提交人希望承认由路易斯安那州立的健康科学中心提供的机构支助, 资助了该项目。

所有的任务都是遵守 #8759 和 #9189 的协议, 如 LSUHSC 的 IRB 办公室批准的。所有动物的工作都是遵守协议 #3285 在 LSUHSC IACUC 办事处批准的。
1. 夹细胞片的播种
注: 见图 1。
<ol><li>种子干细胞在大约80% 融合在组织培养板材 (6 cm 或6井板材)。为每一个特警所需, 种子1常规组织培养井和1井相应大小的聚 (n-丙烯酰胺 (pNIPAAm) 涂层组织培养塑料板材。 注: 因此, 一个6井板的斯瓦特将需要种子细胞在一个6良好的标准组织培养板 (基层) 和一个6井 pNIPAAm 涂层组织培养板 (上层)。pNIPAAm 涂层板可以商业或生产在实验室12,13,14。</li>
	<li>保持干细胞在37摄氏度和 5% CO2 Dulbecco 的改良鹰培养基 (DMEM) 补充10% 血清和1x 青霉素/链霉素.每2天更换一次媒体。</li>
	<li>允许细胞联合, 直到它们成为100% 汇合, 并采取一个纹状模式 (大约6–8天)。 注意: 这些细胞将需要作为一个完整的细胞表功能, 以稳定的浮力脂肪组织。没有足够的汇合细胞将片段后, 与笏播种。</li>
</ol>2. 特警装备的准备工作
<ol><li>准备10毫升整除数1x 的平衡盐溶液 (HBSS);准备足够的需要数量的板块与体积备用。</li>
	<li>
准备用于斯瓦特播种的柱塞装置。使用简单的丙烯酸塑料来构造柱塞, 它由连接在圆圆盘上的茎组成。确保它适合组织培养井的周长 (直径: < 6 厘米为 6 cm 菜, < 3.5 cm 为6井板材; 近似质量: 6.7 g 为 6 cm 盘, 5.1 g 为6井板材)。
	<ol>
<li>准备额外的活塞, 以确保该过程将顺利运行, 以防有任何个别的活塞问题。</li>
		<li>将实验室胶带绕在柱塞盘的边缘, 至少 2x, 以防止明胶溶液渗漏。</li>
		<li>用70% 乙醇喷洒生物安全柜 (BSC)。在平衡计分卡内喷洒15毫升锥形管架, 空, 名额15毫升圆锥管;管将有助于确保放置明胶活塞。</li>
		<li>将每个带包的柱塞彻底喷涂70% 乙醇, 放入机架上的15mL 锥形管。喷涂金属垫圈 (近似质量6.3 克), 并将它们放在机架上, 连同一对钩钳;在斯瓦特播种期间, 垫圈会增加活塞的重量。</li>
		<li>关闭平衡计分卡和打开 UV 光, 以方便干燥和灭菌。 注意: 理想情况下, 在斯瓦特播种之前执行此步骤24小时。或者, 在组织收集/斯瓦特播种的当天进行处理;然而, 在这种情况下, 允许15–45最小的 UV 干燥/灭菌。</li>
	</ol>
</li>
</ol>3. 制备明胶活塞-应用于上细胞片
<ol><li>将水浴加热到摄氏75摄氏度。</li>
	<li>
将0.75 克明胶粉加入10毫升的 1x HBSS, 制备明胶溶液。在通风罩下添加100µL 1 米氢氧化钠, 以平衡溶液的 pH 值。
	<ol>
<li>将10毫升的股票添加到水浴中, 每5分钟用力摇动, 直到粉末溶解成溶液。目的在加热水浴后不久将粉末明胶溶解。</li>
		<li>打开 "平衡计分卡", 关闭 UV 光, 并提高窗扇。喷雾平衡计分卡表面和准备过滤器供应 (5 毫升鲁尔锁注射器和0.2 µm 注射器过滤器)。准备多个过滤器, 以有效地应变足够数量的明胶适用于活塞。</li>
	</ol>
</li>
	<li>当明胶溶液达到均匀一致时, 过滤溶液并将其应用于活塞。用明胶装注射器。将明胶应用于塑料活塞, 通过注射器过滤器 (〜2.5 毫升为6井板, 4.5 毫升为 6 cm 盘) 和允许凝固 (~ 20 分钟)。</li>
	<li>一旦凝胶是固体, 从活塞的边缘打开胶带。用钩钳, 从柱塞的外边缘 (即半月板凸起的边缘) 取出明胶。确保柱塞中心的剩余明胶是完全水平的, 以最大限度地与 ADSC 板接触。</li>
	<li>一旦多余的明胶被除去, 轻轻地将明胶活塞到 pNIPAAm 涂层的 ADSC 板上。用金属垫圈称量活塞。应用活塞时不要剪切细胞板。</li>
	<li>
在室温下将活塞放在电池片上1.5 小时。在冰水浴中孵化板/活塞1.5 小时, 以完成从 pNIPAAm 涂层板表面分离的细胞片。
	<ol>
<li>在冰浴中孵化时要小心;不要让冰浴在孵化过程中污染细胞介质。</li>
		<li>完成孵化后, 清洁底部的 pNIPAAm 涂层, 以消除无菌水。</li>
	</ol>
</li>
</ol>4. 白色脂肪组织加工
<ol><li>当从手术室收集人体脂肪组织时, 将所有样品保存在冰上的无菌容器中, 直到斯瓦特被播种。添加不育的维持培养基, 如磷酸盐缓冲盐水 (PBS), 脂肪组织容器的组织稳定性。</li>
	<li>增加冷细胞培养培养基到1.5 毫升离心管为每个井/菜将播种 (100 µL 为6井板, 200 µL 为 6 cm 菜)。</li>
