Name: characterization of immune cells in human adipose tissue by using flow cytometry
Uploaded: 2018-03-06T15:00:00.000+00:00
Duration: 7 min 32 s
Description: Watch this Scientific Journal Video about Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry at JoVE.com

我们要感谢 J. Gaar 和 m. Vroomen (荷兰马斯特里赫特大学) 的技术支持。此外, 我们还要感谢 k. Verboven、汉森、j. Jocken 和 e Blaak 提供用于建立本议定书和随后的实验的血液和组织活检。

根据《赫尔辛基宣言》, Hasselt 和比利时 Hasselt 大学医学伦理委员会 Jessa 医院批准的研究对象, 从样本中抽取内脏和皮下组织。
1. 试剂的制备
<ol><li>胶原酶溶液<ol>
<li>溶解1克胶原酶 I 在10毫升磷酸盐缓冲盐水 (PBS, 没有钙和镁) 做100毫克/毫升存货解答。准备200µL 整除数和存储在-20 摄氏度。</li>
		<li>在10毫升的 PBS 中溶解1克胶原酶, 制作100毫克/毫升的库存溶液。准备200µL 整除数和存储在-20 摄氏度。</li>
		<li>溶解10毫克的 DNase I 在10毫升的 PBS, 使10毫克/毫升的库存解决方案。准备180µL 整除数和存储在-20 摄氏度。</li>
		<li>添加100µL 胶原酶 I (100 毫克/毫升), 100 µL 胶原酶 XI (100 毫克/毫升), 90 µL DNase I (10 毫克/毫升) 10 毫升 DMEM 火腿的 F12。使胶原酶溶液新鲜的每一个隔离。</li>
	</ol>
</li>
	<li>红细胞溶解缓冲液<ol>
<li>在100毫升超纯水中溶解0.84 克 NH4Cl。</li>
		<li>使用前将 pH 值设置为7.4。在4摄氏度的玻璃瓶中贮存。</li>
		<li>使用前将红细胞溶解缓冲剂放在冰上。</li>
	</ol>
</li>
	<li>缓冲区<ol>
<li>在100毫升的 pbs 中溶解0.5 克牛血清白蛋白 (bsa), 以获得 0.5% BSA pbs。</li>
		<li>在100毫升 0.5% BSA pbs 中溶解65毫克的 nan3以获得 10 mM 南3 0.5% BSA pbs。在4摄氏度的玻璃瓶中贮存溶液。</li>
		<li>在使用前, 在冰上放置一个缓冲器。 警告: NaN3具有剧毒。在处理 NaN3时, 在通风罩中工作, 戴上安全眼镜和防护手套。</li>
	</ol>
</li>
	<li>人 IgG 阻滞<ol>
<li>在10毫升 PBS 中溶解10毫克的人体 IgG 以获得1毫克/毫升。准备100µL 整除数和存储在-20 摄氏度。</li>
		<li>使用前将人体 IgG 块放在冰上。</li>
	</ol>
</li>
</ol>2. SVF 的隔离
<ol><li>切割1克的活检成小块 (2 毫米2) 与手术刀和转移到50毫升离心管 (例如, 猎鹰管)。在样品中加入10毫升胶原酶溶液。 注: 完全关闭管盖, 并打开盖子¼回。</li>
	<li>在37摄氏度的水浴中孵育60分钟, 在柔和的震动下 (60 圈/分钟)。</li>
	<li>用200µM 过滤器过滤产生的悬浮液, 并在新的50毫升离心管中收集样品。在过滤器顶部添加7毫升 PBS, 冲洗过滤器并获得所有电池。</li>
	<li>离心样品在 280 x g 5 分钟在4°c。</li>
	<li>通过吹打去除漂浮脂肪细胞的分数。细胞颗粒是 SVF。 注: 取出脂肪细胞分数以获得 SVF。避免淹没在样本中的整个尖端, 因为这将只删除 PBS, 而不是浮动脂肪细胞。</li>
