作者非常感谢协助他与数控加工尼尔豪斯（WPI的，希金斯机铺）。此外，我们想感谢她与MATLAB编程，以及凯特饮料和约瑟夫Cotnoir（WPI的学术技术中心）的帮助下，阿德里安娜赫拉（WPI的计算和通信中心）为协助与CAMTASIA。梁刘柔芬伯克和Jacleen贝克尔（WPI的学术技术中心）提供的补充录像。这项工作是由美国国立卫生研究院（R15 HL097332），麻省大学医学院WPI的试验研究倡议，美国心脏协会（本科生科研奖学金JZH）和伍斯特理工学院（JZH和机构的暑期大学生研究奖学金启动资金，水利部）。

1。细胞种植模具制造铣1 / 2"的聚碳酸酯中心后直径为2毫米厚片创建15日，圆底，环形井开始了。碾磨渠道6毫米深和3.75毫米宽，清洁和干燥的聚碳酸酯模删除任何铣削过程的塑料碎片。 在10:1（W / W）的基地，固化剂，脱气，以消除所有气泡，倒到聚碳酸酯模混合聚二甲基硅氧烷（PDMS）。德加再次删除任何剩余的气泡，并在60 ° C固化烤箱​​4个小时。 治愈后，小心地取出慢慢剥离聚碳酸酯的PDMS模板，用肥皂和水清洗，并高压灭菌。同时蒸压％琼脂糖（W / V）溶解在贝科的改良Eagle培养基（DMEM）的解决方案。 放置在一个水平面上的PDMS模板和填写熔融AGA<html>第一吹打琼脂糖上升到每个中心后的模具，然后进入它周围的空间吹打。允许琼脂糖凝固（约15分钟），然后反转模具的PDMS琼脂糖释放。剪下多余的琼脂糖，各地各井和12孔板放入琼脂糖井。 2。细胞培养和环播种周围添加细胞培养基（10％FBS和1％青霉素，链霉素的DMEM）琼脂糖模具外，不包括模具的顶部。板放置在孵化器，并允许他们平衡约15分钟，当您准备的细胞。 在90％汇合，trypsinize大鼠主动脉平滑肌细胞（rSMCs）和重悬在5X10 6细胞/毫升的浓度。吸取100μL的细胞悬液到每个环形琼脂糖井，用一个细胞每孔的圆周运动。 内容"&gt;请确保孵化器的货架上水平，然后放置在孵化器板，让他们坐了24小时不受干扰。交换的媒介，此后每隔两天吸从周围以及媒体，并重新填充每口井，直到琼脂糖以及完全淹没。 3。组织环收获和厚度测量在文化的结论，请从琼脂糖模具中心后上方滑动环。 环放置在一个小的Petri菜用磷酸盐缓冲液（PBS），中心样品，并使用数字成像系统获取图像。 使用边缘检测的图像分析程序，并测量其周长周围的4个位置（上，下，左，右）在环的厚度。计算平均厚度为每个环值（T），并使用这个值来计算的横截面积（2π（T / 2）2）。 4。机械测试的戒指拉力试验机（英斯特朗公司每股收益1000），在水平位置。附加1N ± 1mN称重传感器和自定义细线夹具（不锈钢丝弯曲创建）。 两个细线夹具安装在环形样品。延长交手，直到一个5分钟的皮重负载应用到样品。输入截面积（厚度测量计算），并记录测量长度。 前皮负载50千帕的压力，并在10毫米/分钟的速度的8倍之间循环圈。第八周期前，后拉要失败的，在10毫米/分钟。失败是指出作为一个有效的减少了40％，从一个测量到下一个。对于每个环样品，测量的极限拉伸应力（UTS）和失效应变和最大切线模量（MTM）从采集到的数据计算。 5。组织环AS组装编造组织管自定义的持有人管加工轮5厘米聚碳酸酯矩形切口（2厘米× 3厘米）的磁盘。通过这些磁盘上钻螺纹孔，让两个持有人，被拧在了一起。高压灭菌器自定义管人，然后转移到生物安全柜和一个空的培养皿。 用手术剪创建斜面边缘的角度削减1.9毫米直径的硅胶管的两端，然后高压灭菌的硅胶管。正在蒸压管，填补了与媒体的培养皿。 从琼脂糖井的戒指取出，并放置在与媒体的培养皿。地方蒸压在同一培养皿硅胶管使用前湿。 使用镊子放到中心的一环斜面结束的硅胶管，轻轻地滑入硅胶管环。所需的里数与重复农工商超市。在两个方向轻轻推他们先后沿管滑入相互接触的戒指。这种方法可用于收获后，只有一个在文化一天的戒指。 一旦环上，对齐内的聚碳酸酯持有硅胶管和螺杆两部分持有人。将在100毫米的培养皿持有人，并添加55毫升媒体。交换媒介文化的持续时间为每3天。 要删除的组织管，释放聚碳酸酯持有人的硅胶管，并使用镊子滑落到培养皿中填充用PBS的硅胶管和组织管。 6。代表性的成果: 正确执行协议时，细胞聚集形成一个内径等于相应的模具后，直径在24小时内组织环。环边缘通常是平滑的外观和（如果cultur在一个水平面上ED），是统一的，在围绕整个圆周的厚度。组织环易于处理，可从随后的机械和组织学分析井（见Gwyther等，2011 年 8）删除。环组织形态，基质成分，性能和机械性能，因种子细胞的数量和类型而定。 组织环代表从不同类型的细胞，播种条件，文化长度的结果如表1所示。两个MM的ID创建环5X10 5 rSMCs 展出 8类似的鼠细胞，人类平滑肌细胞（HSMC）环比从我们先前公布的2毫米戒指（6.6x10 5 rSMCs）创建一个较高的抗拉强度。容易聚合，形成组织环，其中包含大量的胶原蛋白后仅14天文化（数据未显示）。人类间质干细胞（HMSC）汇总，并形成凝聚力的戒指，但LAcked机械强度和发生在单轴拉伸试验的初始precycling阶段。 <table border="1"><tbody><tr><td></td><td> ñ </td><td>细胞数量</td><td>厚度（mm） </td><td>文化的长度（天） </td><td>悉尼科技大学（千帕） </td><td> MTM（千帕） </td><td>失效应变（毫米/毫米） </td></tr><tr><td> RSMC环</td><td> 6 </td><td> 660000 </td><td> 0.94 ± 0.12 </td><td> 14 </td><td> 97 ± 30 </td><td> 497 ± 91 </td><td> 0.50 ± 0.08 </td></tr><tr><td> RSMC环</td><td> 4 </td><td> 500000 </td><td> 0.53 ± 0.02 </td><td> 7 </td><td> 113 ± 8 </td><td> 189 ± 15 </td><td> 0.88 ± 0.05 </td></tr><tr><td> HSMC环</td><td> 3 </td><td> 750000 </td><td> 0.51 ± 0.05 </td><td> 14 </td><td> 160 ± 30 </td><td> 270 ± 20 </td><td> 0.92 ± 0.08 </td></tr><tr><td> HMSC环</td><td> 3 </td><td> 750000 </td><td> 0.40 ± 0.07 </td><td> 14 </td><td> N / A </td><td> N / A </td><td> N / A </td></tr></tbody></table> 表1。表显示的文化参数和从不同的细胞类型，播种浓度，和文化的持续时间产生的2毫米环的机械性能。 <img alt="图1" src="/files/ftp_upload/3366/3366fig1.jpg" /> 图1:（一）组织环生成过程的示意图。 （二）自定义聚碳酸酯模碾磨环形井。中央邮政局直径为2毫米。 （三）PDMS模板后，这是从聚碳酸酯模去皮。 （四）培养琼脂糖模具聚合组织环与一个2毫米后。 （e）两毫米直径的PBS组织环。比例尺= 6毫米（B，C）和2毫米（D，E）。 <img alt="图2" src="/files/ftp_upload/3366/3366fig2.jpg" /> 图2代表积应力-应变曲线机生产线。CED从单轴拉伸试验。

