作者要感谢布罗根，游客米歇尔·韦弗，和Kristen利珀特。资助这项研究是由美国国立卫生研究院资助R03EB009237，以及美国国立卫生研究院的培训资助T32EB001026-06来自国家生物医学成像和生物工程和T32HL076124-05。

1。组织制备及实验设置<ol><li>收获的猪器官后，立即从屠宰场或研究机构的安乐死，并冲洗掉多余的血液。修剪多余的脂肪和组织的心脏，心房和主动脉完整。修剪掉的脂肪分离主动脉肺动脉。如果有任何削减组织，适当地丢弃。 </li><li>单独把每个心，在冷冻纸和存储所有组织在-80°C冷冻至少24小时，以确保完全冻结。 </li><li>当您准备使用（通常不超过3个月），解冻一个完整的冷冻猪心脏在一夜之间淹没在4 L烧杯中的第1类水在4°C </li><li>的心完全解冻后，拍打心脏干，权衡心，并记录重量。市场权重猪的心脏重约375-450克。 </li><li>连接尺寸18的Masterflex管道将带刺的减速和¼"带刺的减速。油管内的主动脉。将2软管夹或主动脉周围的安全拉链关系，只是下面的头臂干。减速器和油管必须保持高于主动脉瓣，所以可以灌注冠状动脉（图1）。 </li><li>使用30或60毫升的注射器，以填补管道与I类水。插入管内的盒的的Masterflex滚子泵，在其近似中点。淹没的流入端的油管在一个4升的烧杯中，用2.5升的水填充的底部和固定管。 </li><li>在装满水的烧杯中，将心和总理的泵，以消除气泡。如果观察到气泡来自管子插入主动脉，主动脉可能需要额外的关系被重新定位或固定。一个密闭的密封是非常重要的，脱细胞过程（图2）的过程中保持足够的压力。 </li><li>将4升的烧​​杯中含有3升的0.2％Trypsin/0.05％EDTA/0.05％NaN的3溶液搅拌板和预热至37°C，在准备的脱细胞处理过程中。 </li></ol> 2。组织冲洗<ol><li>设置泵的流速为400毫升/分钟，确保了正确的油管尺寸被选定。冲洗心脏I类水15-25分钟。由于泵启动时，心脏肿胀和渗出血从心室。新鲜的溶液应被取代的每隔5-10分钟，或根据需要基于从心脏的血液量。如果血液不从心，必要的调整管和夹子三分结构的费托。 </li><li>停止泵和转移心脏充满2X磷酸盐的一个单独的烧杯中，缓冲液（PBS）。后管被浸没在溶液中，启动泵，增加至700毫升/分钟的流速。心应保持在溶液中15分钟，每5分钟改变溶液。每个解决方案的变化需要泵暂时停止，而移动的组织和油管到新的烧杯中。 </li><li>转移心脏I型水10分钟，并增加至750毫升/分钟的流速。 </li></ol> 3。脱细胞和解决方案灌注<ol><li>转移的心脏的烧杯中含有0.2％的Trypsin/0.05％EDTA/0.05％NaN 3的在37°C.泵的转速增加至1,200毫升/分钟，并启动泵。使用搅拌棒的底部放置在烧杯中，烧杯中的溶液循环。的心脏的0.2％Trypsin/0.05％EDTA/0.05％NaN 3的溶液在37℃下应保持在一共有三个小时。 1小时后，增加泵速度为1500毫升/分钟。一小时后，泵的转速增加至1,800毫升/分钟。该组织正在慢慢增加灌注速度条件的组织和防止血管破裂。的心脏会发胀而近一倍的大小，在此步骤中的协议。该组织将失去其自然发展从心房到顶点的颜色，整个日Ë协议（图3）。 </li><li>每个溶液灌注后，一个两步骤进行漂洗，以除去细胞碎片，化学残余物中，援助细胞裂解。每次漂洗由I型由10分钟的冲洗水，接着用2X PBS溶液，在室温下在10分钟的冲洗。每次洗脸的解决方案，从原来的烧杯中去除，加入冲洗液，循环灌流液在烧杯中被淹没的心。 0.2％Trypsin/0.05％EDTA/0.05％NaN 3的溶液后，灌注在1,900 ml / min的水，然后灌注2X PBS中在1950毫升/分钟。 </li><li>转移心3％的Triton X-100/0.05％EDTA/0.05％NaN 3的在室温下的溶液。泵的转速增加至2,000毫升/分钟，一小时灌注溶液。取出从烧杯中的溶液，并用新鲜的溶液替换，增加泵的转速为2100毫升/分钟，和一个额外的一个半小时，灌注的新鲜溶液，使总的时间3％的Triton X-100/0.05％EDTA/0.05％NaN 3的 2.5小时。 </li><li>在2150毫升/分钟和2倍PBS冲洗组织中的I类水2180毫升/分钟，每次10分钟。 </li><li>在室温下4％脱氧胆酸钠溶液转移心脏。泵的转速增加至2,200毫升/分钟和3小时灌注溶液。 </li><li>在I类水冲洗组织和2X PBS在2200毫升/分钟的各15分钟，改变各溶液5-10分钟后的解决方案。所描述的灌注步骤可能被分割多天通过执行两次冲洗步骤和存储心脏附有管，在4℃下过夜，并淹没在I类水。 </li><li>翌日，执行与I类水冲洗5分钟在750毫升/分钟，随后于1500毫升/分钟，用1X PBS冲洗5分钟。然后继续该协议可以在所描述的适当的溶液中的流速。 </li></ol> 4。消毒和最后的处理<ol><li>的心脏转移到0.1％perac的中的客位酸/ 4％的乙醇溶液和1.5小时为2200毫升/分钟的灌注溶液。 </li><li>组织的最终漂洗都执行在2200毫升/分钟。组织1X PBS灌注15分钟，然后用两个5分钟在I类水清洗。重复这一系列的漂洗一次，以完成该溶液灌注过程。 </li><li>将泵关闭，并从溶液中取出心脏排的心。从主动脉切的联系，删除所有的管道，和一个空的烧杯中，将心排1小时。过量的液体将需要定期排出。奠定了心脏的吸水垫，以便完全释放的心（图4）。 </li><li>后，大部分的水被除去，记录心脏的细胞外基质（C-ECM）的重量。心脏可预期的脱细胞过程中失去其初始重量的大约20-25％。 </li><li>解剖左，右心室，以及用于DNA quantificati室间隔和组织学处理，以确认完成脱细胞的组织（图5）。 </li><li>冷冻干燥前的至少2小时，在-80℃下冻结的C-ECM。 </li></ol>