	<li>并用重悬在5毫升的 PBS 中 SVF 去除胶原酶, 用70µM 过滤器过滤悬浮液, 用5毫升 PBS 冲洗过滤器, 并将样品离心在 280 x g 处, 以5摄氏度为4分钟。</li>
	<li>去除3毫升红细胞溶解缓冲液中的上清和并用重悬颗粒。</li>
	<li>在冰上孵化5分钟。孵化后添加7毫升 PBS。</li>
	<li>离心样品在 280 x g 5 分钟在4°c。</li>
</ol>3. 流式细胞仪分析中的 SVF 染色
<ol><li>将细胞颗粒溶于90µL 4 °c 的内流器缓冲液中, 加入10µL 1 毫克/毫升人 IgG 阻滞剂。在 96 v 型井板的2口井中划分细胞悬浮。将盘子放在冰上, 让人体 IgG 块孵化15分钟。</li>
	<li>在每个样品上添加100µL 的流值器缓冲器, 以5分钟为 280 x 克, 在4摄氏度的情况下清洗和离心板材。在不攻板的情况下, 将盘子倒在一个平滑的运动中, 去掉上清。 注意: 一定要用纸巾把盘子顶部的剩余液体移除, 同时保持盘子颠倒。</li>
	<li>如表 1和表 2中所述, 为巨噬细胞和 DC 子集 (1) 和 T 和 B 单元格子集 (资产管制面板 2) 准备抗体鸡尾酒。在优化抗体浓度后选择了表 1和表 2中描述的卷, 并且足够用于一个增值税或 sAT 示例。 注意: 在 "流控制面板 1" 中, 使用标记 CD303 和 CD141 确认 CD11C+ CD11B低单元格是 dc。但是, 建议将这些标记排除在面板中, 以包括活/死染色。当 PE 通道不使用时, 1 和2都可以与活/死可修复的红死细胞染色试剂盒的生存性染色结合在一起, 因为在面板1中排除 CD303。根据制造商的指示执行生存能力染色。</li>
	<li>并用重悬在29.5 µL 抗体鸡尾酒中的球团, 1 和23µL 抗体鸡尾酒为外地资产管制小组2。在黑暗中的冰上孵化30分钟。</li>
	<li>向每个井中添加150µL 的操作器缓冲区, 并并用重悬单元颗粒以执行第二次清洗步骤。离心板为5分钟在 280 x g 和4°c。把盘子倒过来, 去掉上清。</li>
	<li>在每个井中添加150µL 1% 甲醛溶液来修复细胞。通过吹打与 P200 吸管将细胞悬浮液从各井转移到相应的转流管。在4摄氏度的7天的黑暗中存储资产管制管道。 注: 直接测量也是可能的。在每个井中添加150µL 的吹打, 而不是1% 甲醛, 用 P200 吸管将细胞转移到相应的流化管, 并对细胞进行分析。 注意: 甲醛是非常有毒的。在通风罩中工作时, 要准备甲醛溶液, 避免吸入, 戴上手套和安全眼镜保护。</li>
</ol>4. 流式细胞仪分析
<ol><li>在第一次测量之前, 使用无瑕负控制来设置正散射 (FSC) 和侧散射 (SSC)。根据制造商的指示调整流式细胞仪的电压, 使所有感兴趣的人群都能在 FSC 和 SSC 图中可见, 并且可以区分碎片和活细胞。</li>
	<li>根据制造商的协议, 使用抗体捕捉珠进行多色补偿分析。</li>
	<li>制备荧光减一 (FMO) 控制通过使抗体组合, 但排除一个抗体从混合。对每种抗体都做到这一点, 为1和8抗体混合, 为外地资产管制组2提供6抗体混合。这些 FMO 抗体混合用于染色 SVF, 如上文所述的协议。</li>
	<li>测量所有 FMO 控制, 并设置基于 FMO 控制的浇口策略。使用 FMO 控件检测单元格的可能的自动荧光。 注意: 通过从混合中移除一个抗体, 在该通道中检测到的任何荧光电平都是背景/autofluorescent 信号。因此, 通过比较不同的 FMO 控制外地观察结果, 门可以吸引特定的人群, 以确保 gatings 是基于阳性细胞, 而不是基于自动荧光。</li>
	<li>在将其放进流式细胞仪并开始测量之前, 将 800 rpm 的能量计管涡旋。 注意: 建议在活门中至少有5万个事件, 以确保从每个亚群中测量出足够的细胞。</li>
</ol>

