3D Spheroid Model of Adipose Pathophysiology

脂肪病理生理学 3D 球体模型

基本信息

批准号：
9177098
负责人：
Amol Vijay Janorkar
金额：
$ 35.53万
依托单位：
UNIVERSITY OF MISSISSIPPI MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-05 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9177098
关键词：
3-Dimensional Address Adherent Culture Adipocytes Adipose tissue Affect American Animal Testing Animals Anti-Obesity Agents Biocompatible Cardiovascular system Cell Culture Techniques Cell Shape Cell Size Cells Cellular biology Charge Chemistry Clinical Trials Comorbidity Cues Deposition Development Disease Dose Down-Regulation Drops Elastin Encapsulated Fat-Restricted Diet Fatty Acids Fatty acid glycerol esters Figs - dietary Functional disorder Future Genes Goals Growth Human Hydrogels In Vitro Investigation Lipolysis MMP14 gene Mediating Metabolic Pathway Metabolic stress Methods Modeling Modification Morphology Mus Nutrient Obese Mice Obesity Pathway interactions Patients Peroxisome Proliferator-Activated Receptors Pharmaceutical Preparations Phenotype Physiological Play Population Porosity Preclinical Drug Evaluation Production Regimen Role Scientist Shapes Signal Transduction Stress Surface TNF gene Testing Therapeutic Tissue Engineering Tissue Model Triglycerides Visceral adipocyte differentiation adiponectin clinically relevant copolymer diet and exercise genome-wide in vitro Model in vivo in vivo Model innovation invention lipid biosynthesis monolayer novel novel therapeutics polypeptide response scaffold subcutaneous surface coating

项目摘要

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.

项目概要该提案的目标是开发脂肪组织的体外模型，以实现卓越的肥厚性脂肪细胞的生长并促进代谢应激和信号机制的研究病理培养条件模仿现有的体外脂肪细胞培养条件。模型不是最佳的：2-D 单层培养物不代表 3-D 脂肪形态、3-D 细胞封装模型（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、非粘附涂层）由于在测试的培养基更换期间球体损失，现有的体外不支持长期培养。模型导致脂肪细胞功能受损，无法充分发挥其生长潜力，严重限制了细胞内甘油三酯（脂肪）沉积如何影响脂肪细胞功能的研究该提案使用新型组织直接解决了这一技术限制。工程方法制备生理相关的肥胖体外模型的具体目标是： (1) 通过使用一系列生物相容性类弹性蛋白多肽 (ELP) 和带电聚电解质 (PE) 的共聚物作为涂层这里带正电荷的 PE 促进球体形成，ELP 促进稳定。我们将系统地研究电荷含量和化学性质的影响。我们的首要假设是我们将获得优质的脂肪细胞。通过这种表面修饰方法实现成熟和功能，无需使用细胞大小限制性封装支架，并通过表面束缚实现长期培养 (2) 定义 3-D 球体培养中的脂肪生成机制并确定其功能。通过与 2-D 单层和 3-D 水凝胶培养物进行比较，我们寻求定义更好的模型。在形态学线索（通过 mmp14 的细胞形状）的背景下增强脂肪生成的机制 (3)确定多种代谢的影响压力（脂肪酸和 TNF-α）对脂肪细胞表型、活力和功能的影响 3-D 培养物的稳定性。我们的 ELP-PE 涂层可以显着延长培养时间，这项创新使我们能够暴露。在生理水平上优化开发 3-D 球体培养物以获取营养相关的脂肪酸。最后，通过比较代谢应激 3-D 的功能和全基因组反应球体与来自肥胖动物（小鼠）和人类供体的原代脂肪细胞的球体，我们将重述代谢应激的影响在肥胖进展中占主导地位，我们预计功能上会更好。 3-D 球体模型将成为临床相关的体外脂肪细胞模型，具有发明新脂肪细胞的潜力通过检查对体内类似成熟细胞群的药物和营养治疗来进行治疗。