Name: procurement and decellularization of rat hindlimbs using an ex vivo perfusion-based bioreactor for vascularized composite allotransplantation
Uploaded: 2022-06-09T15:00:00
Description: Watch this Scientific Journal Video about Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation at JoVE.com

图 3A 是在 BioRender.com 中创建的。

从多伦多总医院研究所获得的尸体雄性刘易斯大鼠（300-430g）用于所有实验。对于所有外科手术，使用无菌器械和用品来维持无菌技术（见 材料表）。所有程序均按照多伦多总医院研究所动物护理委员会的指导方针进行，大学健康网络（加拿大安大略省多伦多市）。共有4个后肢被去细胞化。
1.术前准备
<ol><li>准备 50 mL 的 5% 肝素化盐水。从 50 mL 盐?...

<ol>
	<li>Kueckelhaus, 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=Vascularized+composite+allotransplantation:+Current+standards+and+novel+approaches+to+prevent+acute+rejection+and+chronic+allograft+deterioration.">Vascularized composite allotransplantation: Current standards and novel approaches to prevent acute rejection and chronic allograft deterioration.</a> Transplant International. 29 (6), 655-662 (2016).</li><li>Duisit, 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=Bioengineering+a+human+face+graft:+The+matrix+of+identity.">Bioengineering a human face graft: The matrix of identity.</a> Annals of Surgery. 266 (5), 754-764 (2017).</li><li>Zhang, Q., 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=Decellularized+skin/adipose+tissue+flap+matrix+for+engineering+vascularized+composite+soft+tissue+flaps.">Decellularized skin/adipose tissue flap matrix for engineering vascularized composite soft tissue flaps.</a> Acta Biomaterialia. 35, 166-184 (2016).</li><li>Londono, R., Gorantla, V. S., 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=Emerging+implications+for+extracellular+matrix-based+technologies+in+vascularized+composite+allotransplantation.">Emerging implications for extracellular matrix-based technologies in vascularized composite allotransplantation.</a> Stem Cells International. 2016 (10), 1-16 (2016).</li><li>Fleissig, Y. Y., Beare, J. E., LeBlanc, A. J., Kaufman, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+rat+hind+limb+transplant+as+an+experimental+model+of+vascularized+composite+allotransplantation:+Approaches+and+advantages.">Evolution of the rat hind limb transplant as an experimental model of vascularized composite allotransplantation: Approaches and advantages.</a> SAGE Open Medicine. 8, 205031212096871 (2020).</li><li>Tao, 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=Sterilization+and+disinfection+methods+for+decellularized+matrix+materials:+Review,+consideration+and+proposal.">Sterilization and disinfection methods for decellularized matrix materials: Review, consideration and proposal.</a> Bioactive Materials. 6 (9), 2927-2945 (2021).</li><li>Chen, Y., Geerts, S., Jaramillo, M., 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=Preparation+of+decellularized+liver+scaffolds+and+recellularized+liver+grafts.">Preparation of decellularized liver scaffolds and recellularized liver grafts.</a> Methods in Molecular Biology. 1577, 255-270 (2018).</li><li>Ahmed, S., Chauhan, V. M., Ghaemmaghami, A. M., Aylott, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+generation+of+bioreactors+that+advance+extracellular+matrix+modelling+and+tissue+engineering.">New generation of bioreactors that advance extracellular matrix modelling and tissue engineering.</a> Biotechnology Letters. 41 (1), 1-25 (2019).</li><li>Cohen, 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=Generation+of+vascular+chimerism+within+donor+organs.">Generation of vascular chimerism within donor organs.</a> Scientific Reports. 11 (1), 13437 (2021).</li><li>Lupon, 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=Engineering+vascularized+composite+allografts+using+natural+scaffolds:+A+systematic+review.">Engineering vascularized composite allografts using natural scaffolds: A systematic review.</a> Tissue Engineering Part B: Reviews. , (2021).</li><li>Urciuolo, 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=Decellularised+skeletal+muscles+allow+functional+muscle+regeneration+by+promoting+host+cell+migration.">Decellularised skeletal muscles allow functional muscle regeneration by promoting host cell migration.</a> Scientific Reports. 8 (1), 8398 (2018).</li><li>Jank, B. 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=Engineered+composite+tissue+as+a+bioartificial+limb+graft.">Engineered composite tissue as a bioartificial limb graft.</a> Biomaterials. 61, 246-256 (2015).</li></ol>

大鼠后肢可用作VCA5中的实验模型。无细胞支架的组织工程是解决与VCA相关的长期免疫抑制方案缺点的第一步。鉴于存在多种组织，每种组织都具有独特的功能、免疫原性和结构特性，复合移植物的使用带来了额外的挑战。本协议显示了获得无细胞复合大鼠后肢的成功方法。这些支架可以进一步重新细胞化，并代表VCA的概念验证模型。
为了确保成功的采购和?...