<ol>
	<li>Ott, H. C., 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=Regeneration+and+orthotopic+transplantation+of+a+bioartificial+lung.">Regeneration and orthotopic transplantation of a bioartificial lung.</a> Nat. Med. 16, 927-933 (2010).</li><li>Ott, H. C., 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=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nat. Med. , (2008).</li><li>Petersen, T. H., 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+lungs+for+in+vivo+implantation.">Tissue-engineered lungs for in vivo implantation.</a> Science. 329, 538-541 (2010).</li><li>Uygun, B. E., 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=Organ+reengineering+through+development+of+a+transplantable+recellularized+liver+graft+using+decellularized+liver+matrix.">Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.</a> Nat. Med. , (2010).</li><li>Wainwright, 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=Preparation+of+cardiac+extracellular+matrix+from+an+intact+porcine+heart.">Preparation of cardiac extracellular matrix from an intact porcine heart.</a> Tissue Eng. Part C Methods. 16, 525-532 (2010).</li><li>Crapo, P. M., Gilbert, T. W., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+tissue+and+whole+organ+decellularization+processes.">An overview of tissue and whole organ decellularization processes.</a> Biomaterials. 32, 3233-3243 (2011).</li><li>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=Strategies+for+tissue+and+organ+decellularization.">Strategies for tissue and organ decellularization.</a> Journal of cellular biochemistry. , (2012).</li><li>Gilbert, T. W., Sellaro, T. L., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+of+tissues+and+organs.">Decellularization of tissues and organs.</a> Biomaterials. 27, 3675-3683 (2006).</li><li>Akhyari, P., 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+quest+for+an+optimized+protocol+for+whole-heart+decellularization:+a+comparison+of+three+popular+and+a+novel+decellularization+technique+and+their+diverse+effects+on+crucial+extracellular+matrix+qualities.">The quest for an optimized protocol for whole-heart decellularization: a comparison of three popular and a novel decellularization technique and their diverse effects on crucial extracellular matrix qualities.</a> Tissue Eng. Part C Methods. 17, 915-926 (2011).</li><li>Weymann, 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=Development+and+evaluation+of+a+perfusion+decellularization+porcine+heart+model--generation+of+3-dimensional+myocardial+neoscaffolds.">Development and evaluation of a perfusion decellularization porcine heart model--generation of 3-dimensional myocardial neoscaffolds.</a> Circulation journal : official journal of the Japanese Circulation Society. 75, 852-860 (2011).</li><li>Cortiella, J., 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=Influence+of+acellular+natural+lung+matrix+on+murine+embryonic+stem+cell+differentiation+and+tissue+formation.">Influence of acellular natural lung matrix on murine embryonic stem cell differentiation and tissue formation.</a> Tissue Eng. Part A. 16, 2565-2580 (1089).</li><li>Remlinger, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrated+xenogeneic+decellularized+tracheal+matrix+as+a+scaffold+for+tracheal+reconstruction.">Hydrated xenogeneic decellularized tracheal matrix as a scaffold for tracheal reconstruction.</a> Biomaterials. 31, 3520-3526 (2010).</li><li>Sellaro, T. L., Ravindra, A. K., Stolz, D. B., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+hepatic+sinusoidal+endothelial+cell+phenotype+in+vitro+using+organ-specific+extracellular+matrix+scaffolds.">Maintenance of hepatic sinusoidal endothelial cell phenotype in vitro using organ-specific extracellular matrix scaffolds.</a> Tissue Eng. 13, 2301-2310 (2007).</li><li>Wainwright, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Right+ventricular+outflow+tract+repair+with+a+cardiac+biologic+scaffold.">Right ventricular outflow tract repair with a cardiac biologic scaffold.</a> Cells, tissues, organs. 195, 159-170 (2012).</li></ol>

