Name: preparation of decellularized kidney scaffolds in rats
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这项研究得到了釜山国立大学医院的生物医学研究所资助。

这项研究得到了釜山国立医科大学的批准，是按照动物使用和护理的道德准则进行的。（2017-119号证书）。在进行任何动物研究之前，应获得机构批准。 注：建议成功进行腹部器官手术演示和成像的所有手术和麻醉器/设备和试剂均详见表1。
1. 大鼠肾脏收获的准备程序
<ol><li>在准备手术时，将8周大的斯普拉格-道利大鼠（重200-250克）放在暖垫上。在直肠中放?...

<ol>
	<li>Kawai, T., 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=Brief+report:+HLA-mismatched+renal+transplantation+without+maintenance+immunosuppression.">Brief report: HLA-mismatched renal transplantation without maintenance immunosuppression.</a> New England Journal of Medicine. 358 (4), 353-361 (2008).</li><li>Rana, D., Zreiqat, H., Benkirane-Jessel, N., Ramakrishna, S., Ramalingam, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+decellularized+scaffolds+for+stem+cell-driven+tissue+engineering.">Development of decellularized scaffolds for stem cell-driven tissue engineering.</a> Journal of Tissue Engineering and Regenerative Medicine. 11 (4), 942-965 (2017).</li><li>Fu, R. 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=Decellularization+and+recellularization+technologies+in+tissue+engineering.">Decellularization and recellularization technologies in tissue engineering.</a> Cell Transplantation. 23 (4-5), 621-630 (2014).</li><li>Hopkinson, 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=Optimization+of+amniotic+membrane+(AM)+denuding+for+tissue+engineering.">Optimization of amniotic membrane (AM) denuding for tissue engineering.</a> Tissue Engineering. Part C, Methods. 14 (4), 371-381 (2008).</li><li>Shupe, T., Williams, M., Brown, A., Willenberg, B., Petersen, 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=Method+for+the+decellularization+of+intact+rat+liver.">Method for the decellularization of intact rat liver.</a> Organogenesis. 6 (2), 134-136 (2010).</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 (5991), 538-541 (2010).</li><li>Fernandez-Perez, J., Ahearne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+decellularization+methods+on+extracellular+matrix+derived+hydrogels.">The impact of decellularization methods on extracellular matrix derived hydrogels.</a> Scientific Reports. 9 (1), 14933 (2019).</li><li>Naik, A., Griffin, M., Szarko, M., Butler, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+the+decellularization+process+of+an+upper+limb+skeletal+muscle;+implications+for+muscle+tissue+engineering.">Optimizing the decellularization process of an upper limb skeletal muscle; implications for muscle tissue engineering.</a> Artificial Organs. 44 (2), 178-183 (2020).</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 (12), 3233-3243 (2011).</li><li>Mason, C., Dunnill, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+definition+of+regenerative+medicine.">A brief definition of regenerative medicine.</a> Regenerative Medicine. 3 (1), 1-5 (2008).</li><li>Guyette, J. 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=Perfusion+decellularization+of+whole+organs.">Perfusion decellularization of whole organs.</a> Nature Protocols. 9 (6), 1451-1468 (2014).</li><li>Song, J. 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=Regeneration+and+experimental+orthotopic+transplantation+of+a+bioengineered+kidney.">Regeneration and experimental orthotopic transplantation of a bioengineered kidney.</a> Nature Medicine. 19 (5), 646-651 (2013).</li></ol>

各种协议已用于器官和其他组织的去细胞化。最佳的去细胞化协议应保留细胞外基质 （ECM） 的三维结构。一般来说，这种协议包括通过物理处理或离子溶液对细胞膜进行溶解，通过酶处理或洗涤剂将细胞质和细胞核从ECM中分离出来，然后从组织3中去除细胞碎片。物理过程包括刮伤、溶液搅拌、压力梯度、捕捉冻结、非热永久电波和超临界流体2。组织或器官外...

