（BMM）的生物医学材料研究所的研究方案进行了这项研究。 BMM是由荷兰经济事务，农业和创新部共同资助。感谢购买！Hartstichting的财务贡献。

1。文化人类腔小隐细胞<ol><li>隔离腔小隐蚤，根据二次使用材料的指引从供体获得，根据协议等施耐尔9细胞，并储存在液氮中。从2×2毫米在6孔板培养一个捐助者的裁片的部分小隐静脉蚤。使用2枚，每口井。一般来说足够的细胞可以得到以填补约3瓶0.25×10 6个细胞在液氮中。的HVSCs的特点是肌纤维母细胞，显示波形蛋白，不表达结蛋白和亚群表达α平滑肌肌动蛋白10的表达。下一次启动的协议从液氮中解冻的细胞，细胞的数量增加。 </li><li>成的T75组织培养瓶，将细胞从一个小瓶，加入生长培养基（GM），包括先进的DMEM培养液，5毫升，50毫升胎牛血清（FBS），青霉素/链霉素和5毫升L-的glutamax的。更改介质每隔2-3天。 </li><li>细胞生长汇合后约14天。存储0.5×10 6细胞在一个小瓶在液氮中，简称为通道1。 </li></ol> 注：冻结的细胞是没有必要的收获时HVSCs，但仅用于存储。 <ol start="4"><li>从一小瓶将HVSCs，成T175组织培养瓶，并添加GM。更改介质每隔2-3天。细胞生长汇合后约7天。商店3×10 6细胞在液氮中在一个小瓶中，简称为通道2。 </li><li>将细胞从一个小瓶到rollerbottle中添加GM。更改介质每隔2-3天。细胞生长汇合后约8天。一个rollerbottle包含约20-30×10 6细胞。培养细胞通过6。不要使用高于9代细胞的一个通道，因为可能会改变细胞的表型。 </li></o升&gt; 2。工程纤维蛋白为基础的组织结构<ol><li>准备有机硅胶与固化剂（10:1）混合的弹性体。切7×3毫米的矩形段的魔术贴。胶魔术贴到6孔培养板中，以形成交叉，并且留下为3毫米的方形空间的维可牢尼龙搭扣带之间的柔性膜。 </li></ol> 注：仅使用软边魔术贴面对这一侧向上。魔术贴粘合时，只覆盖魔术贴，硅胶胶水，不扩散以及整个胶。由于文化板块有硅膜的底部，使用的东西孔板下方增强柔性膜，以确保易于胶合板。 <ol start="2"><li>聚硅氧烷胶在60℃的烘箱中过夜干燥，以确保胶的硬化。孔中并孵育30分钟，加入70％乙醇消毒。用PBS冲洗3次，并删除所有PBS从WELLS和魔术贴。认在紫外光下30分钟，并保持无菌状态，直到使用。 </li><li>准备组织工程介质（商标），由转基因和130毫克的L-抗坏血酸-2 - 磷酸。 </li><li>添加1毫克/毫升的ε-氨基己酸（ACA）的TM。 ACA是用于文化的第7天，以防止纤维蛋白降解11。或者，可以使用抑肽酶。 </li><li>为了防止丛形成，使纤维蛋白原开幕前达到室温。无团块纤维蛋白原会更容易溶解。溶解纤维蛋白原的浓度为10 mg蛋白/ ml实际TM 7辅以ACA。无菌过滤纤维蛋白原的解决方案，可能需要多个过滤器。贮存，直到用冰。 </li></ol> 注：溶解纤维蛋白原，轻轻混匀，以防止过 ​​多的泡沫形成。 <ol start="6"><li>凝血酶溶解的浓度为10国际单位/毫升TM 7辅以ACA。贮存，直到用冰。 </li> &lt;/ OL&gt; 注意：在冰上存储需要，以防止早期凝胶化，凝血酶和纤维蛋白原。 <ol start="7"><li>胰蛋白酶消化细胞，重悬在通用和计数。 </li><li>使用15×10 6个细胞/ ml，用于播种的血纤维蛋白为基础的组织结构（浓度根据用于组织工程心脏瓣膜7的方法）。包括一个单一的凝胶100微升GOF埃尔，从而为1.5×10 6细胞。将1.5×10 6个细胞在一个离心管中，在尽可能多的离心管中，将要进行的凝胶数量。 </li><li>离心机的细胞在350×g离心7分钟，弃上清。细胞重悬在50μl的凝血酶。 4微升的蓝色荧光聚苯乙烯微球添加这种悬挂。将50微升纤维蛋白原在一个小瓶。用吸管取50微升细胞凝血酶。增加至100μl的体积移液管。移取50微升凝血酶溶液细胞瓶中混合的凝血酶和纤维蛋白原与纤维蛋白原，需要100微升的混合物。 </li></ol> 注意 ：荧光聚苯乙烯微球用作内部参考标记，用于图像分析。当混合凝血酶和纤维蛋白原，防止形成气泡，通过仔细地移液混合物。气泡将导致在纤维蛋白凝胶中的孔。 <ol start="10"><li>移液器凝胶混合物和Velcro带之间。为血纤维蛋白凝胶的典型的胶凝时间，一旦组件混合，20秒的顺序。 </li></ol> 注 ：这样做尽可能快地防止凝胶混合物滴在孔板前。练习使用前细胞和珠。 <ol start="11"><li>在湿润的95％空气/ 5％CO 2培养箱中在37℃下孵育30分钟的悬浮液允许凝胶化之前添加的培养TM与ACA。 </li><li>与ACA的第7天，每2-3天更换TM。凝胶是不够稳定，一个星期后要培养无ACA。第一个星期后更换TM每隔2-3天。加入6毫升TM每口井。第12天细胞产生足够的胶原蛋白的可视化。 </li></ol> 3。应用应变及诱导变化的应变和约束<ol><li>静态应变施加直接的细胞，由于细胞的牵引力和压实12。要诱导胶原蛋白重组，削减组织构建在一个方向从两个尼龙搭扣松动。这将导致在单向约束（ 图1A）。执行第12天，因为结构是不够稳定，胶原蛋白已存入。 </li><li>应用循环应变与柔性膜的培养板的底部通过施加真空，与使用FLEXCELL的FX4000T系统。板放置在基板上，顶部装载职位（其中的一部分，日ê循环荷载装置）。该泵适用于真空到膜上，从而拉膜在矩形后，躺在下面。由于组织十字形附件构造的膜（通过魔术贴）和矩形柱的施加的循环应变，是单轴晶体的（ 图1B）。 </li><li>最初，使用静置培养5天，以实现最初的机械完整性，在第6天开始之前应用循环应变。程序一个环状污垢协议到控制器的真空泵。例如，使用先前建立的间歇循环应变协议，与单轴方向13。由间歇性的菌株，用1赫兹的正弦波，，使劲从0到5％的应变，3小时的期间，交替进行3小时的休息时间。执行此循环应变协议，7天，诱导一个对准胶原组织。 </li><li>通常生产后12天HVSCs对准胶原蛋白的工作单位名词达到一个对齐的胶原蛋白组织后，更改的单轴循环应变方向，是垂直于原始菌株方向。做到这一点，把矩形职位90°。 </li></ol> 4。可视化的细胞与胶原<ol><li>为了形象化活跃，实时胶原蛋白的重塑，标签样本的探针不干扰细胞活力或胶原蛋白的形成。使用探针的荧光染色的细胞的细胞质和胶原蛋白。 </li><li>或者二次谐波的产生，使用共聚焦激光扫描显微镜可用于可视化的胶原蛋白的结构14，自体荧光，激发波长为780纳米和500-550纳米之间的检测。 </li><li>从安装和孵化器中取出培养板中，在3小时的休息期间的循环应变的样品，并将它们传送到共焦激光扫描显微镜的可视化细胞和胶原。 </li></ol>