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作者声明没有利益冲突。

这些方法描述如何分离 SVF 从增值税和 sAT 和量化的相对数量的免疫细胞在这些组织。此外, 这些方法还说明了如何确定特定单元格类型上标记的表达式。
组织免疫细胞的流式细胞术是一种有效的方法, 用于表型免疫状态的组织。组织免疫细胞的量化可以有许多应用。如结果所述, 有可能比较特定免疫细胞在患者组 (例如、瘦肉型与肥胖症) 之间的存在。此外, 通过对同一患者的血液进行流式细胞术, 可以研究循环细胞与组织细胞之间的关联。通过这个应用, 我们能够确定循环单核细胞的特定子集与亲炎性 CD11C+脂肪组织巨噬细胞16有关。
对所描述的协议的调整将扩展其应用, 因为许多可用的荧光抗体使流式细胞仪非常多才多艺。与不同的抗体几乎所有细胞类型可以区分和许多标记的表示可以被发现。此外, 还可以通过 permeabilizing 细胞膜来染色标记 intracellularly, 以允许荧光抗体的细胞内结合。这些特征允许不同的巨噬细胞种群以外的过度简化 M1 和 M2 巨噬细胞亚型。除了测量表面标记的表达, 蛋白质 (即, 细胞因子) 可以被染色 intracellularly 提供有关巨噬细胞功能的信息。此外, Ki67 等扩散标记用于量化扩散率。如所述, 巨噬细胞与 DCs 之间的区别是基于多边居民的 DC 标记水平。一般的巨噬细胞标记, 如 CD68 可以并入巨噬细胞面板 (1)。然而, CD68 需要染色 intracellularly 需要通透的细胞膜, 这是不可取的, 并将延长议定书。其他巨噬细胞标记是子集标记例如 CD163 和 CD206 或 CD11c, 后者被集成在这里提出的巨噬细胞小组。
在我们的外地管制小组中, 没有包括一个区分活细胞和死体细胞的标记, 这是可取的, 因为它允许比使用 FSC 和 SSC 更准确地排除死细胞。经常使用的是脱氧核糖核酸染色活性染料碘碘化物 (PI) 或 4 ', 6-diamidino-2-苯基吲哚 (DAPI) 并且自由胺反应染料例如活/死可修复的死细胞染色套件, 在不同的染料颜色可以使用。但是, 在修复单元格时不能使用 PI 和 DAPI。如《议定书》所述, 活/死可修复的红死细胞生存能力染色可以集成到两个面板中, 而不影响总的外地方案的门控策略。
此外, 数据表示为活细胞的百分比, 这意味着所有数据都是相对的。只有在流式细胞仪中输入确切的已知数目的单元格, 才能确定每个细胞类型的确切数字。在用计数室计算 SVF 分数中的细胞数后, 可以计算出细胞的近似数目。然而, 这个数字将不得不调整, 以用于隔离 SVF 的活检组织的数量, 但这有限制, 当比较瘦与肥胖。类似的肥胖人群中含有较少的脂肪细胞, 因为它们充满了脂质, 并大大扩大。这可能导致对免疫细胞数的低估, 如果提出作为免疫细胞的数量每克的在或每个脂肪。
在人类研究中, 纳入患者通常是在较长时间内进行标准化实验程序的重要意义。为比较患者之间的流式细胞仪数据, 有多种选择。如本协议所述, 在测量前可以对单元格进行固定, 以便在同一天对多个样本进行分析。这也可以通过冷冻 SVF 之前的染色, 这使得染色程序是平等的所有样本, 但细胞的生存能力可能受到影响。最后, 在本研究中, 还使用了荧光珠来安装补偿水平, 并采用了双周细胞仪跟踪珠来规范细胞仪的日常测量。这最后一个选择是最有效的测量样本从一项研究跨越了很长一段时间。
流式细胞术的限制因素一般是荧光的使用。可同时检测到的荧光标签数量因发射谱重叠而受到限制。然而, 通过智能化的流式仪表板开发和使用每个增值税或 sAT 样本的几个抗体鸡尾酒, 这个问题可以解决, 如本议定书所述。FMO 控制是资产管制系统面板开发的一个重要方面。通过使用该面板的所有抗体, 除了一个, 可以欣赏的潜在的自发荧光水平, 当比较 FMO 与整个面板。这样就可以精确地对人群进行浇口, 在设置新的 "流控流" 面板时应执行这些步骤。此外, 新一代的资产管制装置可以检测多达50个参数, 允许同时检测每个单元格的许多特性。另一个与荧光方面有关的问题是细胞的自体荧光, 特别是巨细胞。在细胞的励磁作用下激光 (主要与488毫微米波长励磁), 这些细胞发出一个荧光信号 (主要 < 640 毫微米) 可以重叠与抗体标签的发射光谱17,18。为此, 应测量无瑕细胞, 以确定每个通道中的自发荧光。有了这些知识, 荧光应该被选中, 显示超过 autofluorescent 信号的信号强度。在确定人群的门控策略时, 应考虑到 autofluorescent 的背景信号。因此, 通过本协议的应用和智能化的资产管制面板设计, 可以深入研究表型巨噬细胞子类型。新的不同在巨噬细胞和他们的作用可以描绘。

