D. H. Kim thanks the Department of Bioengineering at the University of Washington for the new faculty startup fund. D. H. Kim is also supported by the Perkins Coie Award for Discovery, the Wallace H. Coulter Foundation Translational Research Partnership Award, the Washington State Life Science Discovery Fund, and the American Heart Association Scientist Development Grant (13SDG14560076). J. Macadangdang and A. Jiao thank the support from the NIH Bioengineering Cardiovascular Training Grant Fellowship. &#160;Additional support for this work comes from the National Institutes of Health (NIH) grant R01HL111197 to M. Regnier.

所有过程都是在室温（〜23℃）下进行，除非另有说明。 1，硅制作的大师<ol><li>清洁硅晶片下的O 2 / N 2气的100％乙醇或二甲苯和干燥。 </li><li>将硅片在旋涂机在2,000-4,000转的转速，产生0.3-0.5微米厚的薄膜。 </li><li>通过使用光刻系统模式与正确的尺寸的光致抗蚀剂膜</li><li>完全浸入所述图案化光致抗蚀剂涂覆硅晶片中的光致抗蚀剂显影溶液的适当体积。 </li><li>冲洗显影光致抗蚀剂涂覆的硅晶片，用去离子水。 </li><li>以形成亚微米尺度的脊与接近垂直的侧壁，深反应离子蚀刻使用蚀刻系统中暴露的硅的阵列。 </li><li>通过将硅晶片等离子灰系统中移除剩余的光刻胶。 </li><li>切硅瓦特afers用金刚石尖的刀插入适当大小的硅大师为后续副本成型。 </li></ol> 2，从硅谷大师制作的PUA模具的注意：要添加到主硅纳米加工量将取决于nanopatterned师傅被复制，以及聚氨酯丙烯酸酯（PUA）的溶液的粘度的地区而异。 <ol><li>清洁硅主表面用100％乙醇或下的O 2 / N 2气二甲苯和干燥。 </li><li>放置硅主图案的一面朝上在培养皿中。 </li><li>对于一个硅主用2厘米×2厘米的表面图案，移液管加入40μlPUA的到图案表面。 </li><li>放置4厘米×4厘米的透明聚酯（PET）薄膜在分配PUA片。 </li><li>向下按PET片材和使用辊或边缘平坦的表面（如卡），以便传播PUA整个图案面片材下方的整个图案覆盖的PUA预聚物。 </li><li>将长约10厘米低于20瓦（115伏）UV光（λ= 365 nm）的硅高手，预聚物和PET为50秒。为了有效，紫外光波长可以310-400纳米之间的任何地方。的光的强度为10-15毫瓦/厘米2在基板的表面上。 </li><li>固化后，用血管钳取出PET薄膜慢。 PUA应该附加到PET膜与硅主纳米图案的负极。 </li><li>治愈PUA / PET奈米紫外光下的，使用前至少12小时。过度曝光是不是一个问题。 </li><li>清洁硅的主人，将PET的另一部影片在主的顶部不加PUA和暴露于UV光（λ= 365 nm）的为50秒，除去PET膜。这将除去未反应的单体。 </li><li>冲洗硅师傅用100％乙醇或二甲苯和干下的O 2 / N 2气体。 </li></ol> 3A。纳米图案聚氨酯聚合物</P&gt; <ol><li>通过放置在臭氧处理室10分钟准备直径为25 mm的圆形玻璃片。 </li><li>将臭氧处理过的玻片上小的PDMS块，方便搬运。 </li><li>用画笔来载玻片涂上薄薄一层表面附着力促进剂。空气干燥的载玻片30分钟。 </li><li>放置在一张打印纸的载玻片上。 </li><li>免除下降10微升聚氨酯（PU）预聚物（NOA 76）到载玻片的中心。