要感谢亚斌吴培养的组织图2给出了，照片是由巴特面包车Overbeeke。 SenterNovem，授予ISO 42022的工作的财政支持。

1。小鼠成肌细胞的祖细胞或C2C12细胞培养<ol><li>隔离细胞根据最初由谢费尔和他的同事10出版和购买适于由Collins 等人 11和博南等12的协议，并在液氮中存储这些。这需要小鼠， 如之C57B1 / 6。在其他实验室所使用的替代方法， 例如李瑛等人发表在杂志的可视化实验的方法13。对于试剂和设备，被称为第7和第8页上的表。从一个鼠标的肌肉，一般情况下，你会得到足够的细胞冷冻保存在液氮中约20瓶。下一步，我们启动协议解冻的细胞从液氮储存。 </li><li>细胞来自1小瓶放置到一个25cm 2的基质胶（1毫克/毫升）涂覆的组织培养烧瓶中，并加入生长培养基（GM）的组成的（335毫升DMEM培养液，100毫升胎儿鲍文先进E无血清（FBS），50毫升HS，5 ml青霉素/链霉素，5毫升L-谷氨酰胺，5毫升鸡胚提取物（CEE））。 </li></ol> 注：涂层在室温2小时，取出Matrigel的愿望。 <ol start="3"><li>继代培养的细胞约为1:3，每3天。 这意味着：在第3天的通道：一个25厘米2至75cm 2的烧瓶中，在第6天：传代细胞的细胞从一个75cm 2的烧瓶中的两个150厘米2培养瓶中（预镀在未涂覆的烧瓶中，用30分钟）。天9转印1150厘米2烧瓶1三重150厘米2烧瓶中，或者如果不可用三个150厘米2烧瓶（如果需要的话preplate再次取决于细胞的表型）。第12天的细胞是成肌肉结构的播种做好准备。 </li></ol> 注： -每三重150厘米2的细胞数，将约4.5×10 6细胞。 - 使用通过LS从不同的隔离，将它们混合播种时，以获得混合的细胞群。 注：如果您选择不与原代细胞，C2C12成肌细胞是一个很好的选择。 2。工程骨骼肌组织<ol><li>准备硅胶水混合的“弹性体”与“固化剂”（10:1）。切+ / - 5毫米见方的段维可牢尼龙搭扣，与一个三角形侧（房子的形状， 图1）。胶入井的6孔培养板中，在约12毫米之间的正方形的空间的维可牢尼龙搭扣。 </li></ol> 注： -仅使用软边的魔术贴，面对这一边向上。 - 确保面对对方的屋顶。 - 仅覆盖魔术贴，不扩散硅胶胶在整个井。 - 对于购买电刺激，它是相关的对准结构，在垂直方向上的阱复数吃（沿长轴方向）。 <ol start="2"><li>发布在真空烘箱中干燥过夜，不加热，主要以除去气泡。由孔中并孵育15分钟加入70％乙醇消毒。用PBS冲洗3倍，并在紫外光下15分钟。卸下所有PBS井和魔术贴和地方的孵化器，直到使用。 </li><li>解冻Matrigel的溶液在冰箱和不久之前作出的3D结构制备的胶原蛋白溶液稀释至所需浓度。用无菌的0.02％的乙酸（最终浓度为3.2毫克/毫升）稀释股票胶原。 </li></ol> 注：把一切都放在冰上。 <ol start="4"><li> Trypsinize细胞，重悬在通用汽车和计数。留在孵化器的离心管中的细胞。 </li><li>加入所需量的胶原蛋白原液你想的管（根据表1）的构造的数字，这种胶原溶液中添加0.5M的NaOH直到浅粉结肠r是表示的pH值为7.5。通过移液向上和向下轻轻混匀，并避免形成气泡。然后加入基质胶和非常轻柔但彻底混合。最后，加入GM到胶原/ Matrigel的混合物的（相应的金额见表1）。 </li></ol> 注： -在冰上进行每一步！基底膜和胶原蛋白很容易在温度升高凝胶。 - 介意的颜色确实是粉红色的！ pH值的增加也将导致快速的凝胶。 - 胶原蛋白的最终浓度：1.6毫克/毫升。 <ol start="6"><li>离心所需量的细胞以1,000 rpm离心5分钟，并除去上清液。 </li></ol> 注： -根据每构建的细胞的数量需要被调整的细胞的活性。通常情况下，750,000和1,250,000之间的细胞数在于细胞每构造。 <ol start="7"><li>使用一些凝胶混合物重悬细胞沉淀和细胞转移到剩余的混合物调匀，但不引入气泡。 </li><li>预热孔板的孵化器和吸管0.35 -0.4毫升的细胞凝胶混合成的维可牢尼龙搭扣第一。然后，开始吸取试剂的混合物从附着位点之间的中心并延伸的维可牢尼龙搭扣。最后，使用剩余的凝胶，两者之间的维可牢尼龙搭扣的锚和移液器各地的维可牢尼龙搭扣件，填补了国内空白。 </li><li>如果仔细检查后5-10分钟的凝胶是足够牢固，能够被转移， 即轻轻拍打菜肴和视觉检查，如果凝胶是刚性的。如果是这样，小心地将菜肴的孵化器。通常，它们可以被拾起后10分钟。然后，1-2小时后，轻轻地覆盖每一个凝胶4毫升的温暖GM。 </li></ol> 注：处理板时，不要剧烈运动。 <ol start="10"><li>更换GM分化培养基（生长培养基没有鸡胚提取物）后24小时，并更换新鲜培养基，每2-3天。在第7天，你应该已获得成熟导向的肌纤维，可以评价用染色横纹发展中的稠合的肌纤维。 </li></ol> 3。电刺激<ol><li>消毒电极从ionoptix板（见表的试剂和设备），在使用前用70％乙醇，随后干燥​​的紫外光照射下在安全柜。放置到培养板与构造的电极板，并从培养板和转移到孵化器的盖覆盖。用适当的电缆连接Ionoptix C-佩瑟。 </li></ol> 注：旁边的肌肉构造（ 图2）平行放置电极。 <ol start="2"><li>应用协议如预期的刺激。 </li></ol> 注：我们一般使用4 V / CM，6ms的脉冲在频率2Hz。改变培养基刺激过程中，每24小时。

