Name: engineering 3d cellularized collagen gels for vascular tissue regeneration
Uploaded: 2015-06-16T14:00:00.000+00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration at JoVE.com

该研究是由自然科学和加拿大工程研究理事会，加拿大卫生研究院和麟德魁北克研究中心。

1.制作静态生物反应器和汇编<ol><li>该水库的制作<ol><li>制备50ml离心管中作为培养基储存器的生物反应器。 </li><li>通过钻两个5毫米直径孔在分别为20毫米的底部和容器的顶部，使两个端口。然后将5毫米长的硅胶管2鲁尔配件。通过孔压入这些鲁尔装配件和密封所有与医用级硅胶胶水连接。 </li><li>插入一个0.22微米的过滤器进入贮存器（ 图1A）的上部端口。 </li><li>插入路厄隔膜进入贮存器（ 图1A）的下端口。 </li></ol></li><li>芯棒帽大会<ol><li>钻在储液管的通风帽的中心的直径为4.5mm的孔，而不会损坏该覆盖疏松孔的过滤膜。 </li><li>准备一个搅拌棒（直径为4.5mm，长度=100毫米），为心轴供构建。 </li><li>准备两个硅锥形塞（长度= 10毫米，中间孔直径= 4.5毫米）。 </li><li>组装如图1B所述心轴和所述帽（心轴帽配合物）。 <ol><li>压装芯轴成孔。插入2挡块在使得盖装配它们之间的心轴。调节心轴的位置，使得它的有效长度为78毫米。 </li><li>适用的引物，然后医用级硅酮胶的表面，将在接触接合所述盖和硅氧烷锥形瓶塞在一起之前。取下盖子的多余的胶水。 </li></ol></li><li>让它在室温下干燥1-3天。 </li></ol></li><li>纱布，夹具制造<ol><li>制备3硅胶管（管1:内径= 6.4Hz毫米，长度= 5毫米;管2:直径= 6.4Hz毫米，长度= 10毫米，管3:直径= 3.1Hz毫米，长度= 12毫米）。 </li><li>组装纱布交手​​的描述d。在图1C中 。 <ol><li>切管1纵向，然后打开它在管2与硅酮胶把它们粘在一起。 </li><li>切无菌手术纱布〜5厘米×7厘米片，然后沿纱布的最长边卷紧的纱布在管3。插入管1管2复合在纱布。 </li><li>添加硅胶胶水粘在一起的纱布中，管1管2络合物和管3的剪切纱布在8毫米的长度。 </li></ol></li></ol></li><li>大会和消毒<ol><li>装配芯轴帽络合物和纱布夹具， 如图2所述。 <ol><li>涂层与医疗级润滑脂（ 图2A）的心轴。将纱布夹在所述心轴（ 图2B）。距离在35毫米从彼此固定值的握把。 </li><li>通过使用一台锯（最终长度=8毫米）除去10毫升注射器的底部部分制备的管状模（ <st荣&gt;图2B）。 </li></ol></li><li>插入模具在纱布握把配备芯棒盖组件（壳体模复合），扣合在硅胶塞（ 图2C）的模具。 </li><li>高压灭菌的容器和外壳模复杂。 注意:插入模具，以避免其脱离时要小心，以对硅胶塞紧紧抱。 </li></ol></li></ol> 2.工程平滑肌细胞胶原凝胶为基础构建和静态成熟<ol><li>工程构造<ol><li>扩大猪主动脉平滑肌细胞（pSMCs）175 平方厘米培养瓶中装有20毫升由Dulbecco改良的Eagle培养基的完全培养基的补充有10％（体积/体积）的猪血清（PS），10％（体积/体积）胎牛血清（FBS），1％（体积/体积）青霉素 - 链霉素（笔链球菌）。 </li><li>在≈90％汇合，分离pSMCs（通道2-4）通过从该培养基烧瓶pSMCs的，加入5毫升的胰蛋白酶溶液（1×磷酸缓冲盐溶液，PBS）中，并孵育10分钟（T = 37℃，5％CO 2，100％湿度）。 </li><li>重悬pSMCs以4×10 6个细胞/中的完全培养基ml的浓度。 </li><li>制备胶原溶液如前所述10。 <ol><li>提取和收集鼠尾腱胶原束在PBS的解决方案。 </li><li>随后转移胶原纤维在丙酮（5分钟），异丙醇70％（体积/体积）（5分钟）和乙酸（0.02 N，48小​​时，4℃）的解决方案。 </li><li>共混在-20℃粘稠溶液并冷冻3天。 </li><li>冻干的冷冻溶液，得到胶原海绵。 </li><li>溶解的胶原海绵成以4克/升和离心机的浓度的乙酸溶液（0.02 N），在29581克力为45分钟。 </li><li>经过消毒以后对透析s过程中的胶原溶液乙酸olutions（0.02 N，1小时），氯仿1％（体积/体积，1小时）和乙酸（0.02 N，无菌溶液每2天更换1周）。 </li><li>收集无菌的胶原溶液（4克/升）在无菌细胞培养罩。 </li></ol></li><li>制备细胞化胶原凝胶， 如图3。 <ol><li>通过在8ml无菌去离子水混合35毫升DMEM中（5×）中加入4ml的HEPES（1N）的，将3ml氢氧化钠（1N）的制备加入50ml无菌缓冲溶液。 </li><li>通过混合的缓冲液和25％50％（体积/体积）的无菌的胶原溶液（4克/乙酸0.02的N L），用25％（体积/体积）制备在一个容器置于冰上细胞和胶原凝胶混合物（体积/体积）中的完全培养基pSMCs的悬浮液。 </li></ol></li><li>测量该混合物的pH值，并确保它是7.0和7.4之间。 </li><li>轻轻倒入9mL的细胞，以及胶原混合成上述外壳/模具复杂的（步骤1.4.3， 图3A-B </strong&gt;）。 </li><li>让它在室温下凝胶化的细胞培养罩（ 图3B）下1小时。 </li></ol></li><li>成熟的生物反应器静<ol><li>取出模具（ 图3C）和转让小心构建进入储，含有35毫升培养介质（ 图3D）的。 </li><li>孵育垂直位置中的构建体（T = 37℃，5％CO 2，100％湿度）进行1周或2周静态成熟。 </li><li>安装Web摄像头的构建前面的孵化器内（以确保绝缘密封）。 </li><li>通过吸旧介质从路厄隔膜端口和重新填充贮存器的新鲜培养基相当量改变培养基，每2天。 </li></ol></li><li>平滑肌细胞的胶原厚度和代谢活性测定基于凝胶的构造在静态文化<ol><li>将扫描激光干涉仪到细胞文化前进重新引擎盖和垂直翻转使用一种精神层面的水平位置。 </li><li>转移反应器进入细胞培养罩和从储存器中删除构建体。 </li><li>转移构建体（仍装在芯棒）到激光束的通路，并把它严格正交相对于光束轴（ 如图4）。 </li><li>读扫描激光干涉仪的屏幕上显示的值，对应于构建体的外径。 </li><li>计算是根据它的外部和内部的直径（ 即心轴直径）的构建体的壁厚。 注:重复步骤2.3.1至2.3.5每个小时的第12小时，然后每24小时。 </li><li>使用将1ml旧培养基用于测定乳酸和葡萄糖浓度与血液气体分析仪（改变培养基，步骤2.2.4当采样）的。 </li><li>使用方法1毫升的新鲜培养基作为用于葡萄糖和乳酸浓度的测量14的基线水平。 注:重复步骤2.3.6和2.3.7之后的培养基变化，每2天。 </li></ol></li><li>构建收获了进一步的机械和生物Ccharacterizations <ol><li>后1周或2周的静态成熟期间，传送静态生物反应器进入细胞培养罩。 </li><li>转移轻轻地从它的心轴（ 补充视频1）成熟的构建体，以含有40毫升新鲜培养基（ 图5和图7A）一个直径100mm的培养皿。 </li></ol></li></ol>该构造在纵向和圆周方向的3.机械表征<ol><li>安装实验设置包括微机械测试仪配备有5或10 N加载细胞和含有PBS中的浴，在37℃，以保持样本吨伪生理条件（ 图6）。 </li><li>平衡负载单元和引伸。 注意:平衡是集成到微机械测试仪包括在重置显示扩展值，并同时没有样品被安装在机器上所显示的负载值的函数。该功能允许定义为测量的基准。 </li><li>安装在管状结构上的机械装置，包括:纵向方向。 注意:直接对整个管状结构进行纵向疲劳试验。使用内部构建的夹紧装置的结构的纱布夹具连接到测力传感器和对于PBS浴的底部。 <ol><li>安装管状构造上夹持装置（ 图7B），继收割过程（第2.4节）。 </li><li>铁氟龙胶带包裹的夹持装置和纱布夹在一起，以防止任何打滑纱布交手的测试过程中。装入样品上的微机械测试仪（ 图7C）。 </li></ol></li><li>安装环状结构上的机械装置，包括:沿圆周方向。 注意:对从所述管状结构剖环状标本进行周疲劳试验。使用两个不锈钢棒作为交手保持标本。 <ol><li>安装在管状构造在塑料管作为支撑打上5毫米间隙（ 图7B），继收获（第2.4节）。 </li><li>切10毫米环从管状结构。 </li><li>测量使用游标卡尺用于进一步分析的样品的长度。 </li><li>安装环状试验件上的微机械测试仪（ 图7C）的不锈钢棒。请务必将试样在酒吧的中心。 注意:塑料管道在步骤3.4.1和一个切割系统， 如图7B </str翁&gt;用于避免在切割过程中的构建体的任何损坏。 </li></ol></li><li>上在纵向或圆周方向上的结构的疲劳试验。 <ol><li>伸展构建到其初始量规长度。 </li><li>保持在这个位置中的伪生理环境10分钟的构建体。 </li><li>申请的初始标距长度（30个循环）的构建体的10％循环应变在5％/秒应变速率。 </li><li>以10％循环应变递增步骤直至样品失败重复步骤3.5.3。 注意:使用伪生理环境的需要考虑到的浮力和夹紧系统，其影响施加载荷的测量的惯性。 </li><li>记录的背景如下: <ol><li>移动负荷帧到初始标距长度。 </li><li>重复步骤3.5.3和3.5.4没有任何样品安装，并保持连接到所述负载单元夹持装置（仅1个周期是requi红色）。 </li></ol></li></ol></li></ol>构建了4管腔内皮注意:下面的收割协议（第2.4节）后，将构建体经受处理，以被安装在旋转壁生物反应器中的进一步的内皮。 <ol><li>旋转壁生物反应器的设计<ol><li>钻在储液管的通风帽的中心的直径为4.5mm的孔，而不会损坏该覆盖疏松孔的过滤膜。 </li><li>压入一个芯棒（直径= 4.5毫米，长度= 40 MM）成孔，并按照步骤1.1.2描述修复芯棒。 </li><li>准备构建外径两个C形硅胶支持= 14毫米;内径= 8毫米）。 </li><li>位置的旋转电机在旋转壁生物反应器的一端，并在另一端（ 图8B）的轴承。 </li></ol></li><li>腔内皮<ol><li>拓展人脐人血管内皮细胞（HUVECs）中为25cm 2培养瓶中用5ml的M199培养基中补充有10％（体积/体积）的PS，10％（体积/体积）胎牛血清，1％（体积/体积）青霉素-链霉素在培养皿恒温箱（T = 37℃，5％CO 2，100％湿度），直到90％汇合的内部。 </li><li>通过在无血清的内皮细胞培养基中的浓缩物的蛋白质混合物稀释至10.5纳克/毫升制备1.5ml每所需最佳细胞粘附构建体中的蛋白质涂布溶液。 </li><li>测量使用游标卡尺构建体的长度。 </li><li>计算管腔体积V和构建体的管腔面积A 为:V =分别中的 D 2 L / 4和A = D L 中 （其中D 中是对应于所述心轴的直径的内径，L是结构的长度）。 </li><li>放置在构造油藏的中心下面的收获过程（第2.4节）。使用C形硅支持修复构建体两端到储存器（ 图8A）。 </li><li>用35ml培养基填充的贮存器。 </li><li>填充构建体（V）与在步骤4.2.2制备的蛋白质涂层溶液的计算腔体积的75％。关闭两个构建体的末端，以避免蛋白质涂布液（ 图8A）中的任何泄漏。 </li><li>组装的细胞培养罩内的旋转壁生物反应器系统。 </li><li>将生物反应器在37℃的培养箱中，并开始在生物反应器的旋转在4.02×10 -5克力1小时，以允许腔涂层， 如图8B所示 。 </li><li>打开构建体的上肢，吸从内腔蛋白涂布溶液。 </li><li>通过从内皮细胞的烧瓶除去培养基并加入3ml胰蛋白酶溶液（1×PBS中）分离人脐静脉内皮（通道2-3）。一世ncubate 5分钟（T = 37℃，5％CO 2，100％湿度）。 </li><li>重悬内皮细胞以4×10 6个细胞/在补充M199培养基ml的浓度。 </li><li>内的细胞培养罩，种子的HUVECs入构建体的内腔1000个细胞/ cm 2的15的密度。关闭构建体的上肢避免内皮细胞溶液的任何渗漏。 </li><li>孵育构建体（T = 37℃，5％CO 2，100％湿度）承载到旋转壁生物反应器（ 图8B）和培养2天以4.02×10 -5克力的恒定旋转。 </li><li>后在无菌条件下培养2天后的收获，建造及2.4节所述准备进行进一步的生物学特性。 </li></ol></li></ol>