吉尔伯特博士的科学咨询委员会在ACell，公司正在做的研究，并在最近成为研究和开发的副总裁。 ACell公司销售膀胱矩阵，在本研究中有没有商业利益。

目前的研究描述的一致和有效的方法去细胞的猪的心脏。该协议是一个修改到先前公布的报告，包括更长的曝光时间，流量和压力增加，提供了更多的可重复的结果。由此产生的脱细胞组织满足所有成功的去细胞组织公布的标准。频繁的解决方案进行了修改，以限制重新引入蜂窝材料的组织，每脱细胞处理剂接触到的持续时间减少到最低限度，以减少对ECM的不利影响。的协议的开始阶段期间，灌注率逐渐增加组织的条件，允许更高的流速，在协议的后期阶段。组织在早期阶段没有空调的心脏，血管破裂，使心脏灌注是不可能的。原中网使用，因为它的效率，并没有声称其优于其他协议。可能令人信服地脱细胞处理剂和灌注率的精确组成的变化，产生的协议具有更好的机械或生物学特性，但对心脏的代理商交付的一般原则是适用的。 保存本机的心脏的三维结构是由于整个脱细胞处理协议执行的几个程序。首先，该组织在抵达时被削减和冻结。凝固促进细胞溶解，并且是重要的预调节组织灌注循环。该组织被彻底检查削减2厘米的完整完好主动脉高级的主动脉瓣。如果有任何心包或心外膜被切断，被丢弃的器官，因为灌流没有达到下游地区的心脏和心脏未完全脱细胞。接下来，该组织完全解冻I型水前使用。水允许组织放宽，因为它解冻并也辅助心脏内除去残余血块。最后，作为管子插入，护理采取确保主动脉瓣保持不变，以便它形成一个水密的密封，从而使周围的油管保持适当的压力，并且该溶液进入冠状动脉。 每脱细胞处理协议完成后，完成了一系列的质量控制措施，以确保完全去除细胞物质。验证协议消除组织学染色的细胞核，目前的研究表明，小于50 ng的DNA是本每毫克干重的组织，并且，任何DNA为大小小于200 bp的6。此前公布的心脏去细胞方法表现出类似的脱细胞DNA染色和定量2,5,9,10。在这些研究中使用类似的酶和清洁剂的治疗完全脱细胞处理完成。然而，在本研究中，暴露于每种化学品的长度增加，有更多的溶液，流速增加。本协议化学漂洗的长度也增加，可能导致更有效地去除从细胞外基质的化学残留物。 Recellularization脱细胞的大鼠心脏与心脏特异性细胞在体外 2,5取得了可喜的成果。全器官灌注脱细​​胞原生血管的维护，这是至关重要的组织在recellularization允许。固有的生长因子，基质蛋白和三维纤维结构，也促进了适当的细胞附着，迁移和信令重组收缩的心肌组织。猪心脏extracellu拉尔矩阵将是更加困难以recellularize由于棚架的大小和数量的细胞所必需的适当的细胞通信和养分运输。然而，切从心脏矩阵补丁在体内应用中可能是有用的。多项研究表明使用站点特定的矩阵重建受损的组织在动物模型中11-13的潜在优势。因此，来自心脏组织的胞外基质支架心肌重建应用中是可取的。心脏组织的可能存在的固有结构优于另一个器官或人造的生物材料是来自于一个ECM支架。一个站点特定的支架可支持宿主细胞的浸润和促进建设性的重塑反应，而不是形成疤痕组织。迄今为止，心脏ECM的补丁已经在体内研究，重建心肌壁14中创建的一个缺陷。未来学生模具将在体外进行检查支架支持矩阵接种和培养的心肌细胞的能力。本文描述的方法也可能适用于人类的心灵去细胞的。 总之，猪的心脏去细胞是可能的方法是直截了当的。续调查这种材料将提供洞察其潜在的临床用途。