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作者有没有透露。

肌肉组织工程具有巨大的潜力作为一种疾病​​模型，药物筛选，在再生医学中的使用和肉类生产。然而，这些应用程序的要求有所不同。我们选择与胶原蛋白和基质胶的组合，因为胶原蛋白可以用于细胞取向和成肌细胞前体细胞，因为需要基底膜衍生的蛋白质的存在下，如在之前的2D研究12确定。此外，纤维蛋白凝胶已在我们的实验室测试，似乎不支持C2C12肌母细胞的祖细胞，胶原蛋白?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> Matrigel的生长因子减少</td><td> Beckton和Dickinson </td><td></td><td></td></tr><tr><td> DMEM（高糖）* </td><td> Gibco公司</td><td> 42430 </td><td></td></tr><tr><td>先进的DMEM </td><td> Gibco公司</td><td> 12491 </td><td></td></tr><tr><td>马血清</td><td> Gibco公司</td><td> 65050-122 </td><td></td></tr><tr><td>胎牛血清</td><td>格雷纳</td><td> 758075 </td><td></td></tr><tr><td> 0.45微米和0.22微米针头式过滤器</td><td> Whatmann（Schleicher和Scheull） </td><td> 10462100 </td><td></td></tr><tr><td> L-谷氨酰胺</td><td> Gibco公司</td><td> 25030024 </td><td></td></tr><tR&gt; <td>青霉素/链霉素</td><td> Gibco公司</td><td> 10378016 </td><td></td></tr><tr><td>两性霉素</td><td> Gibco公司</td><td> 15290-018 </td><td></td></tr><tr><td>文化塑胶</td><td>格雷纳</td><td></td><td>包括培养瓶和吸液管</td></tr><tr><td>鸡胚提取物</td><td>美国生物</td><td> C3999 </td><td></td></tr><tr><td>巴斯德吸管* </td><td> Hilgenberg </td><td></td><td>巴氏吸管，收缩，与棉，顶端开口L：230毫米，尖端直径0.9 - 1.1毫米</td></tr><tr><td>巴斯德吸管* </td><td> Hilgenberg </td><td></td><td>巴氏吸管，收缩，与棉，顶端开口L：尖端直径230毫米，1,4 - 1,6毫米</td></tr><tr><td>巴斯德吸管</td><td> VWR </td><td> 612-1702 </td><td></td></tr><tr><td> I型胶原酶* </td><td>西格玛</td><td> C0130-16 </td><td></td></tr><tr><td> 40微米的细胞过滤* </td><td> BD猎鹰</td><td> 352340 </td><td></td></tr><tr><td> 19G针</td><td></td><td></td><td></td></tr><tr><td>弹性体</td><td>道康宁公司</td><td> 3097358-1004 </td><td>硅橡胶MDX 4-4210＃ </td></tr><tr><td>固化剂</td><td>道康宁公司</td><td></td><td>硅橡胶MDX 4-4210＃ </td></tr><tr><td>尼龙搭扣</td><td>正规商店</td><td></td><td>您可以在正规的商店购买，只能使用柔软的一面</td></tr><tr><td> Ⅰ型胶原，鼠尾</td><td> BD Biosciences公司</td><td> 3544236 </td><td></td></tr><tr><td> C-吴佩慈EP文化步行者</td><td> Ionoptix </td><td></td><td></td></tr><tr><td> 6孔培养板，电刺激</td><td>卡特莱特kton迪金森猎鹰</td><td> BD猎鹰＃353846 </td><td></td></tr><tr><td> C-DISH培养皿电极</td><td> Ionoptix </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> *所需要的隔离的细胞（1.1） ＃在同一个工具包</td></tr></tbody></table>