确保没有气泡存在添加后。 </li><li>地方PUA模具，图案面朝下，在玻璃上滑动。分散的聚氨酯均匀地通过轧制沿PUA模具的橡胶辊筒的载玻片的表面上。打印机纸张会吸收聚合物溢出。 </li><li>瓦的紫外线灯。聚氨酯的聚合反应时间是依赖于紫外光源的功率。 </li><li>从UV光源取下样品和PU涂层载玻片上小心剥离的PUA复合模具。聚氨酯的聚合考虑中complete当PUA模具剥离干净地远离样品和PU载玻片具有虹彩外观。 </li><li>将成品样品干燥器中储存长达一个月。 </li></ol>图3b。纳米图案聚（乳酸 - 乙醇酸）水凝胶<ol><li>大力混炼硅橡胶基地和有机硅弹性体固化剂的比例为10:1创建一个平面的PDMS模具。 </li><li>倒混合的PDMS前体的溶液注入到培养皿中，以使PDMS前体达到5毫米高的盘的边缘（ 即 ，以使PDMS为5毫米厚）。 </li><li>放置培养皿和PDMS前体在干燥器中进行1小时脱气。 </li><li>移动培养皿和PDMS前体到一个65℃的烘箱中至少2小时固化。 </li><li> PDMS固化后，用剃刀切平的PDMS成3厘米×3厘米见方的部分。这些将在后面的图案形成方法中使用的平坦PDMS模具。 </li><li>干净的直径为25 mm的圆形玻璃片由PL约占新交在异丙醇中超声波仪水30分钟。 </li><li>根据O 2 / N 2气体干燥清洁玻片。 </li><li>滴分配100μl的PLGA溶液（15％w / v的PLGA的氯仿溶液）到载玻片上。 </li><li>放置在所分配的PLGA的顶部平坦PDMS模具以吸收溶剂，将获得一个平坦的PLGA表面。通过在PDMS 5分钟的顶部放置200克的重量施加轻微压力（〜10千帕）。 </li><li>慢慢剥离平坦PDMS模具和放置在预热的板（120℃）的玻璃盖5分钟，以除去残留的溶剂和增加PLGA和盖玻璃之间的粘合。 </li><li>将NP PUA模具上的平面的PLGA的顶部，并通过对PUA模具的顶部放置1千克的重量施加恒定的压力（〜100千帕），并加热（120℃），同时在加热板上15分钟。 </li><li>从PLGA盖玻片除去重量，并允许基质冷却至室温。请勿移除PUA模具，直到基质已冷却。 </li><li>一旦基质充分冷却，小心地撕下NP PUA模具，揭示了NP PLGA基质。对NP PLGA基质应该有一个闪光的外观。 </li><li>将成品样品干燥器中储存长达一个月。 </li></ol> 4，细胞种植与文化注：本协议描述可用于新生大鼠心室肌细胞（NRVMs）和H7人类胚胎干细胞衍生的心肌细胞（胚胎干细胞-CMS），但其他细胞来源的文化。 <ol><li>装上ANFS盖玻片（PU或PLGA）为35 mm组织培养聚苯乙烯菜。吸取20μL诺兰光学胶（NOA83H）到培养皿的底部，轻轻将ANFS盖玻片上的NOA的顶部。让胶水散开并覆盖整个盖玻片底部。治愈NOA暴露菜紫外线10分钟。 </li><li>消毒ANFS通过用2ml的5分钟，70％含水乙醇溶液漂洗两次。删除Ë通过THANOL愿望。让ANFS彻底风干〜1下的紫外线杀菌灯（λ= 200-290纳米）在生物安全柜小时。 </li><li>细胞黏附是由隔夜涂ANFS中纤维连接蛋白增强。在去离子水稀释至纤连蛋白5微克/毫升。移液管加入2ml纤维连接蛋白溶液放入盘中。放置在培养箱中在37℃和5％CO 2中过夜（至少6小时）。 </li><li>获得NRVMs，人类胚胎干细胞的CM，或其他感兴趣的心肌细胞，根据先前的协议22。 </li><li>在1,000 rpm离心3分钟，以沉淀细胞离心细胞样品。 </li><li>通过抽吸小心去除上清。确保不打扰颗粒。 </li><li>重悬的细胞在适当的培养基，以4.6×10 6细胞/ ml的浓度。 </li><li>小心吸取200微升细胞悬液到消毒ANFS。确保细胞悬液保持在盖玻片。 </li><li>在37℃和5％的细胞放置在孵化器CO 2 4小时，以允许细胞附着到ANFS。 </li><li>加入2毫升温培养基的相同的条件下，以菜和替换细胞培养箱中培养。 </li><li> 24小时后，取出介质并用2ml的DPBS洗两次，以除去多余的细胞。 </li><li>加入2毫升温培养基的相同的条件下，以菜和替换细胞培养箱中培养。培养细胞至汇合。更换媒体隔日。 </li></ol>