肥胖的特点是低品位的炎症1和浸润炎症免疫细胞在内脏和皮下 (增值税, sAT)。在增值税中积累的亲炎症免疫细胞导致胰岛素抵抗, 这是发展2型糖尿病的主要危险因素2。免疫细胞的先天和自适应免疫系统是发现在肥胖的, 如巨噬细胞, 肥大单元格, 中性白细胞, CD4+和 CD8+ T 细胞, B 细胞3,4,5,6 ,7。这些免疫细胞, 连同内皮细胞, 基质细胞, 脂肪胞祖, 成纤维细胞, 和毛细血管, 构成了 SVF8 , 是在9的促炎物质的主要来源。
通常由西方印迹10、qPCR11和免疫组化11等技术对 AT 的炎症状态进行调查。然而, 当使用这些技术时, 整个在, 脂肪细胞和 SVF, 使用。这使得很难确定在中存在的免疫细胞的数量和子集。免疫细胞有各种细胞标记来定义和分类它们, 如巨噬细胞。巨噬细胞在功能和细胞表面标记表达式12中显示显著的异质性。因此, 它们通常被归类为两个巨噬细胞群: M1 和 M2。M2 巨噬细胞通常被称为交替激活的巨噬细胞12,13并且居住在稀薄, 新陈代谢的正常人14。然而, 在肥胖期间, 一个表型开关发生从 M2 巨噬细胞到 M1 巨噬细胞。这些经典活化 M1 巨噬细胞表达 CD11C12和堆积在死脂肪体形成冠状结构13。它已经表明, CD11C+巨噬细胞在损害胰岛素的作用, 并与胰岛素抵抗在肥胖的人15。为了识别 M1 和 M2 巨噬细胞, 免疫组化是一种选择。该技术提供了有关组织中巨噬细胞位置的信息。但是, 它将限制可用于一个染色的标记的数量。此外, 也难以量化。因此, 为了研究不同的免疫细胞亚群在增值税和 sAT 存款, 我们开发了流式细胞术的方法。这种方法使我们有机会使用多个标记每个细胞与一个流式细胞分析, 以定义细胞子集和计数的每个子集存在的在存款。

