Name: human adipose tissue micro-fragmentation for cell phenotyping and secretome characterization
Uploaded: 2019-10-20T14:00:00
Description: Watch this Scientific Journal Video about Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization at JoVE.com

作者希望感谢爱丁堡大学的克莱尔·克里尔和菲奥娜·罗西在流式细胞学方面的专家协助。我们还要感谢默里菲尔德医院的人员，他们通过提供组织标本做出了贡献。
这项工作得到了英国心脏基金会和利波格姆斯的资助，后者提供了脂肪组织处理试剂盒。人类成人组织样本是经东南苏格兰研究伦理委员会完全伦理许可采购的（参考：16/SS/0103）。

本研究中使用人体组织的道德批准来自东南苏格兰研究伦理委员会（参考：16/SS/0103）。
1. 皮下腹腔脂肪组织收集
注：手动裂隙吸气程序中使用的所有仪器均由微碎片装置的制造商提供。
<ol><li>在整个实验中，保持所有液体、容器、仪器和操作区域的不育。</li>
	<li>将腹肌整形样品（由手术小组采购在无菌袋中，没有任何溶液）放在手术布?...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transnasal+endoscopic+microfractured+fat+injection+in+glottic+insufficiency.">Transnasal endoscopic microfractured fat injection in glottic insufficiency.</a> B-ENT. 11 (3), 229-234 (2015).</li><li>Giori, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+function+in+anal+incontinence+after+micro-fragmented+fat+graft+(Lipogems)+injection:+two+years+follow+up+of+the+first+5+cases.">Recovery of function in anal incontinence after micro-fragmented fat graft (Lipogems) injection: two years follow up of the first 5 cases.</a> CellR4. 3 (2), (2015).</li><li>Tremolada, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+mesenchymal+stem+cells+and+regenerative+adipose+tissue+graft+(Lipogems)+for+musculoskeletal+regeneration.">Adipose mesenchymal stem cells and regenerative adipose tissue graft (Lipogems) for musculoskeletal regeneration.</a> European Journal of Muscoloskeletal Diseases. 3 (2), 57-67 (2014).</li><li>Striano, R. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+fat+grafting:+more+than+a+permanent+filler.">Structural fat grafting: more than a permanent filler.</a> Plastic and Reconstructive Surgery. 118 (3), 108S-120S (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Editorial:+An+update+on+organoid+research.">Editorial: An update on organoid research.</a> Nature Cell Biology. 20 (6), 633 (2018).</li></ol>

本文利用闭合系统装置，将人体脂肪组织物理分馏成显示正常脂肪组织微解剖学的小簇。
手动吸气的人类皮下脂肪组织和盐水溶液被装入一个透明塑料圆柱体，里面装着大弹球式金属球，在设备剧烈手动晃动后，将脂肪破裂成亚毫米碎片。附加的过滤器和出口可消除碎屑、血液和游出脂，MAT 被收集到与设备相连的注射器中。在这里，MAT已经成功地进一步加工免疫组织化学，?...

