Name: a human 3d extracellular matrix-adipocyte culture model for studying matrix-cell metabolic crosstalk
Uploaded: 2019-11-07T15:00:00
Description: Watch this Scientific Journal Video about A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk at JoVE.com

我们感谢丹妮尔·伯杰、玛丽莲·伍德拉夫、西蒙娜·科雷亚和雷莎·盖斯协助学习协调工作。SEM由密歇根大学显微镜和图像分析实验室生物医学研究核心设施执行。该项目由NIH赠款R01DK097449（RWO），R01DK115190（RWO，CNL），R01DK090262（CNL），退伍军人事务功绩补助金I01CX001811（RWO），密歇根州糖尿病研究中心试点和可行性赠款（NIH资助P30-DK020572（RWO），退伍军人管理局 VISN 10 SPARK 试点赠款 （RWO）。扫描电子显微镜由密歇根大学显微镜和图像分析实验室生物医学研究核心设施执行。本手稿图 4 最初发表在贝克等人，J 克林恩多·梅塔布2017;3月1;102 （3）， 1032-1043.doi： 10.1210/jc.2016-2915，经牛津大学出版社许可[https://academic.oup.com/jcem/article/102/3/1032/2836329]转载。有关重复使用此材料的权限，请访问http://global.oup.com/academic/rights。

脂肪组织是从接受选择性减肥手术的人体受试者获得，经机构审查委员会批准。
1. 皮里泊细胞分离和培养试剂制备
<ol><li>在1x磷酸盐缓冲盐水溶液（PBS）中制备2%牛血清白蛋白（BSA）。过滤消毒，并储存在4°C。</li>
	<li>制备II型胶原酶：2mg/mL，在2%BSA中，在1xPBS中。使用前立即做好准备。</li>
	<li>制备红血球 （RBC） 莱氨酸溶液： 1.5M NH4Cl， 100 mM NaHCO3， 10 mM 二?...

<ol>
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ECM-脂肪培养模型为剖析ECM和细胞在听写最终组织表型中的个体作用提供了宝贵的工具。ECM 隔离协议相当可重复，但可以观察到脱细胞化过程中的变异性。第 3 天降脂步骤是协议中的一个关键点。在一夜之间提取时，基质的降脂应用极性溶剂溶液变黄来证明，而基质应从完整脂肪组织的黄橙色特征过渡到半透明/白色 （图 1）。如果这些颜色没有变化，则脱脂可能不完整。降?...

