作者要感谢来自欧洲FP7研究项目NAMDIATREAM（NMP4-LA-2010-246479，http://www.namdiatream.eu）和INTERREG IV法国 - 瑞士NAOMI项目的部分资金。

1，文化与扩展人类的ESC <ol><li>制备含有基因敲除DMEM细胞繁殖培养基（称为PM）的补充有20％敲除血清，1％MEM非必需氨基酸，1％L-谷氨酰胺的200mM，1％青霉素 - 链霉素，和3.5微升β-巯基乙醇。 </li><li>在8ml下午中等解冻人类胚胎（胚胎干细胞）和他们离心5分钟，在115 XG放弃DMSO-辅以冷冻介质。 </li><li>加入1 ml PM培养基含有10mM摇滚抑制剂以阻止细胞凋亡和促进细胞存活时解冻（仅24小时培养）。 </li><li>加碱性成纤维细胞生长因子（bFGF）的PM在所需的浓度（4-10纳克/毫升），用于维护多能性的。 </li><li>板状的人类胚胎干细胞上照射的人包皮的饲养细胞（γHFF）（ 图1A），预先以2×10 4个细胞/ cm 2的浓度接种。 </li><li>媒体每天都在变化，必要时，删除的c部分olonies开始吸用细长的大幅削减巴斯德吸管来区分。 </li><li>每周一次，通过菌落酶： <ol><li>除去培养基并用2ml PBS洗涤菌落。 </li><li>除去PBS，用每温暖的胶原酶IV以及0.5毫升在37℃，5％CO 2孵育他们在1毫克/毫升10分钟。 </li><li>收集菌落片段在2毫升新PM和离心机在115×g离心3分钟。 </li></ol></li><li>或者，菌落可以机械地刮传代： <ol><li>除去培养基并用2ml PBS洗涤菌落。 </li><li>手动刮使用STEMPRO EZPassage辊刮板工具（ 图1B）的菌落。 </li><li>收集菌落片段和离心再进行3分钟，在115×g离心以去除上清液。 </li><li>上γHFF或板碎片处理它们分化成胚状体（EB）。 </li></ol></li></ol> 2，人类胚胎干细胞分化协议<ol><li>制备使用基因敲除DMEM分化培养基（DM）的补充有2％Hyclone公司血清，1.0％MEM非必需氨基酸，1.0％L-谷氨酰胺的200mM，1％青霉素 - 链霉素，3.5微升β-巯基乙醇。 </li><li>除去培养基并用2ml PBS洗涤菌落。 </li><li> EB形成菌落片段的悬挂系统： <ol><li>除去PBS，用每温暖的胶原酶IV以及0.5毫升在37℃，5％CO 2孵育他们在1毫克/毫升10分钟。 </li><li>收集菌落片段在2毫升新DM和离心机在115×g离心3分钟。 </li><li>它们培养在悬浮液中的超低附件6孔板中（每孔2毫升）溶液4天（ 图1C）。 </li><li>收集和板新形成拟胚体上0.1％明胶包被的24孔板（约3的EB /孔）（ 图1D）。 </li></ol></li><li>或者，使用Aggrewell板被迫单细胞聚集成的EB： <ol><li>除去PBS中，使用的Accutase（0.5毫升/孔）分离菌落成单个细胞。 </li><li>离心重悬细胞在糖尿病为1.2×10 6 /毫升。 </li><li>加每Aggrewell室2毫升1天（ 图1B&#39;）。 </li><li>收集新形成拟胚体（ 图1C&#39;）和板他们在0.1％明胶包被的24孔板（约3的EB /孔）。 </li><li>改变糖尿病每隔2-3天，直到跳动的心肌细胞簇（15-30天）的外观。 </li></ol></li></ol> 3，空气液体3D打集群和标签用的HNP的培养<ol><li>识别和手动剖析簇跳动的心肌细胞（ 图1D）的，通常出现2-4周的培养后，用细长的大幅削减巴斯德吸管。 </li><li>每个刀片存款4聚四氟乙烯过滤器将被放置在一个6孔板（ 图1F）。 </li><li>加入1 ml的DM介质对EAC的顶部ħ很好。 </li><li>新增一个解剖上跳动每PTFE过滤器（ 图1E）集群，最大4/well，每盘（ 图1F&#39;）， 即 24集合体。 </li><li>改变DM媒体每隔2-3天。 </li><li>标注集群，删除包含簇跳动的PTFE过滤器，并把它放在3.5厘米玻璃底菜（170微米厚）。 </li><li>加入1 ml的HNP的以50μg/ ml的30分钟的浓度。 </li></ol> 4，非线性光学成像<ol><li>样品制备。后HNP标签（见步骤3.6），聚四氟乙烯过滤器在具有高数值孔径物镜的工作距离兼容的170微米厚的玻璃底培养皿或者转让全差分早HEBS或心脏跳动集群（收缩时的外观解剖）。可替换地，应用跳动簇的玻璃底培养皿预先涂有0.1％明胶的直接涂布。 </li><li>转移样品至一个所有功能于一身的显微镜配备孵育室中确保稳定的温度，湿度和CO 2控制在延长的时间周期。 </li><li>经过初步检查，图像在白光照明下通过宽视场成像的样品，以确定拟胚体分化或将要选择作进一步调查活动的跳动集群内的利益结构。 </li><li>如果从NADH细胞的自体荧光和从的HNP SH，必须同时取得，设定更短的激光激发波长（ 即 720纳米），以匹配分子的吸收。使用中心在半激发波长1窄的带通滤波器的频谱（ 即 ，λ= 360±10nm的）来检测SH，另一个用于检测自体荧光。 </li><li>首先执行快速，分辨率低，三维扫描重建心脏簇的整体形态和簇体积与几个可见HNP内选择一个图像平面秒。 </li><li>如果有必要，随意改变激发波长达到最佳匹配样品的光学性能或避免光漂白，光损伤和形象深入到组织中红外波长（ 即 790纳米），因此更换二次谐波滤波器（ 即 395纳米）。通过最大化的HNP沉积在样品的二次谐波信号优化激光脉冲预压缩机的设置在所选择的工作波长。 </li><li>为了实时监控心脏集群收缩，置显微镜光学扫描仪在最快的模式，如谐振模式，允许高速采集的二维图像。 </li><li>选择设置，图像的对比度和采集速度，如之间的最佳折衷： <ul type="disc"><li>扫描平均：4 </li><li>像素停留时间：0.1微秒</li><li>图像速率：每秒3.75张（fps） </li></ul></li><li>激光功率根据焦光点S调整IZE和扫描速度。 50 mW（在物镜的入射光测定）为0.1微秒像素停留时间和计划的APO 20X NA 0.75物镜保持细胞活力的典型值。 </li></ol> 5，图像分析<ol><li>轴向校准程序。选择一个HNP沉积在裸露的基材。调整焦点（ 即在纳米粒子的轴向位置），用于最大化SH强度。变化以受控的方式在显微镜载物台的轴向位置，以获得与SH强度为HNP的函数的校正曲线从焦平面偏移。这个校准程序，为计划的APO 20X NA 0.75物镜的输出报告图4C。 </li><li>选择存在于所有的视频​​帧的HNP，并确定其周期性波动。这个程序可以很容易地实现自动化，例如通过在一个定义的感兴趣区域寻求信号的最大强度点的自定义代码和连接它们不同的帧之间（写在Matlab R2009 B使用功能的图像处理工具箱的“区域道具&#39;的例子代码提交）。 </li><li>评估出平面的位移，与非连续可追踪的HNP移动沿着光轴，通过将它们的强度调制，在整个不同的帧转换成轴向位移利用在步骤5.1确定的校准曲线相关联的量化。注意，该程序可以访问的轴向移动，但不能用来确定绝对方向（向上或向下）。 </li></ol>

