特雷西C. Grikscheit，埃里克R.巴特尔，和弗雷德里克·G.萨拉所支持的加州再生医学研究所（CIRM），授权号码RN2-00946-1（TCG）和TG2-01168（再培训局，FGS）。艾里逊L.施佩尔是一个大学的外科医生学会爱惜学者。 Yashuhiro虎岛是由一家儿童医院洛杉矶萨班研究所的研究生涯发展奖。

1。类器官单位准备<ol><li>仪器适用于鼠标清扫术（剪刀和镊子）应通过高压灭菌器灭菌。 </li><li>人道安乐死的供体小鼠根据当地IACUC协议。确保该动物是死的，然后再继续。 </li><li>车型中线切口进入腹膜腔。皮瓣可以反映需要改进的曝光。 </li><li>剔骨的小肠和Treitz韧带远端分。分离小肠从肠系膜使用锋利和温和的钝性分离。确定回盲部和分裂小肠5毫米近端。 </li><li>使用剪刀，打开肠道纵向沿antimesenteric边界在培养皿用10毫升4°C，无菌的Hanks缓冲盐溶液（HBSS，Invitrogen公司，卡尔斯巴德CA）/ 1X抗生素抗真菌（抗反，Invitrogen公司）的解决方案。从打开的肠道清除排泄物温和搅拌，然后打开小肠转移到15ml离心管中，用10毫升的4℃下，无菌HBSS / 1X防反。 </li><li>打开肠洗净三次，在10毫升的4℃下，无菌HBSS / 1X在试管中的抗 - 抗。每次洗涤可以进行轻度的15毫升的试管摇动30秒。振荡后，在肠道组织下沉到试管底部。丢弃浮的材料，它是间充质碎片。将洗涤液仔细吸管。 </li><li>鱼糜培养皿中洗涤过的小肠用10ml 4℃，无菌HBSS / 1X抗 - 抗小于1毫米见方的小块，用一个剪刀。收集碎的材料与一个自动移液管，将其放置到试管中。 </li><li> 8分钟，在500rpm离心管。丢弃上清，它含有的脂肪和间充质。 </li><li>消化剁碎，洗涤后的物质用10ml无菌HBSS / 1X防反加0.125毫克/毫升分散酶（Invitrogen公司）和800单位/米L胶原酶1型（沃辛顿，新泽西州莱克伍德）。为了制备40毫升的消化溶液，称出分散酶，胶原酶142毫克的5毫克，并添加无菌HBSS中高达40毫升的体积。准备新鲜每次类器官单位准备的解决方案，并保持在4°C，直到准备使用。从步骤1.8添加的消化溶液直接沉淀。 </li><li>孵育试管含有碎消化溶液在37℃下20分钟，洗涤后的物质。 </li><li>检索试管中，并进一步扰乱消化的组织与10毫升移液管研制。重复20〜50次之间，直到得到均匀的外观。 </li><li>离心试管中，以800rpm的转速进行5分钟。弃上清，其中包含单个细胞。 </li><li>停止4°C，无菌Dulbecco改良的Eagle培养基（DMEM，Invitrogen）中加10％体积/体积的热灭活的胎儿牛血清（HI-FBS，Invitrogen公司）用10毫升的消化反应。