	<li>
切碎脂肪组织。
	<ol>
<li>对于固体脂肪组织段, 请按如下所示进行。<ol>
<li>在无菌 pbs 中清洗大块的脂肪组织 3x, 尽可能多地去除 pbs。</li>
			<li>粗切的组织与镊子和无菌剃刀和删除尽可能多的血管和筋膜 (见讨论更详细)。</li>
			<li>用剃刀切碎的脂肪, 直到切碎的组织采取厚, 液体的一致性。 注: 理想的组织将出现均匀, 没有可见的单独部分的笏, 虽然这并不总是可能的。</li>
		</ol>
</li>
		<li>过程 lipoaspirate 如下所示。<ol>
<li>在平衡计分卡下, 将无菌纱布放在烧杯顶部, 并将烧杯放在较大的烧杯中, 收集多余的液体。</li>
			<li>使用25毫升血清学吸管, 绘制尽可能多的 lipoaspirate, 并将其应用于无菌纱布的表面。将 PBS 直接应用于此表面, 以洗涤 lipoaspirated 脂肪, 清除多余的血液和脂质残留物。</li>
			<li>使用镊子恢复排泄的组织, 转移到一个无菌切碎的表面, 并剁碎 lipoaspirate。</li>
		</ol>
</li>
	</ol>
</li>
	<li>使用无菌剃刀切断 p1000 吸管的远端, 转移切碎的组织;这将减少可能导致脂肪细胞裂解的剪切应力。一旦达到适当的组织一致性, 将所需的碎组织体积转移到每1.5 毫升管 (300–400µL 6 井板, 500–600µL 6 厘米菜)。将碎笏和介质简单地混合在管子里。</li>
	<li>
取基 ADSC 板, 醒酒/吸入介质。更换介质与笏/媒体混合物从每1.5 毫升管。
	<ol>
<li>轻轻地从 pNIPAAm 涂层板上取出明胶活塞, 并将其应用于基 ADSC 板上的笏混合物上。在显微镜下检查 pNIPAAm 涂层板的单层, 以确认细胞脱离。</li>
	</ol>
</li>
	<li>在平衡计分卡下设置一个热块到大约37–40°c。随着活塞仍然到位, 移动板块到热块的表面。加入2–3毫升的温热培养基, 孵化细胞, 促进凝胶融化。</li>
	<li>30分钟后, 轻轻地从板面上取出活塞。更换基部组织培养板的盖子, 移动到细胞培养孵化器。一旦明胶已完全液化37摄氏度, 吸入和取代细胞培养培养基。</li>
	<li>保持斯瓦特在37摄氏度和 5% CO2在苯酚无红 M199 培养基与7µM 胰岛素, 30 µM 地塞米松, 1x 青霉素/链霉素。维护在大约2毫升介质为6井板材和3毫升为 6 cm 菜。每2天更换一次媒体。</li>
</ol>5. 斯瓦特收获
<ol><li>制备胶原酶 (0.5 毫克/毫升胶原酶, 500 nM 腺苷, 在 PBS) 整除数15毫升圆锥管 (大约体积10毫升)。冷冻管, 并储存在-20 摄氏度。</li>
	<li>从细胞中吸取任何培养基, 用 pbs 冲洗 1x, 然后吸入 pbs。在37摄氏度的水浴中解冻胶原酶整除数。 注: 理想情况下, 胶原酶溶液将达到摄氏37摄氏度;紧接着, 添加组织。</li>
	<li>
将斯瓦特板上的所有组织添加到单个整除数。收获斯瓦特使用一个不育细胞刮刀和转移到胶原酶整除数与切断 p1000 吸管提示。将组织直接添加到含有胶原酶溶液的15毫升锥形管中。
	<ol>
<li>或者, 在50毫升圆锥管中孵化组织/胶原酶混合物;增加的表面积将进一步促进酶消化。</li>
	</ol>
</li>
	<li>将取样管放置在一个45°角的孵化轨道振动筛中。孵育在 200 rpm, 在37°c 为30–60分钟。</li>
	<li>将250µm 网格过滤器放入一个新的15毫升锥形管中收集。通过过滤器倒入经消化的脂肪细胞溶液。 注意: 这将允许所有细胞通过过滤纤维组织。</li>
	<li>允许流动通过在室温下坐5分钟, 以允许相分离。 注: 脂肪细胞将漂浮到溶液的顶端, 而脂质基质干细胞 (陶瓷) 将沉淀到较低的阶段。离心5分钟在 500 x g也可以最大化细胞分离或收获周围的干细胞在颗粒。</li>
	<li>
使用切断 p1000 吸管尖端, 转移脂肪细胞 (漂浮层在胶原酶溶液的顶部) 到一个1.5 毫升离心收集管。一次服用250µL, 慢慢地沿着管子的边缘缓慢地收集脂肪细胞。
	<ol>
<li>在收集脂肪细胞的同时, 慢慢地旋转管子, 以最大限度地恢复附着在体内的体细胞。继续绘制更多的细胞, 直到1.5 毫升离心管满。</li>
	</ol>
</li>
	<li>用注射器 (~ 21 克) 从分离的脂肪细胞中取出多余的液体。将针头浸入漂浮的脂肪细胞层下。简单地搅动针, 使任何细胞粘在针轴上, 然后等待脱落的脂肪胞漂浮到顶端。</li>
	<li>慢慢地画出多余的液体, 小心避免意外去除脂肪细胞。使用离心管毕业, 以一贯隔离样本量 (例如,.每个样品0.1 毫升)。</li>
	<li>利用分离细胞进行 DNA/RNA 提取、葡萄糖摄取测定、脂肪测定等.
</li>
</ol>

