作者想感谢杰克Milwid的体外灌注腔设计。这项工作是由来自美国国立卫生研究院，R01DK59766和R01DK084053授予我，R00DK080942 KU，美国国家科学基金会CBET - 0853569 KU和施赖纳斯BEU儿童医院（批准号：8503）的支持。我们也承认支持和施赖纳斯儿童医院。

1。肝脱细胞<ol><li>大鼠肝门静脉插管和18号导尿管收获。让劣势和上腔静脉开放。器官的水分保持在磷酸盐缓冲生理盐水（PBS）的一个10厘米的培养皿中。 </li><li>设置灌注系统，包含8升水库，蠕动泵和泡沫陷阱。 </li><li>填写灌注系统，用磷酸盐缓冲液，保持10分钟运行。填写成10厘米的培养皿中的PBS和减少流量的磷酸盐缓冲生理盐水1毫升/分钟。 </li><li>仔细收获肝脏转移到磷酸盐缓冲生理盐水填充10厘米的培养皿。 </li><li>继续用PBS灌注过夜。 </li><li>开始灌注0.01％（W / V）十二烷基硫酸钠（SDS），蒸馏水5分钟。 </li><li>用PBS灌注1小时。 </li><li>重复步骤1.6和1.7三个次以上，每次增加10，15和20分钟SDS灌注时间。 </li><li>继续灌注24小时为0.01％（W / V）SDS。 </li><li>继续0.1％（W / V）SDS灌注24小时。 </li><li>与0.2％（W ​​/ V）SDS灌注3小时。 </li><li>与0.5％（W / V）SDS灌注3小时。 </li><li>用蒸馏水灌注15分钟。 </li><li> 1％（W / V）蒸馏水海卫在X - 100的灌注30分钟，以消除任何约束的核酸。 </li><li>洗脱细胞肝矩阵（DLM），2小时，用PBS灌注。 </li><li>可选：除位数肺叶切除所有的肺叶。 </li><li>在PBS浸泡在摄氏4度，直到准备使用（图1）在清洁和密封的Petri菜店的DLM的。 </li><li>要消毒的DLM：1）用无菌PBS包含在摄氏4度0.1％（V / V）过氧乙酸和4％（V / V）乙醇和3个小时的孵化的冲洗DLM 2）用无菌PBS的2倍。 3）用无菌PBS含2％的青霉素，链霉素，庆大霉素10ug /毫升和2.5微克/毫升两性霉素B店在摄氏4度相同的解决方案，直到准备使用recellularization实验脱细胞肝。 </li></ol> 2。 Recellularization的脱细胞肝基质<ol><li>设置灌注系统灌注室，蠕动泵和无菌条件下的泡沫陷阱。装满200毫升培养基，例如，高糖DMEM（Sigma公司），10％胎牛血清（Hyclone公司），100 U毫升-1青霉素，100微克毫升-1链霉素（Invitrogen）的灌注系统。 </li><li>将脱细胞肝脏灌注室矩阵和连接的DLM，门静脉插管灌注系统，而泵运行在5毫升/分钟，以避免任何气泡的形成。 </li><li>允许通过30分钟的DLM的灌注介质。 </li><li>隔离与至少90％的可行性的成年大鼠原代肝细胞。 </li><li>停止灌注系统的流量，慢慢注入灌注系统，通过50万肝泡沫陷阱（1-3毫升培养基）。 </li><li>开始于10毫升/分钟的流量循环10分钟的中等。 </li><li>重复步骤2.5和2.6，共有200万个单元格，直到DLM（图2）6 。 </li><li>一旦所有的细胞都灌注到DLM的，收集到4个50 mL离心管和离心10分钟600转的灌流。丢弃上清液，收集到一个单一的管的颗粒。确定细胞，并通过台盼蓝拒确定播种效率的可行性。 </li></ol> 3。Recellularized移植肝脏离体培养<ol><li>成立了灌注系统灌注室，蠕动泵，氧合和无菌条件下（图3）的泡沫陷阱。填写与50毫升培养液灌注系统，例如，威廉姆斯“E（Sigma公司），5％胎牛血清（Hyclone公司），0.5 U毫升-1胰岛素（礼来），20纳克毫升-1 EGF（Invitrogen公司） 14纳克毫升-1胰高血糖素（贝德福德实验室），7.5微克毫升-1氢化可的松（Pharmacia公司），100 U毫升-1青霉素，100微克毫升-1链霉素（Invitrogen公司）。 </li><li>放置在灌注室recellularized肝移植和连接的DLM，门静脉插管灌注系统，而泵运行在5毫升/分钟，以避免任何气泡的形成。 </li><li>无菌关闭灌注室和密封紧密，以避免任何文化过程中的泄漏。 </li><li>灌注系统转移到一个孵化器，是37℃和10％CO 2，增加灌注流量为15毫升/分钟。 </li><li>连接氧合95％O 2和5％的CO 2混合气体罐和气体流量设置为0.5升/分钟。这应该实现一个近似的氧分压tely 400毫米汞柱。 </li><li>文化可能会持续长达10天的每日变动与培养基。培养基可每天进行采样监测肝功能的嫁接，如白蛋白，尿素和总胆汁酸的分泌。在文化时期的结束，recellularized肝移植可能是采样的分子和组织学分析。 </li></ol> 4。代表性的成果： 完成脱细胞的老鼠的肝脏大约需要72小时，使用所描述的协议。结果矩阵保留100％的纤维状的胶原蛋白，50％的粘多糖，只有5％的原生肝DNA（ 表1）6。矩阵的血管结构保存腐蚀铸造和扫描电子显微镜分析（图4）6证明。 DLM的血管内微架构的存在有利于它的复育与96％ 的效率和其在体外培养的后续灌注细胞。 recellularized肝移植可能培养10天的体外 ，并显示适当的肝功能确认通过白蛋白，尿素和总胆汁酸的分泌（图5）6。 <img src="/files/ftp_upload/2394/2394fig1.jpg" alt="图1" /> 图1。肝基质脱细胞在脱细胞过程的结束。 （一）（二）全肝切除后的中位叶。 <img src="/files/ftp_upload/2394/2394fig2.jpg" alt="图2" /> 图2。recellularization的DLM的示意图。 <img src="/files/ftp_upload/2394/2394fig3.jpg" alt="图3" /> 图3。灌注recellularized肝移植的体外培养系统设置。 <img src="/files/ftp_upload/2394/2394fig4.jpg" alt="图4" /> 图4。微血管结构被保留在脱细胞肝基质。腐蚀铸一）正常肝脏B）一个脱细胞肝，门户网站（红色）和静脉（蓝色）血管的图像。扫描电子显微镜图像的DLM的C的）船，D）的部分胆管般的小血管（箭头），比例尺（A，B）5毫米（C，D）20微米。 <img src="/files/ftp_upload/2394/2394fig5.jpg" alt="图5" /> 图5。recellularized肝移植肝特异性功能， 在体外灌注培养。一）白蛋白的分泌（P = 0.5249），B）的尿素产量（P = 0.5271）和C），总胆汁酸的分泌（P = 0.0114）。统计分析实验和控制之间的区别做了10天的文化时期，弗里德曼在= 0.01的测试。误差棒代表SEM（N = 3）。 <table border="1"><tbody><tr><td></td><td> 新鲜livera </td><td> 脱细胞肝矩阵 A </td><td rowspan="2"> P -值 </td><td rowspan="2"> 鲜猪肝％ </td></tr><tr><td></td><td> N = 4 </td><td> N = 8 </td></tr><tr><td> 胶原 </td><td rowspan="2"> 0.07 ± 0.01 </td><td rowspan="2"> 0.08 ± 0.03 </td><td rowspan="2"> 0.56 </td><td rowspan="2"> 114％ </td></tr><tr><td> （每克肝毫克） </td></tr><tr><td> 粘多糖 </td><td rowspan="2"> 73.1 ± 6.7 </td><td rowspan="2"> 34.2 ± 2.9 </td><td rowspan="2"> 0.004 </td><td rowspan="2"> 47％ </td></tr><tr><td> （每克肝毫克） </td></tr><tr><td> 的DNA </td><td rowspan="2"> 14.9 ± 5.6 </td><td rowspan="2"> 0.44 ± 0.08 </td><td rowspan="2"> 3.3 10-5 </td><td rowspan="2"> 2.9％ </td></tr><tr><td> （每克肝毫克） </td></tr></tbody></table> 表1。生化成分的脱细胞肝基质相比，原生肝。 一个数值表示为平均± SEM

