笔者想承认"诱导多能干细胞的行为控制的组织工程支架材料"NSERC发现格兰特402462。

开始之前协议的注释:纤维蛋白原是血源性蛋白，从而适当的安全训练之前，必须处理完成。此协议需要2天才能完成所需的干文化，所以时间适当，以确保它们的播种准备。在计算多少纤维蛋白原权衡，三个35毫米培养皿菜TRIS缓冲液（TBS，pH值7.4），含有110-130毫克的纤维蛋白原溶解于3毫升的TBS将足以产生1 24孔板含400在每口井的微升纤维支架。 1。第一天:纤维蛋白原的解决方案和隔夜透析<ol><li>冻干纤维蛋白原的冰箱，让它坐了20分钟，以允许它来室温。 </li><li>加入3毫升的Tris缓冲液（TBS）每35毫米培养皿。每个板的纤维蛋白原产量2至3毫升的纤维蛋白原溶液浓度从12到20mg / mL和纤维蛋白原总额可以根据这些近似计算。 </li><li>称取约100-130毫克纤维蛋白原（Fg）和TBS电视台目前表面上洒在每一道菜。等待5分钟的纤维蛋白原开始进入解决方案。盖上盖培养皿。 </li><li>孵育培养皿菜在37°C的2个小时，使纤维蛋白原完全进入TBS的解决方案。 </li><li>湿透析管（7000兆瓦切断），用TBS。透析钳倍底端油管钳年底关闭。 </li><li>吸取纤维蛋白原的解决方案，从菜肴到透析管。倍多高端和透析钳钳关闭。 </li><li>成的TBS 4公升的容器中放置透析管含有纤维蛋白原的解决方案。混合溶液搅拌盘设置为低转速（100转）。 </li><li>隔夜透析溶液，搅拌至少12小时板。 TBS的解决方案并不需要通过这段时间内改变。 </li></OL&gt; 2。第2天:聚合纤维蛋白支架和干细胞播种到支架这个过程中描述的所有步骤应执行这些支架，将干细胞培养接种在无菌组织培养引擎盖。 <ol><li>删除到一个适当大小的锥形管（15毫升或50毫升）从透析管和地方的纤维蛋白原的解决方案。 </li><li> 5.0微米的注射器过滤除去溶液中大量杂质的过滤纤维蛋白原的解决方案。根据解决方案的体积，使用多个过滤器可能需要处理的整个解决方案。 </li><li>无菌过滤器的过滤用0.22微米到50毫升锥形管的纤维蛋白原的解决方案。使用适当的大小吸管，确定过滤纤维蛋白原的解决方案，以备后用做计算时的音量。根据解决方案的体积，可能需要使用多个过滤器处理耳鼻喉科愤怒的解决方案。 </li><li>测量用分光光度计在280 nm的纤维蛋白原溶液的吸光度。一个50的稀释因素往往是必要的情况下取得1吸光度。 </li><li>纤维蛋白原解决方案，使用下列公式计算:[毫克/毫升血浆纤维蛋白原浓度的蛋白质目前] =（280×稀释因子）÷1.55，其中1.55是在一个280人纤维蛋白原13消光系数。接下来计算所需的TBS稀释浓度为11.1毫克/毫升的初始音量根据纤维蛋白原的解决方案。终浓度的蛋白质纤维蛋白支架将聚合后的10毫克/毫升。 </li><li>获取无菌凝血酶和氯化钙解决方案。首先，添加15μL的凝血酶溶液（浓度为40 U / ml的 - 在脚手架终浓度将是2个U / mL）的右侧第一排的24孔板中，每口井。接下来，添加15μL50毫米钙CHLoride解决方案，以每口井的左侧。最后，添加270μL的纤维蛋白原溶液的板块。摇板从侧面一边摇晃后到前的解决方案，以确保混合。 </li><li>让行包含聚合混合物在室温下5分钟。棚架变得更加不透明，为他们聚合。 </li><li>重复步骤2.6和2.7，直到所需数量的纤维蛋白支架已聚合。 </li><li>最后5分钟的潜伏期后，放在里面的37℃培养箱板，棚架，以便完全聚合。 </li><li>经过一个小时的潜伏期是完整的，下一步就是种子胚体的脚手架。使用20μLpipettemen，选择个别胚体中的纤维蛋白支架的中心地点。用显微镜检查，每口井包含一个单一的胚体。 </li><li>添加5μL凝血酶溶液（浓度为40 U / ml的）和5μL50毫米氯化钙纤维蛋白原的解决方案，以每孔90μL每个胚体顶部的解决方案。该解决方案应形成泡沫封装胚体。 </li><li>现浇板早在37°Ç孵化器增加一个小时，以确保聚合。 </li><li>孵化后，合适的细胞培养基添加1毫升，每口井的地方板成37°C间孵化器。 </li></ol> 3。代表结果 图1显示了个人以及包含的三维纤维支架文化系统种植胚体。 图2A侧视示意图显示鼠标诱导多能干细胞培养小鼠胚胎成纤维细胞饲养层的代表图像。然后，这些细胞被诱导形成胚体，含有神经祖细胞的集合体，用8天的维甲酸治疗协议（ 图2B）previouslŸ描述14，而图3显示的IPS衍生的胚体经过3天的文化内的三维纤维蛋白支架的外观。已取得了类似的结果，以前用小鼠胚胎干细胞7。此外，这种方法已被用于文化IPS-衍生的胚体制作使用不同的协议，涉及维甲酸和purmorphamine的15三维纤维支架内，展示这个文化系统的多功能性。 <img alt="图1。" src="/files/ftp_upload/3641/3641fig1.jpg" /> 图1。示意图显示的个人以及三维纤维支架种子含胚体的侧视图。 <img alt="图2。" src="/files/ftp_upload/3641/3641fig2.jpg" /> 图2。小鼠诱导多能干细胞（iPS）细胞培养和分化 。这些细胞分化成neur人的表型，iPS细胞悬浮培养产生细胞的集合体称为胚体（EBS）。这些胚，然后用维甲酸诱导神经细胞分化和本议定书以前用来诱导胚胎干细胞的神经分化。一）未分化的小鼠iPS细胞集落培养对小鼠胚胎成纤维细胞饲养层。 b）从小鼠iPS细胞的悬浮培养的胚体维甲酸治疗后第8天，诱导神经细胞分化。比例尺是100微米。 <img alt="图3。" src="/files/ftp_upload/3641/3641fig3.jpg" /> 图3示例结果的小鼠iPS胚状体的三维纤维支架内经过3天的文化机构。细胞迁移和分化的纤维蛋白支架内已经开始。比例尺是500微米。