细胞培养技术应用于功能组织和器官的再生，以取代患病或受损的器官。同源器官移植是目前最常见的不可逆转器官损伤治疗方法：然而，这种方法需要使用免疫抑制，以防止移植器官的排斥。此外，尽管移植免疫学有所进展，但20%的移植接受者可能在5年内出现急性排斥，在移植后10年内，40%的接受者可能失去移植移植或死亡1。
组织工程技术的进步为移植新器官带来了新的范式，无需使用分化干细胞进行免疫排斥。干细胞分化后，需要一种称为合成细胞外基质的脚手架，以促进三维器官的生成，使新的组织在受体内茁壮成长。来自去细胞化原生器官的脚手架具有优势，包括建立细胞和增强干细胞增殖的更有效的环境，尽管这些机制尚未完全阐明2。特别是，肾脏是脚手架生成的合适器官，因为它具有丰富的循环和干细胞建立的利基。此外，由于肾脏结构复杂，很难人工再生肾脏进行器官移植。
在本报告中，我们介绍了一种利用大鼠模型中去细胞化器官开发血管化脚手架的方法，以促进未来用于组织工程目的的动物研究。

<table>
	<tbody>
		<tr>
			<td>1 cc syringe (inject probes and vehicle solutions</td>
			<td>Becton Dickinson</td>
			<td>305217</td>
			<td></td>
		</tr>
		<tr>
			<td>10-0 ethilon for vessel anastomosis</td>
			<td>Ethicon</td>
			<td>9032G</td>
			<td></td>
		</tr>
		<tr>
			<td>25 gauge inch guide needle(for vascular catheters)</td>
			<td>Becton Dickinson</td>
			<td>305145</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 PDS incision closure rat</td>
			<td>Ethicon</td>
			<td>Z316H</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 Prolene incision closure rat</td>
			<td>Ethicon</td>
			<td>8832H</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 silk spool vascular access/ligation in rat</td>
			<td>Braintree Scientific</td>
			<td>SUT-S 110</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 PDS incision closure mouse</td>
			<td>Ethicon</td>
			<td>Z773D</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 Prolene incision closure mouse</td>
			<td>Ethicon</td>
			<td>8831H</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 silk spool vascular access/ligation in mouse</td>
			<td>Braintree Scientific</td>
			<td>SUT-S 106</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors to cut fascia/connective tissue</td>
			<td>Fine Science Tools</td>
			<td>14058-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey needle holder</td>
			<td>Fine Science Tools</td>
			<td>12001-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Kelly Hemostat for rats: muscle clamp to minimize bleeding when cut</td>
			<td>Fine Science Tools</td>
			<td>13018-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethelyne 50 tubing, catheter tubing 100 ft</td>
			<td>Braintree Scientific</td>
			<td>.023&#34; &#215; .038&#8221;</td>
			<td></td>
		</tr>
		<tr>
			<td>Schwartz microserrefine vascular clamps</td>
			<td>Fine Science Tools</td>
			<td>18052-01 (straight)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>18052-03 (curved)</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors to cut skin</td>
			<td>Fine Science Tools</td>
			<td>14002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas-Tubingen Spring scissors for arteriotomy</td>
			<td>Fine Science Tools</td>
			<td>15003-08</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of decellularized kidney scaffolds in rats

组织工程是生物医学领域的前沿学科。细胞培养技术可用于功能组织和器官的再生，以取代患病或受损的器官。需要脚手架，以便于在体内使用分化干细胞产生三维器官或组织。在这份报告中，我们描述了一种使用去细胞化的老鼠肾脏开发血管化脚手架的新方法。这项研究使用了8周大的斯普拉格-道利大鼠，肝素被注射到心脏，以方便流入肾脏血管，使肝素渗透到肾脏血管中。腹腔被打开，左肾被收集。收集的肾脏使用洗涤剂，如Triton X-100和硫酸钠，以去细胞化组织，灌注了9小时。然后用1%青霉素/链霉素和肝素轻轻清洗去除细胞碎片和化学残留物。用去细胞化血管脚手架移植干细胞有望促进新器官的产生。因此，血管化脚手架可能为将来器官移植组织工程奠定基础。

老鼠肾的总形态是深红色（图1A）。去细胞化后，肾脏变得苍白和半透明（图1D）。残余基因组DNA根据制造商的说明，在去细胞化的肾脏脚手架中，并与本地肾脏（对照）进行比较，用商业套件进行评估。定量分析证实，组织基因组DNA在去细胞化后几乎被消灭。在14个病例中，控制的平均DNA含量为115.05 ng/μL，去细胞化脚手架的平均DNA含量为1.96 ng/μL。总?...

Watch this Scientific Journal Video about Preparation of Decellularized Kidney Scaffolds in Rats at JoVE.com

Preparation of Decellularized Kidney Scaffolds in Rats

该协议引入了使用去细胞化的鼠肾开发脚手架的方法。该协议包括去细胞化和再细胞化过程，以确认生物可用性。脱细胞化使用三顿X-100和硫酸钠进行。

在大鼠中准备去细胞化肾脏脚手架

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs ...

