我们是贾尼娜·贝克尔，卡罗琳·比克尔曼和鲁思·尼克尔斯的优秀的技术援助表示感谢。 （ - 德国研究基金会DFG） - LA 2682 / 7-1这项研究是由德意志研究联合会的资助。

根据对实验动物的使用准则卫生研究所进行并遵循机构准则（Landesamt献给Soziales，GESUNDHEIT UND Verbraucherschutz，ABT。Lebensmittel- UNDVeterinärwesen，Zentralstelle，萨尔布吕肯，德国）的所有程序。 1.手术器械的制备<ol><li>保持准备的清扫剪刀，手术钳，小准备剪刀，精细镊和无菌培养皿用15ml Dulbecco改良的Eagle培养基（DMEM; 10％胎牛血清（FCS），100U / mL青霉素，0.1毫克/毫升链霉素）为收获附睾脂肪垫。 </li><li>暴露手术器械至5分钟的消毒溶液。可替换地，它们进行消毒（蒸汽灭菌; 121℃，20分钟）。 </li></ol> 2，动物和麻醉<ol><li>仔细选择的小鼠品系作为研究和指示<html>受调查之中。 注:在本研究中，我们使用的雄性野生型C57BL / 6小鼠作为捐助者的附睾脂肪的收获。的兴趣，广告-MVF也可以从转基因绿色荧光蛋白（GFP）阳性供体动物（C57BL / 6-TG（CAG-EGFP）1Osb / J）22隔离。这证明的主要优点是，片段是通过植入GFP阴性野生型小鼠14之后的GFP免疫组织化学染色容易检测的。 </li><li>用甲苯噻嗪（15毫克/千克）和氯胺酮（75毫克/千克）的腹膜内注射麻醉动物。确保动物深受无响应执行脚趾捏麻醉。作为供体动物脂肪获取后处死眼部润滑剂无法显示。 注:确保镇痛和手术不育是与其中的实验计划的国家和机构各自的指导方针一致。 </li></ol>附睾脂肪垫的乐"&gt; 3。收获<ol><li>动物转移到手术台上。放置在仰卧位的动物外科手术立体显微镜下，用脚趾捏确认深度麻醉。 </li><li>他们录音到手术单固定的爪子并用消毒液消毒腹部。 </li><li>分离游离下面的肌肉层与清扫剪刀的腹部皮肤。 </li><li>与清扫剪刀进行中线剖腹手术和横向展开腹壁的襟翼。 </li><li>双边识别睾丸，附睾和使用细钳子（ 图1）的附睾脂肪垫。 不要伤害肠道结构，防止脂肪垫的粪便污染。 </li><li>用立体显微镜下的小剪刀准备和罚款钳收获附睾脂肪垫。 保持附睾和脂肪之间的几个毫米的安全边际减少意外收获附睾的风险。 注:此过程也可以在没有立体显微镜进行。然而，这可能进一步增加附睾和精索的意外伤害的风险。 </li><li>在37℃下转移附睾脂肪垫到含有15毫升的DMEM的培养皿预加热输送至细胞实验室。 </li><li>经腹主动脉或颈椎脱位切开牺牲动物。 </li></ol> 4.隔离广告的MVF <ol><li>用15mL磷酸盐缓冲盐水（PBS），无菌的14毫升聚丙烯（PP）管，无菌的50姆·埃伦迈尔瓶中，无菌1.5 mL锥形离心管中并灭菌细剪刀制备三种无菌培养皿中。 </li><li>在层流罩下用15ml PBS的三次洗涤脂肪垫在培养皿中。 </li><li>转移脂肪成一个14毫升PP管。用t来确定收获的脂肪组织（以mL）的体积他管的规模。与细剪刀切碎机械的脂肪组织，直到获得均匀的组织悬浮液。 </li><li>通过测量移液管的10mL的手段用胶原酶NB4G（0.5 U / ml的PBS）的两卷转移切碎的组织成为50毫升锥形瓶中。总体积变为三次在步骤4.3测量脂肪组织的体积。在37℃，并用5％CO 2湿润大气条件:在剧烈搅拌下10分钟的孵化器由自动搅拌器（25毫米磁力搅拌棒的大小）来进行组织消化。 </li><li>观察消化的组织的一小部分（10μL），在显微镜下，以判断是否消化可以停止。确定该消化物主要含有下为单细胞"免费"广告MVF，指示适当的点以终止酶促消化处理（ 图2）。 注:此步骤需要与程序的经验和对质量的关键孤立广告MVF。长期在一个单细胞悬液脂肪消化的结果，而广告MVF。 </li><li>中和，用PBS / 20％FCS的两个体积的酶。总体积变为三次细胞 - 悬浮液容器的步骤4.4的体积。转移细胞的容器悬挂放回新PP管中。 </li><li>孵育5分钟将悬浮液在37℃至广告MVF从通过重力剩余的脂肪分离。然后小心地用1mL的精密移液管取出主脂肪上清液。重复这个循环多次使用100μL精密移液管，直到悬挂似乎是不含脂肪。 </li><li>把一个500微米的过滤器上的50mL锥形离心管的顶部。从14毫升PP管用10毫升移液管测量转移的细胞悬浮液容器到所述过滤膜，以除去剩余的脂肪凝块。根据个别广告-MVF的数目传送过滤的悬浮液到新的14毫升PP管分离为计划的实验。 注:有关VITRÒ测定着眼于微血管网络的形成，这可能是有益的，以进一步纯化所述细胞悬浮液容器，以改善在显微镜分析成像质量。为了这个目的，该悬浮液可以另外用20微米过滤器过滤一次，以去除从所述ad-MVF单个细胞收集在过滤器上。 </li><li>离心细胞悬浮液容器（600×g下，5分钟，室温），得到含有广告-MVF的颗粒。 </li><li>离心后，除去直到1毫升留上清液。重悬在此1毫升用Ad-MVF颗粒与悬浮液转移到1.5-mL锥形离心管。 </li><li>离心机的离心管中的ad-MVF悬浮液（600×g下，5分钟），得到颗粒。 </li><li>除去上清液重悬在PBS / 20％FCS的所要求的最终体积的颗粒。 注:每个隔离广告MVF的数量可通过显微计数进行评估。为此，1/10决赛细胞vesse的升悬浮液在PBS中稀释1:10，该稀释的100微升转移到一个96孔板的孔中。然后广告-MVF的呈现容器状形态的整个数目进行计数，并外推到整个分离物。所需的广告MVF浓缩和纯化可以在体外或在 ad-MVF的体内应用单独适应于相应。这可以包括广告-MVF的在胶原凝胶用于微血管网络的形成的体外分析或广告MVF的支架上用于体内植入到组织中的缺陷的播种的嵌入。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