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K., Schmuki, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanosize+and+Vitality:+TiO+2Nanotube+Diameter+Directs+Cell+Fate.">Nanosize and Vitality: TiO 2Nanotube Diameter Directs Cell Fate.</a> Nano Lett. 7, 1686-1691 (2007).</li><li>Entcheva, E., Bien, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+optical+mapping+of+excitation+in+cardiac+cell+networks+with+ultra-high+spatiotemporal+resolution.">Macroscopic optical mapping of excitation in cardiac cell networks with ultra-high spatiotemporal resolution.</a> Progress in Biophysics and Molecular Biology. 92, 232-257 (2006).</li><li>Tung, L., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+arrhythmias+in+tissue+culture.">Optical imaging of arrhythmias in tissue culture.</a> Journal of Electrocardiology. 39, (2006).</li><li>Himel, H. D., Bub, G., Lakireddy, P., El-Sherif, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+arrhythmias+in+the+cardiomyocyte+monolayer.">Optical imaging of arrhythmias in the cardiomyocyte monolayer.</a> Heart Rhythm. 9, 2077-2082 (2012).</li><li>Kim, J., Hayward, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mimicking+dynamic+in+vivo+environments+with+stimuli-responsive+materials+for+cell+culture.">Mimicking dynamic in vivo environments with stimuli-responsive materials for cell culture.</a> Trends in Biotechnology. 30, 426-439 (2012).</li><li>Henderson, D. J., Anderson, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Development+and+Structure+of+the+Ventricles+in+the+Human+Heart.">The Development and Structure of the Ventricles in the Human Heart.</a> Pediatr Cardiol. 30, 588-596 (2009).</li><li>Badie, N., Bursac, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Micropatterned+Cardiac+Cell+Cultures+with+Realistic+Ventricular+Microstructure.">Novel Micropatterned Cardiac Cell Cultures with Realistic Ventricular Microstructure.</a> Biophysj. 96, 3873-3885 (2009).</li><li>Badrossamay, M. R., McIlwee, H. A., Goss, J. A., Parker, K. 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Authors have nothing to disclose.

功能上成熟的心脏组织都在体内和心脏组织工程的体外应用缺乏供。这里所描述的紧凑型荧光灯的纳米加工方法是健壮的技术可以实现蜂窝对准与影响宏观组织的功能，由于该系统的可伸缩性。大区域可以很容易地被图案化，并用于细胞培养。宏观蜂窝对准是在心脏组织工程必要的，以便建立仿生的，因为它影响着心肌38的机械和电气性能的功能组织。 ?...

心血管疾病是发病率和死亡率在世界上领先的原因，并提出对已经紧张的全球卫生系统1,17一个沉重的社会经济负担。心脏组织工程有两个不同的目标：（1）缺血性疾病或心肌病后的再生受损心肌或（2）创建的心脏进行体外药物筛选或疾病模型的高保真度模型。 心脏是一个复杂的器官，必须不断地努力提供血液到身体。心肌细胞和组织的支持密密麻麻的层状结构在整个心脏壁18,19排列成螺旋状图案。心脏是机电也以高度协调的方式有效地排出血液输送到全身21加上20。几个主要障碍仍有待解决，但是，之前巧夺天工的设计，能够可靠体外概括。首先，尽管强劲的心肌细胞分化的方法继续发展22，HPSC-CMS仍然表现出相当不成熟的表现型。其机电性能和形态最接近胎儿的水平23。其次，当保持在传统的培养条件下，无论是干细胞衍生和初级心肌不能组装成原生，组织样结构。相反，细胞变得随机取向，并没有表现出成人的心肌24的带状杆形外观。 细胞外基质（ECM）的环境中与细胞相互作用中起着许多细胞过程11,13,25显著作用。细胞外基质组成的复杂的，定义良好的分子和地形线索显著影响细胞6,26的结构和功能。内部的心脏，细胞排列紧密遵循的基本纳米尺度的ECM纤维2。这些nanotopograph的影响对细胞和组织的功能iCal的线索，然而，还远远没有完全理解。纳米尺度的细胞的生物材料相互作用的初步研究表明，亚微米，沟槽宽度为细胞信号27，粘附28-30，生长31和分化32,33的潜在重要性和影响。然而，由于在显影重现的和可扩展的nanofabricated基板的难度，这样的研究不能再现复杂的体内细胞外基质环境中的多尺度的细胞效应。在这个协议中，一个简单和具有成本效益的纳米加工技术生产的细胞培养支架模仿原生心脏细胞外基质纤维排列进行了描述，从而为广泛的心肌生物材料相互作用的新调查。了解心肌细胞与细胞外基质的纳米级环境如何相互作用可能允许控制细胞行为更加紧密地模仿天然组织功能的能力化。此外，单层细胞是一种简化的实验系统相比，三维结构，但仍然表现出对有见地的调查和功能性筛选2,34-36复杂的多细胞行为。最后，这样的支架可以用于改善细胞的移植物功能时进行再生的目的37植入到心脏。