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作者没有透露。

上面详细介绍该协议产生的多能干细胞培养小鼠胚胎和诱导多能干细胞的三维纤维支架，专门提供了一个方法。这三维生物材料为基础的文化系统，更准确地模仿体内发现的干细胞小生境，作为一个结果，它可以用来筛选生物的线索，以确定对干细胞分化6的影响。我们的观察表明，干胚体细胞在体外培养2周保持完全降解之前，播种时，这些支架。为了进一步提高这些支架的稳定，...

所需设备: 天平 pH计 组织文化的孵化器（37℃，5％CO 2） 搅拌板 分光光度计 无菌组织培养罩 Tris缓冲液（pH值7.4）（需要4升，加上​​足够的溶解纤维蛋白原） 50毫米的氯化钙溶液 无菌锥形管（15或50毫升） 35毫米培养皿 透析管（7,000兆瓦截止） 透析剪辑 5.0微米的针头式过滤器 单独包装的无菌0.22微米针头式过滤器 注射器 24孔组织培养板<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>纤维蛋白原（人） </td><td> calbioc下摆</td><td> 341578 </td><td></td></tr><tr><td>凝血酶（人） </td><td>西格玛</td><td> T7009 </td><td></td></tr></tbody></table>

preparation of 3d fibrin scaffolds for stem cell culture applications

干细胞被发现在三维微环境中自然产生的体内 ，它通常被称为干细胞小生境1。三维生物材料支架内培养干细胞提供了一种方法，准确地模仿这些微环境，提供文化，用聚苯乙烯以及更换工程的组织2的方法比传统的2D方法的优势。虽然已为大多数细胞培养实验中使用的二维组织培养聚苯乙烯，三维生物材料支架可以更密切地复制在体内环境中，使更准确的细胞极性建立和拥有生化和机械性能类似于软组织的微环境。 3一个多种自然派生和合成生物材料支架已被用于支持干细胞生长的3D环境调查。虽然合成支架材料可以合成有一个更大的ŕ安格机械和化学性能，往往有更大的重复性，通常由天然生物​​材料在细胞外基质含有细胞粘附的结合位点，并随时支持细胞培养的结果发现蛋白质和多糖。纤维蛋白支架，通过聚合获得的血浆蛋白纤维蛋白原生产，已被广泛研究了各种组织工程的应用， 在体外和体内 4。这种支架可以修改使用多种方法纳入控释系统提供治疗的因素5。以前的工作表明，这种支架可以用来成功地培养胚胎干细胞，并基于此脚手架的文化系统，可用于筛选内6,7种子的干细胞分化的各种生长因子的影响。 该协议的细节过程polymerizinĞ纤维支架纤维蛋白原使用凝血酶酶活性的解决方案。这个过程需要2天才能完成，包括纤维蛋白原的解决方案，以消除，抑制聚合的柠檬酸隔夜透析的步骤。这些详细的方法依赖于胚胎和诱导多能干细胞培养确定为最佳的纤维蛋白原浓度。其他团体作进一步调查的细胞类型和应用范围广泛的纤维蛋白支架-证明了这种方法的8-12的多功能性。