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最近，出现了增加细胞为基础的"脚手架"组织工程方法来解决一些支架为基础的组织工程方法的局限性的利益。由于细胞衍生的组织创建从细胞和它们产生的矩阵，他们本质上包含的细胞密度高得多，不包含外源性材料，可完全从人体细胞和蛋白质。从人类细胞中的血管移植可以达到大量的机械强度，在缺乏外源性支架（例如，爆破压力3400毫米汞柱相比，1600毫米汞柱为人类隐静脉）12。虽然细胞为基础的血管组织表现出提高细胞密度和机械强度，目前大多数的制造方法（如"表为基础工程"3,4,12"bioprinting"5,13）需要广泛的文化时期（&gt; 3个月）或3D组织建设的专门设备。细胞衍生的组织环方法这里所描述的OD能够快速的细胞自组装，形成一个很短的时间时期内并没有使用专门的设备强大的3D组织结构​​。 该协议的细节的过程中，我们开发创建2 mm的内径大鼠平滑肌细胞衍生的组织环。在本例中，组织培养7天（再培养一个额外的7天环融合和管形成）环。然而，2毫米的老鼠细胞种植后的一天早日（人）平滑肌细胞环可以从水井中删除和凝聚力处理（例如，转移到硅胶管）不够。此外，强大的组织环不同内径（2，4和6毫米），可以用这种方法创建通过简单地改变了原来的聚碳酸酯模后直径 8我们最近还修改了聚碳酸酯的模具设计，使5个2毫米播种要在一个单一的多井琼脂糖室的投井，使用较少的硅橡胶和琼脂糖，适合于6孔板（数据未显示）。后直径的变化，以及播种的宽度，曲率半径的圆形底部，播种井，或播种井的深度都可以进行修改，只需通过改变数控CAD文件的规格聚碳酸酯模具的加工。最后，一​​个单一的聚碳酸酯模可用于制造的PDMS模板的数量不受限制，每个PDMS的模板，可清洗，灭菌和重复使用几十次。 除了改变组织环的大小，我们从许多不同类型的细胞环，包括:原代大鼠平滑肌细胞（细胞应用，R354 - 05），主人体冠状动脉平滑肌细胞（龙沙，CC - 2583），首要人权皮肤成纤维细胞11（乔治销博士的慷慨捐赠，WPI的生物医学工程学系），大鼠肺成纤维细胞（RFL - 6，ATCC CCL - 192），和间质干细胞（龙沙，PT-2501）。这些类型的细胞聚集和围绕中心岗位合同形式组织环;虽然不同的细胞类型的细胞组织的ECM成分，和力学性能的构造不同。播种不同的细胞类型参数必须是经验确定，根据细胞的大小和他们的能力，以总。因此，在这个创建细胞衍生的组织环系统是非常灵活的，该协议可能需要轻微调整优化组织形成与不同类型的细胞。 组织环几何促进单轴拉伸试验容易装载和评估组织的材料特性，描述。使用血管环段测量血管收缩和生理功能也有大量的先例。初步研究表明，可在丝肌动描记设备安装测量的药理反应的细胞衍生的组织环和收缩力发电（数据未显示）。总而言之，能够迅速编造的组织学，机械，生理，生化分析自组装细胞环，表明一个强大的新工具，可用于血管的组织结构和在健康和疾病的功能建模。

