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
1Department of Surgery,University of Michigan Medical School, 2Department of Pediatrics and Communicable Diseases,University of Michigan Medical School, 3Graduate Program in Immunology,University of Michigan Medical School, 4Graduate Program in Cellular and Molecular Biology,University of Michigan Medical School, 5Undergraduate Research Opportunity Program,University of Michigan, 6Department of Surgery,Ann Arbor Veterans Affairs Healthcare System
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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。 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。 制备红血球 （RBC） 莱氨酸溶液： 1.5M NH4Cl， 100 mM NaHCO3， 10 mM 二?…
Representative Results
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?…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 0.25% trypsin-EDTA | Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA | Cat#25200056 | |
| 1.5 mL cryovial tube | Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA | Cat#02-682-557 | |
| 10% Neutral Buffered Formalin | VWR International LLC., Radnor, PA, USA | Cat#89370-094 | |
| 100 µm nylon mesh filter | Corning Inc., Corning, NY, USA | Cat#352360 | |
| 2-Deoxy-D-glucose | Sigma-Aldrich, Inc., St Louis, MO, USA | Cat#D8375 | |
| 2 nM 3,3’-5,Triiodo,L-thyronine sodium salt (T3) | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#T6397 | |
| 24-well tissue culture plates | VWR International LLC., Radnor, PA, USA | Cat#10861-700 | |
| 3-Isobutyl-1-methylxanthine (IBMX) | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#I5879 | |
| 96-well tissue culture plates | VWR International LLC., Radnor, PA, USA | Cat#10861-666 | |
| Antibiotic-Antimycotic Solution (ABAM) | Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA | Cat#15240062 | |
| Biotin | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#B4639 | |
| Bovine Serum Albumin (BSA) | Sigma-Aldrich, Inc., St Louis, MO, USA | Cat#A8806 | |
| Buffer RLT | Qiagen, Hilden, Germany | Cat#79216 | |
| Ciglitizone | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#C3974 | |
| Deoxy-D-glucose, 2-[1,2-3H (N)]- | PerkinElmer Inc., Waltham, MA, USA | Cat#NET328A250UC | |
| Deoxyribonuclease I from bovine pancreas, type II-S | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#D4513 | |
| Dexamethasone | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#D4902 | |
| Dimethyl Sulfoxide | Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA | Cat#BP231 | Flammable, caustic |
| Disodium EDTA | Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA | Cat#BP118 | |
| D-pantothenic acid hemicalcium salt | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#21210 | |
| Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12 | Gibco, ThermoFisher Scientific Inc., Waltham, MA USA | Cat#11320033 | |
| Ethanol | Decon Labs, Inc., King of Prussia, PA, USA | Cat#DSP-MD.43 | Flammable |
| EVE Cell Counting Slides, NanoEnTek | VWR International LLC., Radnor, PA, USA | Cat#10027-446 | |
| Fetal bovine serum (FBS) | Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA | Cat#10437028 | |
| Glutaraldehyde | Sigma-Aldrich, Inc., St Louis, MO, USA | Cat#G5882 | Caustic |
| Hexamethyldisalizane | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#440191 | Flammable, caustic |
| Human insulin solution | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#I9278 | |
| Isopropanol | Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA | Cat#A415 | Flammable |
| Isoproterenol | Sigma-Aldrich, Inc., St Louis, MO, USA | Cat#I5627 | Flammable |
| KCl | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#S25484 | |
| KH2PO4 | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#P5655 | |
| Lipase from porcine pancreas, type VI-S | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#L0382 | |
| MgSO4*7H2O | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#230391 | |
| Na2HPO4 | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#S5136 | |
| NaCl | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#S3014 | |
| NaHCO3 | Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA | Cat#S233 | |
| NH4Cl | Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA | Cat#A661 | |
| Optimal cutting temperature (OCT) compound | Agar Scientific, Ltd., Stansted, Essex, UK | Cat# AGR1180 | |
| Oil Red-O Solution (ORO) | Sigma-Aldrich, Inc., St Louis, MO, USA | Cat#O1391 | |
| Oil Red-O Stain Kit | American Master Tech Scientific Inc., Lodi, CA, USA | Cat#KTORO-G | |
| Osmium tetroxide | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#201030 | Caustic |
| Phenylmethylsulfonyl fluoride (PMSF) | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#93482 | Caustic |
| Phosphate Buffered Saline Solution (PBS) | Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA | Cat#SH3025601 | |
| Ribonuclease A from bovine pancreas, type III-A | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#R5125 | |
| RNAEasy Fibrous Tissue MiniKit | Qiagen, Hilden, Germany | Cat#74704 | |
| Scintillation Fluid | Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA | Cat#SX18 | |
| Scintillation Counter | |||
| Scissors, forceps, sterile | |||
| Sorensen's phosphate buffer | Thomas Scientific, Inc., Swedesboro, NJ | CAS #: 10049-21-5 | |
| T-150 culture flask | VWR International LLC., Radnor, PA, USA | Cat#10062-864 | |
| TaqMan Gene Expression Master Mix | ThermoFisher Scientific Inc., Waltham, MA USA | Cat#4369016 | |
| Temperature-controlled orbital shaker | |||
| Tissue Homogenizer, BeadBug Microtube Homogenizer | Benchmark Scientific | Cat#D1030 | |
| Transferrin | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#T3309 | |
| Triglyceride Determination Kit | Sigma-Aldrich, Inc., St Louis, MO, USA | Cat#TR0100 | |
| Trypan blue stain, 0.4% | VWR International LLC., Radnor, PA, USA | Cat#10027-446 | |
| Type II collagenase | Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA | Cat#17101015 | |
| Whatman Reeve Angel filter paper, Grade 201, 150mm | Sigma-Aldrich, Inc., St Louis, MO, USA | Cat#WHA5201150 |
References
- Frantz, C., Stewart, K. M., Weaver, V. M. The extracellular matrix at a glance. Journal of Cell Science. 123, 4195-4200 (2010).