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作者什么都没有透露。

纳米技术的应用，干细胞研究是一个相对较新的，但迅速扩大的领域。正如关于这一主题的各种评论文章，利用纳米颗粒的可应用来完成不同的任务的研究，从细胞追踪（ 在体外和在体内 ），向细胞内递送的蛋白质和基因，建立至少不仿生蜂窝环境中的特定分化优惠政策刺激/抑制通路19,20。在本协议中所述的方法是有限的，以光学跟踪，虽然采用红外线激发的可能性，导致...

非线性显微镜系统中，由于其固有的三维切片能力，已越来越多地用于触发光稳定的荧光团与双光子吸收带在近红外的需求。仅在过去的几年中，以补充基于荧光的标签（染料，量子点，上转换纳米颗粒），不同的成像方法已被开发利用了一种新的非线性固有的纳米粒子的家庭作为标签， 即发展谐波纳米粒子（的HNP）已对多光子显微镜是专门开发的。这些标签的基础上，无机非中心对称晶体，施加的光学对比度产生激励频率的SH：例如通过转换的近红外脉冲激发光的一小部分（λ= 800纳米）转换成可见光的蓝光（λ/ 2 = 400nm）的。几位作者在最近的过去已经测试了不同的材料，包括铁碘酸铁（IO <suB&gt; 3）3 1，铌酸钾（的KNbO 3）2，铌酸锂（LiNbO 3晶体）3，钛酸钡（BaTiO 3的）4,5，磷酸氧钛钾（KTiOPO4，KTP）6-8，和氧化锌（ZnO ）5,9,10。相比于荧光探针的HNP具有一系列有吸引力的性质，如完全没有漂白和闪烁，窄发射带，激发波长可调谐性（从紫外到红外），取向检索能力和相干光响应的。这些独特的性能已在两次全面审查文件11,12最近解释。在红外光谱区域，它通过最小化散射和吸收增加成像深度的工作，也有可能大大限制了样品光降解13,14。此外，通过的HNP保证了无限的光稳定的信号，使它们成为理想的探针进行长期跟踪的细胞，这是我更是尤为吸引人再生医学应用15。 在这个可视化实验，提供了协议的细节进行体外标记的人类胚胎干细胞的（胚胎干细胞）与未官能化的HNP。胶体悬浮液的合成与制备详见以前的出版物和参考文献16和超出了工作范围。方法通过对多光子显微镜和其分化成心肌集群（如保持长期气 - 液培养）人类胚胎干细胞研究被提出。人类ESC可以让两种不同的方法，或者通过EB形成集落的碎片在悬浮液，或者，迫使单细胞进入胚体使用Aggrewell板聚集，如示于图1A中所谓的类胚体（EB）内，以区分。培养簇跳动心肌细胞对聚四氟乙烯（PTFE）多孔过滤因子ilitates他们的长期维护进行进一步的研究（例如电动作电位测量）。 扫描显微镜的激发源应当能够提供超短脉冲（与脉冲持续时间超过300飞秒较小的样本），以达到执行的HNP的二次谐波成像所需要的峰值功率。例如，用于成像的最常见FSEC源是可调谐的Ti：蓝宝石激光器。可替换地，其他超快激光器可以采用，例如铒离子17，铬镁橄榄石18或Ti：蓝宝石泵浦红外光参量振荡器。显微镜可以配备一个客观与优选相当高的数值孔径。非常重要的是，之前的测量值，并且目标是，每次更换，这是强制性的，通过优化激光脉冲预压缩机的设置在工作，以减少存在于该集合式（镜片）的分散波长选择。此过程中，在协议中详细描述的，确保了激光脉冲是尽可能接近到变换有限的持续时间（ 即最短可能的话）在焦平面和最大限度地提高了样品的非线性响应。 在协议结束时描述的图像分析的目标是识别并跟踪在跳动的心脏簇的节律性收缩相关联的三维运动的HNP。在图像平面中的跟踪纳米粒子被简单地通过确定它们在连续的动画帧的位置来实现。以提取的轴向运动信息，将非线性响应强度作为轴向位移的一个函数的现有校准是必须的。请注意，对于长期测量，样品的轴向位置的有源干涉控制是必需的，以保持校准曲线的有效性在热和/或机械漂移的存在。 这里使用的TRAC的的HNP聚集在ê跳动电池是基于铌酸钾氧化（的KNbO 3），但其他可用的非线性纳米材料中详细Staedler 等[16]的工作进行检讨。 大部分至今调查纳米材料的非线性光学效率是非常类似的。为的KNbO 3的选择基本上是由胶体溶液的稳定性好，并具有良好的生物相容性，对几种人类细胞系，即使在相当高的浓度和较长的论述16次测试的动机。 鉴于用于这项工作的纳米材料的新颖性，的HNP相比荧光灯/荧光生物标记物的主要特征都显示在很短的原始计算机影视动画由作者来实现。

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harmonic nanoparticles for regenerative research

在这个可视化实验，提供了协议的细节进行体外标记的人类胚胎干细胞的（胚胎干细胞）与二次谐波产生的纳米颗粒（的HNP）。后者是最近推出的用于标记的生物样品的多光子成像探测的一个新的家庭。的HNP是能够通过产生二次谐波的非线性光学过程与激发波长没有限制激发光的频率加倍。 多光子为基础的方法进行人类胚胎干细胞分化成心肌集群（如保持长期的气 - 液培养）将详细介绍。尤其是，显示在如何在三维监测跳动心脏组织中的3D最大化激烈二次谐波（SH）分离的HNP排放的证据。所得到的图像的分析来检索三维位移模式也详细描述。

之前评估由共焦成像的打浆作用，进行了仔细表征的PTFE过滤器的非线性光学响应的​​，单独的或的HNP的存在下，在高浓度（1毫克/毫升）。它确保：i）该裸基材双光子激发荧光很弱，不能防止测量相关的生物样本，以及ii）从孤立的HNP了SH排放可以通过外延检测模式的基板很容易获得通过成像（ 图2）。其目的是有参考控件，用于三维位移的量化。 在图3?...

Watch this Scientific Journal Video about Harmonic Nanoparticles for Regenerative Research at JoVE.com

Harmonic Nanoparticles for Regenerative Research

提供用于体外标记的人类胚胎干细胞的二次谐波产生的纳米颗粒协议的细节。方法通过对多光子显微镜和其分化成心肌人类胚胎干细胞群的调查也被提出。

纳米谐波再生研究

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

harmonic-nanoparticles-for-regenerative-research

Research

JoVE Journal

Bioengineering

11.2K 次观看.  University of Geneva. 提供用于体外标记的人类胚胎干细胞的二次谐波产生的纳米颗粒协议的细节。方法通过对多光子显微镜和其分化成心肌人类胚胎干细胞群的调查也被提出。 

视频: Harmonic Nanoparticles for Regenerative Research

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

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

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

构建交付的干细胞壳聚糖微球的胶原凝胶

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

科研

生物工程

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Biofunctionalized普鲁士蓝纳米粒子多式联运分子成像应用

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

新生儿心脏支架：再生研究新矩阵

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

液体中脉冲激光烧蚀制备金属纳米粒子：研究纳米粒子抗菌性能的工具

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

20 mJ, 1 ps Yb:YAG Thin-disk Regenerative Amplifier

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with turn-key performance and microjoule-level pulse energy is used to seed the regenerative chirped-pulse amplifier. The amplifier is placed in airtight housing. It operates at room temperature and exhibits stable operation at a 5 kHz repetition rate, with a pulse-to-pulse stability less than 1%. By employing a 1.5 mm-thick beta barium borate crystal, the frequency of the laser output is doubled to 515 nm, with an average power of 70 W, which corresponds to an optical-to-optical efficiency of 70%. This superior performance makes the system an attractive pump source for optical parametric chirped-pulse amplifiers in the near-infrared and mid-infrared spectral range. Combining the turn-key performance and the superior stability of the regenerative amplifier, the system facilitates the generation of a broadband, CEP-stable seed. Providing the seed and pump of the optical parametric chirped-pulse amplification (OPCPA) from one laser source eliminates the demand of active temporal synchronization between these pulses. This work presents a detailed guide to set up and operate a Yb:YAG thin-disk regenerative amplifier, based on chirped-pulse amplification (CPA), as a pump source for an optical parametric chirped-pulse amplifier.

A protocol for the operation of a high-energy, high-power optical parametric chirped pulse amplifier pump source based on an Yb:YAG thin-disk regenerative amplifier is presented here.

20 mJ，1 ps Yb：YAG薄盘再生放大器

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

3D Microtissues 用于可注射再生疗法和高通量药物筛选

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.