脂肪组织，长期用作重建和整容手术的填充物，最近已成为再生医学越来越流行，曾经被认为是间质干细胞（MSCs）1的来源。分离到单细胞悬浮液中的脂蛋白产生无脂肪细胞的基质血管分数（SVF），在患者中未改变使用，或者更常见的是，在MSCs2中培养数周。
然而，酶分离会破坏组织微环境，使邻近的调节细胞从假定的再生细胞中分离，这些细胞在体外培养中会大大改性。为了避免这种实验性的伪影和随之而来的功能改变，已经尝试处理脂肪组织用于治疗使用，同时保持其原生结构尽可能完整3，4。值得注意的是，机械组织破坏已经开始取代酶分离。为此，完全浸入式封闭系统通过筛过滤和钢大理石诱导的干扰序列，将脂吸气分光成亚毫米、无血和无油组织簇（如利波格姆）。微碎片脂肪组织的自体移植，使用这种封闭系统技术，已经成功地在多种适应症，包括化妆品，骨科，妇产科4，5，6，7，8，9，10，11，12，13。
使用闭合系统装置获得的人类微碎片脂肪组织（MAT）和同源性SVF的比较表明，在培养的血管/基质细胞分布和分泌活性方面，MAT含有更多的围细胞，即假定MSCs14，并分泌较多的生长因子和细胞因子15。
本文阐述了使用闭合系统装置对人皮下脂肪组织的无酶微分泌，以及对这种微缩脂肪组织的进一步处理，用于体外培养、免疫组织化学和FACS分析，为了识别存在的细胞类型和分泌的可溶性因子（图1）。所述方法安全地生成含有可行脂肪组织细胞群的脂肪衍生亚毫米有机物，适合进一步应用和研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4% Buffered paraformaldehyde (PFA)</td>
			<td>VWR chemicals</td>
			<td>9317.901</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% NaCl Solution</td>
			<td>Baxter</td>
			<td>3KB7127</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 555 goat anti-mouse IgG&#160;</td>
			<td>Life Technologies</td>
			<td>A21422</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 647 goat anti-Rabbit IgG</td>
			<td>Life Technologies</td>
			<td>&#160;A21245</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>fisher chemicals</td>
			<td>1158868</td>
			<td></td>
		</tr>
		<tr>
			<td>Antigent Diluent</td>
			<td>Life Technologies</td>
			<td>3218</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Ig, &#954;/Negative Control (BSA) Compensation Plus</td>
			<td>BD Biosciences</td>
			<td>560497</td>
			<td></td>
		</tr>
		<tr>
			<td>Avidin/Biotin Blocking Kit</td>
			<td>Life Technologies</td>
			<td>4303</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSR Fortessa 5-laser flow cytometer&#160;</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Laser 405nm (violet)/375nm (UV) &#8211; filter V450/50 for DAPI and V450 antibodies; Laser 561nm (Yellow-green) &#8211; filter YG582/15 for PE antibodies; Laser 405nm (violet)/375nm (UV) &#8211; filter V710/50 for BV711 antibodies&#160;</td>
		</tr>
		<tr>
			<td>Biotinylated Ulex europaeus lectin</td>
			<td>Vector Laboratories</td>
			<td>Vector-B1065</td>
			<td></td>
		</tr>
		<tr>
			<td>BV711 Mouse IgG1, k Isotype Control</td>
			<td>BD Biosciences</td>
			<td>563044</td>
			<td></td>
		</tr>
		<tr>
			<td>CD146-BV711</td>
			<td>BD Biosciences</td>
			<td>563186</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31-V450</td>
			<td>BD Biosciences</td>
			<td>561653</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34-PE</td>
			<td>BD Biosciences</td>
			<td>555822</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45-V450</td>
			<td>BD Biosciences</td>
			<td>560367</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Life Technologies</td>
			<td>D1306</td>
			<td>stock concentration: 5mg/mL</td>
		</tr>
		<tr>
			<td>Disposable liposuction cannula (LGI 13Gx185 mm &#8211; AR 13/18) &#160;</td>
			<td>Lipogems&#160;</td>
			<td></td>
			<td>provided in the Lipogems surgical kit</td>
		</tr>
		<tr>
			<td>Diva software 306 (v.6.0)</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX without sodium pyruvate</td>
			<td>Life Technologies</td>
			<td>61965026</td>
			<td></td>
		</tr>
		<tr>
			<td>EGMTM-2 Endothelial Cell Growth Medium-2 BulletKitTM</td>
			<td>Lonza&#160;</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum (FCS)</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo (v.10.0)</td>
			<td>FlowJo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount G</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Acros Organics</td>
			<td>410870025</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipogems Surgical Kit</td>
			<td>Lipogems&#160;</td>
			<td>LG SK 60</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti human- NG2</td>
			<td>BD Biosciences</td>
			<td>554275</td>
			<td>stock concentration: 0.5 mg/mL</td>
		</tr>
		<tr>
			<td>PE Mouse IgG1, &#954; Isotype Control</td>
			<td>BD Biosciences</td>
			<td>555749</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystirene round bottom 5 mL tube with cell strainer snap cap&#160;</td>
			<td>BD Biosciences</td>
			<td>352235, 25/Pack</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene round bottom 5 mL tubes</td>
			<td>BD Biosciences</td>
			<td>352003</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti human - PDGFRb</td>
			<td>Abcam</td>
			<td>32570</td>
			<td>stock concentration: 0.15 mg/mL</td>
		</tr>
		<tr>
			<td>Streptavidin conjugated-488</td>
			<td>Life Technologies</td>
			<td>&#160;S32354</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84100-5kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue infiltration cannula (17GX185 mm-VG 17/18)&#160;</td>
			<td>Lipogems&#160;</td>
			<td></td>
			<td>provided in the Lipogems surgical kit</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>fisher chemicals</td>
			<td>BP152-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Type- II Collagenase</td>
			<td>Gibco</td>
			<td>17101-015</td>
			<td></td>
		</tr>
		<tr>
			<td>V450 Mouse IgG1, &#954; Isotype Control</td>
			<td>BD Biosciences</td>
			<td>560373</td>
			<td></td>
		</tr>
		<tr>
			<td>Widefield Zeiss observer</td>
			<td>Zeiss</td>
			<td></td>
			<td>Objective used: Plan-Apo 20x/0.8</td>
		</tr>
		<tr>
			<td>Zeiss Colibri7 LED light source ( LEDs: 385, 475, 555, 590, 630 nm)</td>
			<td>Zeiss</td>
			<td></td>
			<td>DAPI: UV, excitation 385nm; 488: Blue, excitation 475nm;&#160; 555: Green, excitation 555nm;&#160; 647:Red, excitation 630nm&#160;</td>
		</tr>
	</tbody>
</table>

