Name: procurement and perfusion-decellularization of porcine vascularized flaps in a customized perfusion bioreactor
Uploaded: 2022-08-01T13:00:00
Description: Watch this Scientific Journal Video about Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor at JoVE.com

所有涉及动物受试者的程序均已获得大学健康网络机构动物护理和使用委员会（IACUC）的批准，并按照大学健康网络动物资源中心的协议和程序以及加拿大动物护理指南委员会执行。所有实验均使用五头约克郡猪（35-50公斤;约12周龄）。
1. 灌注生物反应器制造
<ol><li>有关灌注生物反应器中使用的所有组件，请参见 图1 。对于组织室的制造，请使用市售的聚...

<ol>
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H., Panje, W. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+tissue+decellularization+used+for+preparation+of+biologic+scaffolds+and+in+vivo+relevance.">Methods of tissue decellularization used for preparation of biologic scaffolds and in vivo relevance.</a> Methods. 84, 25-34 (2015).</li><li>Mendibil, 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=Tissue-specific+decellularization+methods:+Rationale+and+strategies+to+achieve+regenerative+compounds.">Tissue-specific decellularization methods: Rationale and strategies to achieve regenerative compounds.</a> International Journal of Molecular Sciences. 21 (15), 5447 (2020).</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. 28 (3), 677-693 (2022).</li><li>Duisit, J., Maistriaux, L., Bertheuil, N., Lellouch, A. G. <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+tissues+by+perfusion+decellularization/recellularization:+Review.">Engineering vascularized composite tissues by perfusion decellularization/recellularization: Review.</a> Current Transplantation Reports. 8, 44-56 (2021).</li><li>Adil, A., Xu, M., Haykal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recellularization+of+bioengineered+scaffolds+for+vascular+composite+allotransplantation.">Recellularization of bioengineered scaffolds for vascular composite allotransplantation.</a> Frontiers in Surgery. 9, 843677 (2022).</li><li>Phelps, E. A., Garc&#237;a, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+more+than+a+cell:+Vascularization+strategies+in+tissue+engineering.">Engineering more than a cell: Vascularization strategies in tissue engineering.</a> Current Opinion in Biotechnology. 21 (5), 704-709 (2010).</li><li>Pozzo, V., 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+reliable+porcine+fascio-cutaneous+flap+model+for+vascularized+composite+allografts+bioengineering+studies.">A reliable porcine fascio-cutaneous flap model for vascularized composite allografts bioengineering studies.</a> Journal of Visualized Experiments. (181), e63557 (2022).</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=Decellularization+and+recellularization+of+whole+livers.">Decellularization and recellularization of whole livers.</a> Journal of Visualized Experiments. (48), e2394 (2011).</li><li>Uzarski, J. 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=Epithelial+cell+repopulation+and+preparation+of+rodent+extracellular+matrix+scaffolds+for+renal+tissue+development.">Epithelial cell repopulation and preparation of rodent extracellular matrix scaffolds for renal tissue development.</a> Journal of Visualized Experiments. (102), e53271 (2015).</li><li>Sullivan, D. 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=Decellularization+methods+of+porcine+kidneys+for+whole+organ+engineering+using+a+high-throughput+system.">Decellularization methods of porcine kidneys for whole organ engineering using a high-throughput system.</a> Biomaterials. 33 (31), 7756-7764 (2012).</li><li>Choudhury, D., Yee, M., Sheng, Z. L. J., Amirul, A., Naing, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+systems+and+devices:+State-of-the-art.">Decellularization systems and devices: State-of-the-art.</a> Acta Biomaterialia. 115, 51-59 (2020).</li><li>Schilling, B. 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拟议的方案使用低浓度SDS的灌注来使一系列猪源性皮瓣脱细胞。通过此程序，可以使用有利于低浓度 SDS 的方案成功脱细胞，无细胞网膜、阔筋膜张肌和桡骨前臂皮瓣。初步优化实验已确定，当使用组织学技术分析时，2天至5天之间低浓度（0.05%）的SDS能够去除网膜，阔筋膜张肌和桡骨前臂瓣的细胞物质（补充图1，补充图2，补充图3）。这种方法提供了一种简单的方法，可以在短时间?...