<table><tbody><tr><td>Centrifuge</td><td>Hettich Rotanta 460R</td><td>5660</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)-Ham&amp;rsquo;s F12</td><td>Gibco ThermoFisher</td><td>31330-095</td><td></td></tr><tr><td>Collagenase XI from Clostridium histolyticum</td><td>Sigma Aldrich</td><td>C7657-1g</td><td></td></tr><tr><td>Collagenase I from Clostridium histolyticum</td><td>Sigma Aldrich</td><td>C0130-1g</td><td></td></tr><tr><td>DNAse I from bovine pancreas</td><td>Sigma Aldrich</td><td>DN25</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Merck</td><td>1.01145.0500</td><td></td></tr><tr><td>Bovine Serum Albumine</td><td>Sigma Aldrich</td><td>A4503-100</td><td></td></tr><tr><td>NaN&lt;sub&gt;3&lt;/sub&gt;</td><td>Merck</td><td>1.06688.0100</td><td></td></tr><tr><td>200 &amp;micro;M syringe Filcons</td><td>BD Biosciences</td><td>340613</td><td></td></tr><tr><td>70 &amp;micro;M filter</td><td>Greiner Bio-one</td><td>542070</td><td></td></tr><tr><td>Fc block / human IgG</td><td>Sigma Aldrich</td><td>14506</td><td></td></tr><tr><td>Formaldehyde</td><td>Merck</td><td>1.04003.2500</td><td></td></tr><tr><td>CD11b-BV421</td><td>Biolegend</td><td>301324</td><td></td></tr><tr><td>CD19-Fitc</td><td>BD Biosciences</td><td>555412</td><td></td></tr><tr><td>CD3-Fitc</td><td>BD Biosciences</td><td>561807</td><td></td></tr><tr><td>CD66b-Fitc</td><td>BD Biosciences</td><td>555724</td><td></td></tr><tr><td>CD56-Fitc</td><td>BD Biosciences</td><td>562794</td><td></td></tr><tr><td>CD11c-APC-Cy7</td><td>Biolegend</td><td>337218</td><td></td></tr><tr><td>CD45-PE-Cy7</td><td>BD Biosciences</td><td>557748</td><td></td></tr><tr><td>CD19-BV421</td><td>Biolegend</td><td>302234</td><td></td></tr><tr><td>CD3-V500</td><td>BD Biosciences</td><td>561416</td><td></td></tr><tr><td>CD56-APC</td><td>Biolegend</td><td>318310</td><td></td></tr><tr><td>CD4-PerCP-Cy5.5</td><td>Biolegend</td><td>300528</td><td></td></tr><tr><td>CD8-APC-H7</td><td>BD Biosciences</td><td>641400</td><td></td></tr><tr><td>CD303-PE</td><td>Biolegend</td><td>354204</td><td></td></tr><tr><td>CD141-APC</td><td>Biolegend</td><td>344106</td><td></td></tr><tr><td>FACS-Canto II</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>96 v-shape well plate</td><td>Greiner Bio-one</td><td>651101</td><td></td></tr><tr><td>PBS PH 7.4</td><td>Gibco ThermoFisher</td><td>10010023</td><td></td></tr><tr><td>FACS tube 5 mL polystyrene round-bottom tube</td><td>Corning</td><td>352052</td><td></td></tr><tr><td>IgG from human serum</td><td>Sigma Aldrich</td><td>I4506</td><td></td></tr><tr><td>Anti-Rat Ig, &amp;kappa;/Negative Control Compensation Particles Set</td><td>BD Biosciences</td><td>552844</td><td></td></tr><tr><td>Anti-Mouse Ig, &amp;kappa;/Negative Control Compensation Particles Set</td><td>BD Biosciences</td><td>552843</td><td></td></tr><tr><td>LIVE/DEAD Fixable Red Dead Cell Stain Kit</td><td>ThermoFisher</td><td>L23102</td><td></td></tr><tr><td>IKA&amp;nbsp;MS 3 basic shaker</td><td>Sigma Aldrich</td><td>Z645028-1EA</td><td></td></tr></tbody></table>

characterization of immune cells in human adipose tissue by using flow cytometry

在皮下和内脏脂肪组织中的免疫细胞浸润会导致低级别炎症, 导致肥胖相关并发症 (如2型糖尿病) 的发展。为了定量和定性地研究人体内的免疫细胞亚群, 我们开发了一种流式细胞仪方法。基质血管分数 (SVF), 含有免疫细胞, 从皮下和内脏在活检的胶原酶消化分离。脂肪细胞在离心后被除去。用流式细胞仪对 SVF 细胞进行染色, 筛选出多种膜结合标记, 以区分免疫细胞亚群和分析。由于这种方法, 可以检测和量化抗炎巨噬细胞亚群、树突状体细胞 (DCs)、B 细胞、CD4+和 CD8+ T 细胞以及 NK 细胞。此方法提供有关免疫细胞的详细信息, 以及每个特定子集的数量。由于有许多荧光抗体可用, 我们的流式细胞术方法可以调整, 以测量各种其他细胞和细胞内的标记感兴趣。