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作者没有什么可透露的。

本议定书详细介绍了干细胞在三明治人体白色脂肪组织中的应用;人类 ADSC 细胞系可以通过建立完善的协议15进行隔离。然而, 该系统可以适应个性化的研究要求 (如使用 3T3L 1 细胞三明治小鼠笏)。这个过程涉及处理主要的人体组织。应采用标准安全预防措施;作为 BSL-2 病原体 (如HIV、HepC) 处理人体组织。只在平衡计分卡下直接处理组织。根据机构安全标准, 佩戴所有合适的 PPE--强烈建议使用双手套。在重新使用或处置前, 用10% 漂白剂溶液或70% 乙醇溶液对所有用品和废料进行适当的消毒。
斯瓦特文化的第一个关键步骤是采购和维护 pNIPAAm 涂层的组织培养板。这些板材是可利用的在商业上, 或者他们可以通过建立的方法12,13,14生产。这些板块是温度响应细胞培养表面。在临界温度以上 (约32°c), 表面是疏水性和细胞将坚持, 类似于其他处理塑料的组织培养。然而, 在这个临界温度下, 表面变得亲水性, 细胞将与塑料表面分离16。因此, 应注意建立: 1) 在达到完全融合 (即在正常的介质变化期间) 和2中, 给定的细胞类型从单层中分离出的速度如何快速 (即在常规的培养基改变期间), 并为降低温度与明胶的必要时间。柱塞应用于 pNIPPAm 涂覆板, 确保整个细胞片离解。媒体的变化应执行与媒体升温到37°c;如果细胞易于快速离解, 则在平衡计分卡下的热块上进行介质变化设置为37摄氏度。
第二个关键步骤是处理脂肪组织准备斯瓦特播种。我们通过用灭菌钳和灭菌剃刀切碎笏来进行表征。然而, 人的筋膜不能轻易削减与剃刀。最大限度地增加脂肪细胞对筋膜的比率是重要的保留尽可能多的脂肪组织在斯瓦特尽可能在长期的时间。大块完整的筋膜可以防止干细胞和脂肪细胞之间的细胞膜接触, 这是保持斯瓦特在一起的必要条件。过多的筋膜也能产生 "拉线" 效应。虽然一个笏星团可以脱离板块而不影响邻近的星团, 但通过筋膜连接的多个星团可以从单层中提取足够的簇, 使斯瓦特的使用不太好。吸脂, 其中涉及机械化切碎的笏, 可以绕过这个问题, 因为筋膜是在手术期间细碎。然而, 在抽脂过程中, 患者通常接受高剂量的肾上腺素治疗;这种治疗对组织 (和随后的实验) 的可能后果必须在研究期间被占去。
斯瓦特文化的最后一个关键步骤是选择一个实验性的文化媒体。我们使用标准细胞培养配方 (DMEM 10% 只血清, 1x 青霉素/链霉素) 进行了初步的表征实验。然而, 根据现有文献关于最大脂肪细胞转录, 我们使用了一个专门的配方 (7 µM 胰岛素, 30 µM 地塞米松, 1x 青霉素/链霉素在 M199 培养基)17。这一提法改善了早期转录水平, 但随着时间的推移, 水平下降。我们相信这是因为在身体里, 脂肪细胞接触到各种各样的化学信号, 在一天的过程中经常在喂食和饥饿状态之间循环。因此, 通过进一步优化对细胞培养基的补充, 可以改善观察到的转录剖面。
斯瓦特收获 (协议, 5 节) 将脂肪细胞从养殖中分离出来, 减少样品体积, 并消除周围 ADSC 片的干扰。这可能在脂肪细胞的均匀化 (这是必要的蛋白质或核酸隔离), 或功能化验的完整脂肪细胞 (如糖酵解或葡萄糖摄取化验)。另外, 完整的斯瓦特板块可以获得免疫细胞化学染色, 这使得 ADSC 板在整个染色过程中确保笏簇到单层。在这种情况下, 通过标准协议来吸取区域性媒体并修复整个区域性。然而, 我们建议在成像前添加一个玻璃盖玻片, 以扁平化3D 脂肪细胞簇, 并改善产生的图像质量。
我们相信斯瓦特有很大的潜力作为一个药物筛选平台。该技术简单, 重现性好, 并利用传统的组织培养板。因此, 通过将药理活性化合物添加到培养基中进行药物筛选试验是可执行的。脂肪细胞可以收获, 以检查该化合物对基因表达, 表观遗传, 胰岛素敏感性等的影响.在培养基中添加环境毒素同样可以用来检查它们对脂肪细胞的影响。由于斯瓦特允许研究人员跟踪个体的脂肪细胞簇随着时间的推移, 它是独特的适合评估的治疗, 使可见表型改变脂肪细胞。一个突出的例子是人类脂肪细胞 "褐变", 其中一个白色的, 脂肪储存脂肪细胞变得 "褐色" 或类似的热量, multilocular 棕色脂肪18。这是一个潜在的重大临床相关性的现象, 已经证明了在老鼠模型, 但从来没有在人类。在斯瓦特的共同培养的变化也具有巨大的研究潜力。斯瓦特是一种天然的共培养系统, 利用标准细胞培养井。因此, 你可以通过改变夹细胞或利用组织培养膜插入物来维持其他临床相关细胞类型中的脂肪。例如, 肝细胞可以作为上夹细胞片, 在到达脂肪组织之前筛查通过肝脏过滤的药物。同样, 癌细胞可以在斯瓦特的膜上生长, 以研究脂肪细胞是如何促进肿瘤病理的。