创伤性损伤和肿瘤切除可导致大而复杂的软组织缺损。这些缺陷会损害患者的生活质量，导致功能丧失，并导致永久性残疾。虽然自体组织皮瓣移植等技术已被普遍采用，但皮瓣可用性和供体部位发病率问题是主要限制1，2，3。血管化复合同种异体移植（VCA）是一种有前途的替代方案，可将复合组织（例如肌肉，皮肤，脉管系统）作为一个单元转移给受体。然而，VCA需要长期免疫抑制，这会导致药物毒性、机会性感染和恶性肿瘤4，5，6。
组织工程无细胞支架是这些限制的潜在解决方案7。无细胞组织支架可以使用去细胞化方法获得，该方法从天然组织中去除细胞物质，同时保留底层细胞外基质（ECM）微结构。与在组织工程中使用合成材料相比，使用生物衍生支架提供了一种仿生ECM底物，可实现生物相容性和临床翻译的潜力8。脱细胞后，随后用受体特异性细胞对支架进行再细胞化，然后可以产生几乎没有免疫原性的功能性血管化组织9，10，11。通过开发使用灌注脱细胞技术获得无细胞组织的有效方案，可以设计多种组织类型。反过来，基于该技术允许应用于更复杂的组织。迄今为止，已经使用简单的血管化组织（例如啮齿动物 12、猪 13 和人类模型14 的全层筋膜皮瓣）以及猪腹直肌骨骼肌15 研究了血管化软组织的灌注脱细胞化。此外，复杂的血管化组织也已灌注脱细胞，如猪和人耳16，17模型和人全脸移植模型18所示。
在这里，该协议描述了使用生物衍生的ECM支架的血管化游离瓣的去细胞化。我们介绍了三个临床相关皮瓣的去细胞化：1）网膜，2）阔筋膜张量和3）桡骨前臂，所有这些都代表了重建手术中常规使用的主力皮瓣，并且以前从未在组织脱细胞背景下的动物研究中进行过检查。这些生物工程皮瓣提供了一个多功能且易于获得的平台，具有临床应用的潜力，可用于大型软组织缺损修复和重建领域。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >0.2 &#181;m pore Acrodisk Filter</td>
			<td >VWR</td>
			<td >CA28143-310</td>
			<td ></td>
		</tr>
		<tr >
			<td >0.9 % Sodium Chloride Solution (Normal Saline)</td>
			<td>Baxter</td>
			<td>JF7123</td>
			<td></td>
		</tr>
		<tr >
			<td >20 L Polypropylene Carboy</td>
			<td>Cole-Parmer</td>
			<td>RK-62507-20</td>
			<td></td>
		</tr>
		<tr >
			<td >3-0 Sofsilk Nonabsorbable Surgical Tie</td>
			<td>Covidien</td>
			<td>&#160;LS639</td>
			<td></td>
		</tr>
		<tr >
			<td >3-way Stopcock</td>
			<td>Cole-Parmer</td>
			<td>UZ-30600-04</td>
			<td></td>
		</tr>
		<tr >
			<td >Adson Forceps</td>
			<td>Fine Science Tools</td>
			<td>11027-12</td>
			<td></td>
		</tr>
		<tr >
			<td >Antibiotic-Antimycotic Solution, 100X</td>
			<td>Wisent</td>
			<td>450-115-EL</td>
			<td></td>
		</tr>
		<tr >
			<td >Atropine Sulphate 15 mg/30ml</td>
			<td>Rafter 8 Products</td>
			<td>238481</td>
			<td></td>
		</tr>
		<tr >
			<td >BD Angiocath 20-Gauge</td>
			<td>VWR</td>
			<td>BD381134</td>
			<td></td>
		</tr>
		<tr >
			<td >BD Angiocath 22-Gauge</td>
			<td>VWR</td>
			<td>BD381123</td>
			<td></td>
		</tr>
		<tr >
			<td >BD Angiocath 24-Gauge</td>
			<td>VWR</td>
			<td>BD381112</td>
			<td></td>
		</tr>
		<tr >
			<td >Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C4901</td>
			<td>DNAse Co-factor</td>
		</tr>
		<tr >
			<td >DNase I from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr >
			<td >DNA assay (Quant-iT PicoGreen dsDNA Assay Kit)</td>
			<td>Invitrogen</td>
			<td>P7589</td>
			<td></td>
		</tr>
		<tr >
			<td >DPBS, 10X</td>
			<td>Wisent</td>
			<td>311-415-CL</td>
			<td>&#160;without Ca++/Mg++</td>
		</tr>
		<tr >
			<td >Halsted-Mosquito Hemostat</td>
			<td>Fine Science Tools</td>
			<td>13008-12</td>
			<td></td>
		</tr>
		<tr >
			<td >Heparin, 1000 I.U./mL</td>
			<td>Leo Pharma A/S</td>
			<td>453811</td>
			<td></td>
		</tr>
		<tr >
			<td >Ketamine Hydrochloride&#160; 5000 mg/50 ml</td>
			<td>Bimeda-MTC Animal Health Inc.</td>
			<td>612316</td>
			<td></td>
		</tr>
		<tr >
			<td >Ismatec Pump Tygon 3-Stop Tubing</td>
			<td>Cole-Parmer</td>
			<td>RK-96450-40</td>
			<td>Internal Diameter:&#160; 1.85 mm</td>
		</tr>
		<tr >
