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

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

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

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

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

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

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

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

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

定向细胞自我组装制造，为生物力学分析与组织工程细胞衍生的组织环

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

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

Research

JoVE Journal

Bioengineering

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

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

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the importance of 3-dimensional (3D) cellular assemblies in these fields. Artificial scaffolds have often been used to construct 3D cellular assemblies. However, the scaffolds used to construct cellular assemblies are sometimes toxic and may change the properties of the cells. Thus, it would be beneficial to establish a non-toxic method for facilitating cell-cell contact. In this paper, we introduce a novel method for constructing stable cellular assemblies by using optical tweezers with dextran. One of the advantages of this method is that it establishes stable cell-to-cell contact within a few minutes. This new method allows the construction of 3D cellular assemblies in a natural hydrophilic polymer and is expected to be useful for constructing next-generation 3D single-cell assemblies in the fields of regenerative medicine and tissue engineering.

We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.

操纵活细胞构建稳定的3D 蜂窝组件, 无需人工支架

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

科研

生物化学

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 ...

生物工程

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. ...

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

自动点胶机器人技术在细胞表面和指导生物印刷

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

工程血管移植缩放使用3D打印指南和环堆叠方法

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

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 ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

用 3 d-印刷模具制作细胞播种和组织环自组装用的自定义琼脂糖井

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Controlling Electrospun Polymer Morphology for Tissue Engineering Demonstrated Using hepG2 Cell Line

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of electrospun polymers is regarded as a structural imitation of the extracellular matrix (ECM). Hence, electrospun fibers fabricated from biocompatible polymers have been widely investigated by tissue engineering researchers for their potential role as an artificial ECM for guiding tissue growth both in vitro and in vivo. All cells are acutely sensitive to their mechanical environment. This has been demonstrated by the discovery of multiple mechanotransduction pathways intrinsically linked to the cytoskeletal actin filaments. The cytoskeleton acts as a mechanical sensor that can direct the functionality and differentiation of the host cell depending on the stiffness and morphology of its substrate. Electrospun fibers can be tuned both in terms of fiber size and morphology to easily modulate the mechanical environment within a fibrous polymer scaffold. Here, methods for electrospinning polycaprolactone (PCL) for three distinct morphologies at two different fiber diameters are described. The morphological fiber categories consist of randomly oriented fibers, aligned fibers, and porous cryogenically spun fibers, with 1 &#181;m and 5 &#181;m diameters. The methods detailed within this study are proposed as a platform for investigating the effect of electrospun fiber architecture on tissue generation. Understanding these effects will allow researchers to optimize the mechanical properties of electrospun fibers and demonstrate the potential of this technology more thoroughly.

This method provides the means to test different polycaprolactone fiber morphologies and topographies for the purpose of tissue engineering. Small and large fibers are fabricated with random orientations, aligned orientations, and also porous cryogenically electrospun structures and used as platforms for cell culture.

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of ...

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

使用DNA对细胞进行简单、经济、模块化的图案化

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

成纤维细胞衍生的人类工程结缔组织用于筛查应用

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...