engineering skeletal muscle tissues from murine myoblast progenitor cells and application of electrical stimulation

工程的肌肉组织，可用于几个不同的用途，包括用于生产的组织在体外作为一种疾病模型， 如学习压力性溃疡，再生医学和肉类替代1。首次报道的3D肌肉结构已作了很多年前在该领域的先驱，是Vandenburgh和他的同事2,3。在肌肉组织工程取得的进展不仅是广大的生化因子，干细胞和祖细胞知识增益的结果，但研究人员所获得的洞察力，特别是基于物理因素发挥重要作用，在控制细胞的行为，组织的发展。国家的最先进的工程肌肉构造目前包括人口细胞的水凝胶结构。在我们的实验室中，这些通常由鼠成肌细胞的祖细胞，分离出小鼠后肢肌肉或小鼠成肌细胞系C2C12，MIXED与胶原/基底膜的混合物，并镀两个锚固点之间，模仿肌肉韧带。其他细胞可能被视为良好， 例如 ，替代的细胞系，例如L6大鼠成肌细胞4，新生儿肌源性祖细胞5，来自成年肌肉组织从其他物种如人类6甚至诱导式多能性干细胞（iPS细胞）7的细胞。细胞收缩引起的细胞的取向沿长轴的构建体8,9和文化约一个星期后的肌肉祖细胞的分化。此外，应用程序可以增强电刺激分化的过程中，在一定程度上8。由于其有限的尺寸（8×2×0.5 mm）的完整的组织可以使用共焦显微镜分析，监视例如生存能力，分化和细胞对准。根据特定的应用要求的enginee红色的肌肉组织会有所不同， 例如用于再生医学需要向上缩放的组织大小和血管，而作为一个肉替代的词条到其他物种是必要的。

最终产品将是肌肉如在图3中所示的构造。组织的大小将是约8毫米长，2毫米宽，0.5毫米厚。在分化过程中的电刺激将改变的表达，肌球蛋白重链异构体，但不大大提高的分化的方法，诱导分化培养基8，但也可以应用于电刺激可以肌肉的功能的过程结束时，判断是否，因为将能够充分​​发展的肌节的肌肉收缩时的电脉冲。 分化和成熟的也可以使用定量PCR来分析?...

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

工程肌肉组织再生医学具有巨大的潜力，作为疾病模型，也可作为肉类的替代来源。在这里，我们描述的工程的肌肉构造，在这种情况下，从小鼠成肌细胞的祖细胞，通过电脉冲刺激。

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 ...

engineering-skeletal-muscle-tissues-from-murine-myoblast-progenitor

Research

JoVE Journal

Bioengineering

20.8K 次观看.  Eindhoven University of Technology, The Netherlands. 工程肌肉组织再生医学具有巨大的潜力，作为疾病模型，也可作为肉类的替代来源。在这里，我们描述的工程的肌肉构造，在这种情况下，从小鼠成肌细胞的祖细胞，通过电脉冲刺激。

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

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

微组织使用胶原蛋白的模块的制备凝胶含有细胞

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

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 ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