			<td >Ismatec REGLO 4-Channel Pump</td>
			<td>Cole-Parmer</td>
			<td>78001-78</td>
			<td></td>
		</tr>
		<tr >
			<td >Ismatec Tubing Cassettes</td>
			<td>Cole-Parmer</td>
			<td>RK-78016-98</td>
			<td></td>
		</tr>
		<tr >
			<td >Isoflurane 99.9%, 250 ml</td>
			<td>Pharmaceutical Partners of Canada Inc.</td>
			<td>2231929</td>
			<td></td>
		</tr>
		<tr >
			<td >LB Agar Lennox</td>
			<td>Bioshop Canada</td>
			<td>LBL406.500</td>
			<td>Sterility testing agar plates</td>
		</tr>
		<tr >
			<td >Magnesium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td>DNAse Co-factor</td>
		</tr>
		<tr >
			<td >Masterflex L/S 16 Tubing</td>
			<td>Cole-Parmer</td>
			<td>RK-96410-16</td>
			<td></td>
		</tr>
		<tr >
			<td >Midazolam 50 mg/10 ml</td>
			<td>Pharmaceutical Partners of Canada Inc.</td>
			<td>2242905</td>
			<td></td>
		</tr>
		<tr >
			<td >Monopolar Cautery Pencil</td>
			<td>Valleylab</td>
			<td>E2100</td>
			<td></td>
		</tr>
		<tr >
			<td >Normal Buffered Formalin, 10%</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr >
			<td >N&#176;11 scalpel blade</td>
			<td>Swann Morton</td>
			<td>303</td>
			<td></td>
		</tr>
		<tr >
			<td >Papain from papaya latex</td>
			<td>Sigma-Aldrich</td>
			<td>P3125</td>
			<td></td>
		</tr>
		<tr >
			<td >Peracetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>269336</td>
			<td></td>
		</tr>
		<tr >
			<td >Plastic Barbed Connector for 1/4&#34; to 1/8&#34; Tube ID</td>
			<td>McMaster-Carr</td>
			<td>5117K61</td>
			<td></td>
		</tr>
		<tr >
			<td >Plastic Barbed Tube 90&#176; Elbow Connectors</td>
			<td>McMaster-Carr</td>
			<td>5117K76</td>
			<td></td>
		</tr>
		<tr >
			<td >Plastic Quick-Turn Tube Plugs</td>
			<td>McMaster-Carr</td>
			<td>51525K143</td>
			<td>Male Luer</td>
		</tr>
		<tr >
			<td >Plastic Quick-Turn Tube Sockets</td>
			<td>McMaster-Carr</td>
			<td>51525K293</td>
			<td>Female Luer</td>
		</tr>
		<tr >
			<td >Punch Biopsy Tool</td>
			<td>Integra Miltex</td>
			<td>3332</td>
			<td></td>
		</tr>
		<tr >
			<td >Potassium Chloride 40 mEq/20 ml</td>
			<td>Hospira Healthcare Corporation</td>
			<td>37869</td>
			<td></td>
		</tr>
		<tr >
			<td >Povidone-Iodine, 10%</td>
			<td>Rougier</td>
			<td>833133</td>
			<td></td>
		</tr>
		<tr >
			<td >Serological Pipet, 2mL</td>
			<td>Fisher Science</td>
			<td>13-678-27D</td>
			<td></td>
		</tr>
		<tr >
			<td >Snap Lid Airtight Containers</td>
			<td>SnapLock</td>
			<td>142-3941-4</td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium Dodecyl Sulfate Powder</td>
			<td>Sigma-Aldrich</td>
			<td>L4509</td>
			<td></td>
		</tr>
		<tr >
			<td >Surgical Metal Ligation Clips, Small</td>
			<td >Teleflex</td>
			<td >001200</td>
			<td></td>
		</tr>
		<tr >
			<td >Stevens Tenotomy Scissors, 115 mm, straight</td>
			<td >B. Braun</td>
			<td >BC004R</td>
			<td></td>
		</tr>
		<tr >
			<td >TruWave Pressure Monitoring Set</td>
			<td >Edwards Lifesciences</td>
			<td >PX260</td>
			<td></td>
		</tr>
	</tbody>
</table>