VCA是需要复杂手术重建的患者的新兴选择。创伤性损伤或肿瘤切除会导致难以重建的体积组织丢失。VCA 提供多种组织的移植，例如皮肤、骨骼、肌肉、神经和血管，作为从供体到受体的复合移植物1.尽管VCA具有希望的性质，但由于长期免疫抑制方案而受到限制。终身使用此类药物会增加机会性感染、恶性肿瘤和终末器官毒性的风险1，2，3。为了帮助减少和/或消除对免疫抑制的需求，使用VCA脱细胞方法的组织工程支架显示出巨大的前景。
组织脱细胞需要保留细胞外基质结构，同时去除细胞和核内容物。这种脱细胞支架可以用患者特异性细胞重新填充4。然而，保存复合组织的ECM网络是一个额外的挑战。这是由于支架内存在多种组织类型，具有不同的组织密度、结构和解剖位置。本协议提供了一种用于大鼠后肢的手术技术和去细胞化方法。这是将这种组织工程技术应用于复合组织的概念验证模型。这也可以促使随后的努力通过细胞再生再生复合组织。

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	<tbody>
		<tr>
			<td>0.9% Sodium Chloride Injection USP 50 mL</td>
			<td>Baxter Corporation</td>
			<td>JB1308M</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Disposable Serological Pipets</td>
			<td>VWR</td>
			<td>75816-102</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cc Disposable Syringes</td>
			<td></td>
			<td></td>
			<td>Obtained from Research Institution</td>
		</tr>
		<tr>
			<td>3-way Stopcock</td>
			<td></td>
			<td></td>
			<td>Obtained from Research Institution</td>
		</tr>
		<tr>
			<td>5cc Disposable Syringes</td>
			<td></td>
			<td></td>
			<td>Obtained from Research Institution</td>
		</tr>
		<tr>
			<td>70% Isopropyl Alcohol</td>
			<td></td>
			<td></td>
			<td>Obtained from Research Institution</td>
		</tr>
		<tr>
			<td>Acrodisc Syringe Filter 0.2 &#181;m</td>
			<td>VWR</td>
			<td>CA28143-310</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson Forceps, Straight</td>
			<td>Fine Science Tools</td>
			<td>11006-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Angiocatheter 24 G 19 mm (&#190;&#8221;)&#160;</td>
			<td>VWR</td>
			<td>38112</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic Solution (100x) 100 mL</td>
			<td>Multicell</td>
			<td>450-115-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone Cutter</td>
			<td>Fine Science Tools</td>
			<td>12029-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Connectors for&#160; 1/16&#34; to 1/8&#34; Tubes</td>
			<td>McMasterCarr</td>
			<td>5117K52</td>
			<td></td>
		</tr>
		<tr>
			<td>Female Luer to barbed adapter (PVDF) - 1/8&#34; ID</td>
			<td>McMasterCarr</td>
			<td>51525K328</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps with Micro-Blunted Tips</td>
			<td>Fine Science Tools</td>
			<td>11253-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium Injection 10,000 IU/10 mL</td>
			<td>LEO Pharma Inc.</td>
			<td>006174-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Male Luer to barbed adapter (PVDF) - 1/8&#34; ID</td>
			<td>McMasterCarr</td>
			<td>51525K322</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Needle Holder</td>
			<td>WLorenz</td>
			<td>04-4125</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>WLorenz</td>
			<td>SP-4506</td>
			<td></td>
		</tr>
		<tr>
			<td>Peracetic Acid</td>
			<td>Sigma Aldrich</td>
			<td>269336-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic Pump, 3-Channel</td>
			<td>Cole Parmer</td>
			<td>RK-78001-68</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline 1x 500 mL</td>
			<td>Wisent</td>
			<td>311-425-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone Surgical Scrub Solution</td>
			<td></td>
			<td></td>
			<td>Obtained from Research Institution</td>
		</tr>
		<tr>
			<td>Pump Tubing, 3-Stop, Tygon E-LFL</td>
			<td>Cole Parmer</td>
			<td>RK-96450-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Pump Tubing, Platinum-Cured Silicone</td>
			<td>Cole Parmer</td>
			<td>RK-96410-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Blade - #10</td>
			<td>Fine Science Tools</td>
			<td>10010-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle - #3&#160;</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate Reagent Grade: Purity: &#62;99%, 1 kg</td>
			<td>Bioshop</td>
			<td>SDS003.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Suture #6-0</td>
			<td>Covidien</td>
			<td>VS889</td>
			<td></td>
		</tr>
	</tbody>
</table>

procurement and decellularization of rat hindlimbs using an ex vivo perfusion-based bioreactor for vascularized composite allotransplantation