<ol>
	<li>Kim, B. S., Nikolovski, J., Bonadio, J., Smiley, E., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+smooth+muscle+tissues:+regulating+cell+phenotype+with+the+scaffold.">Engineered smooth muscle tissues: regulating cell phenotype with the scaffold.</a> Experimental cell research. 251, 318-328 (1999).</li><li>Heureux, N., McAllister, T. N., de la Fuente, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+blood+vessel+for+adult+arterial+revascularization.">Tissue-engineered blood vessel for adult arterial revascularization.</a> The New England journal of medicine. 357 (14), 1451-1453 (2007).</li><li>Syedain, Z. H., Meier, L. A., Bjork, J. W., Lee, A., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantable+arterial+grafts+from+human+fibroblasts+and+fibrin+using+a+multi-graft+pulsed+flow-stretch+bioreactor+with+noninvasive+strength+monitoring.">Implantable arterial grafts from human fibroblasts and fibrin using a multi-graft pulsed flow-stretch bioreactor with noninvasive strength monitoring.</a> Biomaterials. 32 (3), 714-722 (2011).</li><li>Lee, C., Singla, A., Lee, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+applications+of+collagen.">Biomedical applications of collagen.</a> International journal of pharmaceutics. 221 (1-2), 1-22 (2001).</li><li>Couet, F., Rajan, N., Mantovani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+biomaterials+for+scaffold-based+vascular+tissue+engineering.">Macromolecular biomaterials for scaffold-based vascular tissue engineering.</a> Macromolecular bioscience. 7 (5), 701-718 (2007).</li><li>Christiansen, D. L., Huang, E. K., Silver, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+type+I+collagen:+fusion+of+fibril+subunits+and+the+influence+of+fibril+diameter+on+mechanical+properties.">Assembly of type I collagen: fusion of fibril subunits and the influence of fibril diameter on mechanical properties.</a> Matrix Biology. 19 (5), 409-420 (2000).</li><li>Eppell, S. J., Smith, B. N., Kahn, H., Ballarini, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano+measurements+with+micro-devices:+mechanical+properties+of+hydrated+collagen+fibrils.">Nano measurements with micro-devices: mechanical properties of hydrated collagen fibrils.</a> Journal of the Royal Society, Interface / the Royal Society. 3 (6), 117-121 (2006).</li><li>Lai, V. K., Lake, S. P., Frey, C. R., Tranquillo, R. T., Barocas, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+behavior+of+collagen-fibrin+co-gels+reflects+transition+from+series+to+parallel+interactions+with+increasing+collagen+content.">Mechanical behavior of collagen-fibrin co-gels reflects transition from series to parallel interactions with increasing collagen content.</a> Journal of biomechanical engineering. 134 (1), 011004 (2012).</li><li>Seliktar, D., Nerem, R. M., Galis, Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Matrix+Metalloproteinase-2+in+the+Remodeling+of+Cell-Seeded+Vascular+Constructs+Subjected+to+Cyclic+Strain.">The Role of Matrix Metalloproteinase-2 in the Remodeling of Cell-Seeded Vascular Constructs Subjected to Cyclic Strain.</a> Annals of Biomedical Engineering. 29 (11), 923-934 (2001).</li><li>Rajan, N., Habermehl, J., Cot&#233;, M. -. F., Doillon, C. J., Mantovani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+ready-to-use,+storable+and+reconstituted+type+I+collagen+from+rat+tail+tendon+for+tissue+engineering+applications.">Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications.</a> Nature protocols. 1 (6), 2753-2758 (2006).</li><li>Kumar, V. A., Caves, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen-Based+Substrates+with+Tunable+Strength+for+Soft+Tissue+Engineering.">Collagen-Based Substrates with Tunable Strength for Soft Tissue Engineering.</a> Biomaterials science. 1 (11), 1193-1202 (2013).</li><li>Li, S., Van Den Diepstraten, C., D&#8217;Souza, S. J., Chan, B. M. C., Pickering, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+smooth+muscle+cells+orchestrate+the+assembly+of+type+I+collagen+via+alpha2beta1+integrin,+RhoA,+and+fibronectin+polymerization.">Vascular smooth muscle cells orchestrate the assembly of type I collagen via alpha2beta1 integrin, RhoA, and fibronectin polymerization.</a> The American journal of pathology. 163 (3), 1045-1056 (2003).</li><li>Badylak, S. F., Freytes, D. O., Gilbert, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+as+a+biological+scaffold+material:+Structure+and+function.">Extracellular matrix as a biological scaffold material: Structure and function.</a> Acta biomaterialia. 5 (1), 1-13 (2009).</li><li>Engbers-Buijtenhuijs, P., Buttafoco, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+characterisation+of+vascular+grafts+cultured+in+a+bioreactor.">Biological characterisation of vascular grafts cultured in a bioreactor.</a> Biomaterials. 27 (11), 2390-2397 (2006).</li><li>Cheung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+human+umbilical+vein+endothelial+cells+(HUVEC).">Isolation and culture of human umbilical vein endothelial cells (HUVEC).</a> Current protocols in microbiology. 4, 4B (2007).</li><li>Seifu, D. G., Purnama, A., Mequanint, K., Mantovani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-diameter+vascular+tissue+engineering.">Small-diameter vascular tissue engineering.</a> Nature reviews. Cardiology. , (2013).</li><li>Weinberg, C., Bell, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+blood+vessel+model+constructed+from+collagen+and+cultured+vascular+cells.">A blood vessel model constructed from collagen and cultured vascular cells.</a> Science. 231 (4736), 397-400 (1986).</li><li>Meghezi, S., Chevallier, P., Mantovani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+Mechanical+Properties+of+Collagen+Scaffolds+Should+Be+Tested+in+a+Pseudo-Physiological+Environment.">Why Mechanical Properties of Collagen Scaffolds Should Be Tested in a Pseudo-Physiological Environment.</a> Advanced Materials Research. 409, 158-163 (2011).</li><li>Tirella, A., Liberto, T., Ahluwalia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riboflavin+and+collagen:+New+crosslinking+methods+to+tailor+the+stiffness+of+hydrogels.">Riboflavin and collagen: New crosslinking methods to tailor the stiffness of hydrogels.</a> Materials Letters. 74, 58-61 (2012).</li><li>Madhavan, K., Belchenko, D., Tan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+genipin+crosslinking+and+biomolecule+conditioning+in+collagen-based+biopolymer:+Potential+for+vascular+media+regeneration.">Roles of genipin crosslinking and biomolecule conditioning in collagen-based biopolymer: Potential for vascular media regeneration.</a> Journal of biomedical materials research. Part A. , 16-26 (2011).</li><li>Meghezi, S., Couet, F., Chevallier, P., Mantovani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+a+pseudophysiological+environment+on+the+elastic+and+viscoelastic+properties+of+collagen+gels.">Effects of a pseudophysiological environment on the elastic and viscoelastic properties of collagen gels.</a> International journal of biomaterials. 2012, 319290 (2012).</li></ol>