Watch this Scientific Journal Video about Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications at JoVE.com

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

这项工作的具体编制三维纤维支架plutipotent干细胞培养和分化。这种支架可用于筛选各种生物化合物对干细胞的行为的影响，以及修改为包含药物输送系统。

制备纤维蛋白三维支架干细胞培养应用

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

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25.1K 次观看.  University of Victoria. 这项工作的具体编制三维纤维支架plutipotent干细胞培养和分化。这种支架可用于筛选各种生物化合物对干细胞的行为的影响，以及修改为包含药物输送系统。

视频: Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

静电纤维组织工程的后期处理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

科研

生物工程

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

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

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 ...

Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ECM also participates in cell migration, proliferation, and differentiation. Fibroblasts are the main type of cells modifying ECM fiber arrangement and production. In cancer, CAFs (cancer associated fibroblasts) are in permanent activation status, participating in ECM remodeling, facilitating tumor cell migration, and stimulating tumor-associated angiogenesis, among other pro-tumorigenic roles. The objective of this method is to create a three-dimensional matrix with a fiber composition that is similar to in vivo matrices, using immortalized fibroblasts or human primary CAFs. Fibroblasts are cultured in pre-treated cell culture plates and grown under ascorbic acid stimulation. Then, fibroblasts are removed and matrices are blocked for further cell seeding. In this ECM model, fibroblasts can be activated or modified to generate different kinds of matrix, whose effects can be studied in cell culture. 3D matrices are also shaped by cell signals, like degradation or cross-linking enzymes that might modify fiber distribution. In this context, angiogenesis can be studied, along with other cell types such as epithelial tumor cells.

The aim of this method is to obtain fibroblast-derived 3D matrices as a natural scaffold for subsequent cellular assays. Fibroblasts are seeded in a pre-treated culture plate and stimulated with ascorbic acid for matrix generation. Matrices are decellularized and blocked to culture relevant cells (e.g., endothelial cells).

纤维细胞衍生的三维矩阵系统适用于内皮管形成测定

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ...

癌症研究

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

组织工程:建设多细胞三维支架的层状细胞片交货

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

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

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

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

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

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

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

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials induce an inflammatory response upon implantation, which is the driver of subsequent resorption of the material and the regeneration of new tissue. This strategy, also known as in situ tissue engineering, is pursued to obtain cardiovascular replacements such as tissue-engineered vascular grafts. Both the inflammatory and the regenerative processes are determined by the local biomechanical cues on the scaffold (i.e., stretch and shear stress). Here, we describe in detail the use of a custom-developed bioreactor that uniquely enables the decoupling of stretch and shear stress on a tubular scaffold. This allows for the systematic and standardized evaluation of the inflammatory and regenerative capacity of tubular scaffolds under the influence of well-controlled mechanical loads, which we demonstrate on the basis of a dynamic co-culture experiment using human macrophages and myofibroblasts. The key practical steps in this approach&#8212;the construction and setting up of the bioreactor, preparation of the scaffolds and cell seeding, application and maintenance of stretch and shear flow, and sample harvesting for analysis&#8212;are discussed in detail.

The goal of this protocol is to execute a dynamic co-culture of human macrophages and myofibroblasts in tubular electrospun scaffolds to investigate material-driven tissue regeneration, using a bioreactor which enables the decoupling of shear stress and cyclic stretch.

评估流和拉伸下生物材料的炎症和再生能力的多奎生物反应器

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

工程学

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

逐步在泰瑟尔脚手架上播种，研究发芽血管

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, ...