严重创伤和组织丢失的患者需要复杂的手术重建。血管化复合同种异体移植（VCA）是一种不断发展的重建途径，用于将多个组织作为复合亚单位转移。尽管VCA具有前景，但由于恶性肿瘤、终末器官毒性和机会性感染的风险增加，长期免疫抑制需求是一个重大限制。无细胞复合支架的组织工程是减少免疫抑制需求的潜在替代方案。本文描述了大鼠后肢的获取及其随后使用十二烷基硫酸钠（SDS）的去细胞化。所提出的采购策略基于股总动脉。构建了基于机器灌注的生物反应器系统，用于后肢 离体 脱细胞。成功进行了灌注脱细胞，导致后肢呈白色半透明状外观。观察到整个后肢的完整、可灌注的血管网络。组织学分析显示，所有组织隔室的核内容物去除和组织结构的保存。

采购方案成功地分离和插管股总动脉以进行后续灌注步骤。 图1A，B 中的代表性解剖图像显示了距分叉点足够距离的股血管的切口位置和暴露。 图2 示出了制备生物反应器和灌注回路所需的装置。通过观察组织的白色半透明外观来确定去细胞化的终点。 离体 机灌注系统成功地实现了大鼠后肢的灌注去细胞化。维持单通道闭合系统电?...

Watch this Scientific Journal Video about Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation at JoVE.com

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

我们描述了复合大鼠后肢的手术技术和去细胞化过程。使用低浓度十二烷基硫酸钠通过 离体 机灌注系统进行脱细胞。

使用 离体 灌注生物反应器进行大鼠后肢的获取和去细胞化血管化复合同种异体移植

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...