procurement and perfusion-decellularization of porcine vascularized flaps in a customized perfusion bioreactor

大量软组织缺损会导致功能缺陷，并会极大地影响患者的生活质量。虽然手术重建可以使用自体游离皮瓣移植或血管化复合同种异体移植（VCA）进行，但这种方法也有缺点。供体部位发病率和组织可用性等问题限制了自体游离皮瓣移植，而免疫抑制是 VCA 的重要限制。使用去细胞化/再细胞化方法的重建手术中的工程组织代表了一种可能的解决方案。使用去除天然细胞物质同时保留底层细胞外基质（ECM）微结构的方法生成去细胞化组织。然后，这些无细胞支架随后可以用受体特异性细胞重新细胞化。
该协议详细介绍了用于在猪模型中实现无细胞支架的获取和脱细胞方法。此外，它还提供了灌注生物反应器设计和设置的描述。皮瓣包括猪网膜、阔筋膜张肌和桡骨前臂。通过低浓度十二烷基硫酸钠（SDS）洗涤剂的 离体 灌注进行脱细胞，然后在定制的灌注生物反应器中进行DNase酶处理和过氧乙酸灭菌。
成功的组织脱细胞的特征是肉眼下皮瓣呈白色不透明外观。无细胞瓣显示组织学染色时没有细胞核，DNA 含量显着降低。该协议可以有效地用于生成具有保留ECM和血管微结构的脱细胞软组织支架。这种支架可用于随后的细胞再生研究，并具有在重建手术中的临床转化潜力。

这种使血管化猪瓣脱细胞的方案依赖于离子基洗涤剂SDS通过定制灌注生物反应器中的皮瓣脉管系统的灌注。在脱细胞之前，根据猪模型的主要供应血管采购和插管三个血管化皮瓣。采购后立即冲洗瓣，以保持专利，可灌注脉管系统，以便成功脱细胞。使用密封的卡扣盖容器，设计了一种定制的生物反应器，以允许在封闭环境中进行瓣灌注。使用连接到组织室的两个蠕动泵以单程方式实现生物反应?...