用流式细胞仪测量了 SVF 和 sAT 的分离。流式细胞仪测量根据细胞标记 (图 1A和1B) 生成显示不同细胞群的地块。首先, 通过绘制正散射宽度 (fsc-w) 和正散射区 (fsc A), 可以消除细胞团聚体的进一步分析, 将单个细胞作为低 FSC W。接下来, 选择活细胞, 并分别使用 FSC a 和侧散射区 (SSC a), 将正确大小和复杂度的细胞排除在细胞碎片之外。死细胞是小的, 因此可见作为一个独特的人口与一个小的 FSC-a。接下来, 使用泛白细胞标记 CD45 (面板1和 2,图 1A) 选择免疫细胞。为了分析巨噬细胞, 其他免疫细胞, 如 T 细胞 (CD3), B 细胞 (CD19), 中性粒细胞 (CD66b+ CD11b+) 和 NK 细胞 (CD56) 被排除在进一步的分析, 通过使用不同的抗体针对这些细胞, 但具有相同的fluorochrome。其余细胞的进一步细分是基于 CD11b 和 CD11c 表达。这导致以下人群: CD11b+ CD11c+巨噬细胞, CD11b+ CD11c-巨噬细胞, CD11b低/ CD11c+ DCs (1,图 1B)。平均荧光强度的测量允许量化 CD303 (浆 dc 标记) 和 CD141 (dc 标记) 的表达式, 在 CD11b+ CD11c+巨噬细胞和 CD11b低/ CD11c+ DCs 中。在 CD11b低/ CD11c+单元格中, 两个标记的表达式都较高, 确认 CD11b低/ CD11c+单元格为 dc (图 1C)。
CD45+单元格 (图 1A) 分别使用 CD3 和 CD19 划分为 T 细胞和 B 细胞。t 细胞被细分为 t 辅助细胞 (CD4+) 和细胞毒性 T 细胞 (CD8+)。最后, CD3-CD19 单元格被绘制成使用标记 CD56 (2,图 1D) 来量化 NK 细胞。每个门中的单元格数是量化的, 可用于计算所有活单元格类型的百分比 (表 3)。
可以计算每个科目的活细胞百分比, 允许计算一组中所有科目的平均值, 例如瘦或肥胖的男性, 显示特定免疫细胞的丰度,即, 赞成炎症的 CD11b+CD11c+内脏中的巨噬细胞 (图 2)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57319/57319fig1.jpg"> 图1。内脏脂肪组织的门控策略.(A) 具有正向散射宽度强度 (fsc W) 和正向散射区域强度 (fsc A) 的所有事件 (黑色) 的资产管制计划图包含一个只选择单个单元格 (红色) 的门, 后跟一个基于 FSC-a 和侧散射区域强度 (SSC a) 的外地跟踪图。包含选择活细胞 (浅绿色) 的门。下图与 SSC-A 和 CD45 荧光强度包含一个门选择所有 CD45+ (免疫) 细胞 (蓝色)。(B) CD19、CD3、CD66b 和 CD56 荧光强度与 CD11b 荧光强度相比较的图元, 以及选择所有 CD19、CD3、CD66b 和 CD56 阴性 (褐色) 的细胞以进一步分裂为人群的门。进一步细分的下一个情节基于 CD11b 和 CD11c 荧光强度。显示的闸门包含 CD11b+ CD11c+巨噬细胞 (深绿色)、CD11b+ CD11c-巨噬细胞 (紫色) 和 CD11b低/- CD11c+树突状细胞 (蓝色)。(C) 数量的 CD11b+、CD11c+或 CD11b低/- CD11c+单元格 (Y 轴), 它们显示了 CD303 和 CD141 的荧光强度 (X 轴) 的水平, 并相应地量化了平均值荧光强度 (多边基金会)。(D) 根据 CD3 和 CD19 荧光强度 (包括选择 T 细胞 (洋红)、B 细胞 (深绿色) 和非 autofluorescent 单元格 (绿色) 为两个 CD3 的 CD45, 显示以前的+填充 (蓝色) 的资产管制计划图和 CD19。下面的图是基于 CD4 和 CD8 荧光的, 选择 CD4+ (淡绿色) 和 CD8+ (洋红) T 单元格的门。皮下脂肪组织采用相同的门控策略。皮下脂肪组织采用相同的门控策略。此数字已从武泰et . 中进行了修改。16<a href="//cloudflare.jove.com/files/ftp_upload/57319/57319fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57319/57319fig2.jpg"> 图2。肥胖的增值税含有更多的促炎巨噬细胞.CD11b+ CD11c+巨噬细胞的数量显示为瘦身和肥胖男子增值税中所有活细胞的百分比。所有数据都是指电子扫描电镜;n = 20 为精益和 n = 31 为肥胖。**p ≤0.01 与精益。<a href="//cloudflare.jove.com/files/ftp_upload/57319/57319fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>目标</td>
			<td>定义目标</td>
			<td>呈现在</td>
			<td>Fluorochrome</td>
			<td>数量</td>
			<td>克隆</td>
		</tr>
<tr>
<td>CD11B</td>
			<td>髓素整合素标记</td>
			<td>粒细胞, 单核/巨噬, 树突状干细胞低和自然杀伤细胞</td>
			<td>BV421</td>
			<td>2.5 µL</td>
			<td>ICRF44</td>
		</tr>
<tr>
<td>CD19</td>
			<td>常见 B 细胞抗原</td>
			<td>b 细胞向 blastoid b 淋巴细胞和血浆 b 细胞发育的研究</td>
			<td>fitc</td>
			<td>3µL</td>
			<td>HIB19</td>
		</tr>
<tr>
<td>CD3</td>
			<td>普通 T 细胞抗原</td>
			<td>t 淋巴细胞, 自然杀伤细胞和胸腺</td>
			<td>fitc</td>
			<td>3µL</td>
			<td>UCHT1</td>
		</tr>
<tr>
<td>CD66B</td>
			<td>癌胚抗原 (CEA) 样糖蛋白家族成员</td>
			<td>粒</td>
			<td>fitc</td>
			<td>5µL</td>
			<td>G10F5</td>
		</tr>
<tr>
<td>CD56</td>
			<td>重糖化黏附蛋白</td>
			<td>自然杀伤细胞与自然杀伤细胞</td>
			<td>fitc</td>
			<td>5µL</td>
			<td>B159</td>
		</tr>
<tr>
<td>CD303</td>
			<td>II. 型跨膜糖蛋白</td>
			<td>浆树突状细胞</td>
			<td>体育</td>
			<td>1µL</td>
			<td>201A</td>
		</tr>
<tr>
<td>CD141</td>
