兴趣的领域。 </li><li>调整两个摄像头，从样品的距离，并根据选定的面板校准相机的高度之间的距离。每个校准面板是不同的，因此的的阿拉米斯用户手册将进行磋商，以确定这些距离。 </li><li>打开在阿拉米斯新项目，选择"文件"，然后"新建项目"。下一步点击"传感器"标签，然后选择"校准"，然后"全面校准"。 </li><li>阿拉米斯软件现在走过的步骤，用户校准ţ<html>他相机。 </li><li>随着镜头光圈完全开放，注重校准面板上的相机，相机上的固定螺丝松动和旋转镜头。一旦集中，重新拧紧螺丝和关闭光圈。 </li><li>校准过程中采取的第一个图像。移动或旋转校准面板，根据计算机上的示范，直到集中在计算机屏幕上的图像。采取的第二个形象。重复这个过程中，为校正图像的其余部分。 </li><li>一旦所有的校准图像，阿拉米斯图像分析软件，将计算的校准设置。如果校准偏差小于0.04，在校准过程中，应反复。相机或摄像机之间的距离的焦点所做的任何调整将呈现在校准过程中无效。 </li><li>删除校准面板，并放置到刺的软管接头手绘乳胶船只。 </li></ol>标题"&gt; 3。前测，以避免过多的背景噪声<ol><li>确定所需的测试帧每秒的数量。每秒增加帧会产生更均匀的应变结果。 </li><li>调整快门速度小于每秒1帧，所以，没有红色上显示的图像。 </li><li>以5个图像。 </li><li>新增启动点上的图像序列，并计算测试。 </li><li>按住"Ctrl"，单击"样品中心，以观察背景噪声。如果前测的噪音超过75是显微校准过程中，必须重做。 </li></ol> 4。支架部署<ol><li>选择所需的测试过程中采取的图像数量。 200张图片，将足够的支架扩张。 </li><li>逐步乳胶船只插入导管，导管的使用标记的指标，以指导支架置入，直到它到达中央位置。 </li><li>开始服用阿拉米斯图像。 </li><li>球囊扩张式支架，气球的压力逐渐增加，扩大支架，直到气球完全展开;然后逐渐下降的压力为零，气球，气球瘪撤回导管。 </li><li>自扩张支架，逐步消除鞘，直到支架完全展开，然后逐步收回导管。 </li></ol> 5。图像分析<ol><li> 应变历史上的船只的具体点 <ol><li>按住"Ctrl"键并点击感兴趣的领域，创建一个阶段点。 </li><li>选择所需的应变类型，即在X，Y，XY的，大应变，小应变，或米塞斯应变应变。 </li><li>在右下角的情节将显示在测试期间选定点的应变。 </li></ol></li><li> 沿着船只的具体路径的空间应变 <ol><li>铬eate多级点线，通过点击"节"标签，然后在"创造节"。选择X轴在Y上的图像平行线为零。这将创建一个阶段点的行号。 </li><li>创建多段线后，在右下角的情节上会显示一个情节线系列。每一行代表的应变沿节的长度时的一个实例。 </li></ol></li><li> 创建最适合的缸分析船只的扩张速度和半径 <ol><li>在上部的工具栏，选择"原始"，然后选择"最合适的缸"。 </li><li>选择使用合适的工具栏上的"选择通过表面"工具的一小部分。 </li><li>该的阿拉米斯软件将产生一个三维的最合适的气缸。 </li><li>图像，然后可以循环通过观察如何乳胶船只的直径不同。 </li></ol></li><li> 评估之间的距离两点<ol><li>根据"分析"选项卡上单击"点到点的距离"。 </li><li>选择上选择两个点的图像进行分析所需的长度。 </li><li>图像，然后可以通过骑自行车来观察随着时间的推移，两点之间的距离变化。 </li></ol></li></ol> 6。代表结果株支架支撑血管壁向外扩大，将一般左右支架的位置。 图1是一个应变映射在球囊扩张支架，以及主要的应变历史的一个特定点的反冲过程的例子。 图1中的黑点为参考点，由高速摄像机，用来捕捉和跟踪这些参考点的位移的管道。参考点的记录运动的基础上，该软件将被用来计算株导管或NY其他有针对性的对象。也称为最大主应变，主要应变，计算公式如下: <img alt="式（1）" src="/files/ftp_upload/3945/3945eq1.jpg" />它是明确的，植入的支架导致非均匀的容器表面上的应变分布。这可以解释完约束乳胶导管和支架的网状结构的反冲加载。这株字段对应支架的反冲作为红十字标志在图1的底部形象标识，在初始阶段。历史的一个特定点10的主要应变曲线显示支架植入术的区分阶段。球囊扩张时约10至12秒后，气球的通缩发生在12和14秒之间的支架反冲。 <img alt="图1" src="/files/ftp_upload/3945/3945fig1.jpg" /> 图1。 </strong&gt;实验装置（顶部）;非均匀应变分布的支架导管表面（中）;在10点（底部）的主要应变历史。