脂肪组织是肥胖的主要器官, 其在美国1的直接每年医疗费用为1470亿美元和2100亿美元。脂肪组织的积聚也会导致其他主要死因, 如心脏病、II 型糖尿病和某些类型的癌症2。体外培养模型对代谢研究和药物开发是必不可少的, 但目前脂肪组织的研究模式有很大的不足。脂肪细胞是脆弱的, 浮力的, 和晚期分化, 不会坚持细胞培养塑料, 因此不能使用传统的细胞培养方法培养。自二十世纪七十年代以来, 一些方法被用来克服这些障碍, 包括使用玻璃盖玻片, 天花板文化, 悬浮培养, 细胞外基质3,4,5,6,7. 然而, 这些方法的特点是细胞死亡和分化, 通常只用于为期两周的研究期。此外, 这些模型不尝试重述本机脂肪微环境, 因为它们不维持完整的 ECM, 脂肪细胞和基质支持细胞之间的相互作用, 也不是在体内相互施加的收缩力单元. 笏。
在缺乏金标准的原发脂肪培养方法的情况下, 脂肪的研究主要依赖于分化前脂肪细胞 (diffAds)。DiffAds 是 multilocular, 黏附和新陈代谢活跃。相比之下, 原发性白脂肪细胞单房性, nonadherent, 并表现出相对较低的新陈代谢。目前的脂肪培养模型未能概括健康成熟脂肪组织的生理, 可能是缺乏 FDA 批准的药物直接靶向脂肪细胞的主要因素。事实上, 缺乏生理的体外器官模型是一个主要的问题, 在大多数器官和疾病。
美国国立卫生研究院 (NIH) 在其立场文件中宣布创建其 Microphysiological 系统 (MPS) 计划, 报告说, 在所有人类药物临床试验中, 2013 的成功率仅为第二阶段和50% 阶段的18%。临床试验8。MPS 方案的设计是为了直接解决在体外单一的能力, 以模型人类生理学。NIH 将松龙定义为由人类主要或干细胞组成的多细胞3D 结构中的培养系统, 这些细胞概括了器官功能。与简化模型的同类, 永生化细胞培养, 松龙应准确模型细胞, 药物细胞, 药物药物, 和器官-药物相互作用9。与短期初级文化方法不同的是, NIH 标准规定了4周文化8的 MPS 的可持续性。MPS 计划的进一步细节可以在 NIH 的 RFAs (#RFA TR-18-001)10中找到。
我们开发了一种简单、新颖、适应性强、价格低廉的脂肪 MPS, 称为 "夹心白脂肪组织" (斯瓦特)11。我们克服了脂肪细胞的自然浮力的 "夹" 切碎的原始脂肪组织之间的脂质基质干细胞 (干细胞) (图 1)。由此产生的3D 构造概括细胞接触和当地脂肪微环境的周围成熟脂肪体与自然脂肪细胞支持的人群。斯瓦特已经通过展示8周的生存能力、对外源信号的反应、脂肪分泌物和植入成为动物模型来证实。