细胞外基质（ECM）不仅为组织提供机械支架，而且与驻留在细胞中的细胞进行复杂的串扰，调节组织平衡所需的各种过程，包括细胞增殖，分化、信令和代谢1.虽然健康的ECM在维持正常组织功能中起着至关重要的作用，但功能失调的ECM与多种疾病有牵连2。
脂肪组织在代谢性疾病的发病机制中起着重要的作用。肥胖与过度的脂肪细胞肥大和细胞缺氧、脂肪细胞细胞代谢缺陷、脂肪组织内质视网膜和氧化应激和炎症有关。虽然了解不足，但这些复杂的过程合谋损害脂肪组织营养缓冲能力，导致脂肪组织营养溢出，多组织毒性，以及系统性代谢疾病3，4， 5.脂肪组织衰竭的先后事件和具体机制被人们难以理解，但脂肪组织ECM的改变被牵连其中。ECM组合物在人类和小鼠肥胖的脂肪组织内发生改变，ECM蛋白的沉积增加，脂肪组织ECM中与人类代谢疾病相关的定性生化和结构差异，包括2型糖尿病和高脂血症6，7，8，9，10，11。
尽管有这些观察，脂肪组织ECM在调解脂肪组织功能障碍中的作用没有明确界定。部分原因在于缺乏可处理的实验模型，允许解剖ECM和脂肪细胞在调节最终脂肪组织功能方面的具体作用。ECM-脂肪培养至少在两个方面更好地模拟了原生脂肪组织的体内环境。首先，ECM培养提供了类似于原生脂肪组织的分子环境，包括原生胶原蛋白、乙基蛋白和标准二维培养中不存在的其他基质蛋白。其次，由于塑料基板12的弹性降低，二维塑料的培养物通过机械效应改变脂肪细胞代谢，ECM培养消除这种作用。
在再生和重建医学和组织工程13、14中研究了通过分离ECM从脱细胞脂肪和其他组织分离来设计生物支架的方法。15，16，17，18.我们之前已经发表了方法，其中我们调整这些方法，以开发一个体外3D模型的人ECM-脂肪细胞培养，使用ECM和脂肪细胞干细胞（preadipo细胞）派生自人类内脏脂肪组织11。在本文中，我们将详细介绍这些方法。人体脂肪组织的去细胞化过程是一个为期四天的过程，涉及机械和酶处理，以去除细胞和脂质，留下一个生物支架，保持其衍生组织的特征。去细胞化ECM支持人类单体细胞的增生分化，当用脂肪细胞重组时，保持完整脂肪组织的微结构、生化和疾病特异性特性，并参与代谢原生脂肪组织的功能特征。这种基质可以单独研究或与细胞重新播种，允许研究脂肪组织的细胞和细胞外成分之间的相互作用和串扰。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% trypsin-EDTA</td>
			<td>Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA</td>
			<td>Cat#25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL cryovial tube</td>
			<td>Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA</td>
			<td>Cat#02-682-557</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>VWR International LLC., Radnor, PA, USA</td>
			<td>Cat#89370-094</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;m nylon mesh filter</td>
			<td>Corning Inc., Corning, NY, USA</td>
			<td>Cat#352360</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Deoxy-D-glucose</td>
			<td>Sigma-Aldrich, Inc., St Louis, MO, USA</td>
			<td>Cat#D8375</td>
			<td></td>
		</tr>
		<tr>
			<td>2 nM 3,3&#8217;-5,Triiodo,L-thyronine sodium salt (T3)</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#T6397</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture plates</td>
			<td>VWR International LLC., Radnor, PA, USA</td>
			<td>Cat#10861-700</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#I5879</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture plates</td>
			<td>VWR International LLC., Radnor, PA, USA</td>
			<td>Cat#10861-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic Solution (ABAM)</td>
			<td>Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA</td>
			<td>Cat#15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#B4639</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich, Inc., St Louis, MO, USA</td>
			<td>Cat#A8806</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer RLT</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>Cat#79216</td>
			<td></td>
		</tr>
		<tr>
			<td>Ciglitizone</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#C3974</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxy-D-glucose, 2-[1,2-3H (N)]-</td>
			<td>PerkinElmer Inc., Waltham, MA, USA</td>
			<td>Cat#NET328A250UC</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas, type II-S</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#D4513</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA</td>
			<td>Cat#BP231</td>
			<td>Flammable, caustic</td>
		</tr>
		<tr>
			<td>Disodium EDTA</td>
			<td>Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA</td>
			<td>Cat#BP118</td>
			<td></td>
		</tr>
		<tr>
			<td>D-pantothenic acid hemicalcium salt</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#21210</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12</td>
			<td>Gibco, ThermoFisher Scientific Inc., Waltham, MA USA</td>
			<td>Cat#11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs, Inc., King of Prussia, PA, USA</td>
			<td>Cat#DSP-MD.43</td>
			<td>Flammable</td>
		</tr>
		<tr>
			<td>EVE Cell Counting Slides, NanoEnTek</td>
			<td>VWR International LLC., Radnor, PA, USA</td>
			<td>Cat#10027-446</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA</td>