没有资金，从组织或机构收到的潜在利益冲突。

间的血管组织工程师社区，巨大努力已经完成，以重现负责血管16的机械稳定性中膜层。自温伯格和贝尔17的开创性工作，胶原已被广泛地用作支架，因为其生物相容性，非免疫原性性能和可用性的血管组织工程。然而，使用胶原的表示为研究者一个很大的挑战，因为这种材料是不容易处理，由于固有缺乏机械刚度。支架在制备过程中的操作可能会损坏支架，损害它们供进一步使用。 在这项工作中所描述的技术允许:i）至工程师细胞化胶原凝胶成筒状的几何形状; ii）向工程师生物组织足够强短静态成熟期间（1或者2周）后进行处理; 3）为在2个方向，例如管状形状的生物体组织的自编码扩频通信的机械和粘弹性质。在凝胶细胞在胶原基质重塑中发挥关键作用。在成熟期间，收缩平滑肌细胞导致了凝胶，得到的构建体具有更高的机械稳定性，可以在纵向和圆周方向进行评估的压实。此后，内皮细胞接种在腔侧的构建体产生的均匀的和可行的内皮，从而表现出胶原凝胶血管组织工程应用的适用性。 在这项工作中提出的生物反应器是专门设计在静态成熟提供用于细胞生长的最佳环境。此外，对于该构建体的机械性能和粘弹性能表征开发的装置被设计，目的是减少固有的操作的任何潜在损害这种微妙的材料。因此，静态生物反应器中装有一个0.22微米的过滤器和过滤膜在盖（步骤1.1.2， 图1A的 和B），所允许的贮存器和培养箱内培养介质之间的气体交换，同时保持无菌培养环境。鲁尔隔膜在底部用作端口培养基取样和静态培养期间改变。一些关键的步骤都在构建制造和表征加以考虑。所有操作（在步骤2.1.1和在后续步骤中进行的）可能改变系统的无菌性是在无菌生物罩进行。细胞和胶原凝胶混合物制备物在冰上处理以延缓胶凝过程（步骤2.1.4至2.1.7）。在步骤2.1.7，任何气泡之前凝胶包埋在混合物是潜在的应力集中区域，可以妥协的S盈利能力的构造。因此，去除这种气泡，需要微微晃动组装或使用医疗真空3分钟的degasing在无菌条件下。最后，该夹具被用于维持心轴中心在凝胶化过程中，管状模具的轴线和用于允许在收获（去除心轴的，第2.4节），对于内皮细腻操纵构建体设计的，并且为便于安装到机械系统（纵向测试）。 本协议提出的胶原凝胶结构加固的基础上校董会自然收缩的内在潜能的原始易于处理的替代方法。的胶原基质加固常用技术涉及使用可对细胞-基质相互作用18的有害作用的物理和化学交联剂- 20。在给出的制造技术这项工作中允许引导此细胞驱动的重塑过程，得到一种组织工程化构建体与靶向的机械性能，没有任何物理或化学处理。 的水合胶原凝胶的机械和粘弹性能表征是一个巨大的挑战。从这个角度来看，本协议描述为评估管状软组织的力学性能的原始简单而有效的方法。这种表征可以执行不仅在圆周方向上，而且在纵向上，直接在整个管状结构。在机械特性，温度，水环境，pH和离子强度是一些已知显着影响生物组织21的机械行为的环境因素。因此，目前的工作表明，在一个高的初始设置和协议，用于生物组织的机械特性重现的伪生理环境（37℃和pH 7.4的盐水溶液）。尽我们所知，这种表征从未在别处报道。 总之，在此工作提出的技术证明细胞与胶原血管组织工程应用直接混合的高电位。这与机械特性和内皮过程方法一起构成高多价协议。因此，通过该组窗口和同时保持相同的基本原理的协议的轻微的修改，对于工程血管组织当量主要要求是可以解决诸如快速和简单的处理，包括内皮，并有可能被移植到大范围的软组织中具有各种长度和直径。此外，不同的贴壁细胞类型，细胞外基质蛋白和几何形状的成型可以考察了一些有针对性的applicati的项，如工程腱，皮肤移植，心脏修补，神经等。虽然该构建体的机械性能是令人鼓舞的，它们比那些天然组织的更低。在此背景下，我们坚信，在很短的静态成熟期是朝着动态的刺激到生物反应器中的一个关键步骤，从而导致更高的结构完整性和机械稳定性。然而，有可能迅速产生组织工程细胞化的基于胶原构建体适合机械和组织学分析，使本文所述的有用且有希望的工具来生长和重建，或甚至在洞察细胞和细胞外基质之间的相互作用的静态生物反应器作为一个模型用于治疗和药物递送系统。