在这项研究中，我们提出了广告MVF的隔离一套行之有效的协议。从小鼠脂肪组织获取广告MVF是几个关键步骤的手续很简单。小鼠表现出不同的皮下和腹腔内脂肪堆积。如先前针对大鼠描述于广告-MVF的隔离最合适的脂肪源是附睾脂肪垫，由于它们的尺寸，均匀的结构和最少的污染具有更大的血管11，12。相反，在小鼠皮下脂肪沉积要小得多，更粘附到周围的组织。然而，附睾脂肪垫切除熊附睾和精索的意外伤害的风险，导致精子与收获的组织的污染。因此，附睾，睾丸和精索的标识是在此过程中非常重要的。此外，脂肪垫应该是rapidlÿ收获，以尽量减少离体组织损伤后处理。最后，应该考虑到，有在鼠附睾脂肪垫23血管主要依赖于年龄的变化。根据我们的经验，使用6到12个月大的雄性供体小鼠提供足量的广告MVF的体外和体内研究。然而，它也可以是可能的ad-MVF从年轻小鼠，表现出较小的但高度血管化的脂肪垫24隔离。 收获的附睾脂肪垫的酶促消化关键判定孤立广告-MVF的最终质量。太短的论述到胶原酶导致从周围脂肪细胞的ad-MVF的不完全分离。在另一方面，长时间的消化生成的ad-MVF的破坏，单细胞悬浮液，所谓的基质血管级分（SVF）。的SVF是异构含有细胞群米esenchymal干细胞，内皮细胞，周细胞，巨噬细胞和许多其他细胞类型中通过旁分泌和分化机制25的再生潜能。利用广告MVF的相比，基于SVF-方法具有相当大的优势。广告-MVF的完全功能性微血管形态学允许它们在体内实验直接注入，而无需耗时和复杂的体外培养。此外，SVF的分离通常需要用胶原酶的组织的多达90分26的消化。相反，组织降解不应超过用于广告-MVF的分离10分钟。这支持的广告MVF 9术一步法应用的想法。为了这个目的，广告MVF可以自动地从自体脂吸出物中分离，并转移回患者的组织缺陷，而不必离开手术室。这是一个现实的果阿升考虑的事实，SVF的从脂吸出物的自动分离已经在整形外科27，28，29的临床上建立的程序。 最近，我们已经证明了广告MVF对骨14和皮肤15替代品在体内的血管有很大的影响。有趣的是，我们发现它有利的是不从广告MVF删除单个细胞中分离的，因为它们可以提供有助于广告MVF 13的高容量血管更生理环境。因此，我们只广告-MVF的用于去除较大的非消化的脂肪凝块的隔离期间使用的500微米的过滤器。此外，我们发现，在植入皮肤广告MVF诱导淋巴管生成替代15。因此，广告MVF也可有前途的lymphat积木IC组织工程和淋巴水肿治疗。 对于这些实验结果到临床实践的成功翻译，合乎逻辑的下一步是澄清人体脂肪组织是否同样适用于广告MVF的隔离啮齿动物脂肪。在此背景下，一个关键的问题是脂肪分离大量的广告MVF所需要的量。描述协议本文约40000的ad-MVF导致每毫升脂肪组织。因此，3毫升脂肪组织有必要prevascularize真皮皮肤代替15的1 平方厘米。因此，该方法可能不适合较大的伤口或病情严重的患者的治疗是可行的，由于可用的脂肪组织的用于广告MVF隔离有限。 旁的组织工程和再生医学的体内应用潜力的广谱，广告MVF也适用于血管生成的PROC 体外建模esses。例如，老鼠广告MVF已经在三维胶原凝胶培养研究新的微血管网络30成膜。类似的方法已被用于血管生成31中，以评估新血管生长13或间质基质重塑的可视化复杂的成像技术。 总之，这些结果表明，广告MVF不仅有为的血管和淋巴管单位为今后的治疗策略，而且对血管生成和血管生理学领域的基础研究。