human adipose tissue micro-fragmentation for cell phenotyping and secretome characterization

在过去十年中，脂肪组织移植已广泛应用于整形手术和矫形外科，以提高组织再生和/或再生。因此，采集和处理人类脂肪组织的技术已经发展，以便快速有效地获得大量的组织。其中，封闭系统技术代表一种创新和易于使用的系统，可以在短时间内和在同一干预（操作内）中收获、处理和重新注入精制脂肪组织。脂肪组织通过抽脂、洗涤、乳化、冲洗和机械切碎收集成0.3至0.8 mm的细胞簇。 机械碎片脂肪组织的自体移植在不同治疗中显示出显著疗效适应症，如美容医学和手术，骨科和普通外科。微碎片脂肪组织的特征表明，在脂肪细胞簇中存在完整的小血管;因此，血管内壁不受干扰。这些簇在血管内细胞中富集（即间质干细胞（MSC）祖先），体外分析表明，与酶衍生的MSCs相比，参与组织修复和再生的生长因子和细胞因子的释放增加。这表明，微碎片脂肪组织的优越治疗潜力是由较高的假定MSC频率和增强的分泌活性解释。这些添加的围细胞是否直接导致更高的生长因子和细胞因子生产尚不清楚。这种临床认可的程序允许移植假定的MSCs，而无需扩大和/或酶治疗，从而绕过了GMP指南的要求，并降低了基于细胞的治疗的成本。

手动脂吸气的机械分离导致产生微碎片脂肪组织（MAT），由包围微血管网络的脂肪细胞聚集体组成（图3）。明胶嵌入和冷冻MAT的免疫荧光分析突出了这一结构，显示了血管网络标记的内皮细胞标记Ulex europaeus凝胶蛋白1（UEA-1）受体主要由小毛细血管组成（图 4.表达NG2或PDGFR+的围细胞呈正态分布，内皮细胞（图4）...

Watch this Scientific Journal Video about Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization at JoVE.com

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

在这里，我们介绍人类脂肪组织无酶微分裂使用封闭系统装置。这种新方法允许获得适合体内移植、体外培养以及进一步细胞分离和表征的脂肪组织的亚毫米簇。

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. ...