保利的静电纤维支架（甘油 - 十二双）工程神经组织从小鼠胚胎干细胞

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

最大等长收缩力的测量通过透骨骼肌纤维生成

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living components. Due to their inherent advantages, biorobots are gaining interest as alternatives to traditional fully artificial robots. Various studies have focused on harnessing the power of biological actuators, but only recently studies have quantitatively characterized the performance of biorobots and studied their geometry to enhance functionality and efficiency. Here, we demonstrate the development of a self-stabilizing swimming biorobot that can maintain its pitch, depth, and roll without external intervention. The design and fabrication of the PDMS scaffold for the biological actuator and biorobot followed by the functionalization with fibronectin is described in this first part. In the second part of this two-part article, we detail the incorporation of cardiomyocytes and characterize the biological actuator and biorobot function. Both incorporate a base and tail (cantilever) which produce fin-based propulsion. The tail is constructed with soft lithography techniques using PDMS and laser engraving. After incorporating the tail with the device base, it is functionalized with a cell adhesive protein and seeded confluently with cardiomyocytes. The base of the biological actuator consists of a solid PDMS block with a central glass bead (acts as a weight). The base of the biorobot consists of two composite PDMS materials, Ni-PDMS and microballoon-PDMS (MB-PDMS). The nickel powder (in Ni-PDMS) allows magnetic control of the biorobot during cells seeding and stability during locomotion. Microballoons (in MB-PDMS) decrease the density of MB-PDMS, and enable the biorobot to float and swim steadily. The use of these two materials with different mass densities, enabled precise control over the weight distribution to ensure a positive restoration force at any angle of the biorobot. This technique produces a magnetically controlled self-stabilizing swimming biorobot.

In this two-part study, a biological actuator was developed using highly flexible polydimethylsiloxane (PDMS) cantilevers and living muscle cells (cardiomyocytes), and characterized. The biological actuator was incorporated with a base made of modified PDMS materials to build a self-stabilizing, swimming biorobot.

心肌肌细胞基致动器和自稳定Biorobot  - 第1部分

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living ...

Cardiac Muscle Cell-based Actuator and Self-stabilizing Biorobot - Part 2

In recent years, hybrid devices that consist of a living cell or tissue component integrated with a synthetic mechanical backbone have been developed. These devices, called biorobots, are powered solely by the force generated from the contractile activity of the living component and, due to their many inherent advantages, could be an alternative to conventional fully artificial robots. Here, we describe the methods to seed and characterize a biological actuator and a biorobot that was designed, fabricated, and functionalized in the first part of this two-part article. Fabricated biological actuator and biorobot devices composed of a polydimethylsiloxane (PDMS) base and a thin film cantilever were functionalized for cell attachment with fibronectin. Following functionalization, neonatal rat cardiomyocytes were seeded onto the PDMS cantilever arm at a high density, resulting in a confluent cell sheet. The devices were imaged every day and the movement of the cantilever arms was analyzed. On the second day after seeding, we observed the bending of the cantilever arms due to the forces exerted by the cells during spontaneous contractions. Upon quantitative analysis of the cantilever bending, a gradual increase in the surface stress exerted by the cells as they matured over time was observed. Likewise, we observed movement of the biorobot due to the actuation of the PDMS cantilever arm, which acted as a fin. Upon quantification of the swimming profiles of the devices, various propulsion modes were observed, which were influenced by the resting angle of the fin. The direction of motion and the beating frequency were also determined by the resting angle of the fin, and a maximum swim velocity of 142 &#181;m/s was observed. In this manuscript, we describe the procedure for populating the fabricated devices with cardiomyocytes, as well as for the assessment of the biological actuator and biorobot activity.

In this study, a biological actuator and a self-stabilizing, swimming biorobot with functionalized elastomeric cantilever arms are seeded with cardiomyocytes, cultured, and characterized for their biochemical and biomechanical properties over time.

心肌肌细胞基致动器和自稳定Biorobot  - 第2部分

In recent years, hybrid devices that consist of a living cell or tissue component integrated with a synthetic mechanical backbone have been developed. ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

微流控生物工程血管化组织和细胞器解体

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

组织工程应用中植物组织去细胞的两种方法

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells&#039; Cardiomyogenic Potential

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

同时进行机电刺激增强细胞心肌成功能的潜能

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...