			<td>血栓</td>
			<td>单核细胞/巨噬细胞低, 树突状细胞亚群</td>
			<td>apc</td>
			<td>1µL</td>
			<td>M80</td>
		</tr>
<tr>
<td>CD11C</td>
			<td>I 型跨膜糖蛋白;整合素αx</td>
			<td>单核/巨噬细胞, 树突状, 颗粒, 自然杀伤细胞, B 和 T 细胞的子集</td>
			<td>APC-Cy7</td>
			<td>0.5 µL</td>
			<td>Bu15</td>
		</tr>
<tr>
<td>CD45</td>
			<td>常见白细胞抗原</td>
			<td>所有人类白细胞包括淋巴细胞、单核细胞、粒粒、嗜酸性粒和胸腺</td>
			<td>PE-Cy7</td>
			<td>1µL</td>
			<td>HI30</td>
		</tr>
<tr>
<td>缓冲区</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>7.5 µl</td>
			<td>-</td>
		</tr>
</tbody></table>表1。用于anel 1 的抗体鸡尾酒, 用于识别巨噬细胞亚群和树突状体细胞种群. 所描述的抗体量用于分析一个样本。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>目标</td>
			<td>定义目标</td>
			<td>呈现在</td>
			<td>Fluorochrome</td>
			<td>数量</td>
			<td>克隆</td>
		</tr>
<tr>
<td>CD19</td>
			<td>常见 B 细胞抗原</td>
			<td>b 细胞向 blastoid b 淋巴细胞和血浆 b 细胞发育的研究</td>
			<td>BV421</td>
			<td>1µL</td>
			<td>HIB19</td>
		</tr>
<tr>
<td>CD3</td>
			<td>普通 T 细胞抗原</td>
			<td>t 淋巴细胞, 自然杀伤细胞和胸腺</td>
			<td>V500</td>
			<td>3µL</td>
			<td>UCHT1</td>
		</tr>
<tr>
<td>CD56</td>
			<td>重糖化黏附蛋白</td>
			<td>自然杀伤细胞与自然杀伤细胞</td>
			<td>apc</td>
			<td>5µL</td>
			<td>HCD56</td>
		</tr>
<tr>
<td>CD4</td>
			<td>I 型跨膜糖蛋白</td>
			<td>t 辅助细胞, 胸腺, 单核/巨噬, II. 型自然杀伤 T 细胞</td>
			<td>PerCP-Cy5。5</td>
			<td>1µL</td>
			<td>RPA-T4</td>
		</tr>
<tr>
<td>CD8</td>
			<td>二硫化物连接分子络合物的α亚基</td>
			<td>细胞毒性 T 细胞, 胸腺, 自然杀伤细胞子集</td>
			<td>APC-H7</td>
			<td>2µL</td>
			<td>SK1</td>
		</tr>
<tr>
<td>CD45</td>
			<td>常见白细胞抗原</td>
			<td>所有人类白细胞包括淋巴细胞、单核细胞、粒粒、嗜酸性粒和胸腺</td>
			<td>PE-Cy7</td>
			<td>1µL</td>
			<td>HI30</td>
		</tr>
<tr>
<td>缓冲区</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>10µl</td>
			<td>-</td>
		</tr>
</tbody></table>表2。用于流式检测的抗体鸡尾酒面板 2以确定 T 和 B 单元格的数量. 所描述的抗体量用于分析一个样本。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>巨噬细胞面板</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>门名</td>
			<td># 单元格</td>
			<td>% 的活</td>
		</tr>
<tr>
<td>单个单元格</td>
			<td>183054</td>
			<td></td>
		</tr>
<tr>
<td>生活</td>
			<td>10477</td>
			<td>100</td>
		</tr>
<tr>
<td>CD45</td>
			<td>4100</td>
			<td>39.13</td>
		</tr>
<tr>
<td>转储-
</td>
			<td>771</td>
			<td>7.36</td>
		</tr>
<tr>
<td>CD11C- CD11B+
</td>
			<td>430</td>
			<td>4。1</td>
		</tr>
<tr>
<td>CD11C+ CD11B+
</td>
			<td>104</td>
			<td>0.99</td>
		</tr>
<tr>
<td>CD11C+ CD11B低/
</td>
			<td>15</td>
			<td>0.14</td>
		</tr>
<tr>
<td>T 细胞, B 细胞, NK 细胞板</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>门名</td>
			<td>#Cells</td>
			<td>% 的活</td>
		</tr>
<tr>
<td>单个单元格</td>
			<td>34616</td>
			<td></td>
		</tr>
<tr>
<td>生活</td>
			<td>3728</td>
			<td>100</td>
		</tr>
<tr>
<td>CD45+
</td>
			<td>1589</td>
			<td>42.62</td>
		</tr>
<tr>
<td>NK 细胞</td>
			<td>29</td>
			<td>0.78</td>
		</tr>
<tr>
<td>CD3+
</td>
			<td>953</td>
			<td>25.56</td>
		</tr>
<tr>
<td>CD4+
</td>
			<td>601</td>
			<td>16.12</td>
		</tr>
<tr>
<td>CD8+
</td>
			<td>328</td>
			<td>8。8</td>
		</tr>
<tr>
<td>CD19+
</td>
			<td>20</td>
			<td>0.54</td>
		</tr>
</tbody></table>表3。不同细胞类型在增值税中的免疫细胞丰度。每个门中的单元格数, 以及基于生存单元总量的不同单元格类型的百分比。