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>胰蛋白酶</td><td> Gibco公司</td><td> 15090 </td><td></td></tr><tr><td> EDTA </td><td>费舍尔</td><td> BP120-500 </td><td></td></tr><tr><td> NaN的3 </td><td>西格玛</td><td> S2002-500G </td><td></td></tr><tr><td>的Triton X-100的</td><td>西格玛</td><td> X100-1L </td><td></td></tr><tr><td> 10X PBS </td><td>费舍尔</td><td> BP399-20 </td><td></td></tr><tr><td>脱氧胆酸钠</td><td>西格玛</td><td> D6750-500G </td><td></td></tr><tr><td>过氧乙酸</td><td> Pfaltz和Bauer </td><td> P05020 </td><td> 35％CAS＃79-21-0 </td></tr><tr><td>乙醇</td><td> Pharmco </td><td> 111000200 </td><td></tD&gt; </tr><tr><td>的Masterflex泵驱动器</td><td>科尔帕默</td><td> SI-07524-50 </td><td></td></tr><tr><td>的Masterflex管</td><td>科尔帕默</td><td> 96400-18 </td><td>尺寸18 </td></tr><tr><td>刺减速机</td><td>科尔帕默</td><td> EW-30612-20 </td><td></td></tr><tr><td> 4L烧杯</td><td> Fisher Scientific则</td><td> 02-540T </td><td></td></tr></tbody></table>

procedure for decellularization of porcine heart by retrograde coronary perfusion

灌注的整体器官脱细胞最近获得了为手段，以创建特定的细胞外基质支架，组织工程领域的兴趣，同时很大程度上保留了原生架构的支架。至目前为止，这种方法已经被使用在各种不同的器官系统，包括心脏，肺，肝1-5。没有一个方便的血管网组织往前脱细胞方法都依赖于长时间暴露的组织的解决方案的清洁剂，酸或酶处理，作为一个装置，以除去细胞和核的组件，从周围的细胞外环境6-8。然而，这些方法的铰链的有效性的解决方案的能力后，通过扩散渗透到组织。相比之下，通过自然器官灌注血管系统有效地减少了扩散距离，便利的交通decellularizati代理商进入组织和细胞成分的组织。在这里，我们描述一个的方法完全decellularize通过一个完整的猪心脏冠状动脉逆行灌注。该协议得到充分脱细胞的心肌细胞外基质（ECM）的支架的三维结构体的心脏完好无损。我们的方法，使用了一系列的耦合与高渗和低渗漂洗，以帮助在裂解和去除细胞的酶，洗涤剂，和酸。所使用的协议胰蛋白酶溶液分离后跟的Triton X-100和脱氧胆酸钠的解决方案，以帮助除去多孔材料中的基质细胞。所描述的协议也使用大于2升/分钟的长时间的灌注速度。高流速，加上与解决方案的变化允许运输的代理到组织没有细胞碎片污染和确保有效地冲洗该组织。从National Instruments，简称NI所描述的方法删除了所有核材料维生素E猪心脏组织，创建特定于站点的心脏ECM支架，可用于各种各样的应用。