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			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">500g Weights</td>
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			<td class="xl65" style="width:66pt" width="88">PX1835-2</td>
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			<td class="xl65" height="22" style="height:16.5pt;width:66pt" width="88">Hot Plate</td>
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			<td class="xl65" style="width:66pt" width="88">PC-420D</td>
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			<td class="xl65" height="22" style="height:16.5pt;width:66pt" width="88">Sonicator</td>
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			<td class="xl65" style="width:66pt" width="88">B2510MTH</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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capillary force lithography for cardiac tissue engineering

心血管疾病仍然是全世界死亡1的首要原因。心脏组织工程拥有许多承诺能够提供突破性的医学发现与开发用于心脏再生和体外筛选分析功能组织的目标。但是，要创建心脏组织的高保真模型的能力已经证明，很难。心脏的细胞外基质（ECM）是一种复杂的结构，包括生物化学和生物力学信号从微至纳米尺度2。局部机械负载条件和细胞-细胞外基质相互作用的最近被确认为在心脏组织工程3-5关键部件。 心脏ECM的很大一部分是由排列的胶原纤维与纳米级直径的显著影响组织结构和机电耦合2。不幸的是，很少有甲肝病毒方法Ë能够模仿的ECM纤维的组织下来到纳米尺度。在纳米材料制备技术的最新发展，但是，已经启用了模仿的心脏6-9 ECM的体内结构和基底刚度线索可伸缩支架的设计和制造。 这里，我们提出的发展2再现的，具有成本效益且可扩展的纳米图案的过程，使用生物相容的聚合物的聚（丙交酯-共-乙交酯）（PLGA）8和聚氨酯（PU）基聚合物心肌细胞的功能对应。这些各向异性nanofabricated基质（ANFS）模仿的良好组织，对准组织相关的ECM，并且可以用于研究纳米形貌对细胞的形态和功能10-14中的作用。 使用nanopatterned（NP）硅原型为模板，聚氨酯丙烯酸酯（PUA）的模具制造。这PUA模具，然后用PA通过紫外线辅助或溶剂介导的毛细管力光刻（CFL），分别为15,16 ttern聚氨酯或PLGA水凝胶。简要地，PU或PLGA的预聚物是滴分配到一个玻璃盖玻片和PUA模具被放置在顶部。用于UV辅助CFL，拾音器，然后暴露于UV辐射（λ= 250-400纳米）进行固化。对于溶剂介导的节能灯相比，PLGA是用热（120℃）和压力（100千帕）压花。固化后，将模具中的PUA被剥离，留下一个ANFS用于细胞培养。主细胞，如新生大鼠心室肌细胞，以及人多能干细胞衍生的心肌细胞，可以维持在ANFS 2。

图1是在生产过程的两个制造方法的示意图。由于由纳米级形貌光的衍射，纳米图案应导致虹彩表面到ANFS 图2描述了这种虹彩表面上形成良好的图案25毫米NP-PU盖玻片（图2A）与800nm ​​的脊和槽宽度（图2B）。该ANFS的彩虹外观会有所不同略有不同的脊和沟槽宽度。 接种细胞到ANFS后，细胞应该开始在与基板的凸条平行的方向上...

Watch this Scientific Journal Video about Capillary Force Lithography for Cardiac Tissue Engineering at JoVE.com

Capillary Force Lithography for Cardiac Tissue Engineering

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

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JoVE Journal

Bioengineering

12.3K 次观看.  University of Washington. In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

视频: Capillary Force Lithography for Cardiac Tissue Engineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

心肌组织工程生物衍生的注射材料的制备

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

科研

生物工程

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

弹性PGS的动脉组织工程支架

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

等离子体表面图案光刻技术的蜂窝网络创作

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

心肌在心脏组织工程中的纤维蛋白凝胶的封装

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

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 ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

一个人的3D组织工程<em&gt;在体外</em&gt;肿瘤测试系统

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

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. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

温敏纳米结构表面的组织工程准备

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

用于内耳组织工程的 Decellularizing 小鼠耳蜗协议

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...