血管组织工程设想旨在设计容器制作，包括基于合成的支架，细胞片基组织工程血管（TEBVs），和细胞外基质（ECM）的移植物不同的策略组件基于TEBVs。在这些方法中，合成的聚合物表现出良好的机械性能，但都有一个共同的缺点，因为它们缺乏生物活性1。该细胞片基方法允许生产工程化血管代用品具有高机械性能的，但需要产生这种移植物的时间是约28 周 3。 ECM的天然生物聚合物，如胶原，弹性蛋白，纤维蛋白3或它们的组合，仍然是组织工程支架的黄金标准材料。这主要是为这些材料具有大致良好的生物相容性，同时能够诱导功能性细胞应答4-5的原因。在这些生物聚合物S，I型胶原蛋白是细胞外基质中的许多组织如皮肤，血管和腱的最丰富的和主要的承重蛋白之一。大量的工作已经进行了6胶原蛋白的力学性能- 8，但也出现了只有少数研究对胶原凝胶在静态成熟细胞重塑。蜂窝重塑是指诱导细胞可能影响胶原纤维网络9的稳定胶原基质的结构修饰。作为天然的支架，相对大量的I型胶原的可以分离，灭菌，并从不同的来源，例如鼠尾腱10存储。了解胶原蛋白细胞相互作用和细胞化胶原支架（结构）的有关整体力学行为是组织建设的一个重要步骤。基于胶原的TEBVs可以通过直接混合细胞与胶原被加工凝胶制备期间和进一步模塑成特定的形状，例如管状的和平面11。凝胶内的血管细胞增殖和改造I型胶原12。因此，这种方法绕过需要，表示在支架用于组织工程应用的发展显著问题之一特定的大孔。然而，胶原凝胶的主要缺点是它们的低的机械性能相比，合成材料13。 在这项研究中，与细胞的均匀分布的活组织工程改造的胶原质与细胞的一步法直接混合。 "静态生物反应器"被用于1周或2周的细胞化的胶原凝胶的静态成熟的（没有施加外部动态机械约束）。在培养过程中，胶原基质重塑发生，从而提供了结构增强的结构。此外，这些结构如下:R伊迪被转移到一个旋转壁生物反应器和均匀的内皮达到了。另外，在此工作的特定机械测试协议还提出，以提供一个合适的新方法在表征的管状软组织的机械性能。 总之，本工作提出用于体外快速制造和是强大到足以进行处理不仅对生物和机械表征，同时也为进一步的机械调节在一个动态的生物反应器，它被认为是一种重要的血管组织的成熟的方法步骤中组织的再生。