<table><tbody><tr><td>10x HBSS</td><td>Thermofisher</td><td>14185052</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-aldrich</td><td>G9391</td><td></td></tr><tr><td>Collagenase</td><td>Sigma-aldrich</td><td>C5138</td><td></td></tr><tr><td>Adenosine</td><td>Sigma-aldrich</td><td>A9251</td><td></td></tr><tr><td>DMEM</td><td>Thermofisher</td><td>11995065</td><td></td></tr><tr><td>M199 Media</td><td>Thermofisher</td><td>11043023</td><td>Phenol red-free&amp;nbsp;</td></tr><tr><td>250 &amp;micro;m Mesh Filter</td><td>Pierce</td><td>87791</td><td></td></tr><tr><td>0.2 &amp;micro;m Syringe Filter</td><td>Celltreat</td><td>229747</td><td></td></tr><tr><td>5 mL Luer-Lok syringes&amp;nbsp;</td><td>BD</td><td>309646</td><td></td></tr><tr><td>Metal Washers</td><td></td><td></td><td>These are simple metal washers and can be bought at any hardware store. They simply add leight weight to the backs of the plugers to ensure even contact between cells and gelatin, while being easy to stock and sterilize. Approximate mass: 6.3 g&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Heated Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Incubated Orbital Shaker</td><td>VWR</td><td>10020-988</td><td>Samples should be pitched at 45&amp;deg; angle to facilitate collagenase digestion</td></tr><tr><td>Heat Block</td><td></td><td></td><td>Set to 37-40 &amp;deg;C and placed under Biosafety Cabinet</td></tr><tr><td>Water Bath</td><td></td><td></td><td>Set to ~75 &amp;deg;C</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Specialized Plastics&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Upcell Dishes 6cm of 6-multiwell</td><td>Nunc</td><td>174902&amp;nbsp; or 174901</td><td>These are commerically available pNIPAAm-coated dishes which can be used to grow the upper sheet of ADSCs. Alternatively, pNIPAAm-coated plates can be produced in-lab. diameter: &amp;lt;6 cm for 6 cm dish, &amp;lt;3.5 cm for 6-well plate; approximate mass: 6.7g for 6 cm dish, 5.1 g for 6-well plate</td></tr><tr><td>Plastic Plunger Apparatus</td><td></td><td></td><td>These can be fashioned to fit within desired pNIPAAm-coated plastics (multiwell plates, petri dishes). They are comprised of a simple stem attached to a circular disk. They can be produced in-lab or by any facility that can fashion acrylic plastics</td></tr></tbody></table>

a microphysiologic platform for human fat: sandwiched white adipose tissue

白色脂肪组织 (笏) 在调节体重和日常健康方面起着至关重要的作用。 尽管如此, 现有的主要养殖模式仍存在重大限制, 所有这些模型都未能忠实地重述脂肪微环境或延长笏的生存能力超过两周。 缺乏可靠的初级文化模型严重阻碍了笏代谢和药物开发的研究。为此, 我们利用 NIH 的 microphysiologic 系统标准, 为 "斯瓦特" (夹心白脂肪组织) 的主要文化开发了一个新的平台。我们克服脂肪细胞的自然浮力的夹碎笏簇之间的薄层之间的脂质基质干细胞。 在这个构造中, 笏样本在八周的文化中是可行的。 斯瓦特保持完整的 ECM, 细胞对细胞的接触, 和物理压力的体内笏条件;此外, 斯瓦特还保持了强健的转录剖面, 对外源化学信号的敏感性, 以及整个组织功能。斯瓦特是一种简单、可重复、有效的原发性脂肪培养方法。 潜在的, 它是一个广泛适用的平台, 研究在笏生理学, 病理生理学, 新陈代谢和药物发展。