重悬PELLEt和动摇管。 </li><li>离心试管中，以800rpm的转速进行5分钟。仔细取出上清液，用自动移液管，直到最后几滴。最后几滴使用一次性塑料移液管，以避免再悬浮的颗粒。 </li></ol> 2。载入聚羟基乙酸支架<ol><li>表格2毫米长，5毫米外径圆柱支架从非织造布聚乙二醇酸（板材厚度2毫米，60毫克CM-3容重，孔隙率&gt; 95％，康考迪亚纤维，考文垂RI）参考文献。 4。 </li><li>修剪用70％乙醇在蒸馏水中制备用剪刀一次性1000微升的移液管尖端的前端2毫米。 </li><li>成4 - 孔培养板中，将支架。装入类器官的单位上的支架与1000微升的移液管，第一入管腔，然后到的外表面。使用的钳子，以确保涂层的管腔。不要破坏或破开的圆柱形状的聚合物。 </李&gt; </ol> 3。植入主机鼠标<ol><li>使用同系主机鼠标，在相同的背景下，如果有捐助。否则，采用免疫功能低下的非肥胖型糖尿病/重症联合免疫缺陷或NOD / SCID动物（杰克逊实验室，萨克拉门托CA）。 </li><li>异氟醚诱导全身麻醉。剃须，准备和悬垂鼠标的腹部。 </li><li>使一个5毫米的正中切口，获取入口至腹膜腔。仔细剔骨的大网膜。将加载到大网膜和聚合物包装与组织。不要撕大网膜。 </li><li>用5-0 monocryl缝合固定聚合物网膜。轻轻地更换网膜包裹的聚合物进入它的解剖位置。 </li><li>关闭腹壁切口用4-0薇乔缝线层。执行封的肌肉和照顾，不损害以下的切口腹腔脏器。使用间断缝合皮肤。 </li><li>管理术后镇痛2毫克/千克酮洛芬（Ketofen，Fort Dodge动物保健），在无菌水作为相邻切口皮下水疱。动物应每天进行评价，如果动物展示疼痛或痛苦的迹象，一个额外剂量的酮洛芬，可以在手术后第2天给药。术后第3天，应完全康复的动物，没有证据的疼痛或痛苦。如果疼痛或痛苦继续在手术后第3天，这被认为是不正常的和应该在的的IACUC和动物保健设施协议。 </li><li>允许鼠标休养，组织工程小肠生长四周。给动物自由采食啮齿动物的食物（实验室的饮食营养，5001，PMI圣路易斯MO）和水Septra 200毫克/ 40毫克每毫升（5高科医药品，鬼哭神嚎NY）在1:100稀释。 </li></ol> 4。收成<ol><li>呼玛植入4周后，nely安乐死的宿主动物。 </li><li>重新打开原切口，并反映在皮肤头侧，以方便到腹腔。 </li><li>打开的肌肉层，并确定组织工程作为一个全球组织结构。 </li><li>以粘连，腹腔脏器的结构，使用的是锋利的解剖。 </li><li>固定结构在福尔马林后石蜡安装的，或用新鲜的实时PCR或分离蛋白等生化分析的组织。 </li></ol>