<table><tbody><tr><td>CellTreat 50 ml Bio-Reaction tubes</td><td>CELLTREAT Scientific Products</td><td>229-475</td><td>Centrifuge tube</td></tr><tr><td>Male luer with lock ring x 1/8&quot; hose barb. PP. 25/pk</td><td>Cole Parmer</td><td>RK-45503-04</td><td>Luer fittings for &quot;gas-exchange port&quot;</td></tr><tr><td>Female luer x 1/8&quot; hose barb adapter. PP. 25/pk</td><td>Cole Parmer</td><td>RK-45500-04</td><td>Luer fittings for the &quot;medium sampling port&quot;</td></tr><tr><td>Masterflex platinum-cured silicone tubing L/S 17. 25 ft.</td><td>Cole Parmer</td><td>RK-96410-17</td><td>Tube 1 and 2 for the gauze grippers</td></tr><tr><td>Masterflex platinum-cured silicone tubing L/S 16. 25 ft.</td><td>Cole Parmer</td><td>RK-96410-16</td><td>Tube 3 for the gauze grippers</td></tr><tr><td>Silastic Medical adhesive silicone, type A</td><td>Dow Corning</td><td>-</td><td>Silicon glue for the fabrication of the static bioreactor</td></tr><tr><td>Polyvent 4 Vessel venting filters</td><td>Whatman</td><td>6713-0425</td><td>Filter for &quot;gas exchange port&quot;</td></tr><tr><td>Rod. PP. stirring. 8&amp;rsquo;&amp;rsquo;</td><td>Scienceware</td><td>377660008</td><td>Mandrel</td></tr><tr><td>Stopper silicone rubber 00 PK12</td><td>VWR</td><td>59590-084</td><td>Stopper for the insertion of the mandrel to the vented cap of the centrifuge tube</td></tr><tr><td>Krytox PFPE/PTFE Greases</td><td>Dupont</td><td>GPL 202</td><td>Medical grade grease for covering the mandrel</td></tr><tr><td>Trypsin-EDTA (0.5%)</td><td>Gibco</td><td>15400-054</td><td>Cell culture</td></tr><tr><td>Xiameter RTV-4130-J base and curing agent</td><td>Dow Corning</td><td>-</td><td>C-shaped silicone support for endothelialization</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium, DMEM, high glucose, pyruvate</td><td>Gibco (Life Technology)</td><td>11995-065</td><td>Cell culture</td></tr><tr><td>Pure acetone (99%)</td><td>Laboratoire Mat Inc.</td><td>AP0102</td><td>Chemical for collagen extraction</td></tr><tr><td>Isopropyl alcohol (HPLC grade, 99.9%)&amp;nbsp;</td><td>Fisher Scientific</td><td>AC610080040</td><td>Chemical for collagen extraction</td></tr><tr><td>0.02 N acetic acid (glacial acetic acid, HPLC grade, 99%)</td><td>Fisher Scientific</td><td>FL070494</td><td>Chemical for collagen extraction</td></tr><tr><td>Chloroform solution (99%)&amp;nbsp;</td><td>Laboratoire Mat Inc.</td><td>CR 0179</td><td>Chemical for collagen extraction</td></tr><tr><td>Hepes</td><td>Sigma-Aldrich</td><td>163716</td><td>Chemical for construct preparation</td></tr><tr><td>NaOH</td><td>Laboratoire Mat Inc.</td><td>SR-0169</td><td>Chemical for construct preparation</td></tr><tr><td>LaserMike 136</td><td>LaserMike</td><td>Series 183B</td><td>Scanning laser interferometer&amp;nbsp;</td></tr><tr><td>ElectroPulse MicroTester</td><td>Instron Corporation</td><td>-</td><td>Micromechanical Tester</td></tr><tr><td>HyClone Media M199/EBSS, 500&amp;nbsp;ml</td><td>GE Healthcare Life Sciences</td><td>SH30253.01</td><td>Component of cell culture medium</td></tr><tr><td>Fetal bovine serum HI - 500&amp;nbsp;ml</td><td>Gibco</td><td>SH 30396.03</td><td>Component of cell culture medium</td></tr><tr><td>Porcine serum (PS)</td><td>Sigma-Aldrich</td><td>P9783</td><td>Component of cell culture medium</td></tr><tr><td>Penicillin-Streptomicin</td><td>Gibco</td><td>15140-122</td><td>Component of cell culture medium</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Fisher Scientific</td><td>BP661-50</td><td>Saline solution</td></tr><tr><td>Tissue culture flask T17CN Vent Cap Red</td><td>Sarstedt Inc.</td><td>83.1812.002</td><td>Cell culture</td></tr><tr><td>ColorpHast- pH-indicator strips (pH = 6.5-10.0)</td><td>EMD</td><td>9583</td><td>pH measurements</td></tr><tr><td>Matrigel Basement Membrane Matrix Growth Factor Reduced, 5 ml vial</td><td>BD Biosciences - Discovery Labware</td><td>356230</td><td>Concentrate protein mixture for endothelialization process</td></tr><tr><td>LifeCam VX-3000</td><td>Microsoft</td><td>-</td><td>Thickness measurement</td></tr><tr><td>Biochemical analyzer, DxC600</td><td>Beckman Coulter Unicell Synchron</td><td>-</td><td>Glucose and lactate concentrations measurements</td></tr><tr><td>Collagen fibers</td><td>Rat tails</td><td>-</td><td>Collagen was extracted in the laboratory</td></tr><tr><td>Porcine smooth muscle cells (pSMCs)</td><td>Porcine aortas</td><td>-</td><td>pSMCs were isolated in the laboratory</td></tr><tr><td>Human umbilical vein endothelial cells (HUVECs)</td><td>Human umbilical veins</td><td>-</td><td>HUVECs were isolated in the laboratory</td></tr></tbody></table>

engineering 3d cellularized collagen gels for vascular tissue regeneration

合成材料是已知的时血管代用品植入以引发临床并发症如炎症，狭窄，和感染。胶原已被广泛地用于各种各样的生物医学应用，被认为是一个有效的替代合成材料由于其固有的生物相容性（ 即 ，低抗原性，炎症和细胞毒性的响应）。然而，有限的力学性能和胶原凝胶的相关低手的能力，阻碍了其作为支架材料的血管组织工程中使用。因此，这背后的工作的基本原理是第一个工程细胞化胶原凝胶成筒状的几何形状和第二提升平滑肌细胞的胶原基质的驱动重组，以获得组织僵硬足以进行处理。 这里描述的策略是基于胶原和平滑肌细胞（构建）的三维cyli直接装配ndrical几何与使用一模制技术。这个过程需要一个成熟期间，在此期间，结构是在静态条件下的生物反应器培养（没有施加外部动态机械约束）处理1周或2周。 "静态生物反应器"提供了一种监测和控制无菌环境（pH，温度，气体交换，营养供应和废物清除）到构建体。在培养期间，厚度的测量进行评估胶原基质的细胞驱动的重塑，以及葡萄糖消耗和乳酸生产速率进行测定，以监测细胞的代谢活性。最后，机械和粘弹性能进行了评估对于所得的管状结构。为此目的，特定协议和聚焦诀窍（操纵，握持，在水合环境下工作，等等）被开发来表征的工程化组织。