自然对整个猪心脱细胞的效果的不同而不同的大小的差异，压力，和船只安排。因此，确切的成分派生的细胞外基质支架是不一样的，从心脏到心脏。所描述的协议的完成将产生一个心脏出现白色或半透明状，表明细胞物质的损失。然而，它已被广泛接受的组织可以被视为"脱细胞"的基础上，结合数更多的量化参数8。一个成功的脱细胞协议会产生一个矩阵，每毫克的组织（图6）的双链DNA与小于50毫微克。为了避免矩阵植入后的宿主免疫反应，剩余的DNA还应该包含少于200个碱基对（图7）。为了证实这些发现，苏木精和曙红染色显​​示的情况下，核染色有代表性的部分心室，室间隔（图8）。 Masson三色进一步证实心脏肌肉束和胶原网络保留（图9）的损失。 <img src="/files/ftp_upload/50059/50059fig1.jpg" alt="图1"/> 图1。管的正端插入本机的心脏主动脉。必须保证该管与软管夹或ZIP以上的主动脉瓣的关系，以确保通过冠状动脉灌注。 <img src="/files/ftp_upload/50059/50059fig2.jpg" alt="图2"/> 图2。心被淹没在水中，在4L的烧杯中，气泡，必须从管中取出。如果观察到气泡新兴从主动脉附近的油管，额外的关系必须被用来固定管到t<html>他主动脉，以保​​持足够的压力，在组织中。 <img src="/files/ftp_upload/50059/50059fig3.jpg" alt="图3"/> 图3。解决方案是通过冠状动脉灌注，心脏就会失去其天然色彩，发展从心房到顶点的左右冠状动脉的心脏和本地化。 <img src="/files/ftp_upload/50059/50059fig4.jpg" alt="图4"/> 图4的消毒和冲洗步骤的协议完成后，管子被除去，被放置在一个吸收垫允许多余的水排出的心脏的心脏。这可以确保准确的测量称量的组织时，也允许组织之前放松切片。 PG"ALT ="图5"/&gt; 图5。左心室（LV），右心室（RV），室间隔都从脱细胞心脏组织学处理，冷冻和冷冻干燥，DNA定量。 <img src="/files/ftp_upload/50059/50059fig6.jpg" alt="图6" fo:content-width="4.5in" fo:src="/files/ftp_upload/50059/50059fig6highres.jpg"/> 图6。使用一个小型绿色分析DNA含量的定量分析。 CECM心中的心室从一个DNA含量显着减少相比，本地心室。这个协议所观察到的值等于或低于50毫微克/毫克标准脱细胞组织观察的DNA。 <img src="/files/ftp_upload/50059/50059fig7.jpg" alt="图7"/> 图7的DNA片段的大小，所确定的ethidiu米溴铵胶，有少量DNA脱细胞心室相比，一个膀胱矩阵（UBM）的标准。 <img src="/files/ftp_upload/50059/50059fig8.jpg" alt="图8" fo:content-width="5in" fo:src="/files/ftp_upload/50059/50059fig8highres.jpg"/> 图8。苏木精和曙红染色显示完整的转移核材料的脱细胞处理协议完成后，从心室。 <img src="/files/ftp_upload/50059/50059fig9.jpg" alt="图9" fo:content-width="3in" fo:src="/files/ftp_upload/50059/50059fig9highres.jpg"/> 图9。Masson三色染色A）本机和B）脱细胞心室。

Watch this Scientific Journal Video about Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion at JoVE.com

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

迅速，彻底清除细胞成分通过一个完整的猪心脏逆行灌注的方法。这种方法产生一个站点特定的心脏细胞外基质支架，它有可能用于在多个临床应用。

脱细胞猪心逆行灌注的程序

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

procedure-for-decellularization-porcine-heart-retrograde-coronary

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

23.8K 次观看.  McGowan Institute for Regenerative Medicine. 迅速，彻底清除细胞成分通过一个完整的猪心脏逆行灌注的方法。这种方法产生一个站点特定的心脏细胞外基质支架，它有可能用于在多个临床应用。

视频: Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

整个肝脏的脱细胞和Recellularization

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

科研

生物工程

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

龙工程的程序

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

A Decellularization Methodology for the Production of a Natural Acellular Intestinal Matrix

Successful tissue engineering involves the combination of scaffolds with appropriate cells in vitro or in vivo. Scaffolds may be synthetic, naturally-derived or derived from tissues/organs. The latter are obtained using a technique called decellularization. Decellularization may involve a combination of physical, chemical, and enzymatic methods. The goal of this technique is to remove all cellular traces whilst maintaining the macro- and micro-architecture of the original tissue.
Intestinal tissue engineering has thus far used relatively simple scaffolds that do not replicate the complex architecture of the native organ. The focus of this paper is to describe an efficient decellularization technique for rat small intestine. The isolation of the small intestine so as to ensure the maintenance of a vascular connection is described. The combination of chemical and enzymatic solutions to remove the cells whilst preserving the villus-crypt axis in the luminal aspect of the scaffold is also set out. Finally, assessment of produced scaffolds for appropriate characteristics is discussed.

