宋佳Schrepfer，莱纳德Conradi和Arne汉森收到由德意志研究联合会（DFG; SCHR992/3-1，SCHR992/4-1，二氧化碳858/1-1，医管局3423/3-1）的资金。这项工作是由来自欧盟托马斯Eschenhagen（EUGeneHeart和Angioscaff）的赠款支持。

1。东隧代和培养条件。 制造的纤维蛋白为基础的东隧，其他地方4所描述的。总之，生成纤维蛋白为基础的东隧植入的目的，主结构是包含从新生儿的ROSA /荧光素酶LEW转基因大鼠心脏，牛纤维蛋白原和基质胶细胞编制。琼脂糖压铸模具准备使用特氟隆垫片放置在6孔培养皿。琼脂糖凝固后，垫片被拆除，硅胶柱机架上培养皿放置到模具达到的职位。要生成东隧，mastermix简要地混合与凝血酶的计算量和入模吸管。纤维蛋白原聚合后，坚持的职位EHTs硅架轻轻从琼脂糖铸造模具，转移到新的6孔细胞培养皿中，并保持在37 ° C，7％的CO 2细胞培养箱培养使用定制细胞培养液中。 东隧则留在细胞培养条件下，直到一天十个。在这段时间内，新生鼠心脏细胞开始拉长，互连和硅职位之间沿磁力线对齐。在植入时，自发连贯的收缩进行了观察，偏转硅职位。 2。东隧植入东隧植入或者免疫功能的棕色挪威（BN）大鼠，或免疫缺陷的裸鼠大鼠进行调查的异基因免疫反应。 BN和裸大鼠，体重100 - 150克，均购自德国查尔斯河（Sandhofer WEG 7，D - 97633 Sulzfeld）和常规条件下，饲喂标准鼠议员和水广告libidum内。免疫缺陷的老鼠被安置在无菌床上用品，笼子，并使用无菌水。德国的伦理审查委员会审查和批准了动物的程序。所有手术器械消毒后才能使用。 植入过程如下： 大鼠anesthesized与异氟醚使用感应室（2.5-3％）。在下沉气流表或者非循环罩，可减少人类接触到异氟醚。在手术过程中保持体温。 <ol><li>剃须腹部和应用眼膏，以防止在麻醉过程中干燥的眼睛。 </li><li>将大鼠在它的后面，并置于它的鼻子和嘴口罩，以保持麻醉。 </li><li>使用优碘，然后80％的乙醇消毒腹部。棉签清洁脏，剃光区域中心向外移动走向头发地区。 </li><li>检查反射捏后脚要确保充分麻醉大鼠。 </li><li>垂坠的动物和执行中线腹部切口，分离两个步骤的皮肤和肌肉，打开腹部。用于皮肤和肌肉开放之间的热珠消毒。 </li><li>放置在一个温暖的生理盐水滋润无粉手套的肠子。折叠的肠子周围的手套，以防止水分流失。 </li><li>取出的脂肪组织，暴露脾。 </li><li>可视化和传播更大的钳大网膜。 </li><li>小心剪去东隧暂停硅职位和转移的大网膜上。 </li><li>周围环绕的东隧更大omentus和修复使用两个单7-0普理灵缝线（爱惜康，日本东京，德国） </li><li>下一步行动的肠子放回腹部。 </li><li>与预热的无菌生理盐水冲洗腹部。 </li><li>关闭的墙壁用6-0普理灵运行缝线（爱惜康，日本东京，德国）的腹部肌肉层。 </li><li>使用5-0普理灵（爱惜康，日本东京，德国），运行缝合关闭皮肤。 </li><li>虽然仍然是在麻醉大鼠皮下，注射5毫克/公斤卡洛芬。 </li><li>为了提供这种类型的过程足够的镇痛作用，镇痛药（如安乃近）增加了对疼痛3天移植后用药的饮用水（50毫克每100毫升安乃近）。 </li></ol> 3。东隧以下植入的生物发光成像 IVIS生物成像平台（精诺真，阿拉米达，美国加利福尼亚州）是用于非侵入性活体生物发光成像和使用IVIS生活图片（精诺真）软件包对结果进行分析。成像是每天2个小时，术后开始执行。 麻醉与异氟醚（2.5 - 3％）的大鼠，使用感应室。 <ol><li>消毒腹部和胸部区域广泛使用普罗沃碘，下次使用80％乙醇，重复此步骤三次。 </li><li>注射D -荧光素（精诺真; 375毫克/公斤体重）IP </li><li>麻醉大鼠放入IVIS成像室（可同时分析多达4个大鼠）。 </li><li>评估信号强度的LUC +内东隧细胞在光子/秒/厘米2 / steridianin的利益地区在基准率（ROI）（120分钟植入后）的单位和天1次，2，3，5，7，10，并在周2 ， 3和4。请注意峰值信号（D -荧光素注射后20分钟左右出现）。 </li></ol>创建一个图表（Excel）中显示的信号强度随着时间的推移。 4。劳保局结果的相关性和酶联免疫斑点法东隧移植后的细胞在体内的免疫反应，在免疫功能正常的国民和免疫缺陷的裸鼠大鼠进行了研究。收件人动物脾脏东隧移植后5天收获隔离收件人脾。 ELISPOT检测使用丝裂霉素抑制，刺激和5 × 10 6收件人脾EHTs（Sigma公司Aldrich公司，圣路易斯，密苏里州，美国）作为响应者的细胞，根据制造商的协议（BD Biosciences公司）使用IFN -γ和IL - 4涂板调查TH1，和Th2反应分别。干扰素-γ-和IL - 4点显着的免疫活性，模型相比，免疫缺陷模型（IFN -γ：P = 0.001; IL - 4：P = 0.023;学生的t -检验）。 5。代表性的成果： <img src="/files/ftp_upload/2605/2605fig1.jpg" alt="图1" /> 图1。劳保局结果和酶联免疫斑点法的相关性）在免疫布朗挪威和免疫缺陷的裸鼠大鼠体内的免疫反应的细胞进行研究后，东隧植入。收件人脾东隧植入后5天收获。干扰素？和IL - 4表达ELISPOT作为响应者细胞的刺激和5x106收件人脾使用丝裂霉素抑制东隧的检测评估。 Th1和Th2反应显着高于免疫缺陷模型，IFNγ和IL - 4的产量分别在免疫功能的模型。 B）luc +的EHTs移植到更大的omentus或免疫功能正常BN大鼠或免疫缺陷的裸鼠大鼠。纵向随后由劳保局细胞存活。动物率，拒绝了移植EHTs。所有BN大鼠迅速拒绝了东隧移植的细胞，而T细胞缺陷的老鼠存活。