斯瓦特的生存能力最初是由序列 brightfield 成像的单个笏簇 (n = 12) 大约7.6 周。在这段时间内, 簇仍然固定在单层上。微小的形态学变化观察到个别脂肪细胞翘曲轻微或移位的位置。然而, 脂肪细胞并没有成为 multilocular 随着时间的推移, 表明缺乏分化, 也没有表现出任何明显的迹象死亡, 如细胞起泡, 裂解, 或分裂 (图 2)。两周大的特勤组的脂肪细胞特征证实为尼罗红 (NR)。清除单房性脂肪细胞簇的 NR 染色表明, 这些确实是脂肪细胞与完整的膜, 而不是工件, 囊肿, 或死脂肪细胞。在周围的 ADSC 片中没有 NR 染色表明, 这些细胞没有积累脂质, 因此不分化为脂肪血统本身 (图 3a)。碘碘化染色是在53天老斯瓦特进行。在高级 timepoints 脂肪细胞簇内的污点被排除, 这表明缺乏死亡 (图 3b)。油红色 O (破产) 染色是执行51天老斯瓦特与脂染色定位在固定脂肪团簇, 表明, 斯瓦特脂肪细胞维持活力与完整的脂肪细胞, 即使在先进的 timepoints (图 3c)。
免疫细胞化学 (ICC) 的表现, 以表明预期的蛋白质表达和本地化的成熟脂肪细胞标记在斯瓦特。完整的斯瓦特板块染色的抗体抗 perilipin (ab3526, 1:200 稀释), PPARγ2 (sc-166731, 1:100 稀释), FABP4 (ab92501, 1:1, 000 稀释)。12天老斯瓦特的共焦图像显示了预期的 perilipin 周围脂滴表面的定位 (图 4a)。荧光显微镜的3天老斯瓦特与血脂计数器染色 BODIPY 显示 FABP4 本地化内的细胞质 (图 4b), 和重叠信号从 PPARG 和 DAPI 染色显示 PPARG 本土化的核心 (图 4c)。
在多学科组织中对斯瓦特转录剖面进行评估 (n = 4 个捐献者)。收集后, 部分脂肪组织被用于基线 RNA 提取 (d0), 而其余的用于种子特警板块。这些板块随后在斯瓦特文化中收获了24小时, 14 天, 28 天。采用一步法定量反向转录聚合酶链反应 (RT qPCR) 测定转录水平。研究了六项主要脂肪细胞基因: PPARG、FABP4、LPL、CEBPA、HSL 和 ADIPOQ (图 5)。ACTB 被用作内部控制, 转录水平表示为与主语匹配的基线 (d0) 表达式的百分比。在斯瓦特, 所有基因都表现出强健的转录, 尽管随着时间的推移, 水平呈下降趋势。我们认为, 随着我们在讨论部分中阐述的进一步的媒体优化, 观察到的转录剖面可以得到改进。
在转录剖面使用的相同培养基上评价基底内分泌功能。用酶联免疫吸附法 (ELISA) (ab100581) 测定了从斯瓦特板上清液和瘦素水平。斯瓦特显示的基础瘦素分泌物在 1, 14 和28天 (图 6a)。ADSC 板被维护了与斯瓦特板材平行作为控制 (即, ADSC 板料没有夹笏)。在这些控制中无法检测到分泌的瘦素 (未显示数据)。
为了确定斯瓦特是否能够维持一段时间内诱导反应的能力, 脂肪被检查 (n = 4)。为了评估诱导性脂解能力, 新收集的笏 (d0) 的一部分被切碎, 脂肪细胞被酶隔离 (类似于在协议5节的斯瓦特收获), 而其余的则用于种子特警板块。以100µM 佛司可林、1µM 肾上腺素或控制缓冲器为 HBSS, 在300µL 2% 只不含脂肪酸的牛血清白蛋白中培养出3小时的孤立脂肪细胞。用游离甘油试剂测定了收集培养基中甘油的含量。特警队随后在1和5天收割, 脂解的能力也以同样的方式进行了评估。化学刺激在所有三 timepoints 中显著提高甘油释放量 (图 6b): 治疗脂肪细胞的甘油释放量在 d0 笏增加了 82, 46% (p = 0.01), 1 天斯瓦特由 577 @ 387% (p = 0.02), 五天特警队 348 @ 343% (p =0.03). 平均甘油水平在被刺激的脂肪细胞是非常相似的在所有三 timepoints: 主要笏5.2 和 0.8, 1 天斯瓦特 4.4 @ 1.9 和5天斯瓦特 4.8 @ 1.8 µg 甘油/毫克总蛋白。在新收集的笏 (d0) 中, 与斯瓦特收获细胞相比, 平均甘油水平 (即基底脂肪) 相对较高。这可能是由于在外科手术切除、运送到实验室和切碎术期间 d0 组织的压力增加。然而, 这些实验表明, 与新鲜收集的笏相比, 斯瓦特1或5天的脂解能力没有减少。
斯瓦特被证明是能够移植和恢复的小鼠模型 (n = 4) 作为一个复杂的, 整个组织功能的证明。如果研究者希望利用斯瓦特在体外操作笏, 然后将组织移植到动物模型中, 这也是该平台的一个可能应用。斯瓦特养殖10天, 并利用标准协议收获;从斯瓦特板块分离出来的脂肪细胞被装入1毫升注射器 (没有针头)。在免疫缺陷性 eGFP 小鼠的背侧皮肤下进行切口 (点头。Cg-Prkdcscid Il2rgtm1Wjl Tg (CAG-EGFP) 1 定向刨花板/SzJ)。这创造了一个小的背部皮下口袋, 其中特警培养脂肪细胞注射注射器, 然后口袋被缝合关闭。移植后10天, 小鼠被牺牲。移植明显可见, 易于恢复 (图 7a)。恢复的移植和小鼠对侧脂肪库固定, 石蜡嵌入, 切片。免疫组织化学在切片的幻灯片上进行, 主要抗体抗 GFP (A10262, 1:100 稀释) 和人 PLIN (sc-390169, 1:100 稀释)。实验验证了所选择的抗 PLIN 抗体对人体 PLIN 的染色, 而不是老鼠。从恢复的移植脂肪细胞大约50–120µm 的大小, 这是预期的范围, 人类脂肪, 是完全阴性的 GFP (图 7b)。然而, 大约31% 的脂肪细胞是阳性的人 PLIN, 表明成功的植入到宿主。小鼠脂肪库中的脂肪细胞大小20–40µm, 这与小鼠脂蛋白是一致的, 同时染色完全阳性的 GFP 和完全阴性的人类 PLIN (图 7c)。ADSC 床单 (没有夹布笏) 被刮从细胞培养皿和移植到小鼠作为控制。然而, 这些没有产生任何可恢复的组织 (数据没有显示)。这表明, 恢复脂肪细胞来自成熟的培养的斯瓦特, 而不是结果的分化的人类干细胞。