我们已经建立了一个用于肢体去细胞化的小动物模型。这对于促进血管化复合组织的去细胞化和再细胞化的概念验证研究很有用。免疫抑制方案是血管化复合同种异体移植的主要局限性之一。使用去细胞化可以降低组织免疫原性。首先，使用10号手术刀片沿着腹股沟韧带进行皮肤切口，从外侧移动到内侧，同时用Adson镊子固定周围的皮肤。当下面的脂肪暴露出来时，用钝性解剖仔细解剖脂肪，并定位上腹部血管。使用微型剪刀，解剖近端，并在腹股沟韧带水平暴露下面的股神经、动脉和静脉。在解剖显微镜下，识别股血管，并使用细镊子解剖近端动脉和静脉，以从动脉网络的分叉点获得足够的长度。然后，使用 6-0 缝合线分别结扎股静脉和动脉。接下来，在不破坏结扎的股血管的情况下，在后肢的其余部分进行圆周解剖，并使用骨切割器将股骨横断中段。为了完全隔离后肢，使用微型剪刀横断结扎的远端结扎的股骨血管，并在夹层显微镜下使用24号血管导管插管股动脉。然后，用肝素化盐水冲洗，直到观察到股静脉明显流出。通过将一条缝合线绑在空心血管周围的另一条缝合线中，固定套管，确保套管靠近，以防止阻塞分叉点。之后，浸没获得的后肢和PBS，直到去细胞化。要构建生物反应器，请放置灭菌室，并在入口、出口和补充管路处拧上三个一次性三通旋塞阀，确保补给口的旋塞阀在其其余两个端口处盖上以防止泄漏。将先前制造的硅胶管连接到入口和出口管路的旋塞阀上。然后，将蠕动管连接到硅胶管。将暗盒固定在蠕动管上后，将其放在蠕动泵上。接下来，从上述步骤将其中一个硅胶管连接到出口管的蠕动管末端。在另一端，连接一毫升血清移液器，并将末端与血清移液器连接在废储液瓶中。接下来，将连接到血清移液管的管端悬挂到洗涤剂储液槽中，并立即用封口膜密封洗涤剂储液器烧瓶的开口。将0.25%SDS从一升玻璃罐中加入生物反应器室的中途水平。然后，使用Adson Shorts取出采购的后肢，并小心地将其悬挂到生物反应器室中。使用两对爱信钳，将后肢的空心部分引导至入口线，同时用一对镊子握住套管。使用另一对镊子扭转并将入口管固定到套管上。固定后，根据需要从一升玻璃罐中添加更多 0.25% SDS 以完全浸没肢体，确保出口也浸没在生物反应器储液器中，并保持一致的流出。然后，将一次性注射器过滤器连接到生物反应器室盖子上的通风口，并将盖子固定在生物反应器上，确保腔室从四面八方密封。要从管道中去除空气并灌注回路，请使用新的一次性 10 毫升注射器使用入口管路处的三通旋塞阀从洗涤剂储液器中抽取洗涤剂。抽出后，使用相同的流体插入出口管路处的三通旋塞阀中。接下来，按下并将带有管道的盒式磁带固定到蠕动泵中。然后，使用电源按钮打开蠕动泵。在蠕动泵屏幕上，使用箭头键进入第二个选项卡，以输入流速为输送方式设置第一个通道的灌注速率，并将灌注速率设置为每分钟1毫升，确保根据设备设置正确流动方向。校准蠕动泵，以确保通过入口管路输送的流体量和/或从出口管路输送的流体量在两者之间一致流动，确保管道ID设置为1.85毫米。通过机器灌注开始脱细胞，通过按键盘上的电源按钮，以每分钟一毫升的速度对入口和出口管路进行脱细胞。监控并确保入口和出口管路的流量持续不断。使用1升0.25%STS，根据需要通过补充端口补充生物反应器储液器。然后，寻找组织的白色半透明外观，这将在第五天出现，表明大鼠后肢的去细胞化。确认脱细胞后，用PBS和1%抗生素 - 抗真菌剂储液器替换洗涤剂储液器，并用封口膜密封烧瓶的开口。以每分钟一毫升的速度开始PBS和1%抗生素 - 抗真菌药灌注，并持续两天。用200毫升0.1%寄生酸，4%乙醇储液罐替换PBS和1%抗生素 - 抗真菌剂储液器，并以每分钟1毫升的速度开始灌注，持续两小时。在生物安全柜下，使用两对无菌 Adson 镊子断开后肢与入口线的连接，一对镊子扭曲入口线，另一对镊子握住套管，确保套管不被拉动以防止脱节。将肢体储存在装有PBS和1%抗生素 - 抗真菌剂的500毫升玻璃罐中，温度为4摄氏度，直到进一步使用。去细胞化的股动脉和静脉都显示出所有层和周围结缔组织的核含量损失，因为天然血管中缺乏蓝色染色的细胞核。股静脉和动脉的内膜介质和外膜维持在脱细胞血管中。股神经显示组织结构保存，包括神经内膜。骨显示出整体结构保留，骨细胞染色核丢失，脱细胞后从周围的内膜和骨膜层丢失。皮肤显示表皮和真皮细胞丢失。真皮显示保留的胶原纤维类似于天然皮肤组织。最后，骨骼肌的横视图显示位于肌内膜外围的细胞核丢失。肌原纤维含量在脱细胞后保留在各自的分册内。还使用PicoGreen进行了DNA定量，其中股血管，神经，皮肤，肌肉和骨骼的DNA含量显着降低。按照该方案，可以进一步表征脱细胞肢体。这涉及使用组织学和生化方法检查细胞外基质，以及使用成像评估血管。脱细胞化的茎也可以使用组织特异性细胞进行再细胞化。

Research

JoVE Journal

Bioengineering

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Decellularization and Recellularization of Whole Livers

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

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Operation of a Benchtop Bioreactor

一个台式生物反应器的操作

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

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

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

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 ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

微流控生物工程血管化组织和细胞器解体

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Decellularization and Recellularization Methodology for Human Saphenous Veins

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 ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

大鼠体内部分肝工程的外科技术基础

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

A Reliable Porcine Fascio-Cutaneous Flap Model for Vascularized Composite Allografts Bioengineering Studies

Vascularized Composite Allografts (VCA) such as hand, face, or penile transplant represents the cutting-edge treatment for devastating skin defects, ...

使用 离体 灌注生物反应器进行大鼠后肢的获取和去细胞化血管化复合同种异体移植

使用离体灌注生物反应器进行大鼠后肢的获取和去细胞化血管化复合同种异体移植