Preclinical Drug Testing in Scalable 3D Engineered Muscle Tissues

Accurately modeling healthy and disease conditions in vitro is vital for the development of new treatment strategies and therapeutics. For cardiac and skeletal muscle diseases, contractile force and kinetics constitute key metrics for assessing muscle function. New and improved methods for generating engineered muscle tissues (EMTs) from induced pluripotent stem cells have made in vitro disease modeling more reliable for contractile tissues; however, reproducibly fabricating tissues from suspended cell cultures and measuring their contractility is challenging. Such techniques are often plagued with high failure rates and require complex instrumentation and customized data analysis routines. A new platform and device that utilizes 3D EMTs in conjunction with a label-free, highly-parallel, and automation-friendly contractility assay circumvent many of these obstacles. The platform enables facile and reproducible fabrication of 3D EMTs using virtually any cell source. Tissue contractility is then measured via an instrument that simultaneously measures 24 tissues without the need for complex software analysis routines. The instrument can reliably measure micronewton changes in force, allowing for dose-dependent compound screening to measure the effect of a drug or therapeutic on contractile output. Engineered tissues made with this device are fully functional, generating twitch and tetanic contractions upon electrical stimulation, and can be analyzed longitudinally in culture over weeks or months. Here, we show data from cardiac muscle EMTs under acute and chronic dosing with known toxicants, including a drug (BMS-986094) that was pulled from clinical trials after patient fatalities due to unanticipated cardiotoxicity. Altered skeletal muscle function in engineered tissues in response to treatment with a myosin inhibitor is also presented. This platform enables the researcher to integrate complex, information-rich bioengineered model systems into their drug discovery workflow with minimal additional training or skills required.

This protocol provides methods for generating 3D engineered cardiac and skeletal muscle tissues and describes their use in preclinical drug screening modalities. The described methods utilize a magnetic sensing system to facilitate the simultaneous assessment of 24 tissues in parallel.

可扩展的3D工程肌肉组织中的临床前药物测试

Accurately modeling healthy and disease conditions in vitro is vital for the development of new treatment strategies and therapeutics. For cardiac and ...

3D Human Myocardial Tissue Generation Using Melt Electrospinning Writing of Polycaprolactone Scaffolds and hiPSC-Derived Cardiac Cells

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and regenerative medicine. This protocol describes the stepwise fabrication of 3D myocardial tissues with advanced mimicry of native cardiac structure by combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds with fibrin hydrogels and human induced pluripotent stem cell (hiPSC)-derived cardiac cells. The process involves embedding a mixture of cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (hiPSC-CFs) within a fibrin matrix to create mini-tissues, with structural support provided by MEW-generated scaffolds. These fibrillar scaffolds are fabricated at the micro- to nanoscale, allowing for precise control over fiber architecture, which plays a key role in organizing cell distribution and alignment. Meanwhile, the fibrin matrix promotes cell viability and mimics the extracellular environment. Characterization of the generated tissues reveals well-organized sarcomeres within hiPSC-CMs, along with stable contractile activity. The tissues demonstrate consistent spontaneous beating as early as two days post-seeding, with sustained functionality over time. The combination of hiPSC-CFs with hiPSC-CMs enhances the structural integrity of the tissues while supporting long-term cell viability. This approach offers a reproducible, adaptable, and scalable method for creating biomimetic cardiac tissue models, providing a versatile platform for preclinical drug testing, mechanistic studies of cardiac disease, and potential regenerative therapies.

A reproducible method is presented to generate 3D myocardial tissues combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds and fibrin hydrogels with hiPSC-derived cardiomyocytes and fibroblasts. This technique offers precise control over scaffold architecture and can be applied in preclinical drug testing and cardiac disease modeling.

制备纤维蛋白三维支架干细胞培养应用

摘要

探索更多视频

此视频中的章节

静电纤维组织工程的后期处理

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

纤维细胞衍生的三维矩阵系统适用于内皮管形成测定

组织工程:建设多细胞三维支架的层状细胞片交货

适应静电过程中提供三个独特的环境为一个三层次

评估流和拉伸下生物材料的炎症和再生能力的多奎生物反应器

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

逐步在泰瑟尔脚手架上播种，研究发芽血管

可扩展的3D工程肌肉组织中的临床前药物测试

使用聚己内酯支架和 hiPSC 衍生的心肌细胞的熔融静电纺丝写入生成 3D 人体心肌组织

制备纤维蛋白三维支架干细胞培养应用

摘要

探索更多视频

此视频中的章节

静电纤维组织工程的后期处理

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

纤维细胞衍生的三维矩阵系统适用于内皮管形成测定

组织工程:建设多细胞三维支架的层状细胞片交货

适应静电过程中提供三个独特的环境为一个三层次

评估流和拉伸下生物材料的炎症和再生能力的多奎生物反应器

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

逐步在泰瑟尔脚手架上播种，研究发芽血管

可扩展的3D工程肌肉组织中的临床前药物测试

使用聚己内酯支架和 hiPSC 衍生的心肌细胞的熔融静电纺丝写入生成 3D 人体心肌组织

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