3D Spheroid Model of Adipose Pathophysiology

项目总结 这项提议的目标是发展脂肪组织的体外模型，允许上层肥大 脂肪细胞的生长和促进代谢应激和信号机制的研究 模拟进行性肥胖的病理培养条件。存在脂肪细胞的体外培养 模型并不是最优的：2-D单层培养不代表3-D脂肪形态，3-D细胞 囊化模型(例如，水凝胶)由于压缩而限制了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、非粘附性涂层)所做的 不支持长期培养，因为在媒体更换过程中会出现球体丢失。因此，存在于体外的 模型会导致功能受损的脂肪细胞不能充分发挥其生长潜力，这是严重的 限制各种细胞内甘油三酯(脂肪)沉积如何影响脂肪细胞功能的研究 肥胖的发展。这项提议使用一种新的纸巾直接解决了这一技术限制 工程学方法。制备与生理相关的肥胖症体外模型的具体目的是： (1)通过使用一系列的 生物相容弹性蛋白样多肽(ELP)与带电聚电解质(PE)的共聚物涂层 底物。在这里，带正电的PES促进了球体的形成，ELP促进了稳定 球体的表面系绳。我们将系统地研究电荷含量和化学成分的影响。 关于三维椭球体的组织。我们最重要的假设是，我们将获得更好的脂肪细胞 通过这种表面修饰方法实现3D培养而不使用 细胞大小限制性包裹支架，通过表面拴系实现长期培养 椭球体。(2)明确三维球体培养中的成脂机制，并从功能上确定 通过与2-D单层和3-D水凝胶培养进行比较，获得了更好的模型。我们试图定义 在形态线索的背景下促进脂肪生成的机制(通过MMP14的细胞形状 途径)调节PPAR-，脂肪形成的关键效应因子。(3)确定多重代谢的影响 应激(脂肪酸和肿瘤坏死因子-)对脂肪细胞的表型、活性和功能的影响。3D培养的稳定性 在我们的ELP-PE涂层的顶部，允许更长的培养周期。这项创新使我们能够揭露 在生理水平上对营养相关的脂肪酸进行最佳的3-D球状培养。 最后，通过比较新陈代谢应激3-D的功能和全基因组反应 肥胖症动物(小鼠)和人类供体的原代脂肪细胞的球体，我们将扼要介绍 代谢应激在进展性肥胖中起主导作用。我们期待着功能上的卓越 3-D球体模型是一种临床相关的体外脂肪细胞模型，具有发明新的潜力 通过检查药物和营养治疗对体内类成熟细胞群体的治疗作用。

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.