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作者宣称，他们有没有竞争经济利益。

描述的模型系统研究细胞和胶原细胞填充的纤维蛋白结构具有很大的潜力（重新）组织（德容格等15），如用于组织工程的目的。通过使用作为初始细胞载体的血纤维蛋白，纤维蛋白降解后，组织细胞和内源性矩阵只创建。以这种方式，细胞受到刺激进行反应的应变，无论是静态的或环状的性质，通过施加收缩力16,17，检测边界12，刚度或显示菌株避免的。 <p class...

心血管组织有一个突出的承载功能。在特定的内容和组织中的细胞外基质的胶原纤维向承重性能和支配整体组织强度1。在组织工程机械空调的构造被使用-通常包括（循环）使劲方案是-以增强组织的组织和机械性能2,3。充分的了解应变诱导胶原蛋白组织创建与预定义的胶原蛋白结构的组织的复合组织的几何形状还没有被实现。这主要是由于我们有限的知识，在发展组织的胶原蛋白的重塑。现有的车型主要使用静态应变4-6胶原重塑，最终净结果信息。在这里，我们提供了一个高度可调的模型系统，它允许在一个实时的方式，在三维胶原蛋白（重）组织的研究，受到影响的情况下的静态或环状的应变。组织结构的纤维蛋白为主，确保所有构造中的胶原蛋白是内源性的。在构建体的细胞和胶原组织的可视化，和一个内部参考系统使我们能够移居时间推移分析细胞和胶原结构。在这个协议中，我们将描述人类腔小隐的细胞（HVSCs），因为这些细胞被称为增强细胞外基质生产和改造能力在工程心血管组织7矩阵和我们既定的用途，根据使用的模型系统的容格等[8]的工作

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Culture plastic</td>
			<td>Greiner</td>
			<td></td>
			<td>Includes culture flasks and pipettes</td>
		</tr>
		<tr>
			<td>Advanced DMEM</td>
			<td>Gibco</td>
			<td>12491</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Greiner</td>
			<td>758075</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Gibco</td>
			<td>35050-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastomer and curing agent</td>
			<td>Dow Corning Corporation</td>
			<td>3097358-1004</td>
			<td>Silastic MDX 4-4210#</td>
		</tr>
		<tr>
			<td>Velcro</td>
			<td>Regular store</td>
			<td></td>
			<td>You can buy this at a regular store, only use the soft side</td>
		</tr>
		<tr>
			<td>Bioflex culture plates</td>
			<td>Flexcell Int</td>
			<td>BF-3001U</td>
			<td>Untreated</td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid 2-phosphatase</td>
			<td>Sigma</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>&#949;-Amino Caproic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>D7754</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine thrombin</td>
			<td>Sigma</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine fibrinogen</td>
			<td>Sigma</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 syringe filter</td>
			<td>Whatmann (Schleicher and Scheul)</td>
			<td>10462100</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene microspheres</td>
			<td>Invitrogen</td>
			<td>F-8829</td>
			<td>Blue fluorescent, 10 &#956;m diameter</td>
		</tr>
		<tr>
			<td>Flexcell FX-4000T</td>
			<td>Flexcell Int</td>
			<td></td>
			<td>Includes rectangular loading posts</td>
		</tr>
		<tr>
			<td>Cell Tracker Orange</td>
			<td>Invitrogen Molecular Probes</td>
			<td>C2927</td>
			<td></td>
		</tr>
		<tr>
			<td>CNA35-OG488</td>
			<td></td>
			<td></td>
			<td>Cordially provided by the Laboratory for Macromolecular and Organic Chemistry, Department of Biomedical Engineering, Eindhoven University of Technology</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>LSM 510 Meta laser scanning microscope and Two-Photon-LSM mode</td>
		</tr>
		<tr>
			<td>Amphotericin</td>
			<td>Gibco</td>
			<td>15290-018</td>
			<td>Needed for cell isolation</td>
		</tr>
	</tbody>
</table>

engineering fibrin-based tissue constructs from myofibroblasts and application of constraints and strain to induce cell and collagen reorganization