利用这个系统，我们可以收集人类脂肪组织的亚毫米聚类，其中再生细胞的整个微环境是完好无损的。整个微血管系统也是完好无损的，因此我们可以使用这种微器官进行体内移植，用于体外培养和细胞分离，以进一步进行表征。在研究实验室中使用该系统的主要理由是，该工艺是完全无酶的，因此它允许快速、高效地处理人体脂肪组织，同时保持微结构，并且它将成为完全完好无损的组织微创体。因此，我们可以直接研究一种细胞治疗产品，它已被证明在临床上非常有用。这种封闭式系统技术是一种易于使用的技术，用于术内收获、加工和再注射精制脂肪组织，可成功应用于美容骨科、产学科和妇科。这种方法允许进一步研究脂肪对组织再生的影响。该程序将由博士后研究员比安卡·韦扎尼和博士生马里奥·戈麦斯-萨拉萨尔进行演示。在收获后 16 小时内的无菌条件下工作，将包皮成形术样品放在手术布上，皮肤朝上。使用一次性组织渗透管将50至100毫升37摄氏度的0.9%氯化钠溶液注射到皮下脂肪组织中。将 10 毫升 Luer 锁注射器连接到一次性抽脂管，并在样品边缘附近的皮肤区域抓住样品，小心地将该管引入脂肪组织。一旦管管在组织内，提取柱塞，并在脂肪组织样品中径向移动管。在吸脂过程中，必须牢固地处理脂肪组织。如果收集没有足够的脂肪，请考虑执行额外的盐水注射。当注射器充满脂吸酸盐时，小心地从组织中取出管，用新的空注射器更换整个注射器，直到总共收获了 60 毫升的吸气剂。对于吸脂物的微碎块，请从包装上取出设备，并验证主装置是否连接到废纸袋。将废物收集袋放在地面上，并确保阀门固定在工艺单元盖上。刺穿袋连接端口，将输入线的端子尖峰连接到盐水袋中，并放置高于加工单元的盐水袋。验证连接到电路管的所有五个夹具是否打开，将处理单元放在垂直位置，允许盐水溶液填充处理单元，并确认流量到达废袋。摇动加工单元以清除任何气泡，将装置放置到垂直位置，蓝色盖朝上。关闭输入管路旁边的夹紧，将吸脂剂注射器连接到蓝色输入盖的自封阀，开始注射脂吸气剂。保持加工单元垂直，缓慢向下推注射器柱塞，直到所有吸脂剂已转移到装置。当所有脂吸气剂都已注入时，打开输入夹以恢复盐水流量，并大力摇动加工单元至少两分钟，定期检查盐水溶液流入废袋。当加工单元中的盐水溶液变为透明时，将带灰色盖的装置向上放置，并关闭位于靠近蓝色和灰色盖的排水管上的夹子。加工过的组织将漂浮在顶部。接下来，用盐水溶液填充 10 毫升 Luer 锁注射器，然后将注射器连接到蓝色盖的装载阀。将空的 10 毫升 Luer 锁注射器与柱塞完全插入灰帽阀上。从蓝色盖子将盐水牢固地注入加工单元，检查连接到灰色阀的注射器是否因此被加工组织填充。当所有盐水被注射后，小心地取出两个注射器并重复盐水输液，直到收集所有加工过的组织。在加工结束时，取出所有注射器，关闭所有夹子，分离盐水袋，并按照当地协议处置设备。收集所有加工组织后，将微碎脂肪组织转移到无菌容器中，并将等量的消化介质添加到容器中。将密封容器放在37摄氏度的摇水浴中，设置为每分钟120次旋转45分钟，在孵化结束时用同等体积的阻尼溶液停止消化。顺序过滤样品，首先通过100微米细胞过滤器，然后通过70微米细胞滤株过滤，然后以200倍g将过滤的悬浮液离心5分钟。我们在室温下将颗粒悬浮在大约5毫升的红细胞解液缓冲液中15分钟。用等量的阻尼溶液停止解，并通过 40 微米电池滤株过滤溶液。然后通过离心收集微碎脂肪组织细胞，我们将颗粒悬浮在新鲜阻块溶液中，用面粉混合进行计数。分离和抗体染色后，微碎脂肪细胞可被带到流动细胞仪进行细胞分析和/或分拣。手动吸脂物的机械分离导致微碎片脂肪组织的生产，这种组织由包裹微血管网络的脂肪细胞的聚集体组成。嵌入低温冷冻微碎脂肪组织中的明胶的免疫荧光分析突出了结构，显示了以内皮细胞标记 Ulex Europaeus Agglutinin 1 受体为标志的血管网络，主要由小毛细血管状血管组成。值得注意的是，表达神经元-胶质抗原2或血小板衍生生长因子受体β的周利源是正向分布的，是内皮细胞。通过排除CD31阳性内皮和CD45阳性造血细胞，可以在MAT中识别CD146阳性CD34阴性造血和CD146阳性和CD34阳性造发细胞的存在。微碎脂肪组织可以通过免疫组织化学染色、体外培养或分泌物分析进一步加工，以更深入地了解脂肪组织生物学。这项技术允许研究各种组织在自然，本地，三维排列作为一种小器官，这是小到足以移植在小鼠或长期培养。我们主要利用这个系统来实验不同的器官特异性微环境。我们的脂肪组织捐赠者不被称为可传播感染的携带者。然而，在没有任何进一步筛查的情况下，所有人体组织应被视为潜在污染，并据此处理。

Research

JoVE Journal

Biology

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

科研

生物学

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

三维白色脂肪组织结构的全装染色可视化

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

Y-27632 丰富成人皮肤组织中人类黑色素细胞的产量

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

通过甲基异硫清除和三D成像探索脂肪组织结构

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

培养的人间充质干细胞细胞外囊泡的分离、表征和治疗应用

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

Adipose Tissue-Derived Stem Cell Isolation and Characterization

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats (Video) | JoVE 

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

Semi-Automated Isolation of Murine White Adipose Tissue

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

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator (Video) | JoVE 

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 ...