<ol>
	<li>Fischman, D. L., Leon, M. B., Baim, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+randomized+comparison+of+coronary-stent+placement+and+balloon+angioplasty+in+the+treatment+of+coronary+artery+disease.+Stent+Restenosis+Study+Investigators.">A randomized comparison of coronary-stent placement and balloon angioplasty in the treatment of coronary artery disease. Stent Restenosis Study Investigators.</a> N. Engl. J. Med. 331, 496-501 (1994).</li><li>Abul Hasan Muhammad Bashar, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Properties+of+Various+Z-Stent+Designs:+An+Endovascular+Stent-Grafting+Perspective.">Mechanical Properties of Various Z-Stent Designs: An Endovascular Stent-Grafting Perspective.</a> Artificial Organs. 27, 714-721 (2003).</li><li>Nuutinen, J. u. h. a. -. P. e. k. k. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+and+in+vitro+degradation+of+bioabsorbable+self-expanding+braided+stents.">Mechanical properties and in vitro degradation of bioabsorbable self-expanding braided stents.</a> Journal of Biomaterials Science -- Polymer Edition. , 255-266 (2003).</li><li>C. Schulz, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+stent+symmetry+and+vascular+injury+determine+experimental+restenosis.">Coronary stent symmetry and vascular injury determine experimental restenosis.</a> Heart. 83, 462-467 (2000).</li><li>Jiménez, J. M., Davies, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamically+Driven+Stent+Strut+Design.">Hemodynamically Driven Stent Strut Design.</a> Annals of Biomedical Engineering. 1483, (2009).</li><li>Johnston, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mechanical+Properties+of+Endovascular+Stents:+An+In+Vitro+Assessment.">The Mechanical Properties of Endovascular Stents: An In Vitro Assessment.</a> Cardiovascular Engineering: An International Journal. 10, 128-135 (2010).</li><li>Mejia, J. u. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+effect+of+stent+strut+profile+on+shear+stress+distribution+using+statistical+moments.">Evaluation of the effect of stent strut profile on shear stress distribution using statistical moments.</a> Biomedical Engineering Online. , 1-10 (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ARAMIS User Manual. , (2009).</li><li>Chesler, N. C., Thompson-Figueroa, J., Millburne, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+Mouse+Pulmonary+Biomechanics.">Measurements of Mouse Pulmonary Biomechanics.</a> Journal of Biomechanical Engineering. 126, 309-314 (2004).</li><li>de Korte, C. L., Sierevogel, M. J., Mastik, F., Strijder, C., Schaar, J. A., Velema, E., Pasterkamp, G., Serruys, P. W., van der Steen, A. F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Atherosclerotic+Plaque+Components+With+Intravascular+Ultrasound+Elastography+In+Vivo+A+Yucatan+Pig+Study.">Identification of Atherosclerotic Plaque Components With Intravascular Ultrasound Elastography In Vivo A Yucatan Pig Study.</a> Circulation. 105, 1627-1630 (2002).</li></ol>

没有利益冲突的声明。

立体声光学表面应变测量系统用于测量在平面议案变形，表面不接触标本本地菌株。该系统采用两个高速光学相机随机对比把表面上的模式构建与精度高解决表面株的准确测量每个点的议案，采取图片。 应当指出的是，所需的对比模式需要坚持的表面充分，足以提供准确的测量。此外，有针对性的取样面积需要有良好的照明，无眩光，相机来区分的对比格局的变动。否则，?...

<table border="1"><tbody><tr><td> 使用的设备 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>阿拉米斯相机系统</td><td>墨西哥湾:光学测量技术</td><td></td><td></td></tr><tr><td>的Palmaz创世记肝胆支架</td><td> Cordis公司</td><td> PG5910B </td><td>球囊扩张式支架</td></tr><tr><td>的Z-MED球囊扩张导管</td><td>贝朗医疗公司</td><td> PDZ336 </td><td>球囊扩张导管</td></tr></tbody></table>

monitoring the wall mechanics during stent deployment in a vessel

临床试验报告各1支架设计不同的再狭窄率。据推测，动脉壁支架致应变浓度导致组织损伤，从而启动再狭窄2-7。这一假说需要进一步调查，包括更好地量化，对动脉支架植入后的非均匀应变分布。非接触式表面应变支架动脉的测量方法，提出了这项工作。阿拉米斯立体声光学表面应变测量系统使用两个光学高速摄影机捕捉每个参考点的运动，并解决了三维株，变形，表面8,9。作为部署成一个随机对比它的外表面喷涂或绘制图案的乳胶血管网状支架，表面应变记录在每一个瞬间变形。计算的应变分布，然后可以用来了解劳"CAL病变反应，验证计算模型，并制订进一步的体内研究的假设。