Watch this Scientific Journal Video about Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor at JoVE.com

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

该协议描述了通过在定制的灌注生物反应器中通过皮瓣脉管系统灌注十二烷基硫酸钠洗涤剂来手术获取和随后的血管化猪皮瓣的脱细胞。

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 ...

该方法描述了三个血管化猪瓣的手术获取及其随后的灌注脱细胞。该方法可以帮助使用组织去细胞化和再细胞化方法再生猪瓣。该技术的主要优点是其多功能性，可以在易于组装的生物反应器设置中实现各种血管化猪瓣的大量脱细胞。这里描述的技术可以广泛应用于其他血管化皮瓣，作为在重建手术中使用猪皮瓣的潜在组织工程解决方案。首先，通过测量大约 50 厘米的 LS16 铂涂层硅胶管来制作流入和流出管。将一端连接到黄色 3 停止泵管，另一端连接到无菌 2 毫升血清移液管，以将灌注液从洗涤剂储液槽输送到生物反应器室中。将腔室和管道高压灭菌在单独包装的无菌包装中。对于网膜瓣获取，将一头 12 周龄麻醉的约克夏猪置于仰卧位，然后以通常的无菌方式准备腹部。进行剑脐剖腹进入腹部并活动胃头，并将网膜放入手术区域。从大曲率的左侧开始，左胃表皮动脉与脾动脉连接，使用直史蒂文斯肌腱切开剪刀将左胃表皮动脉和静脉骨骼化。然后用手术扎带或夹子分别结扎和分割两者。沿着胃的较大曲率从左到右进行，结扎并分开从网膜产生的短血管分支，提供胃的更大曲率。继续动员大网膜，直到遇到右胃表皮血管和胃十二指肠动脉的交界处。然后结扎并分割右胃表皮动脉和静脉，以释放网膜瓣，并在以后进行皮瓣插管。对于张筋膜张张肌皮瓣的获取，将猪置于侧卧位。标记从髂前上棘 （ASIS） 到髌骨外侧的内皮瓣边界，以及尾部约 8-10 厘米的后边界。使用手术刀在髌骨远端边缘做一个锋利的切口。通过皮下组织加深切口以到达股直肌上方的筋膜，并沿着标记的边界继续切口朝向ASIS。要单独隔离筋膜瓣，请从下面的筋膜层中去除覆盖的皮肤成分。然后将小穿支血管结扎或烧灼到皮肤上。返回皮瓣远端边界，切开深筋膜。将筋膜从下面的股外侧肌向 ASIS 移动。将椎弓根追踪到股深部血管后，将股骨外侧回旋动脉和供应筋膜瓣的静脉骨骼化。然后结扎并分开近端的皮瓣血管，在那里它们连接深股血管并允许以后插管。对于桡向前臂皮瓣采购，在伸展的猪前臂的桡骨上标记一个 3 x 3 厘米的正方形。在近端皮瓣边缘的中点和肘前窝之间画一条线，以表示桡动脉蒂的大致病程。通过触诊确认动脉病程。接下来，使用手术刀在远端切开皮肤，深度可达臂前筋膜。用肌腱切开剪刀钝性解剖，直到遇到桡动脉远端及其静脉。然后用手术绑带或夹子结扎并分割动脉和静脉。继续在标记的皮肤方块的桡骨和尺骨边缘进行皮肤切口。然后用手术刀片或烧灼将皮瓣从下面的桡骨上移开，从尺骨向桡骨方向延伸，同时将皮瓣向肘前窝近端抬起。使用肌腱切开剪刀，将桡动脉和静脉在肘前窝近端骨骼化，分别在那里它们连接肱动脉和静脉。从周围的纤维脂肪组织中解剖血管以分离血管蒂。然后分别结扎和分割动脉和静脉，以释放桡骨前臂皮瓣。接下来，用 20 至 24 号血管导管插管，血管用 3-0 丝带固定。将肝素化盐水冲洗至皮瓣动脉插管中，直到观察到清晰的静脉流出。在层流生物安全柜中工作时，将襟翼放入生物反应器组织室中。带有无菌肝素化盐水的灌注管，以去除残留空气并布置瓣，以允许皮瓣动脉套管和公鲁尔连接器之间的连接。然后使用无菌止血器将动脉插管拧到连接器上。然后通过旋塞阀冲洗肝素化的盐水，以检查鲁尔连接是否没有泄漏，并且翻转器是否可以注入静脉流出的证据。关闭组织室盖后，将带有黄色 3 停止泵管的流入管连接到组织室的流入旋塞阀。此外，将在线压力传感器传感器连接到流入的 3 通旋塞阀上，以进行灌注压力监测。接下来，打开流入蠕动泵，在初始屏幕上，使用箭头移动到第三个选项卡，将管道 ID 设置为 1.85 毫米。然后从第二个选项卡设置灌注速率。作为输送方式的输入速率设置为每分钟 2 毫升。确保屏幕上显示的流向正确。使用兼容的盒式磁带将三止泵管装入蠕动泵。将流出泵设置设置为每分钟 4 毫升的速率。