组织工程集中在组织和器官的替代品维持， 体内同行1,2恢复或增强的不可操作的功能的制造。工程化组织构建的命运很大程度上取决于适当的血管3。这些结构中的微血管网络应与小动脉，毛细血管和小静脉分层组织，使吻合到收件人的血管4后有效血液灌注。这种网络的产生是在组织工程中的主要挑战之一。为了这个目的，实验血管策略广谱已经出台，在过去二十年里5，6。 血管生成的方法刺激接受者微血管长入工程做卷烟由结构或物理化学骨架修饰的手段，如生长的掺入因子7的UE。然而，对于大的三维结构的血管形成，血管生成依赖性策略显着通过开发微血管8的生长速度缓慢的限制。 与此相反，prevascularization的概念旨在用于功能微血管网络的生成中之前将其植入9组织构建体。常规prevascularization涉及形成血管的细胞，如内皮细胞，周细胞或干细胞10，支架内的共培养物。后微血管网络的形成，预血管化构建体可以然后被植入到组织中的缺陷。值得注意的是，此方法prevascularization难以在临床环境中应用，因为它是基于复杂的和耗时的体外&lt;/ em&gt;的程序，由主要监管障碍限制9。因此，仍然需要新颖的prevascularization策略，更适合广泛的临床应用的发展。 这种prevascularization策略可能是来自脂肪组织的微血管片段（广告-MVF）的应用。广告-MVF表示可以在大量从大鼠11,12和小鼠13的脂肪组织收获效的血管形成单元。它们由小动脉，毛细管，和小静脉血管区段，其表现出与管腔的生理微血管形态和稳定血管周围细胞14,15。这种独特的功能使得广告MVF接种支架的直接植入组织缺损无预培养。在那里，广告MVF快速重新装配到功能性微血管网络。此外，广告-MVF表示富间充质干细胞16，其可额外地有助于其撞击再生能力的源。因此，广告-MVF正越来越多地在组织工程14，15，17，18，19，20，21的不同领域中使用。 广告MVF的隔离最初已经建立大鼠11，12。在此，我们描述了一种协议，它允许从附睾脂肪垫鼠广告-MVF的标准化隔离。这可以提供进一步的见解成通过使用转基因小鼠模型基础的ad-MVF功能的分子机制。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5-mL conical microcentrifuge tube</td>
			<td>VWR, Kelsterbach, Germany</td>
			<td>700-5239</td>
			<td></td>
		</tr>
		<tr>
			<td>100-&#181;L precision pipette</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>4920000059</td>
			<td></td>
		</tr>
		<tr>
			<td>10-mL measuring pipette</td>
			<td>Costar, Corning Inc., New York, USA</td>
			<td>4488</td>
			<td></td>
		</tr>
		<tr>
			<td>14-mL PP tubes</td>
			<td>Greiner bio-one, Frickenhausen, Germany</td>
			<td>187261</td>
			<td></td>
		</tr>
		<tr>
			<td>1-mL precision pipette</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>4920000083</td>
			<td></td>
		</tr>
		<tr>
			<td>500-&#181;m filter (pluriStrainer 500 &#181;m)</td>
			<td>HISS Diagnostics, Freiburg, Germany</td>
			<td>43-50500-03</td>
			<td></td>
		</tr>
		<tr>
			<td>50-mL conical centrifuge tube</td>
			<td>Greiner bio-one, Frickenhausen, Germany</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>50-mL Erlenmeyer flask</td>
			<td>VWR, Kelsterbach, Germany</td>
			<td>214-0211</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Greiner bio-one, Frickenhausen, Germany</td>
			<td>65518</td>
			<td></td>
		</tr>
		<tr>
			<td>cell detachment solution (Accutase)</td>
			<td>eBioscience, San Diego, CA USA</td>
			<td>00-4555-56</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Charles River, Cologne, Germany</td>
			<td>027</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6-Tg(CAG-EGFP)1Osb/J mice</td>
			<td>The Jackson Laboratory, Bar Harbor, USA</td>
			<td>003291</td>
			<td></td>
		</tr>
		<tr>
			<td>CD117-FITC</td>
			<td>BD Biosciences, Heidelberg, Germany</td>
			<td>553373</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31-PE</td>
			<td>BD Biosciences, Heidelberg, Germany</td>
			<td>553354</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase NB4G&#160;</td>
			<td>Serva Electrophoresis GmbH, Heidelberg, Germany</td>
			<td>17465.02</td>
			<td>Lot tested by manufacturer for enzymatic activity</td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>Braun Aesculap AG &#38;CoKG, Melsungen, Germany</td>
			<td>BC 601</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA-binding dye (Bisbenzimide H33342)</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle medium (DMEM)&#160;</td>
			<td>PAN Biotech, Rickenbach, Germany</td>
			<td>P04-03600</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Biochrom GmbH, Berlin, Germany</td>
			<td>S0615</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>S&#38;T AG, Neuhausen, Switzerland</td>
			<td>FRS-15 RM-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissors</td>
			<td>World Precision Instrumets, Sarasota, FL, USA</td>
			<td>503261</td>
			<td></td>
		</tr>
		<tr>
			<td>Dermal skin substitute (Integra)</td>
			<td>Integra Life Sciences, Sain Priest, France</td>
			<td>62021</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine&#160;</td>
			<td>Serumwerk Bernburg AG, Bernburg, Germany</td>
			<td>7005294</td>
			<td></td>
		</tr>
		<tr>
			<td>M-IgG2akAL488&#160;&#160;</td>
			<td>eBioscience, San Diego, CA USA</td>
			<td>53-4724-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Octeniderm (disinfecting solution)</td>
			<td>Sch&#252;lke &#38; Mayer, Norderstedt, Germany</td>
			<td>118211</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>A2213</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Greiner bio-one, Frickenhausen, Germany</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Lonza Group, Basel, Switzerland</td>
			<td>17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>pluriStrainer 20-&#181;m (20 &#181;m filter)</td>
			<td>HISS Diagnostics, Freiburg, Germany</td>
			<td>43-50020-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat-IgG2akFITC</td>
			<td>BD Biosciences, Heidelberg, Germany</td>
			<td>553988</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat-IgG2akPE</td>
			<td>BD Biosciences, Heidelberg, Germany</td>
			<td>553930</td>
			<td></td>
		</tr>
		<tr>
			<td>Small preparation scissors</td>
			<td>S&#38;T AG, Neuhausen, Switzerland</td>
			<td>SDC-15 R-8S</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical forceps</td>
			<td>Braun Aesculap AG &#38;CoKG, Melsungen, Germany</td>
			<td>BD510R</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape (Heftpflaster Seide) 1.25 cm</td>
			<td>Fink &#38; Walter GmbH, Mechweiler, Germany</td>
			<td>1671801</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine&#160;</td>
			<td>Bayer Vital GmbH, Leverkusen, Germany</td>
			<td>1320422</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-SMA-AL488</td>
			<td>eBioscience, San Diego, CA USA</td>
			<td>53-9760-82</td>
			<td>Intracellular labeling additionally requires Cytofix/Cytoperm (BD Biosciences, Heidelberg, Germany; #554722)</td>
		</tr>
	</tbody>
</table>

isolation of murine adipose tissue-derived microvascular fragments as vascularization units for tissue engineering

功能性微血管网络的生存和工程化组织构建整合至关重要的意义。为此，一些血管生成和prevascularization战略已经确立。然而，大多数的基于细胞的方法包括耗时的体外用于微血管网络的形成步骤。因此，它们不适合于术中的一步程序。脂肪组织来源的微血管片段（广告-MVF）代表有希望的血管形成单元。它们可以从脂肪组织容易地分离，并表现出的功能性微血管形态。此外，他们很快重新组合成在体内植入后新的微血管网络。此外，广告MVF已被证明诱导淋巴管生成。最后，它们都具有丰富的间充质干细胞，它可以进一步促进其高血管潜在来源。在以前的研究中，我们已经证明了显着的vascularizati在工程化骨和皮肤代用品广告MVF的能力。在本研究中，我们对广告MVF从小鼠脂肪组织中的酶隔离的标准化协议的报告。

在本研究中，我们进行六次广告-MVF分离程序与脂肪组织从7至12个月大的雄性野生型C57BL / 6小鼠（平均体重:35±1克）。 图1示出的鼠附睾脂肪垫，随后机械和酶的ad-MVF隔离收获。对于脂肪的收获所需的时间为30分钟，并为广告-MVF的隔离为120分钟。总的来说，该过程带着150分钟。 我们收获1.2±0.1毫升每供体动物的脂肪组织的。的脂肪该量允许每毫升42000±2000广告-MVF的分离。所述分离的广告-MVF的平均长度为42±1微米（ 图2A）。 如图2B酶消化的持续时间是由微观控制的方法来确定。广告-MVF表现出与分层VES典型微血管形态SEL片段（ 图2C）。 通过流式细胞仪的方式，我们还表征了广告MVF。为了这个目的，广告-MVF进一步在细胞剥离溶液消化30分钟成单个细胞15。流式细胞术分析显示，广告MVF含有26±2％CD31阳性内皮细胞，17±2％α平滑肌肌动蛋白（SMA）阳性血管周围细胞，和9±1％的细胞为阳性的间充质干细胞标记CD117（ 图3A-C）。接种上的真皮皮肤替代物，这是固定的，并立即用于组织学分析切片，之后较大的广告-MVF主要定位在植入物的表面（ 图3D）上。然而，毛细血管段也可在其孔内检测。种子广告MVF的免疫组织化学染色进一步揭示了与小动脉，小静脉生理微血管配置和毛细管样片段（ 图3E）。 <img alt="图1" src="/files/ftp_upload/55721/55721fig1.jpg" /> 图1: 广告MVF隔离。 A）继中线剖腹，附睾脂肪垫（星号）标识。脂肪组织收获的小剪刀制备和动物由腹主动脉（箭头）的切口处死。 B）用细剪刀脂肪垫的机械切碎直至组织悬浮液出现均匀。 C）对于酶消化，胶原酶被添加到组织悬浮液中。 D）将细胞-悬浮液容器中温育，将上清液脂肪层被丢弃。 E）离心后，将含有广告-MVF的沉淀物重新悬浮，并且可以用于不同的应用，如microvasc的体外分析ular网络形成或广告MVF的支架上用于体内植入到组织缺损（F）播种。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55721/55721fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55721/55721fig2.jpg" /> 图2: 广告MVF的形态特征。 A）新鲜分离的广告MVF的长度分布。平均值±平均值的标准误差的（N = 6）。细胞-悬浮液容器涂抹B）显微图像具有大（箭头），中型（双箭头）和小（黑色箭头）的ad-MVF以及单细胞（白色箭头）。比例尺= 110微米。更高的放大倍率显示，广告MVF表现出成熟的微血管形态与分层微血管段（C， <stro纳克&gt;小图b的）。比例尺:C = 50微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55721/55721fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55721/55721fig3.jpg" /> 图3:流式细胞术，组织学和广告MVF的免疫组化特征。 AC）流动新鲜分离的ad-MVF示出CD31阳性内皮细胞（A），α-SMA阳性细胞的血管周围（B）和CD117阳性间充质干细胞（C）的级分的流式细胞分析。适当同种型相同的对照用于调整阈值电平。平均值±平均值的标准误差的（N = 6）。 D）苏木精和接种的真皮皮肤替代的曙红染色切片。一些毛细血管段位于植入物＃39; S孔（箭头）。与小静脉（双箭头）或小动脉（箭头）形态学较大微血管是局部在其表面上。比例尺=40μm左右。虚线=植入物的边界。 E）通过免疫组织化学检测内皮细胞标记物CD31（红）和血管周细胞标记物α-SMA（绿色）的手段的ad-MVF的表征。细胞核用DNA结合的荧光染料（蓝色）染色。相比具有较大内腔（双箭头）小静脉片段时小动脉的ad-MVF（箭头）的特征在于一个较厚的α-SMA阳性细胞层。箭头=毛细管广告MVF。比例尺= 25微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55721/55721fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering at JoVE.com

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

我们提出了一个协议，以分离代表有希望的血管单元脂肪组织来源的微血管片段。它们能够迅速地分离，不体外处理要求，因此，可以用于在组织工程的不同领域一步法prevascularization。

小鼠脂肪组织来源的微血管片段作为血管化单元的分离组织工程

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

isolation-murine-adipose-tissue-derived-microvascular-fragments-as

Nanyang Technological University

Research

JoVE Journal

Bioengineering

12.3K 次观看.  Saarland University. 我们提出了一个协议，以分离代表有希望的血管单元脂肪组织来源的微血管片段。它们能够迅速地分离，不体外处理要求，因此，可以用于在组织工程的不同领域一步法prevascularization。 

视频: Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

心肌组织工程生物衍生的注射材料的制备

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

科研

生物工程

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

静电纤维组织工程的后期处理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

工程骨骼肌组织中的小鼠成肌细胞的内皮祖细胞与应用电刺激

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

小鼠模型肠组织工程

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

温敏纳米结构表面的组织工程准备

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

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 ...