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57909/57909fig1.jpg"> 图 1: 斯瓦特方法.(a) 斯瓦特的示意图显示将一张 eGFP 标记的陶瓷从 pNIPAAm 涂层的组织培养皿转移到第二个未标记的陶瓷上, 在标准的组织培养皿上生长。碎, 第一人类笏被夹在2陶瓷床单之间。(b) 荧光显微镜演示了由 (a) 所述技术所产生的 Brightfield、GFP 和合并通道。刻度条 = 100 µm. (c) 一名代表的数码照片, 5 天老特警在标准的6井板培养。笏的大容积稳定地固定在井底。图是由刘等 al修改的。11<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57909/57909fig1large.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/57909/57909fig2.jpg"> 图 2: 特警队在8周的文化中仍然可行.55天内单个斯瓦特星团的串行 brightfield 成像显示, 没有与细胞死亡相一致的形态学变化。刻度条 = 100 µm. 11<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57909/57909fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57909/57909fig3.jpg"> 图 3: 斯瓦特在超过50天的时间内仍然可行.(a) 尼罗河红染色表明对斯瓦特养殖15天的染色限制, 表明存活的脂肪细胞。周围的基质细胞没有染色。图的比例为40X 倍, 缩放条 = 100 µm. (b) 53 天内培养的斯瓦特 (PI) 染色显示完全的 PI 排斥, 表明没有脂肪细胞死亡;刻度条 = 100 µm. (c) 51 天老特警队的油红 O 染色表明, 中性脂对脂肪细胞的限制, 表明脂肪细胞膜的长期稳定性。图是由刘等 al修改的。11<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57909/57909fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57909/57909fig4.jpg"> 图 4: 免疫细胞化学表明, 斯瓦特染色阳性的成熟脂肪干细胞标记与预期的定位.(a) 共聚焦显微镜2周老斯瓦特发现人 PLIN + 单房性细胞与局部化的脂滴表面。刻度条 = 100 µm. (b) 3 天老特警的荧光显微术揭示了人类 FABP4+ 细胞的定位到细胞质和 (c) PPARγ + 细胞与细胞细胞核定位。刻度条 = 100 µm. 图是由刘等人修改的。11<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57909/57909fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57909/57909fig5.jpg"> 图 5: 斯瓦特保持关键的脂肪细胞识别基因表达.在1、14、28天后, qPCR 的培养和基因表达中, 特勤组进行了胶原酶消化。qPCR 表明, 1 天的老14天的特警队保持高水平的 (a) PPARG, (b) FABP, (c) HSL, (d) CbEBPA, (e) LPL 和 (f) ADIPOQ。ACTB 被用作参考基因。错误条 = 标准错误。图是由刘等 al修改的.11.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57909/57909fig5large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57909/57909fig6.jpg"> 图 6: 斯瓦特基部分泌脂肪因子, 并对儿茶酚胺刺激生理反应.(a) 24 h 基瘦素分泌物的 ELISA 定量显示在培养28天后无显著变化。(b) 肾上腺素和佛司可林肾上腺素刺激, 在1和5天的培养中, 在刚收割的主要笏 (1.7x) 和主题匹配特警队中产生显著的酵反应。1天特警队对儿茶酚胺刺激反应, 6.3 倍以上的基础糖酵解, 而5天斯瓦特显示3.3 倍以上的基础糖酵解增加。在斯瓦特, 受刺激的糖酵解与新鲜的笏 (p = 0.53, n = 4) 相比没有显著的差异。错误条 = 标准错误。图是修改的刘等。11.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57909/57909fig6large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57909/57909fig7.jpg"> 图 7: 斯瓦特可以在体内reimplanted, 证明整个组织功能的保存.(a) 斯瓦特在移植后10天内容易被可视化成免疫缺陷的老鼠 (星号)。如果斯瓦特没有成功植入, 10 天后, 坏死的斯瓦特将被液化。(b) 被恢复的斯瓦特的部分显示了大的, 单房性脂肪细胞 (直径50至120µm), eGFP-。31.3% 的脂肪细胞是人类 PLIN +。人类 PLIN + 脂肪细胞倾向于小, 这将适合于脂肪移植的临床观察, 在人类中, 小脂肪细胞更可能存活的转移。缩放条 = 100 µm, 200X 放大倍数。(c) 从同一小鼠身上取出的对侧脂肪垫显示出更小的脂肪细胞 (20–40µm), 是人类的 PLIN。缩放条 = 100 µm, 200X 放大倍数。图被修改了从刘等。11.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57909/57909fig7large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>