			<td>Cat#10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma-Aldrich, Inc., St Louis, MO, USA</td>
			<td>Cat#G5882</td>
			<td>Caustic</td>
		</tr>
		<tr>
			<td>Hexamethyldisalizane</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#440191</td>
			<td>Flammable, caustic</td>
		</tr>
		<tr>
			<td>Human insulin solution</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA</td>
			<td>Cat#A415</td>
			<td>Flammable</td>
		</tr>
		<tr>
			<td>Isoproterenol</td>
			<td>Sigma-Aldrich, Inc., St Louis, MO, USA</td>
			<td>Cat#I5627</td>
			<td>Flammable</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#S25484</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipase from porcine pancreas, type VI-S</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#L0382</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4*7H2O</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#230391</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#S5136</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA</td>
			<td>Cat#S233</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA</td>
			<td>Cat#A661</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal cutting temperature (OCT) compound</td>
			<td>Agar Scientific, Ltd., Stansted, Essex, UK</td>
			<td>Cat# AGR1180</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red-O Solution (ORO)</td>
			<td>Sigma-Aldrich, Inc., St Louis, MO, USA</td>
			<td>Cat#O1391</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red-O Stain Kit</td>
			<td>American Master Tech Scientific Inc., Lodi, CA, USA</td>
			<td>Cat#KTORO-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#201030</td>
			<td>Caustic</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#93482</td>
			<td>Caustic</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline Solution (PBS)</td>
			<td>Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA</td>
			<td>Cat#SH3025601</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribonuclease A from bovine pancreas, type III-A</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#R5125</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAEasy Fibrous Tissue MiniKit</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>Cat#74704</td>
			<td></td>
		</tr>
		<tr>
			<td>Scintillation Fluid</td>
			<td>Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA</td>
			<td>Cat#SX18</td>
			<td></td>
		</tr>
		<tr>
			<td>Scintillation Counter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, forceps, sterile</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorensen&#39;s phosphate buffer</td>
			<td>Thomas Scientific, Inc., Swedesboro, NJ</td>
			<td>CAS #: 10049-21-5</td>
			<td></td>
		</tr>
		<tr>
			<td>T-150 culture flask</td>
			<td>VWR International LLC., Radnor, PA, USA</td>
			<td>Cat#10062-864</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Master Mix</td>
			<td>ThermoFisher Scientific Inc., Waltham, MA USA</td>
			<td>Cat#4369016</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature-controlled orbital shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Homogenizer, BeadBug Microtube Homogenizer</td>
			<td>Benchmark Scientific</td>
			<td>Cat#D1030</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma-Aldrich, Inc. St Louis, MO, USA</td>
			<td>Cat#T3309</td>
			<td></td>
		</tr>
		<tr>
			<td>Triglyceride Determination Kit</td>
			<td>Sigma-Aldrich, Inc., St Louis, MO, USA</td>
			<td>Cat#TR0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue stain, 0.4%</td>
			<td>VWR International LLC., Radnor, PA, USA</td>
			<td>Cat#10027-446</td>
			<td></td>
		</tr>
		<tr>
			<td>Type II collagenase</td>
			<td>Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA</td>
			<td>Cat#17101015</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Reeve Angel filter paper, Grade 201, 150mm</td>
			<td>Sigma-Aldrich, Inc., St Louis, MO, USA</td>
			<td>Cat#WHA5201150</td>
			<td></td>
		</tr>
	</tbody>
</table>