<ol>
	<li>Grikscheit, T. C., Siddique, A., Ochoa, E. R., 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=Tissue-engineered+small+intestine+improves+recovery+after+massive+small+bowel+resection.">Tissue-engineered small intestine improves recovery after massive small bowel resection.</a> Ann. Surg. 240, 748-754 (2004).</li><li>Wales, P. W., Christison-Lagay, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+bowel+syndrome:+epidemiology+and+etiology.">Short bowel syndrome: epidemiology and etiology.</a> Sem. Ped. Surg. 19, 3-9 (2010).</li><li>Evans, G. S., Flint, N., Somers, A. S., 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=The+development+of+a+method+for+the+preparation+of+rat+intestinal+epithelial+cell+primary+cultures.">The development of a method for the preparation of rat intestinal epithelial cell primary cultures.</a> J. Cell Sci. 101, 219-231 (1992).</li><li>Sala, F. G., Matthews, J. A., Speer, A. 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=A+multicellular+approach+forms+a+significant+amount+of+tissue-engineered+small+intestine+in+the+mouse.">A multicellular approach forms a significant amount of tissue-engineered small intestine in the mouse.</a> Tiss. Eng. Part A. 17, 1841-1850 (2011).</li><li>Speer, A. L., Sala, F. G., Matthews, J. A., Grikscheit, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+tissue-engineered+stomach+demonstrates+epithelial+differentiation.">Murine tissue-engineered stomach demonstrates epithelial differentiation.</a> J. Surg. Res. 171, 6-14 (2011).</li><li>Haxhija, E. Q., Yang, H., Spencer, A. U., 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=Intestinal+epithelial+cell+proliferation+is+dependent+on+the+site+of+massive+small+bowel+resection.">Intestinal epithelial cell proliferation is dependent on the site of massive small bowel resection.</a> Pediatr. Surg. Int. 23, 379-390 (2007).</li><li>Zhao, L., Cheng, Z., Dhall, D., 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=A+novel+corrective+pullthrough+surgery+in+a+mouse+model+of+Hirschsprung's+disease.">A novel corrective pullthrough surgery in a mouse model of Hirschsprung's disease.</a> J. Pediatr. Surg. 44, 759-766 (2009).</li><li>Petrosyan, M., Guner, Y. S., Williams, 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=Current+concepts+regarding+the+pathogenesis+of+necrotizing+enterocolitis.">Current concepts regarding the pathogenesis of necrotizing enterocolitis.</a> Ped. Surg. Int. 25, 309-318 (2009).</li><li>Shew, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+concerns+in+malrotation+and+midgut+volvulus.">Surgical concerns in malrotation and midgut volvulus.</a> Ped. Radiol. 39, S167-S171 (2009).</li><li>Sampietro, G. M., Corsi, F., Maconi, G., 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=Prospective+study+of+long-term+results+and+prognostic+factors+after+conservative+surgery+for+small+bowel+Crohn's+disease.">Prospective study of long-term results and prognostic factors after conservative surgery for small bowel Crohn's disease.</a> Clin. Gastroenterol. Hepatol. 7, 183-191 (2009).</li><li>Klempnauer, J., Grothues, F., Bektas, H., Pichlmayr, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+results+after+surgery+for+acute+mesenteric+ischemia.">Long-term results after surgery for acute mesenteric ischemia.</a> Surgery. , 121-239 (1997).</li><li>Fitzgibbons, S. C., Jones, B. A., Hull, M. A., 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=Relationship+between+biopsy-proven+parenteral+nutrition-associated+liver+fibrosis+and+biochemical+cholestasis+in+children+with+short+bowel+syndrome.">Relationship between biopsy-proven parenteral nutrition-associated liver fibrosis and biochemical cholestasis in children with short bowel syndrome.</a> J. Ped. Surg. 45, 95-99 (2010).</li><li>Spencer, A. U., Kovacevich, D., McKinney-Barnett, 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=Pediatric+short+bowel+syndrome:+the+cost+of+comprehensive+care.">Pediatric short bowel syndrome: the cost of comprehensive care.</a> Am. J. Clin. Nutr. 88, 1552-1559 (2008).</li><li>Kato, T., Tzakis, A. G., Selvaggi, G., 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=Intestinal+and+multivisceral+transplantation+in+children.">Intestinal and multivisceral transplantation in children.</a> Ann. Surg. 243, 756-766 (2006).</li><li>Reyes, J., Bueno, J., Kocoshis, S., 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=Current+status+of+intestinal+transplantation+in+children.">Current status of intestinal transplantation in children.</a> J. Ped. Surg. 33, 243-254 (1998).</li></ol>