该协议使得在老鼠模型中使用肾脏开发血管化脚手架成为可能。肾脏是最适合分化干细胞脚手架的器官。这是一个非常成功的肾脏去细胞化协议。它有效地去除核，对 ECM 超结构的破坏最小。该技术可应用于移植医学和组织工程。示范手术的将是金珠孝，一个MD/博士的整形外科副教授从我的实验室。首先，将八周大的斯普拉格·道利老鼠放在暖垫上，并在直肠中放置直肠温度计探头，以监测核心温度。给老鼠麻醉后，将其置于超原位置，用胶带将四肢固定在桌子上。用杀菌肥皂剃光腹部并清洁腹部。涂抹2%贝他丁至少一到两分钟，然后用70%乙醇溶液擦除。重复此序列三次。用无菌的、有芬芳的窗帘覆盖手术场。做一个垂直的腹部切口，并暴露左肾，输尿管，腹主动脉，和劣质静脉卡瓦。在切割脚踏车之前解剖组织。通过将PBS与每毫升肝素约10个单位混合，为每只大鼠准备50毫升的灌注溶液。将等量 8% PFA 与 PBS 混合，以制作 4% PFA 解决方案。疯狂地伸展垂直腹部切口，在切割时确保剪刀远离器官，以避免损伤。继续切口通过肋骨笼，然后通过抬起胸骨切开隔膜。用 Allis 夹子将松动的皮肤皮瓣从道路上拉开，并通过用钳子撕裂任何结缔组织来释放心脏。打开 PBS 线，确保针头进入左心室之前，它流动。用钝钳轻轻握住心脏，并用血压器控制针头。针头的插入不应超过四分之一英寸。在用针头和血浆支撑心脏的同时，找到合适的中庭，用虹膜切除术剪刀穿过心脏。将血化体放在大鼠身上，确保针头仍位于心脏内。如果肾脏和肝脏中仍有可见的血液，请继续灌注 PBS 四分钟或更长时间。用腹部主动脉和劣质静脉卡瓦收获左肾。利盖特尿管，胸动脉，优越的静脉卡瓦，和腹部主动脉的分支。将器官在 10 厘米的培养皿中保持 DPBS 中的水分。用23口径导管调节腹部主动脉和劣质静脉卡瓦。要去除残留血液，将管子与围骨泵连接起来，在 5 rpm 和 37 摄氏度下用 500 毫升 DPBS 和每毫升肝素 16 个单位清洗器官 90 分钟。要去细胞化肾脏，用1%Triton X-100灌注3小时，然后用0.75%SDS溶液6小时，持续压力为40毫升汞。要去除残留的 SDS，在蒸馏水中用 1% 青霉素将样品灌注 18 小时，然后用无菌 DPBS 和每毫升肝素 16 个单位 90 分钟。老鼠肾的总形态是深红色。去细胞化后，肾脏变得苍白和半透明。残余基因组DNA在去细胞化的肾脏脚手架中被量化，与原生肾脏相比，证实组织基因组DNA在去细胞化后几乎被消除。在14个病例中，控制的平均DNA含量为每微升115.05毫微克，去细胞化脚手架的平均DNA含量为每微升1.96纳米。总共有98.3%的DNA被去除。维持了三维结构，在皮质帕奇马中保留了细胞球状体。将针插入左心室时，应插入不超过四分之一英寸。任何进一步的插入可能会从另一边出来。我们相信，这项技术将在组织工程领域开辟新的前景。

Research

JoVE Journal

Medicine

Acute Myocardial Infarction in Rats

大鼠急性心肌梗死

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

科研

医学

Orthotopic Small Bowel Transplantation in Rats

大鼠原位小肠移植

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Orthotopic Liver Transplantation in Rats

大鼠原位肝移植

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

血管功能在慢性肾脏疾病的评估

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Murine Kidney Transplant Technique

小鼠肾脏移植技术

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

常温<em&gt;离体</em&gt;肾脏的灌注肾的移植保护移植前

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

Histological Quantification of Chronic Myocardial Infarct in Rats

大鼠慢性心肌梗塞的组织学定量分析

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

小鼠肾小球疾病模型肾脏功能的评价

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

板基检测血液中一氧化氮的 vasp 磷酸化

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

One-anastomosis Gastric Bypass (OAGB) in Rats

One-anastomosis Gastric Bypass OAGB in Rats

大鼠胃旁路 (oagb) 的一次吻合

The goal of this protocol is to set up a preclinical model of bariatric surgery and, more specifically, OAGB in obese rats. Based on this preclinical ...