Watch this Scientific Journal Video about A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue at JoVE.com

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

白色脂肪组织 (笏) 在其目前的主要培养模式中存在严重缺陷, 阻碍了药理学的发展和代谢研究。在这里, 我们提出一个协议, 以产生一个脂肪 microphysiological 系统的夹笏之间的基质细胞。该构造为原笏培养提供了一个稳定、适应性强的平台。

人体脂肪 Microphysiologic 平台: 夹心白色脂肪组织

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health.  Still, there are significant limitations to available primary ...

a-microphysiologic-platform-for-human-fat-sandwiched-white-adipose

Nanyang Technological University

Research

JoVE Journal

Behavior

6.2K 次观看.  Louisiana State University Health Sciences Center. 白色脂肪组织 (笏) 在其目前的主要培养模式中存在严重缺陷, 阻碍了药理学的发展和代谢研究。在这里, 我们提出一个协议, 以产生一个脂肪 microphysiological 系统的夹笏之间的基质细胞。该构造为原笏培养提供了一个稳定、适应性强的平台。

视频: A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

人网膜和皮下脂肪组织 CD34+CD31+ 内皮细胞的分离、扩增和脂肪诱导

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

科研

免疫与感染

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

生物工程

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 Subcutaneous Adipose Tissue Sampling Using a Mini-Liposuction Technique

Studies on adipose tissue are useful in understanding metabolic and other conditions. Human subcutaneous adipose tissue is accessible. With appropriate training and strict adherence to aseptic technique, subcutaneous adipose samples can be safely and efficiently obtained in a non-clinical setting by researchers. Following the administration of local anesthetic lateral to the umbilicus, a 14 G needle attached to a 5 or 10 mL syringe is inserted through the skin into the subcutaneous tissue. Under suction, the syringe is moved in a reciprocating, slicing motion to isolate fragments of adipose tissue. Withdrawing the plunger is enough to ensure that adipose tissue fragments are aspirated through the needle into the syringe. A single biopsy can collect about 200 mg of tissue. This biopsy technique is very safe for both participants and research staff. Following the biopsy, participants can resume most everyday activities, although they should avoid swimming and overly strenuous activities for 48 h to avoid excessive bleeding. Participants can safely undergo 2 biopsies within a single day, meaning that the technique can be applied in before-after acute intervention studies.

The manuscript and associated video demonstrate a percutaneous biopsy technique to obtain samples of subcutaneous adipose tissue from areas surrounding the umbilicus. This method is a low-risk and efficient way to investigate a range of parameters (e.g., gene or protein expression, enzyme activity, lipid content) within adipose tissue.

使用微型吸脂技术进行人体皮下脂肪组织取样

Studies on adipose tissue are useful in understanding metabolic and other conditions. Human subcutaneous adipose tissue is accessible. With appropriate ...

医学

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

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

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 ...

生物学

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. ...

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

利用膜成熟腺细胞聚合培养（MAAC）的人类成熟腺细胞的分离与培养

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent 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 ...

<meta charset="utf8"></head><body>人 SATMVEC：分离和脂肪细胞串扰研究

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk.
hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function.
hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

<meta charset="utf8"></head><body>作者聚焦：通过皮下脂肪组织微血管研究洞察心脏代谢疾病

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.