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>

<ol><li>Langer, R., Vacanti, J. P. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed?term=((%22Science%22%5BJournal%5D)%20AND%205110%5BIssue%5D)%20AND%20Langer%5BAuthor%5D">Tissue engineering.</a> Science. 260 (5110), 920-926 (1993).</li>
<li>Khademhosseini, A., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+decade+of+progress+in+tissue+engineering%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">A decade of progress in tissue engineering.</a> Nat Protoc. 11 (10), 1775-1781 (2016).</li>
<li>Novosel, E. C., Kleinhans, C., Kluger, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vascularization+is+the+key+challenge+in+tissue+engineering%5BTitle%5D)+AND+%22Adv+Drug+Deliv+Rev%22%5BJournal%5D)">Vascularization is the key challenge in tissue engineering.</a> Adv Drug Deliv Rev. 63 (4-5), 300-311 (2011).</li>
<li>Rouwkema, J., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vascularization+and+Angiogenesis+in+Tissue+Engineering%3A+Beyond+Creating+Static+Networks%5BTitle%5D)+AND+%22Trends+Biotechnol%22%5BJournal%5D)">Vascularization and Angiogenesis in Tissue Engineering: Beyond Creating Static Networks.</a> Trends Biotechnol. 34 (9), 733-745 (2016).</li>
<li>Laschke, M. W., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vascularization+in+tissue+engineering%3A+angiogenesis+versus+inosculation%5BTitle%5D)+AND+%22Eur+Surg+Res%22%5BJournal%5D)">Vascularization in tissue engineering: angiogenesis versus inosculation.</a> Eur Surg Res. 48 (2), 85-92 (2012).</li>
<li>Sarker, M., Chen, X. B., Schreyer, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+approaches+to+vascularisation+within+tissue+engineering+constructs%5BTitle%5D)+AND+%22J+Biomater+Sci+Polym+Ed%22%5BJournal%5D)">Experimental approaches to vascularisation within tissue engineering constructs.</a> J Biomater Sci Polym Ed. 26 (12), 683-734 (2015).</li>
<li>Frueh, F. S., Menger, M. D., Lindenblatt, N., Giovanoli, P., Laschke, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+and+emerging+vascularization+strategies+in+skin+tissue+engineering%5BTitle%5D)+AND+%22Crit+Rev+Biotechnol%22%5BJournal%5D)">Current and emerging vascularization strategies in skin tissue engineering.</a> Crit Rev Biotechnol. 20, 1-13 (2016).</li>
<li>Utzinger, U., Baggett, B., Weiss, J. A., Hoying, J. B., Edgar, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large-scale+time+series+microscopy+of+neovessel+growth+during+angiogenesis%5BTitle%5D)+AND+%22Angiogenesis%22%5BJournal%5D)">Large-scale time series microscopy of neovessel growth during angiogenesis.</a> Angiogenesis. 18 (3), 219-232 (2015).</li>
<li>Laschke, M. W., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prevascularization+in+tissue+engineering%3A+Current+concepts+and+future+directions%5BTitle%5D)+AND+%22Biotechnol+Adv%22%5BJournal%5D)">Prevascularization in tissue engineering: Current concepts and future directions.</a> Biotechnol Adv. 34 (2), 112-121 (2016).</li>
<li>Baiguera, S., Ribatti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelialization+approaches+for+viable+engineered+tissues%5BTitle%5D)+AND+%22Angiogenesis%22%5BJournal%5D)">Endothelialization approaches for viable engineered tissues.</a> Angiogenesis. 16 (1), 1-14 (2013).</li>
<li>Wagner, R. C., Kreiner, P., Barrnett, R. J., Bitensky, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biochemical+characterization+and+cytochemical+localization+of+a+catecholamine-sensitive+adenylate+cyclase+in+isolated+capillary+endothelium%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Biochemical characterization and cytochemical localization of a catecholamine-sensitive adenylate cyclase in isolated capillary endothelium.</a> Proc Natl Acad Sci U S A. 69 (11), 3175-3179 (1972).</li>