<ol>
	<li>Kulig, K. M., Vacanti, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+tissue+engineering.">Hepatic tissue engineering.</a> Transpl Immunol. 12, 303-310 (2004).</li><li>Lutolf, M. P., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biomaterials+as+instructive+extracellular+microenvironments+for+morphogenesis+in+tissue+engineering.">Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering.</a> Nature biotechnology. 23, 47-55 (2005).</li><li>Dahl, S. L., Koh, J., Prabhakar, V., Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+native+and+engineered+arterial+scaffolds+for+transplantation.">Decellularized native and engineered arterial scaffolds for transplantation.</a> Cell Transplant. 12, 659-666 (2003).</li><li>Schechner, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engraftment+of+a+vascularized+human+skin+equivalent.">Engraftment of a vascularized human skin equivalent.</a> FASEB J. 17, 2250-2256 (2003).</li><li>Gilbert, T. W., Sellaro, T. L., Badylak, S. F., F, S. <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>Uygun, B. E. <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></ol>

这里描述灌注脱细胞方法产生一个全肝支架具有相同的总体结构和原生肝血管微架构。支架有一个类似的原生的肝细胞外基质组成。 recellularization方法实现高效率和细胞的细胞仍然在测试期间在体外培养可行和功能的支架复育。到recellularized移植肝非实质细胞此外，我们设想，可作为一个平台，用于研究药物代谢，再生和发展，如各种肝功能的recellularized肝移植。此外，可能会发现recellulariz...

A correction was made to <a href="http://www.jove.com/Details.php?ID=2394">Decellularization and Recellularization of Whole Livers</a>. There was an error with an author's name. The author's last name had a typo and was corrected to:
Nima Saeidi
instead of:
Nima Saedi.

Erratum: Decellularization and Recellularization of Whole Livers

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<td>Sodium dodecyl sulfate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>L4390</td>
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<td>Triton X-100</td>
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<td>Sigma-Aldrich</td>
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<td>Masterflex L/S Digital Drive</td>
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decellularization and recellularization of whole livers

肝脏是一个复杂的器官，这就需要不断提供营养和氧气和废物清除灌注为了生存 1 。努力重建或模仿肝脏显微结构与向上的方法，利用组织工程和微细加工技术的理由都没有成功至今，由于这一设计挑战。此外，用于创建为肝组织工程中的应用支架的合成生物材料由于缺乏特定的细胞结合的图案，将促使正常的细胞功能2，在诱导组织再生和修复在很大程度上受到了限制。脱细胞原生组织，如血管3和皮肤4另一方面发现在组织工程中的许多应用，并提供了一个切实可行的解决办法的一些挑战。脱细胞原生基质的优点是，它保留了，在一定程度上，原来的组成和微观结构，从而增强细胞附着和重组5。 在这项工作中，我们描述的方法来执行灌注脱细胞的肝脏，这样一个完整的肝bioscaffold保留主要血管的结构得到。此外，我们描述recellularize与成人原发性肝细胞的这些bioscaffolds，创造了肝移植，体外功能的方法，和体内移植所需的船只。

Watch this Scientific Journal Video about Decellularization and Recellularization of Whole Livers at JoVE.com

Decellularization and Recellularization of Whole Livers

灌注脱细胞是一种新型的的技术来生产全肝支架，保留器官的细胞外基质成分和微。在这里，准备灌注脱细胞与肝细胞和后续复育的整个器官支架的方法描述。使用这种技术，可以生成功能和移植肝移植。

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 ...

decellularization-and-recellularization-of-whole-livers

Research

JoVE Journal

Bioengineering

21.9K 次观看.  Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children. 灌注脱细胞是一种新型的的技术来生产全肝支架，保留器官的细胞外基质成分和微。在这里，准备灌注脱细胞与肝细胞和后续复育的整个器官支架的方法描述。使用这种技术，可以生成功能和移植肝移植。

视频: Decellularization and Recellularization of Whole Livers

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

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 ...

科研

生物工程

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 ...

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

胎儿和成人心脏组织外植体作为三维体外研究平台的可比较去角质

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

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 ...

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells.
This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor.
Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor.

在定制灌注生物反应器中获取和灌注-脱细胞猪血管化皮瓣

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction ...