Name: isolation of murine adipose tissue-derived microvascular fragments as vascularization units for tissue engineering
Uploaded: 2017-04-30T13:00:00.000+00:00
Duration: 7 min 9 s
Description: Watch this Scientific Journal Video about Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering at JoVE.com

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><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-&amp;micro;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-&amp;micro;m filter (pluriStrainer 500 &amp;micro;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&amp;nbsp;</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 &amp;amp;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&#039;s modified Eagle medium (DMEM)&amp;nbsp;</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&amp;amp;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&amp;nbsp;</td><td>Serumwerk Bernburg AG, Bernburg, Germany</td><td>7005294</td><td></td></tr><tr><td>M-IgG2akAL488&amp;nbsp;&amp;nbsp;</td><td>eBioscience, San Diego, CA USA</td><td>53-4724-80</td><td></td></tr><tr><td>Octeniderm (disinfecting solution)</td><td>Sch&amp;uuml;lke &amp;amp; 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-&amp;micro;m (20 &amp;micro;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&amp;amp;T AG, Neuhausen, Switzerland</td><td>SDC-15 R-8S</td><td></td></tr><tr><td>Surgical forceps</td><td>Braun Aesculap AG &amp;amp;CoKG, Melsungen, Germany</td><td>BD510R</td><td></td></tr><tr><td>Tape (Heftpflaster Seide) 1.25 cm</td><td>Fink &amp;amp; Walter GmbH, Mechweiler, Germany</td><td>1671801</td><td></td></tr><tr><td>Xylazine&amp;nbsp;</td><td>Bayer Vital GmbH, Leverkusen, Germany</td><td>1320422</td><td></td></tr><tr><td>&amp;alpha;-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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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 ...

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Universidad San Sebastian

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JoVE Journal

Bioengineering

12.4K 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

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

弹性PGS的动脉组织工程支架

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

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 ...

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for the design of personalized microtissues for health screening and drug testing. Current strategies are often quite complex and fail to accurately fully depict the multicellular and functional properties of thermogenic adipose tissue. Microvascular fragments, small intact microvessels comprised of arteriole, venules, and capillaries isolated from adipose tissue, serve as a single autologous source of cells that enable vascularization and adipose tissue formation. This article describes methods for optimizing culture conditions to enable the generation of three-dimensional, vascularized, and functional thermogenic adipose tissues from microvascular fragments, including protocols for isolating microvascular fragments from adipose tissue and culture conditions. Additionally, best practices are discussed, as are techniques for characterizing the engineered tissues, and sample results from both rodent and human microvascular fragments are provided. This approach has the potential to be utilized for the understanding and development of treatments for obesity and metabolic disease.

Here, we present a detailed protocol outlining the use of microvascular fragments isolated from rodent or human fat tissue as a straightforward approach to engineer functional, vascularized beige adipose tissue.

微血管碎片血管化产热脂肪组织的三维培养

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for ...

Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. Accordingly, techniques for harvesting and processing human adipose tissue have evolved in order to quickly and efficiently obtain large amounts of tissue. Among these, the closed system technology represents an innovative and easy-to-use system to harvest, process, and re-inject refined fat tissue in a short time and in the same intervention (intra-operatively). Adipose tissue is collected by liposuction, washed, emulsified, rinsed and minced mechanically into cell clusters of 0.3 to 0.8 mm. Autologous transplantation of mechanically fragmented adipose tissue has shown remarkable efficacy in different therapeutic indications such as aesthetic medicine and surgery, orthopedic and general surgery. Characterization of micro-fragmented adipose tissue revealed the presence of intact small vessels within the adipocyte clusters; hence, the perivascular niche is left unperturbed. These clusters are enriched in perivascular cells (i.e., mesenchymal stem cell (MSC) ancestors) and in vitro analysis showed an increased release of growth factors and cytokines involved in tissue repair and regeneration, compared to enzymatically derived MSCs. This suggests that the superior therapeutic potential of microfragmented adipose tissue is explained by a higher frequency of presumptive MSCs and enhanced secretory activity. Whether these added pericytes directly contribute to higher growth factor and cytokine production is not known. This clinically approved procedure allows the transplantation of presumptive MSCs without the need for expansion and/or enzymatic treatment, thus bypassing the requirements of GMP guidelines, and reducing the costs for cell-based therapies.

Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and characterization.

用于细胞表征和分泌特征的人类脂肪组织微分裂

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. ...

生物学

从血管壁分离和磁性分离小鼠 CD34+ 干细胞

Immunomagnetic Isolation of the Vascular Wall-Resident CD34+ Stem Cells from Mice

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury and repair. Establishing a stable and efficient method to culture functional murine CD34+ VW-SCs is essential for further investigating the mechanisms involved in the proliferation, migration, and differentiation of these cells under various physiological and pathological conditions. The described method combines magnetic bead screening and flow cytometry to purify primary cultured resident CD34+ VW-SCs. The purified cells are then functionally identified through immunofluorescence staining and Ca2+ imaging. Briefly, vascular cells from the adventitia of the murine aorta and mesenteric artery are obtained through tissue block attachment, followed by subculturing until reaching a cell count of at least 1 &#215; 107. Subsequently, CD34+ VW-SCs are purified using magnetic bead sorting and flow cytometry. Identification of CD34+ VW-SCs involves cellular immunofluorescence staining, while functional multipotency is determined by exposing cells to a specific culture medium for oriented differentiation. Moreover, functional internal Ca2+ release and external Ca2+ entry is assessed using a commercially available imaging workstation in Fura-2/AM-loaded cells exposed to ATP, caffeine, or thapsigargin (TG). This method offers a stable and efficient technique for isolating, culturing, and identifying vascular wall-resident CD34+ stem cells, providing an opportunity for in vitro studies on the regulatory mechanisms of VW-SCs and the screening of targeted drugs.

作者聚焦:使用磁珠筛选偶联流式细胞术增强血管壁驻留小鼠 CD34+ 干细胞的分离和表征

This study has established a stable and efficient method for the isolation, culture, and functional determination of vascular wall-resident CD34+ stem cells (CD34+ VW-SCs). This easy-to-follow and time-effective isolation method can be utilized by other investigators to study the potential mechanisms involved in cardiovascular diseases.

从小鼠中免疫磁性分离血管壁驻留的 CD34+ 干细胞

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury ...

医学

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

从不同的脂肪库分离和鉴定血管内皮细胞，用于下游应用

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

小鼠白色脂肪组织的半自动分离

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

作者聚焦：使用组织解离剂从小鼠白色脂肪组织中半自动分离基质血管组分  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

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

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此视频中的章节

弹性PGS的动脉组织工程支架

静电纤维组织工程的后期处理

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

微血管碎片血管化产热脂肪组织的三维培养

用于细胞表征和分泌特征的人类脂肪组织微分裂

从小鼠中免疫磁性分离血管壁驻留的 CD34⁺ 干细胞

从小鼠中免疫磁性分离血管壁驻留的 CD34⁺ 干细胞

从小鼠中免疫磁性分离血管壁驻留的 CD34⁺ 干细胞

从不同的脂肪库分离和鉴定血管内皮细胞，用于下游应用

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分