a human 3d extracellular matrix-adipocyte culture model for studying matrix-cell metabolic crosstalk

细胞外基质（ECM）在调节组织平衡、与细胞进行串扰和调节细胞功能的多个方面方面发挥着核心作用。ECM在肥胖的脂肪组织功能中起着特别重要的作用，脂肪组织ECM沉积和成分的变化与小鼠和人类的代谢疾病有关。允许解剖ECM和细胞在促成全球组织表型中的作用的可移植体外模型是稀疏的。我们描述了人类ECM-脂肪细胞培养的一个新的3D体外模型，它允许研究ECM和脂肪细胞在调节脂肪组织代谢表型方面的具体作用。人体脂肪组织脱细胞以分离ECM，随后重新填充了板细胞，然后在ECM中分化成成熟的脂肪细胞。此方法创建代谢活性的 ECM-脂肪细胞结构，并保留组织和患者的特征，从中派生它们。我们用这个系统来演示人类脂肪组织中疾病特异性的ECM-脂肪细胞串扰。此培养模型提供了一个工具，用于解剖 ECM 和脂肪细胞在促进全球脂肪组织代谢表型中的作用，并允许研究 ECM 在调节脂肪组织平衡中的作用。

脂肪组织ECM的制备，用板菌细胞播种，以及体外分化成成熟的脂肪细胞，导致组织中清晰的顺序形态变化，从而能够对整个方案的进展进行视觉评估（图1）.用于播种ECM的Preadipo细胞使用胶原酶从单独的VAT样品中分离（图2）。在加工的每个阶段扫描ECM-脂肪细胞结构的电子显微镜显示ECM的去细胞化，以及随后重新播种和分化时含?...

Watch this Scientific Journal Video about A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk at JoVE.com

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

我们描述了一个3D人类细胞外基质-脂肪细胞体外培养系统，它允许解剖基质和脂肪细胞的作用，促进脂肪组织代谢表型。

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 ...