没有利益冲突的声明。

我们提出了一个协议，用于组织工程在小鼠小肠类器官单位支架的方法。最关键的步骤是类器官单位准备。必须小心，以充分清洗和机械处理的组织，但等于必须小心不overdigest，或overtriturate的类器官单位消化后进行（步骤1.11）。类器官单位如果做到这一点，可以减少到单细胞，这可能会丢失在步骤1.12的上清液，并不大可能生存产生组织工程小肠，作为肠干细胞小生境将不被保存在这的情况下。...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> HBSS </td><td> Gibco公司</td><td> 114170-112 </td><td></td></tr><tr><td>抗生素抗真菌100X的</td><td> Invitrogen公司</td><td> 15240-062 </td><td></td></tr><tr><td>中性蛋白酶</td><td> Gibco公司</td><td> 17105-041 </td><td></td></tr><tr><td>胶原酶1 </td><td>沃辛顿</td><td> LS004194 </td><td></td></tr><tr><td> DMEM高糖1X </td><td> Gibco公司</td><td> 11995-065 </td><td></td></tr><tr><td>热灭活FBS </td><td> Invitrogen公司</td><td> 16140-071 </td><td></td></tr><tr><td> Biofelt 100％PGA </td><td>康医疗</td><td> FELT01-1005 </td><td>对于聚合物的制备参考文献。4 </td></tr><tr><td>聚-L-乳酸</td><td> Durect </td><td> B6002-1 </td><td>对于聚合物的制备参考文献。 4 </td></tr><tr><td> I型胶原蛋白，鼠尾</td><td> Sigma-Aldrich公司</td><td> C3867-1VL </td><td>对于聚合物的制备参考文献。 4 </td></tr><tr><td>苯酮苯丙酸100毫克/毫升</td><td> Fort Dodge动物保健</td><td> 71-KETOI-100-50 </td><td></td></tr><tr><td> LabDiet 5001啮齿动物（译文） </td><td> LabDiet </td><td> 5001 </td><td></td></tr><tr><td> septra 200毫克/ 5毫升，USP 40毫克每</td><td>高科医药品</td><td> 50383-824-16 </td><td></td></tr><tr><td>异氟醚，USP </td><td>凤凰制药</td><td> 57319-507-06 </td><td></td></tr></tbody></table>

tissue engineering of the intestine in a murine model

组织工程化小肠（TESI）已经成功地被用于拯救Lewis大鼠大量小肠切除后，在40天内恢复到术前的权重。1在人类中，大量小肠切除会导致短肠综合征，功能性吸收不良国家赋予重大的发病率，死亡率和医疗费用，包括肠外营养的依赖，肝功能衰竭和肝硬化，需要multivisceral器官移植。本文中，我们描述和记录我们的协议在小鼠模型中创建组织工程小肠一个多细胞类器官单位支架的方法。类器官单位是多细胞聚集来自肠道粘膜与间充质元素，包含3之间的关系，保留了肠道干细胞小生境。在目前和未来的研究中，我们的技术过渡到的鼠标将允许利用转基因工具，可以在这个物种在TESI形成的过程进行调查。5免疫功能低下小鼠品系的可用性也将允许我们应用该技术对人体肠道组织和优化形成的人TESI作为小鼠异种移植前的过渡到人类。我们的方法，采用良好生产规范（GMP）试剂和材料已经被批准用于人类患者，因此提供了一个重要的方法，依赖于脱细胞动物组织的优势。这种方法的最终目标是将其翻译给人类作为再生医学的短肠综合征的治疗策略。

图1示出了一个整体的协议架构的记录在这里。此协议的最终结果是一个球形或球状结构的组织工程小鼠小肠的管腔，粘膜，粘膜下层，和周围肌层。 图2A示出一个典型的地球比较起始聚合物支架。 图2B中显示的相同的构造与急剧双瓣壳，以揭示其管腔。 图3演示了一个苏木精/伊红染色的石蜡安装截面一个典型的成功构建体，4周后的潜伏期。在?...

Watch this Scientific Journal Video about Tissue Engineering of the Intestine in a Murine Model at JoVE.com

Tissue Engineering of the Intestine in a Murine Model

本文及随附视频我们的协议，用于生成在小鼠小肠组织工程，使用类器官单位支架的方法。

小鼠模型肠组织工程

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

tissue-engineering-of-the-intestine-in-a-murine-model

Research

JoVE Journal

Bioengineering

14.8K 次观看.  Keck School of Medicine of the University of Southern California. 本文及随附视频我们的协议，用于生成在小鼠小肠组织工程，使用类器官单位支架的方法。

视频: Tissue Engineering of the Intestine in a Murine Model

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 ...

科研

生物工程

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 ...

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

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

工程骨骼肌组织中的小鼠成肌细胞的内皮祖细胞与应用电刺激

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

组织工程生物反应器的双轴机械装载设计

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

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 ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

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. ...

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 ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

在小型动物模型的动静脉（AV）循环研究血管生成和血管组织工程

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

用运动调节血清治疗韧带结构：转化组织工程模型

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...