在开发胶原组织的胶原蛋白含量和组织可以影响局部组织的菌株和组织约束。组织工程师的目标是利用这些原则来创建与预定义的胶原蛋白结构组织。胶原重构，以控制最终的组织结构的确切相关的过程的充分理解，但是，是缺乏的。特别是（重新）胶原纤维的方向响应在组织机械负荷条件的变化知之甚少。我们开发了一个体外模型系统，包括双向约束肌成纤维细胞的种子纤维蛋白结构，进一步阐明胶原蛋白（重）响应我的方向）恢复双轴单轴静载荷条件及ii）循环单轴载荷双向约束在装载方向，使用FLEXCELL FX4000T装载装置之前和之后的变化的构造。时间推移共聚焦成像到visualize胶原蛋白（重新）方向，一个非破坏性的方式。 细胞和胶原组织中的构造可以进行实时可视化，和一个内部参考系统使我们能够移居时间推移分析细胞和胶原结构。可以调整的模型系统的各个方面，如健康和患病细胞的细胞来源或使用。可以使用添加剂，以进一步阐明相关机制胶原重构，例如，通过添加蛋白酶或阻断整合。该构建体的形状和大小可以很容易地适应特定需要的，从而导致在一个高度可调的模型系统来研究细胞和胶原蛋白组织（重）。

这个模型系统允许种子培养成肌纤维细胞纤维蛋白凝胶。 图1A显示了一个组织首先培养静态双轴约束下的。组织的约束通过切割纤维蛋白凝胶从两个约束被释放，以创建单轴静态约束，和组织压块和重塑之后（ 图1A）。对于循环应变，组织培养以及静态双轴约束下。 5天之后，可以应用于周期性单轴拉伸（ 图1B）。为了诱导胶原蛋白的重新取向，单轴应变的?...

Watch this Scientific Journal Video about Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization at JoVE.co

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

这个模型系统开始从填充的成肌纤维细胞的血纤维蛋白凝胶，可用于研究内源性的胶原蛋白（重）非破坏性的方式组织实时。系统的模型是非常可调谐的，因为它可以用不同的细胞来源，培养基添加剂，可以容易地适应具体需要。

肌成纤维细胞和约束的应用工程纤维蛋白的​​组织结构和应变诱导细胞和胶原蛋白（RE）组织

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

engineering-fibrin-based-tissue-constructs-from-myofibroblasts

Research

JoVE Journal

Bioengineering

10.8K 次观看.  Eindhoven University of Technology. 这个模型系统开始从填充的成肌纤维细胞的血纤维蛋白凝胶，可用于研究内源性的胶原蛋白（重）非破坏性的方式组织实时。系统的模型是非常可调谐的，因为它可以用不同的细胞来源，培养基添加剂，可以容易地适应具体需要。 

视频: Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

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

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

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 ...

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, ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

工程骨骼肌组织中的小鼠成肌细胞的内皮祖细胞与应用电刺激

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

肿瘤微环境基质与应用癌细胞侵袭组织工程

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

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. ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

用运动调节血清治疗韧带结构：转化组织工程模型

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

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.

重组胶原蛋白 i. 肽载体在生物反应器中的应用及其作为细胞传递系统的潜力

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

一种新型的腱绦缝合技术，采用组织工程胶原蛋白移植物修复大肌腱缺损

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

用于打印 3D 细胞-拉丹胰腺组织构造的胰腺组织衍生细胞外基质生物墨水

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

肌成纤维细胞和约束的应用工程纤维蛋白的​​组织结构和应变诱导细胞和胶原蛋白（RE）组织

肌成纤维细胞和约束的应用工程纤维蛋白的组织结构和应变诱导细胞和胶原蛋白（RE）组织