我们的协议表明，在没有经典致病介质的情况下，ECM 可促进致病性分化，并且是首次证明 ECM 对细胞代谢的疾病特异性调控。我们的技术提供了一个工具，用于解剖 ECM 和脂肪细胞在脂肪组织代谢中的作用，并允许研究 ECM 在调节脂肪组织平衡中的作用。与标准 2D 细胞培养相比，我们的 3D 模型允许在 2 型糖尿病背景下对脂肪细胞和脂肪组织 ECM 之间的相声进行更有力的检查和解剖。我们建议从较小的样本开始，以测量其细胞化水平。请记住，细胞的生长以及组织去细胞化程度的变异性。重要的是要观察组织之前和之后的每一步，以确保组织变得去细胞化，并了解样品之间的潜在差异。从手术获得内脏脂肪组织或增值税后，在50毫升锥形管中加入5-10克组织至15-25毫升冷冻缓冲液，准备储存。将样品完全浸入零下80摄氏度，直到去细胞化。对于预细胞分离，将两克新鲜样品的完好增值税放入20毫升胶原酶II型溶液中，然后将无菌剪刀插入管子中，彻底切碎组织。一旦被切碎到细浆中，在37摄氏度的轨道摇床上，在胶原酶溶液中孵育60分钟。孵育后，通过100微米尼龙网过滤消化结果，放入一个新鲜的50毫升锥形管中。消化的应为具有适量粘度和少量未消化组织的残留链的黄橙色液体。将样品以270倍g离心10分钟，取出上流液，用移液器将细胞颗粒重新用在两毫升1X RBC解液中。在25摄氏度下孵育溶液1分钟，然后加入10毫升15%FBS DMEM F12，以270倍g离心10分钟。要准备 ECM，请通过定期轻轻手动搅拌将先前冻结的 VAT 样品在预热水浴中孵育 20 分钟，将以前冻结的 VAT 样品冻结/解冻至 37 摄氏度。解冻后，将组织转回零下80摄氏度20分钟，并重复冷冻/解冻过程三次，以解冻结束。使用无菌钳将 VAT 样品转移到含有 15-25 毫升酶溶液 1 的新鲜 50 毫升圆锥形管中，确保样品完全浸入。然后在37摄氏度下孵育过夜，同时在130 RPM下摇晃。第二天，用15-25毫升的冲洗缓冲液洗样品三次，每次洗涤后倒出溶液。将样品转移到一个新鲜的50毫升管与15-25毫升的酶溶液2，并孵育过夜。使用冲洗缓冲液重复洗涤，将样品转移到含有 15-25 毫升极性溶剂萃取溶液的新鲜管中。在摇动时孵育样品过夜。使用极性溶剂溶液孵育后，检查样品是否去除脂质非常重要。如果未去除大部分脂质，则可能需要重复此步骤。可以使用较短的孵育时间。孵育后，将样品转移到含有15-25毫升冲洗缓冲液的新鲜管子中，并在摇床上清洗样品三次。然后用70%乙醇溶液洗样品三次，每次洗涤后倒掉乙醇。用储存溶液清洗样品一次，然后使用无菌钳子转移到含有 15-25 毫升存储溶液的新鲜管子中，确保将其完全浸浸。然后，样品可以在四摄氏度下储存长达一个月。异丙醇和乙醇是易燃的，必须在室温下处理，并处理在易燃废物中。任何人类废物都必须与通常的生物危害废物分开处理。将存储的 ECM 碎片转移到 24 孔板的单个孔中，无菌钳子可添加计划下游测定所需的尽可能多的碎片。在轨道摇床上用500微升70%乙醇洗涤三次，然后用PBS洗涤三次，以补充水分。使用无菌剪刀将 ECM 切割成 100 毫克碎片。将每个碎片放入24井板的一个井中，并在25摄氏度下孵育板15分钟，让多余的PBS从碎片中挤出。然后用移液器小心地从井中去除剩余的 PBS。通过将细胞直接移入 ECM，用 20 微升的预细胞悬浮液为每个片段播种。将移液器尖端放入 ECM 中，轻轻将移液器尖端排出到矩阵中心，确保电池悬架不会溢出，最终位于井底。如果单元悬架从 ECM 溢出，则从该位置拆下移液器尖端，并将其插入 ECM 的其他位置。细胞播种后，在37摄氏度下孵育 ECM 40 分钟。用 500 微升生长介质填充每一个井，以覆盖种子 ECM 碎片。在37摄氏度和5%的二氧化碳下培养细胞72小时。72 小时后，小心吸气生长介质，而不干扰 ECM 碎片。添加 500 微升的分化介质和培养细胞 14 天，每两到三天更换一次培养。使用光显微镜检查差异。细胞会积累脂质，变成棕色-黄色的颜色，并变得更加球形，因为他们区分。脂肪组织ECM的制备，与预细胞的播种，并在体外分化为成熟的脂肪细胞监测与扫描电子显微镜，揭示了ECM的去细胞化和随后的脂质含有脂肪在重新播种和分化后重新捕获。脱细胞 ECM 的胶原蛋白 1 特异性免疫组织化学显示维持胶原蛋白微结构，而活体和固定 3D ECM-adipocy 细胞构造的油红-O 染色显示脂质含有脂肪细胞。使用定量实时PCR对 ECM 中分化的脂肪细胞进行筛选，结果表明，这些基因相对于在 ECM 培养的未分化前细胞，在分化细胞中得到调节。显示了成绩单水平的全部差异。来自糖尿病和非糖尿病受试者的ECM和脂肪细胞的四种组合受到代谢表型的诊断。在糖尿病组织的构造中发现脂解功能的葡萄糖吸收受损。重要的是，发现非糖尿病ECM可挽救糖尿病脂肪细胞中胰岛素刺激的葡萄糖吸收和脂解能力。分离的预核细胞、去细胞化组织和重新播种的 ECM 的污染并不少见。在整个协议中必须努力维护无菌。

Research

JoVE Journal

Bioengineering

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

一个简单的悬滴细胞生成三维球体文化协议

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

科研

生物工程

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

旋转的人类细胞培养的细胞培养系统：作为一种模式的滋养细胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

3D水凝胶支架的关节软骨细胞培养及软骨生成

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

人类乳腺上皮细胞的激素反应3D文化模式

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

3D 动态培养系统构建多层间充质干细胞片材

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

利用大分子挤出的人类食性增大疤痕的体外模型

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

维持静止人类造血干细胞的文化方法

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...