- Berrier, A. L., Yamada, K. M. Cell-matrix adhesion. Journal of Cell Physiology. 213 (3), 565-573 (2007).
- Trayhurn, P. Hypoxia and adipose tissue function and dysfunction in obesity. Physiology Reviews. 93 (1), 1-21 (2014).
- O’Rourke, R. W., Lumeng, C. N. Obesity heats up adipose tissue lymphocytes. Gastroenterology. 145 (2), 282-285 (2013).
- Engin, A. The Pathogenesis of Obesity-Associated Adipose Tissue Inflammation. Advances in Experimental Medicine and Biology. 960. 960, 221-245 (2017).
- Dankel, S. N., et al. COL6A3 expression in adipocytes associates with insulin resistance and depends on PPARγ and adipocyte size. Obesity (Silver Spring). 22 (8), 1807-1813 (2014).
- Divoux, A., et al. Fibrosis in human adipose tissue: composition, distribution, and link with lipid metabolism and fat mass loss. Diabetes. 59, 2817-2825 (2010).
- Lackey, D. E., et al. Contributions of adipose tissue architectural and tensile properties toward defining healthy and unhealthy obesity. American Journal of Physiology, Endocrinology, and Metabolism. 306 (3), E233-E246 (2014).
- Muir, L. A., et al. Adipose tissue fibrosis, hypertrophy, and hyperplasia: correlations with diabetes in human obesity. Obesity (Silver Spring). 24 (3), 597-605 (2016).
- Spencer, M., et al. Adipose tissue macrophages in insulin-resistant subjects are associated with collagen VI and fibrosis and demonstrate alternative activation. American Journal of Physiology, Endocrinology, and Metabolism. 299 (6), E1016-E1027 (2010).
- Baker, N. A., et al. Diabetes-specific regulation of adipocyte metabolism by the adipose tissue extracellular matrix. Journal of Clinical Endocrinology and Metabolism. 102 (3), 1-12 (2017).
- Pellegrinelli, V., et al. Human adipocyte function is impacted by mechanical cues. Journal of Patholology. 233 (2), 183-195 (2014).
- Flynn, L. E. The use of decellularized adipose tissue to provide an inductive microenvironment for the adipogenic differentiation of human adipose-derived stem cells. Biomaterials. 31 (17), 4715-4724 (2010).
- Perea-Gil, I., et al. In vitro comparative study of two decellularization protocols in search of an optimal myocardial scaffold for recellularization. American Journal Translational Research. 7 (3), 558-573 (2015).
- Porzionato, A., et al. Decellularized omentum as novel biologic scaffold for reconstructive surgery and regenerative medicine. European Journal of Histochemistry. 57 (1), e4 (2013).
- Tebyanian, H., et al. A Comparative Study of Rat Lung Decellularization by Chemical Detergents for Lung Tissue Engineering. Open Access Macedonian Journal of Medical Sciences. 5 (7), 859-865 (2017).
- Crapo, P. M., Gilbert, T. W., Badylak, S. F. An overview of tissue and whole organ decellularization processes. Biomaterials. 32 (12), 3233-3243 (2011).
- Wang, L., Johnson, J. A., Zhang, Q., Beahm, E. K. Combining decellularized human adipose tissue extracellular matrix and adipose-derived stem cells for adipose tissue engineering. Acta Biomaterials. 9 (11), 8921-8931 (2013).
- Booth, A. J., et al. Acellular normal and fibrotic human lung matrices as a culture system for in vitro investigation. American Journal of Respiratory and Critical Care Medicine. 186 (9), 866-876 (2012).
- Parker, M. W., et al. Fibrotic extracellular matrix activates a profibrotic positive feedback loop. Journal of Clinical Investigation. 124 (4), 1622-1635 (2014).
- Baker, N. A., Muir, L. A., Lumeng, C. N., O’Rourke, R. W. Differentiation and Metabolic Interrogation of Human Adipocytes. Methods in Molecular Biology. 1566, 61-76 (2017).
- O’Rourke, R. W., et al. Hexosamine biosynthesis is a possible mechanism underlying hypoxia’s effects on lipid metabolism in human adipocytes. PLoS One. 8 (8), e71165 (2013).
- Tchkonia, T., et al. Fat depot-specific characteristics are retained in strains derived from single human preadipocytes. Diabetes. 55 (9), 2571-2578 (2006).
- Tchoukalova, Y. D., et al. Sex- and depot-dependent differences in adipogenesis in normal-weight humans. Obesity (Silver Spring). 18 (10), 1875-1880 (2010).
Cite This Article
Flesher, C. G., Baker, N. A., Strieder-Barboza, C., Polsinelli, D., Webster, P. J., Varban, O. A., Lumeng, C. N., O’Rourke, R. W. A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk. J. Vis. Exp. (153), e60486, doi:10.3791/60486 (2019).