这项工作中描述了含有血管细胞工程管状的基于胶原构建体的制造。后已经1小时的早期胶凝，细胞和 - 胶原混合物直接组装在一个3D管状几何形状，与外部直径等于相应的模具的直径（大约14毫米）。所有沿静态成熟，测量显示迅速减少管状细胞化结构的外径的，如表1所示。1天静态培养后缩水的约60％的初始值的细胞化胶原凝胶的直径，和近85 7天内（ 补充视频2）％。构建物内的SMC是负责观察到的收缩和相关的机械加固，因为这种现象不会发生在非细胞化胶原支架。需要注意的是任何类型的（热，生化，机械或其他）的无梯度施加。细胞驱动Ñ ​​压实导致以更大的胶原密度的材料，可以被处理和制服，以机械募捐（ 补充画3和4）。 到涉及细胞驱动的重构的整体机械和粘弹性能，疲劳试验是在所述构建体（ 补充画5和6）进行。这些测试包括在循环的构造（30次），在不同的恒定应变（10％，20％，和初始标距长度的30％），并记录应力作为构建体的机械请求随时间的响应。代表性的结果对于一个构建示于图9 的构建体经受在纵向方向（75千帕）更高的应力比当经受相同的应变范围（30％应变）的圆周方向（16千帕）。同时，在每个周期中，应力峰值达到了TArgeted最大应变随时间而减少。此行为是典型的由这些基于胶原的构建体显示出高的粘弹性性质。 在细胞化构建体的生物学活性在静态成熟进行了评估。因此，平滑肌细胞的代谢活性通过测量静态培养过程中的葡萄糖消耗和乳酸生产的评价。培养基进行取样，每2天，葡萄糖和乳酸浓度使用血气分析仪测定。在葡萄糖消耗和乳酸生产相结合，所述构建体的重要收缩的不断增加，证明了平滑肌细胞活性的所有沿静态培养（ 图10）。 增加的机械稳定性由于电池驱动的重构允许构建体的操作和随后的内皮化过程。对内皮化构建体马松染色进行表现出高度同质内皮。平滑肌细胞表现出纺锤状形的形态，并通过壁出现均匀地分散，而内皮细胞出现很好的普及，在腔侧（ 图11）。 <img alt="图1" src="/files/ftp_upload/52812/52812fig1highres.jpg" width="700" /> 图1:静态生物反应器组件静态生物反应器包括一个修正的50ml离心管（A）和心轴配备盖（B）的。管担任介质贮存，并装有一个端口为0.22μm的过滤器，用于气体交换，和一个隔膜，对于中采样和改变。心轴存在于通风帽结构允许在管状的制造。纱布-夹具（C）的设计和制造，以支持构建体在芯棒的凝胶化。此外，这些把持构建静态熟化后进行处理，也可以固定到机械设备允许的。心轴的外直径为4.7毫米。 <img alt="图2" src="/files/ftp_upload/52812/52812fig2highres.jpg" width="700" /> 图2:装配静态生物反应器的灭菌前组装的生物反应器的阶段。纱布-握把被安装在以固定距离心轴（A）。一种模具插入（B）和紧密固定在硅胶塞（℃）。心轴的外直径为4.7毫米。 <img alt="图3" src="/files/ftp_upload/52812/52812fig3highres.jpg" width="700" /> 图3:在无菌条件下，构建体的制作将细胞和胶原混合物倒入壳体模复<html> （A）中，并让凝胶1小时，在室温（B）中 。之后，模具中取出（C）中，静态生物反应器的组装（D）和一个储存器内传送的构建体在培养箱静态成熟（T = 37℃，5％CO 2，100％湿度）。心轴的外直径为4.7毫米。 <img alt="图4" src="/files/ftp_upload/52812/52812fig4highres.jpg" width="700" /> 图4:该构建体的厚度/外径的测量的激光扫描干涉仪被用来执行该构建体的外部直径的测量。将构建体放置在激光束的通路，并产生一个阴影。阴影的宽度，对应于构建体的外径，然后测量并显示在屏幕上。 e_content"FO:保持together.within页="总是"&gt; <img alt="图5" src="/files/ftp_upload/52812/52812fig5highres.jpg" width="500" /> 图5:收获结构的外观形态（a）在静态成熟2周凝胶和（B）后细胞驱动的重塑之后。 <img alt="图6" src="/files/ftp_upload/52812/52812fig6highres.jpg" width="500" /> 图6:实验装置进行机械表征它包括微机械测试仪配备有5或10 N加载细胞和含有PBS，在37℃，以保持在样品中的伪生理条件的浴的。 <img alt="图7" src="/files/ftp_upload/52812/52812fig7highres.jpg" width="700" /> 图7:试样制备F<html>或机械刻划。样品采集（A）和制剂（B），用于在纵向进行疲劳试验和圆周方向（C）。心轴的外直径为4.7毫米。 <img alt="图8" src="/files/ftp_upload/52812/52812fig8highres.jpg" width="600" /> 图8:旋转壁生物反应器（A）中的管状结构组装在具有C形硅支撑的帮助下，储存器的中心。两个构建体的四肢被关闭，以避免所述的HUVECs溶液的任何渗漏。 （B）中的构建体培养在培养箱中（T = 37℃，5％CO 2，100％湿度）中的旋转在2天4.02×10 -5克力。 2 / 52812fig9highres.jpg"WIDTH ="700"/&gt; 图9:机械表征对构建纵向（A）进行疲劳试验和细胞驱动的重构周后（B）方向的结果<a href="https://www.jove.com/files/ftp_upload/52812/52812fig9highres.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <img alt="图10" src="/files/ftp_upload/52812/52812fig10highres.jpg" width="600" /> 图10:在胶原凝胶内平滑肌细胞的代谢活性的葡萄糖消耗率和乳酸产生率的测量是与血液气体分析仪上进行，每​​2天，将培养基改变之后。新鲜培养基用作葡萄糖和乳酸浓度的测量的基线水平。 图11:流明内皮管状结构的径向横截面的组织学图像。管状结构的Masson的腺毛染色培养静态1周（A）和2周（B）中 。管状结构（C）的H＆E染色。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 时间 </td><td> 厚度（mm） </td><td> 压实（％） </td></tr><tr><td> 0小时</td><td> 4.83±0.02 </td><td> 0±0 </td></tr><tr><td> 2小时</td><td> 4.26±0.02 </td><td> 12±0 </td></tr><tr><td> 4小时</td><td> 4.21±0.03 </td><td> 13±1 </td></tr><tr><td> 6小时</td><td> 4.06±0.10 </td><td> 16±2 </td></tr><tr><td> 12小时R </td><td> 3.16±0.07 </td><td> 35±1 </td></tr><tr><td> 1天</td><td> 2.08±0.11 </td><td> 57±2 </td></tr><tr><td> 1周</td><td> 0.68±0.07 </td><td> 86±1 </td></tr><tr><td> 2周</td><td> 0.36±0.00 </td><td> 93±0 </td></tr></tbody></table> 表1:构建体直径的静态成熟期间快速压实壁厚的构建体和压缩率的静态培养时间的函数。压实通过测定该管状结构，用扫描激光干涉仪（系列183B，LaserMike 136）的外直径测量。 24小时后，该构建体压缩至57％的成型体的尺寸的±2％。数据表示为平均值±标准差（n = 3）。的存在和活平滑肌细胞的活性是唯一负责这样大的变化。 苏pplemental视频1:非改装管状胶原凝胶的收获<a href="https://www.jove.com/files/ftp_upload/52812/JoVE_Video1.mov" target="_blank">请点击此处观看该视频。</a> 补充视频2:管状胶原凝胶电池驱动的压实 <a href="https://www.jove.com/files/ftp_upload/52812/JoVE_Video2.m4v" target="_blank">请点击此处观看该视频。</a> 补充视频3:非改装管状胶原凝胶的操作 <a href="https://www.jove.com/files/ftp_upload/52812/JoVE_Video3.mov" target="_blank">请点击此处观看该视频。</a> 补充视频4:细胞，肾小管改造胶原凝胶的操作 <a href="https://www.jove.com/files/ftp_upload/52812/JoVE_Video4.mov" target="_blank">，请点击此处查看次</a>是视频。 补充视频5:纵向疲劳试验（30％）的细胞，肾小管改造胶原凝胶 <a href="https://www.jove.com/files/ftp_upload/52812/JoVE_Video5.mov" target="_blank">请点击此处观看该视频。</a> 补充视频6:圆周疲劳试验（30％）的细胞，肾小管改造胶原凝胶 <a href="https://www.jove.com/files/ftp_upload/52812/JoVE_Video6.mov" target="_blank">请点击此处观看该视频。</a>