<ol>
	<li>Eschenhagen, T., Zimmermann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+myocardial+tissue.">Engineering myocardial tissue.</a> Circ Res. 97, 1220-1231 (2005).</li><li>van der Bogt, K. E., Schrepfer, S., Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+transplantation+of+adipose+tissue-+and+bone+marrow-derived+mesenchymal+stem+cells+in+the+infarcted+heart.">Comparison of transplantation of adipose tissue- and bone marrow-derived mesenchymal stem cells in the infarcted heart.</a> Transplantation. 87, 642-652 (2009).</li><li>Hakamata, Y., Murakami, T., Kobayashi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Firefly+rats"+as+an+organ/cellular+source+for+long-term+in+vivo+bioluminescent+imaging.">"Firefly rats" as an organ/cellular source for long-term in vivo bioluminescent imaging.</a> Transplantation. 81, 1179-1184 (2006).</li><li>Hansen, A., Eder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Drug+Screening+Platform+Based+on+Engineered+Heart+Tissue.">Development of a Drug Screening Platform Based on Engineered Heart Tissue.</a> Circ Res. , (2010).</li></ol>

对心肌修复东隧技术的临床实施之前，根本问题，如细 ​​胞和基质成分或移植物存活的免疫原性植入后需要在活体生物发光成像实验模型进行评估。一个有用的新技术监测细胞的存活，移植后。我们成功地生成纤维蛋白为基础的东隧，含有荧光素酶（LUC）一个既定的协议4适应的心脏细胞。这些东隧植入受体鼠的大网膜。腹腔注射的特异底物（D -荧光素），产生的信号被检测到使...