<table border="1"><tbody><tr><th>试剂/设备名称</th><th>公司</th><th>目录＃ </th></tr><tr><td>聚二甲基硅氧烷（PDMS） </td><td>道康宁</td><td> Sylgard 184 </td></tr><tr><td>琼脂糖</td><td>龙沙</td><td> 50000 </td></tr><tr><td> DMEM培养液</td><td> Mediatech </td><td> 15 - 017 - CV </td></tr><tr><td>胎牛血清（FBS） </td><td>临时机场管理局</td><td> A05 - 201 </td></tr><tr><td>青霉素/链霉素</td><td> Mediatech </td><td> 30 - 002 - CI </td></tr><tr><td>数字成像系统</td><td> DVT的公司</td><td>型号630 </td></tr><tr><td>单轴试验机</td><td>英斯特朗</td><td> ElectroPuls E1000 </td></tr><tr><td>边缘检测软件</td><td> DVT的公司</td><td>框架2.4.6 </td></tr></tbody></table>

directed cellular self-assembly to fabricate cell-derived tissue rings for biomechanical analysis and tissue engineering

每年，数以十万计的患者进行冠状动脉搭桥手术在美国大约这些患者的三分之一不适合自体捐赠者的船只因病情恶化或以前收获。血管组织工程的目标是开发出适合这些旁路移植的替代来源。此外，血管组织工程可能证明是有价值的生活血管模型来研究心血管疾病。已探索工程血管的几个有前途的方法，与许多最近的研究重点发展和基于细胞的方法的分析。2-5此中，我们提出了一个方法来迅速自我组装成三维组织，可用于环细胞体外模型的血管组织。 要做到这一点，平滑肌细胞的悬浮液接种到圆底环形琼脂糖井。琼脂糖非粘接性能允许日Ë细胞定居，周围以及中心的职位总量和合同，形成一个有凝聚力的组织环。6,7这些戒指可以收获的机械，生理，生化，或病理分析前几天的培养。我们已经表明，这些细胞衍生的组织环产量在100-500千帕的极限抗拉强度超过其他组织工程血管培养结构类似的持续时间（&lt;30千帕）报告的值 。9,10我们的研究结果表明，强大的细胞衍生的血管组织环发电可以实现的一个短的时间内内，并提供机会直接和定量评估细胞和细胞衍生矩阵（CDM）血管的组织结构和功能的贡献。

Watch this Scientific Journal Video about Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering at JoVE.com

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

本文概述了一个通用的方法来创建由细胞来源的组织细胞自我组装环。平滑肌细胞接种到环形琼脂糖井总与合同内形成强大的三维（3D）组织的7天。毫米波规模的组织环，有利于力学性能测试，并作为组织大会的组成部分。

定向细胞自我组装制造，为生物力学分析与组织工程细胞衍生的组织环

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

directed-cellular-self-assembly-to-fabricate-cell-derived-tissue

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

18.9K 次观看.  Worcester Polytechnic Institute. 本文概述了一个通用的方法来创建由细胞来源的组织细胞自我组装环。平滑肌细胞接种到环形琼脂糖井总与合同内形成强大的三维（3D）组织的7天。毫米波规模的组织环，有利于力学性能测试，并作为组织大会的组成部分。

视频: Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

电场控制的二维和三维环境中的神经祖细胞定向迁移

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

科研

生物工程

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

一个人的3D组织工程<em&gt;在体外</em&gt;肿瘤测试系统

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

三明治般的微环境来驾驭细胞/材料相互作用

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

温敏纳米结构表面的组织工程准备

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

定向细胞自我组装制造，为生物力学分析与组织工程细胞衍生的组织环

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