Watch this Scientific Journal Video about Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration at JoVE.com

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

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. ...

engineering-3d-cellularized-collagen-gels-for-vascular-tissue

Research

JoVE Journal

Bioengineering

20.8K 次观看.  Laval University. 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.

视频: Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

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

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

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

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

定向细胞自我组装制造，为生物力学分析与组织工程细胞衍生的组织环

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

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

工程双层凝胶控制升序分化

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

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 ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

三维仿生技术:新型Biorubber创建自定义微观和宏观尺度架构的胶原凝胶

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

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 ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

重组胶原蛋白 i. 肽载体在生物反应器中的应用及其作为细胞传递系统的潜力

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

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

管状结构组织工程的核心/壳印刷支架

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create tissues by adding layer upon layer of printable biomaterials, termed bioinks, and cells in an orderly and automatic manner, which allows for creating highly intricate structures that traditional tissue engineering techniques cannot achieve. Thus, 3D bioprinting is an appealing in vitro approach to mimic the native vasculature complex structure, ranging from millimetric vessels to microvascular networks. 
Advances in 3D bioprinting in granular hydrogels enabled the high-resolution extrusion of low-viscosity extracellular matrix-based bioinks. This work presents a combined 3D bioprinting and sacrificial mold-based 3D printing approach for fabricating engineered vascularized tissue flaps. 3D bioprinting of endothelial and support cells using recombinant collagen-methacrylate bioink within a gelatin support bath is utilized for the fabrication of a self-assembled capillary network. This printed microvasculature is assembled around a mesoscale vessel-like porous scaffold, fabricated using a sacrificial 3D printed mold, and is seeded with endothelial cells. 
This assembly induces the endothelium of the mesoscale vessel to anastomose with the surrounding capillary network, establishing a hierarchical vascular network within an engineered tissue flap. The engineered flap is then directly implanted by surgical anastomosis to a rat femoral artery using a cuff technique. The described methods can be expanded for the fabrication of various vascularized tissue flaps for use in reconstruction surgery and vascularization studies.

Engineered flaps require an incorporated functional vascular network. In this protocol, we present a method of fabricating a 3D printed tissue flap containing a hierarchical vascular network and its direct microsurgical anastomoses to rat femoral artery.

使用3D打印技术制造工程血管瓣

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create ...