Watch this Scientific Journal Video about Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry at JoVE.com

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

本文介绍了一种用流式细胞仪从脂肪组织中分离免疫细胞来分析脂肪组织免疫细胞含量的方法和后续分析。

应用流式细胞仪对人体脂肪组织免疫细胞的表征

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

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Research

JoVE Journal

Medicine

17.4K 次观看.  MUMC. 本文介绍了一种用流式细胞仪从脂肪组织中分离免疫细胞来分析脂肪组织免疫细胞含量的方法和后续分析。

视频: Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

脂肪组织免疫细胞的分离

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

科研

免疫与感染

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

基于图像的流式细胞仪技术来评估变化粒细胞功能<em&gt;在体外</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

通过成像流式细胞仪人单核细胞来源的树突状细胞的特征:两个单核细胞分离方法的比较

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

高维流式细胞术用于解剖种植体组织的免疫功能分析

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

生物工程

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

非酒精性脂肪性肝炎合并2型糖尿病的一种先进的小鼠模型

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

从小鼠腹内脂肪库分离成脂肪和纤维炎症基质细胞亚群

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while suppressing unwanted reactions to environmental antigens. Lungs host several populations of innate and adaptive immune cells that provide immune surveillance but also mediate protective immune responses. These cells, which keep the healthy pulmonary immune system in balance, also participate in several pathological conditions such as asthma, infections, autoimmune diseases, and cancer. Selective expression of surface and intracellular proteins provides unique immunophenotypic properties to the immune cells of the lung. Consequently, flow cytometry has an instrumental role in the identification of such cell populations during steady-state and pathological conditions. This paper presents a protocol that describes a consistent and reproducible method to identify the immune cells that reside in the lungs of healthy mice under steady-state conditions. However, this protocol can also be used to identify changes in these cell populations in various disease models to help identify disease-specific changes in the lung immune landscape.

In this study, we present an effective and reproducible protocol to isolate the immune populations of the murine respiratory system. We also provide a method for the identification of all innate and adaptive immune cells that reside in the lungs of healthy mice, using a 9-color-based flow cytometry panel.

流式细胞术分析鉴定鼠肺先天免疫细胞和适应性免疫细胞

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

从人内脏脂肪组织中分离出活的脂肪细胞和基质血管组分，适用于 RNA 分析和巨噬细胞表型分析

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

医学

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. ...