<li>Wagner, R. C., Matthews, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+isolation+and+culture+of+capillary+endothelium+from+epididymal+fat%5BTitle%5D)+AND+%22Microvasc+Res%22%5BJournal%5D)">The isolation and culture of capillary endothelium from epididymal fat.</a> Microvasc Res. 10 (3), 286-297 (1975).</li>
<li>Laschke, M. W., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adipose+tissue-derived+microvascular+fragments%3A+natural+vascularization+units+for+regenerative+medicine%5BTitle%5D)+AND+%22Trends+Biotechnol%22%5BJournal%5D)">Adipose tissue-derived microvascular fragments: natural vascularization units for regenerative medicine.</a> Trends Biotechnol. 33 (8), 442-448 (2015).</li>
<li>Laschke, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vascularisation+of+porous+scaffolds+is+improved+by+incorporation+of+adipose+tissue-derived+microvascular+fragments%5BTitle%5D)+AND+%22Eur+Cell+Mater%22%5BJournal%5D)">Vascularisation of porous scaffolds is improved by incorporation of adipose tissue-derived microvascular fragments.</a> Eur Cell Mater. 24, 266-277 (2012).</li>
<li>Frueh, F. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adipose+tissue-derived+microvascular+fragments+improve+vascularization%2C+lymphangiogenesis+and+integration+of+dermal+skin+substitutes%5BTitle%5D)+AND+%22J+Invest+Dermatol%22%5BJournal%5D)">Adipose tissue-derived microvascular fragments improve vascularization, lymphangiogenesis and integration of dermal skin substitutes.</a> J Invest Dermatol. 137 (1), 217-227 (2017).</li>
<li>McDaniel, J. S., Pilia, M., Ward, C. L., Pollot, B. E., Rathbone, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+and+multilineage+potential+of+cells+derived+from+isolated+microvascular+fragments%5BTitle%5D)+AND+%22J+Surg+Res%22%5BJournal%5D)">Characterization and multilineage potential of cells derived from isolated microvascular fragments.</a> J Surg Res. 192 (1), 214-222 (2014).</li>
<li>Nakano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+autotransplantation+of+microvessel+fragments+on+experimental+random-pattern+flaps+in+the+rat%5BTitle%5D)+AND+%22Eur+Surg+Res%22%5BJournal%5D)">Effect of autotransplantation of microvessel fragments on experimental random-pattern flaps in the rat.</a> Eur Surg Res. 30 (3), 149-160 (1998).</li>
<li>Nakano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Successful+autotransplantation+of+microvessel+fragments+into+the+rat+heart%5BTitle%5D)+AND+%22Eur+Surg+Res%22%5BJournal%5D)">Successful autotransplantation of microvessel fragments into the rat heart.</a> Eur Surg Res. 31 (3), 240-248 (1999).</li>
<li>Shepherd, B. R., Hoying, J. B., Williams, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microvascular+transplantation+after+acute+myocardial+infarction%5BTitle%5D)+AND+%22Tissue+Eng%22%5BJournal%5D)">Microvascular transplantation after acute myocardial infarction.</a> Tissue Eng. 13 (12), 2871-2879 (2007).</li>
<li>Pilia, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transplantation+and+perfusion+of+microvascular+fragments+in+a+rodent+model+of+volumetric+muscle+loss+injury%5BTitle%5D)+AND+%22Eur+Cell+Mater%22%5BJournal%5D)">Transplantation and perfusion of microvascular fragments in a rodent model of volumetric muscle loss injury.</a> Eur Cell Mater. 28, 11-23 (2014).</li>
<li>Laschke, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adipose+tissue-derived+microvascular+fragments+from+aged+donors+exhibit+an+impaired+vascularisation+capacity%5BTitle%5D)+AND+%22Eur+Cell+Mater%22%5BJournal%5D)">Adipose tissue-derived microvascular fragments from aged donors exhibit an impaired vascularisation capacity.</a> Eur Cell Mater. 28, 287-298 (2015).</li>
<li>Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T., Nishimune, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%27Green+mice%27+as+a+source+of+ubiquitous+green+cells%5BTitle%5D)+AND+%22FEBS+Lett%22%5BJournal%5D)">'Green mice' as a source of ubiquitous green cells.</a> FEBS Lett. 407 (3), 313-319 (1997).</li>