Watch this Scientific Journal Video about Monitoring the Wall Mechanics During Stent Deployment in a Vessel at JoVE.com

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

动脉支架诱导应变分布的特点是使用光学表面应变测量系统。这个可视化技术是用于获取到主机上的船只的影响支架植入术的见解。

在一个容器的支架部署在监测的墙力学

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

monitoring-the-wall-mechanics-during-stent-deployment-in-a-vessel

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.2K 次观看.  University of Nebraska-Lincoln. 动脉支架诱导应变分布的特点是使用光学表面应变测量系统。这个可视化技术是用于获取到主机上的船只的影响支架植入术的见解。

视频: Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

磁共振弹性成像方法评价组织工程构造增长

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

科研

生物工程

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

质量流式细胞术:协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

一种多功能自动化平台微尺度细胞刺激实验

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180&#176; Curved Artery Test Section

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and tortuosity). Secondary flow structures are vortical flow patterns that occur in curved arteries due to the combined action of centrifugal forces, adverse pressure gradients and inflow characteristics. Such flow morphologies are greatly affected by pulsatility and multiple harmonics of physiological inflow conditions and vary greatly in size-strength-shape characteristics compared to non-physiological (steady and oscillatory) flows 1 - 7.
Secondary flow structures may ultimately influence the wall shear stress and exposure time of blood-borne particles toward progression of atherosclerosis, restenosis, sensitization of platelets and thrombosis 4 - 6, 8 - 13. Therefore, the ability to detect and characterize these structures under laboratory-controlled conditions is precursor to further clinical investigations.
A common surgical treatment to atherosclerosis is stent implantation, to open up stenosed arteries for unobstructed blood flow. But the concomitant flow perturbations due to stent installations result in multi-scale secondary flow morphologies 4 - 6. Progressively higher order complexities such as asymmetry and loss in coherence can be induced by ensuing stent failures vis-&#224;-vis those under unperturbed flows 5. These stent failures have been classified as &#34;Types I-to-IV&#34; based on failure considerations and clinical severity 14.
This study presents a protocol for the experimental investigation of the complex secondary flow structures due to complete transverse stent fracture and linear displacement of fractured parts (&#34;Type IV&#34;) in a curved artery model. The experimental method involves the implementation of particle image velocimetry (2C-2D PIV) techniques with an archetypal carotid artery inflow waveform, a refractive index matched blood-analog working fluid for phase-averaged measurements 15 - 18. Quantitative identification of secondary flow structures was achieved using concepts of flow physics, critical point theory and a novel wavelet transform algorithm applied to experimental PIV data 5, 6, 19 - 26.

Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

在180°弧形动脉试验段的二次流动结构下游的型号IV型支架失败的实验研究

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

适应静电过程中提供三个独特的环境为一个三层次<em&gt;在体外</em&gt;的气道壁模型

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

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

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

使用简单搅拌容器在藻酸盐珠中的哺乳动物细胞包封

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

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved through the contraction of the ciliary body and relaxation of zonule fibers, which results in the thickening of the lens necessary for near vision. Here, we present a novel, simple method in which accommodation is replicated by tensing the zonules connected to the lens capsule via a manual lens stretcher (MLS). This method monitors the radial stretching achieved by a lens when subjected to a consistent force and allows for a comparison of accommodating lenses, which can be stretched, to non-accommodating lenses, which cannot be stretched. Importantly, the stretcher couples to the zonules directly, and not to the sclera of the eye, thus only requiring the lens, zonules, and ciliary body rather than the entire globe sample. This difference can significantly decrease the cost of acquiring donor cadaver lenses by about 62% compared to acquiring an entire globe.

We present an efficient method of studying lens accommodation by using a manual lens stretcher. The protocol mimics physiological accommodation by pulling the zonules connected around the lens capsule, thereby, stretching the lens.

用人工晶状体担架模拟透镜体的力学性能

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved ...

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

A step by step protocol for nondestructive and long-period monitoring the process of vascular remodeling and scaffold degradation in real-time culture of biodegradable polymeric scaffold-based tissue-engineered blood vessels with pulsatile stimulation using optical coherence tomography is described here.

基于光学相干断层扫描的可降解支架组织工程血管发育的无损监测

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

一种三肽稳定的油酸纳米乳液

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

在符合标准的基质上对人类胚胎干细胞菌落的几何结构进行模式化，以控制组织级力学

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...