3D Spheroid Model of Adipose Pathophysiology

项目摘要 该建议的目标是开发脂肪组织的体外模型，其允许上级肥大性脂肪组织。 脂肪细胞的生长，并促进研究代谢应激和信号传导机制， 病理性培养条件模拟进行性肥胖。现有的体外脂肪细胞培养 模型不是最佳的：2-D单层培养不能代表3-D脂肪形态，3-D细胞 封装模型（例如，水凝胶）限制分化脂肪细胞的体积， 应力和有限的孔隙率，以及3-D“无支架”模型（例如，悬滴，非粘附涂层） 不支持长期培养，因为在培养基更换过程中球体丢失。因此，现有的体外 模型导致功能受损的脂肪细胞不能达到其全部生长潜力， 限制了对全范围的细胞内甘油三酯（脂肪）沉积如何影响脂肪细胞功能的研究， 肥胖症的发展。该提议使用新型组织直接解决了该技术限制 工程方法。制备肥胖症的生理学相关体外模型的具体目的是： (1)通过使用一个阵列，创建脂肪细胞培养的稳定的、表面束缚的3-D球体模型。 作为涂层的生物相容性弹性蛋白样多肽（ELP）和带电聚电解质（PE）的共聚物 印刷受体.在这里，带正电荷的PE促进球状体形成，而ELP促进稳定的 球体的表面束缚。我们将系统地研究电荷含量和化学性质的影响 三维球体组织的研究我们的首要假设是，我们将实现上级脂肪细胞 通过这种表面改性方法实现3-D培养，而不使用 细胞大小限制性包封支架，并通过表面栓系实现长期培养， 球状体(2)明确三维球体培养中脂肪形成的机制， 通过与2-D单层和3-D水凝胶培养物进行比较，获得了上级模型。我们试图定义 在形态学线索（通过MMP 14的细胞形状）的背景下增强脂肪形成的机制 途径）调节脂肪形成的关键效应物PPAR-beta。(3)确定多种代谢的影响 应激（脂肪酸和TNF-α）对脂肪细胞表型、活力和功能的影响。三维培养的稳定性 在我们的ELP-PE涂层上添加一层可显著延长培养时间。这项创新使我们能够揭露 最佳发展的三维球体文化，营养相关的脂肪酸在生理水平。 最后，通过比较代谢应激3D的功能和全基因组反应， 与来自肥胖动物（小鼠）和人类供体的原代脂肪细胞的球状体相比，我们将概括 代谢压力的影响在进行性肥胖中占主导地位。我们希望功能上的上级 3-D球体模型是一种临床相关的体外脂肪细胞模型，有可能发明新的 通过检查对体内样成熟细胞群的药物和营养治疗来确定治疗方法。

PROJECT SUMMARY The goals of this proposal are to develop in vitro models of adipose tissue that allow a superior hypertrophic growth of adipocytes and facilitate investigation of metabolic stresses and signaling mechanisms during pathological culturing conditions mimicking those of progressing obesity. Existing in vitro adipocyte culture models are not optimal: 2-D monolayer culture does not represent the 3-D adipose morphology, 3-D cell encapsulation models (e.g., hydrogels) restrict the volume of differentiating adipocytes due to compressive stress and limited porosity, and 3-D “scaffold-free” models (e.g., hanging drop, non-adherent coatings) do not support long term culture due to spheroid loss during media changes. Consequently, the existing in vitro models result in functionally impaired adipocytes that do not reach their full growth potential, seriously limiting the study of how a full range of intracellular triglyceride (fat) deposition affects adipocyte function in the development of obesity. This proposal directly addresses this technical limitation using a novel tissue engineering approach. Specific Aims to prepare physiologically relevant in vitro models of adiposity are to: (1) Create the stable, surface-tethered 3-D spheroid model of adipocyte culture by using an array of copolymers of biocompatible elastin-like polypeptide (ELP) and charged polyelectrolytes (PE) as coating substrates. Here the positively-charged PEs encourage spheroid formation and ELP encourages stable surface-tethering of spheroids. We will systematically investigate the effect of charge content and chemistry on 3-D spheroid organization. Our overarching hypothesis is that we will achieve superior adipocyte maturation and functionality by this surface modification method that achieves 3-D culture without using a cell-size restrictive encapsulation scaffold and achieves long term culture through surface-tethering of spheroids. (2) Define the mechanism of adipogenesis in 3-D spheroid culture and determine the functionally superior model by comparing against 2-D monolayer and 3-D hydrogel cultures. We seek to define the mechanism of enhanced adipogenesis in the context of morphological cues (cell shape through the mmp14 pathway) regulating PPAR-, a key effector of adipogenesis. (3) Determine the effects of multiple metabolic stresses (fatty acids and TNF-) on adipocyte phenotype, viability, and function. The stability of 3-D culture atop our ELP-PE coatings allows a substantially longer culture period. This innovation allows us to expose the optimally developing 3-D spheroid cultures to nutritionally relevant fatty acids at physiological levels. Finally, by comparing the functional and genome-wide responses of the metabolically stressed 3-D spheroids to those of primary adipocytes from obese animal (mice) and human donors, we will recapitulate the effects of metabolic stresses predominant in progressing obesity. We expect the functionally superior 3-D spheroid model to be a clinically relevant in vitro adipocyte model with the potential to invent novel therapeutics by examining drug and nutrient treatments on an in vivo-like mature cell population.