<li>Honek, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+age-related+insulin+sensitivity+by+VEGF-dependent+vascular+plasticity+in+adipose+tissues%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Modulation of age-related insulin sensitivity by VEGF-dependent vascular plasticity in adipose tissues.</a> Proc Natl Acad Sci U S A. 111 (41), 14906-14911 (2014).</li>
<li>Cho, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Angiogenic+role+of+LYVE-1-positive+macrophages+in+adipose+tissue%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Angiogenic role of LYVE-1-positive macrophages in adipose tissue.</a> Circ Res. 100 (4), e47-e57 (2007).</li>
<li>Han, S., Sun, H. M., Hwang, K. C., Kim, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adipose-Derived+Stromal+Vascular+Fraction+Cells%3A+Update+on+Clinical+Utility+and+Efficacy%5BTitle%5D)+AND+%22Crit+Rev+Eukaryot+Gene+Expr%22%5BJournal%5D)">Adipose-Derived Stromal Vascular Fraction Cells: Update on Clinical Utility and Efficacy.</a> Crit Rev Eukaryot Gene Expr. 25 (2), 145-152 (2015).</li>
<li>Chen, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+Differentiation+of+Adipose-Derived+Stem+Cells+from+Porcine+Subcutaneous+Adipose+Tissues%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues.</a> J Vis Exp. (109), e53886(2016).</li>
<li>Guillaume-Jugnot, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autologous+adipose-derived+stromal+vascular+fraction+in+patients+with+systemic+sclerosis%3A+12-month+follow-up%5BTitle%5D)+AND+%22Rheumatology+%28Oxford%29%22%5BJournal%5D)">Autologous adipose-derived stromal vascular fraction in patients with systemic sclerosis: 12-month follow-up.</a> Rheumatology (Oxford). 55 (2), 301-306 (2016).</li>
<li>Tissiani, L. A., Alonso, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Prospective+and+Controlled+Clinical+Trial+on+Stromal+Vascular+Fraction+Enriched+Fat+Grafts+in+Secondary+Breast+Reconstruction%5BTitle%5D)+AND+%22Stem+Cells+Int%22%5BJournal%5D)">A Prospective and Controlled Clinical Trial on Stromal Vascular Fraction Enriched Fat Grafts in Secondary Breast Reconstruction.</a> Stem Cells Int. , 2636454(2016).</li>
<li>Calcagni, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+novel+treatment+of+SVF-enriched+fat+grafting+for+painful+end-neuromas+of+superficial+radial+nerve%5BTitle%5D)+AND+%22Microsurgery%22%5BJournal%5D)">The novel treatment of SVF-enriched fat grafting for painful end-neuromas of superficial radial nerve.</a> Microsurgery. , (2016).</li>
<li>Hoying, J. B., Boswell, C. A., Williams, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Angiogenic+potential+of+microvessel+fragments+established+in+three-dimensional+collagen+gels%5BTitle%5D)+AND+%22In+Vitro+Cell+Dev+Biol+Anim%22%5BJournal%5D)">Angiogenic potential of microvessel fragments established in three-dimensional collagen gels.</a> In Vitro Cell Dev Biol Anim. 32 (7), 409-419 (1996).</li>
<li>Kirkpatrick, N. D., Andreou, S., Hoying, J. B., Utzinger, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+imaging+of+collagen+remodeling+during+angiogenesis%5BTitle%5D)+AND+%22Am+J+Physiol+Heart+Circ+Physiol%22%5BJournal%5D)">Live imaging of collagen remodeling during angiogenesis.</a> Am J Physiol Heart Circ Physiol. 292 (6), H3198-H3206 (2007).</li>
</ol>

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...

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?...

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

Research

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

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 ...

科研

生物工程

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 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 ...

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

摘要

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在组织工程由内在血管化

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