Name: a microphysiologic platform for human fat: sandwiched white adipose tissue
Uploaded: 2018-08-15T14:00:00.000+00:00
Duration: 8 min 14 s
Description: Watch this Scientific Journal Video about A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue at JoVE.com

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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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoprotein+lipase+regulation+by+insulin+and+glucocorticoid+in+subcutaneous+and+omental+adipose+tissues+of+obese+women+and+men.">Lipoprotein lipase regulation by insulin and glucocorticoid in subcutaneous and omental adipose tissues of obese women and men.</a> Journal of Clinical Investigation. 92 (5), 2191-2198 (1993).</li><li>Keipert, S., Jastroch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brite/beige+fat+and+UCP1+-+is+it+thermogenesis?.">Brite/beige fat and UCP1 - is it thermogenesis?.</a> Biochimica et Biophysica Acta. 1837 (7), 1075-1082 (2014).</li></ol>

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 PP...

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?...

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

Research

JoVE Journal

Behavior

6.3K 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

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

本地化识别，切除脂肪小鼠油库，并

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

科研

医学

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 &#176;C) or cold-activated (CA) (17 &#176;C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 &#176;C) and cold conditions (17 &#176;C) in order to map brown adipose tissue (BAT) in adult human subjects.

人类的褐色脂肪组织的仓库的正电子发射断层扫描/计算机断层扫描自动分段及注册磁共振图像

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in ...

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 ...

生物工程

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

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

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

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 ...

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. ...

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.

人体脂肪 Microphysiologic 平台: 夹心白色脂肪组织

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此视频中的章节

本地化识别，切除脂肪小鼠油库，并

人类的褐色脂肪组织的仓库的正电子发射断层扫描/计算机断层扫描自动分段及注册磁共振图像

微血管碎片血管化产热脂肪组织的三维培养

Adipo: 三维脂肪组织成像的组织清除方法

三维白色脂肪组织结构的全装染色可视化

用于细胞表征和分泌特征的人类脂肪组织微分裂

通过甲基异硫清除和三D成像探索脂肪组织结构

研究人类皮下脂肪组织中的脂肪内皮细胞/脂肪细胞串扰

研究人类皮下脂肪组织中的脂肪内皮细胞/脂肪细胞串扰

研究人类皮下脂肪组织中的脂肪内皮细胞/脂肪细胞串扰