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
Summary
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。
Abstract
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。
Introduction
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</ em>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。
Protocol
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…
Representative Results
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?…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 1.5-mL conical microcentrifuge tube | VWR, Kelsterbach, Germany | 700-5239 | |
| 100-µL precision pipette | Eppendorf, Hamburg, Germany | 4920000059 | |
| 10-mL measuring pipette | Costar, Corning Inc., New York, USA | 4488 | |
| 14-mL PP tubes | Greiner bio-one, Frickenhausen, Germany | 187261 | |
| 1-mL precision pipette | Eppendorf, Hamburg, Germany | 4920000083 | |
| 500-µm filter (pluriStrainer 500 µm) | HISS Diagnostics, Freiburg, Germany | 43-50500-03 | |
| 50-mL conical centrifuge tube | Greiner bio-one, Frickenhausen, Germany | 227261 | |
| 50-mL Erlenmeyer flask | VWR, Kelsterbach, Germany | 214-0211 | |
| 96-well plate | Greiner bio-one, Frickenhausen, Germany | 65518 | |
| cell detachment solution (Accutase) | eBioscience, San Diego, CA USA | 00-4555-56 | |
| C57BL/6 mice | Charles River, Cologne, Germany | 027 | |
| C57BL/6-Tg(CAG-EGFP)1Osb/J mice | The Jackson Laboratory, Bar Harbor, USA | 003291 | |
| CD117-FITC | BD Biosciences, Heidelberg, Germany | 553373 | |
| CD31-PE | BD Biosciences, Heidelberg, Germany | 553354 | |
| Collagenase NB4G | Serva Electrophoresis GmbH, Heidelberg, Germany | 17465.02 | Lot tested by manufacturer for enzymatic activity |
| Dissection scissors | Braun Aesculap AG &CoKG, Melsungen, Germany | BC 601 | |
| DNA-binding dye (Bisbenzimide H33342) | Sigma-Aldrich, Taufkirchen, Germany | B2261 | |
| Dulbecco's modified Eagle medium (DMEM) | PAN Biotech, Rickenbach, Germany | P04-03600 | |
| Fetal calf serum (FCS) | Biochrom GmbH, Berlin, Germany | S0615 | |
| Fine forceps | S&T AG, Neuhausen, Switzerland | FRS-15 RM-8 | |
| Fine scissors | World Precision Instrumets, Sarasota, FL, USA | 503261 | |
| Dermal skin substitute (Integra) | Integra Life Sciences, Sain Priest, France | 62021 | |
| Ketamine | Serumwerk Bernburg AG, Bernburg, Germany | 7005294 | |
| M-IgG2akAL488 | eBioscience, San Diego, CA USA | 53-4724-80 | |
| Octeniderm (disinfecting solution) | Schülke & Mayer, Norderstedt, Germany | 118211 | |
| Penicillin/Streptomycin | Biochrom, Berlin, Germany | A2213 | |
| Petri dish | Greiner bio-one, Frickenhausen, Germany | 664160 | |
| Phosphate-buffered saline (PBS) | Lonza Group, Basel, Switzerland | 17-516F | |
| pluriStrainer 20-µm (20 µm filter) | HISS Diagnostics, Freiburg, Germany | 43-50020-03 | |
| Rat-IgG2akFITC | BD Biosciences, Heidelberg, Germany | 553988 | |
| Rat-IgG2akPE | BD Biosciences, Heidelberg, Germany | 553930 | |
| Small preparation scissors | S&T AG, Neuhausen, Switzerland | SDC-15 R-8S | |
| Surgical forceps | Braun Aesculap AG &CoKG, Melsungen, Germany | BD510R | |
| Tape (Heftpflaster Seide) 1.25 cm | Fink & Walter GmbH, Mechweiler, Germany | 1671801 | |
| Xylazine | Bayer Vital GmbH, Leverkusen, Germany | 1320422 | |
| α-SMA-AL488 | eBioscience, San Diego, CA USA | 53-9760-82 | Intracellular labeling additionally requires Cytofix/Cytoperm (BD Biosciences, Heidelberg, Germany; #554722) |
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Cite This Article
Frueh, F. S., Später, T., Scheuer, C., Menger, M. D., Laschke, M. W. Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering. J. Vis. Exp. (122), e55721, doi:10.3791/55721 (2017).