<ul><li> LEW - TG（GT（ROSA）26Sor - LUC）11Jmsk大鼠（NBPR大鼠号码0299，http://www.anim.med.kyoto-u.ac.jp/NBR/） </li><li>牛纤维蛋白原（原液：200毫克/毫升加抑肽酶5微克/毫升纤维蛋白原在NaCl 0.9％，Sigma公司F4753） </li><li>基质胶（BD生物科学356235） </li><li> DMEM（F0415 Biochrome） </li><li>马血清（Gibco公司26050） </li><li>青霉素/链霉素（GIBCO 15140） </li><li>胰岛素（Σ- Aldrich公司I9278） </li><li>抑肽酶（西格玛爱秩序A1153） </li><li> D -荧光素（Biosynth L - 8220） </li><li>酶联免疫斑点（ELISPOT）检测板（Hassa Laborbedarf，S2EM004M99） </li></ul>

bioluminescence imaging for assessment of immune responses following implantation of engineered heart tissue (eht)

心脏组织工程的各种技术已经在过去几十年，包括使用本机或生物人工肝的支架材料，如纤维蛋白或胶原蛋白凝胶心肌细胞塌陷1心肌细胞单层堆放的脚手架战略追求。这些概念的目的是恢复心脏功能受损（如心肌梗死后），或作为实验模型（如预测毒理学和药物筛选或疾病建模）。精确监测细胞工程心脏组织（东隧）植入后的生存现在已经成为可能使用在体内的生物发光成像技术（BLI） 2。在这里，我们描述的纤维蛋白为基础的东隧从无处不在表达萤火虫荧光素酶（罗萨/荧光素酶LEW TG; 3）的转基因鼠应变的一代。植入的到不同的大鼠株的大网膜，以评估东隧植入受体生物体的免疫反应。 BLI和酶的酶联免疫斑点技术（ELISPOT）确认的评估免疫反应的可用性，BLI产生的结果比较。

Watch this Scientific Journal Video about Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT at JoVE.com

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

这个视频演示的使用<em&gt;在体内</em&gt;生物发光成像研究植入后大鼠的心脏组织工程（东隧）的免疫反应。

生物发光成像评估的免疫反应，植入心脏组织工程（东隧）

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

bioluminescence-imaging-for-assessment-immune-responses-following

Research

JoVE Journal

Bioengineering

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

9.6K 次观看.  University Heart Center Hamburg. 这个视频演示的使用在体内生物发光成像研究植入后大鼠的心脏组织工程（东隧）的免疫反应。

视频: Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

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

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

协议相对液力评估的三单张聚合物阀

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

手术技术组织工程血管移植和后续的植入<em&gt;在体内</em&gt;监控

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

组织工程血管在小鼠模型的生成和嫁接

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

极化神经组织工程三维蚕丝胶原蛋白为基础的模式

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

人工程心脏组织的收缩自动分析心脏药物安全筛选

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Custom Engineered Tissue Culture Molds from Laser-etched Masters

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue shape. Manipulating tissue shape on the micrometer to centimeter scale can direct cell alignment, alter effective mechanical properties, and address limitations related to nutrient diffusion. In addition, the vessel in which a tissue is prepared can impart mechanical constraints on the tissue, resulting in stress fields that can further influence both the cell and matrix structure. Shaped tissues with highly reproducible dimensions also have utility for in vitro assays in which sample dimensions are critical, such as whole tissue mechanical analysis.
This manuscript describes an alternative fabrication method utilizing negative master molds prepared from laser etched acrylic: these molds perform well with polydimethylsiloxane (PDMS), permit designs with dimensions on the centimeter scale and feature sizes smaller than 25 &#181;m, and can be rapidly designed and fabricated at a low cost and with minimal expertise. The minimal time and cost requirements allow for laser etched molds to be rapidly iterated upon until an optimal design is determined, and to be easily adapted to suit any assay of interest, including those beyond the field of tissue engineering.

Herein we present a rapid, facile, and low-cost method for fabricating custom polydimethylsiloxane molds that can be used for producing hydrogel-based engineered tissues with complex geometries. We additionally describe results from mechanical and histological assessments conducted on engineered cardiac tissues produced using this technique.

由激光蚀刻大师定制的组织培养模具

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

小鼠模型中生物合成组织工程气管移植的育苗与植入

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...