用黄色的3停止泵管将流出管连接到组织室的流出旋塞阀。然后将三停泵管装入蠕动泵，并通过按下每个泵上的电源按钮开始两个泵的流量。在组织室中开始用肝素化盐水对皮瓣进行灌注脱细胞15分钟，以去除任何残留的血栓。完成后，用500毫升十二烷基硫酸钠或SDS脱细胞溶液替换残留的盐水以浸没皮瓣。接下来，将流入管转移到SDS溶液中并大量组织。SDS 去细胞化后，吸出剩余的 SDS，然后用 PBS 灌注皮瓣一天。SDS灌注脱细胞后，依次在DNA溶液中灌注瓣两个小时，然后在PBS中灌注一天，然后将流入管转移到蒸馏水中的过氧乙酸和乙醇中，通过灌注三个小时对皮瓣进行灭菌。完成后，无菌取下皮瓣，并将它们浸入两次含有 1% 抗生素抗真菌剂的 PBS 洗涤液中，每次 15 分钟。将翻盖储存在 4 摄氏度，直到准备好重新细胞化。使用 3 至 10 毫米打孔活检工具获取组织样本以评估脱细胞皮瓣。在手动控制下冲洗的分离脱细胞皮瓣显示有证据显示网膜、阔筋膜张肌和桡骨前臂皮瓣的自由引流静脉插管流出的证据。自体网膜、阔筋膜张肌和桡骨前臂皮瓣的总体形态在采购后立即呈现粉红色。相比之下，去细胞化组织的特征是白色或不透明的外观。用苏木精和伊红对天然组织的组织学检查显示存在蓝色细胞核。在脱细胞皮瓣中，组织学染色显示细胞物质丢失而没有蓝色核染色，表明无细胞组织支架。DNA含量的额外定量显示，与天然组织相比，无细胞支架中的DNA显着减少。最重要的考虑因素是在采购期间使用的手术技术，注意避免损坏皮瓣，以便随后进行灌注脱细胞。灌注脱细胞后，所得的皮瓣可以用组织特异性细胞群重新细胞化，以便再生天然组织区室。

Research

JoVE Journal

Bioengineering

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

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

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

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  ...

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 ...

Engineered Vascularized Muscle Flap

工程化血管肌瓣

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

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 ...

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue

成像引导的生物反应器，用于生成生物工程气道组织

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in ...

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, ...

A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation

一种摩擦试验-用于研究滑膜关节生物力学、机械生物学和物理调节的生物反应器装置

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication ...

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

使用3D打印技术制造工程血管瓣

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

Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture

多流灌注生物反应器集成了出口分馏功能，用于动态细胞培养

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This ...