The article describes a methodology for the production of an acellular matrix from rat intestine. The derivation of intestinal scaffolds is important for future applications in tissue engineering, stem cell biology and drug testing.

一脱细胞方法对天然脱细胞基质肠的生产

Successful tissue engineering involves the  combination of scaffolds with appropriate cells in vitro or in vivo.  Scaffolds may be synthetic, ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

非人灵长类龙脱细胞和Recellularization使用专门的大型器官生物反应器

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

在振荡压力灌流器程序大鼠肝脏的脱细胞

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

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

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

组织工程应用中植物组织去细胞的两种方法

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

Decellularization and Recellularization Methodology for Human Saphenous Veins

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and poor patency. Tissue engineering offers a novel solution to generate personalized grafts with a natural extracellular matrix containing the recipient&#39;s cells using the method of decellularization and recellularization. We show a detailed method for performing decellularization of the human saphenous vein and recellularization by perfusion of peripheral blood. The vein was decellularized by perfusing 1% Triton X-100, 1% tri-n-butyl-phosphate (TnBP) and 2,000 Kunitz units of deoxyribonuclease (DNase). Triton X-100 and TnBP were perfused at 35 mL/min for 4 h while DNase was perfused at 10 mL/min at 37 &#176;C for 4 h. The vein was washed in ultrapure water and PBS and then sterilized in 0.1% peracetic acid. It was washed again in PBS and preconditioned in endothelial medium. The vein was connected to a bioreactor and perfused with endothelial medium containing 50 IU/mL heparin for 1 h. Recellularization was performed by filling the bioreactor with fresh blood, diluted 1:1 in Steen solution, and adding endocrine gland-derived vascular endothelial growth factors (80 ng/mL), basic fibroblast growth factors (4 &#181;L/mL), and acetyl salicylic acid (5 &#181;g/mL). The bioreactor was then moved into an incubator and perfused for 48 h at 2 mL/min while maintaining glucose between 3 - 9 mmol/L. Later, the vein was washed with PBS, filled with endothelial medium and perfused for 96 h in the incubator. Treatment with Triton X-100, TnBP and DNase decellularized the saphenous vein in 5 cycles. The decellularized vein looked white in contrast to normal and recellularized veins (light red). The hematoxylin &#38; eosin (H&#38;E) staining showed the presence of nuclei only in normal but not in decellularized veins. In the recellularized vein, H&#38;E-staining showed the presence of cells on the luminal surface of the vein.

Here, we describe a protocol for the saphenous vein decellularization using detergents and recellularization by perfusion of the peripheral blood and the endothelial medium.

人隐静脉的去细胞和 Recellularization 方法学研究

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and ...

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

倒置方向下整个人的心脏在加压袋内的去细胞化

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

Isolation and Decellularization of a Whole Porcine Pancreas

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an allogeneic or xenogeneic source with human cells. A demonstration of methods for the efficient dissection, decellularization, and recellularization of porcine pancreas might benefit the field. Akin to human pancreases, porcine pancreases have a special anatomical arrangement with three lobes (splenic, duodenal, and connection) rounded by the duodenum and small intestine. The duodenal lobe of the pancreas connects to the duodenum by several small blood vessels. Tissue engineering of the pancreas is complicated because of its exocrine and endocrine nature. In this paper, we show a detailed protocol to dissect the whole porcine pancreas and decellularize it with detergents while saving its structure and some extracellular matrix components. To achieve complete perfusion, the aorta is chosen as inlet and the portal vein as outlet. The other blood vessels (hepatic artery, splenic vein, splenic artery,&#160;mesenteric artery and vein tree) and bile duct are ligated. To prevent the formation of thrombus, the pig is heparinized and, immediately after dissection, the organ is flushed with cold heparin. To inhibit the action of exocrine enzymes, the pancreas decellularization is set at 4 &#176;C. The decellularization is performed by perfusion of Triton X-100, sodium deoxycholate, and deoxyribonuclease, with an intermittent and final extensive washing. With a successful decellularization, the pancreas appears white, and a histological evaluation with hematoxylin and eosin shows an absence of nuclei with a preserved extracellular matrix structure. Thus, the proposed method can be used to successfully dissect and decellularize whole porcine pancreas.

Tissue engineering of the whole pancreas is a challenge because of its exocrine and endocrine functions. We show a method for the dissection of an intact porcine pancreas and the process of successful decellularization by perfusion of detergents Triton X-100, sodium deoxycholate, and deoxyribonuclease.

全猪胰腺的分离与去细胞

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.