3D Culture of Adipose Tissue for Screening Obesity-related Drugs

描述(申请人提供)：我们开发了一种新颖的3D生物反应器技术，允许脂肪细胞的长期培养，这是传统2D细胞培养方法无法实现的。在这项研究中，我们将利用我们的技术来快速有效地筛选药物对人类脂肪组织功能的影响。我们将检测肥胖和非肥胖患者的脂肪细胞功能。我们将研究的参数之一是细胞因子/脂肪因子的表达。有了生物反应器技术，我们能够快速、轻松地分析细胞因子在介质中的日常表达。新药可能针对脂肪细胞的细胞因子表达。研究表明，肥胖涉及的细胞因子行为包括但不限于：单核细胞趋化蛋白-1(MCP-1)、肿瘤坏死因子-1(肿瘤坏死因子-1)、IL-8(巨噬细胞分泌的促炎细胞因子)。同样令人感兴趣的是抗炎细胞因子的表达，如IL-10(白细胞介素10)。虽然这些介体已经在2D体外培养中使用人类和小鼠的脂肪组织进行了检测，但改进的实验系统是必要的，以允许开发高通量的药物发现分析方法。因此，这项建议的具体目标是：1)从40-60岁的男性和女性患者(非肥胖患者和肥胖患者)中分离和鉴定人类脂肪干细胞；2)开发一种新型的、多隔室的中空纤维3D灌注生物反应器技术，用于在3D生物反应器中培养ASC；3)利用3D灌注生物反应器系统作为工具，研究药物疗法对脂肪功能的影响。总而言之，模拟天然脂肪组织的3D培养系统中的细胞-细胞接触代表着对目前旨在开发高通量药物发现测试的培养皿技术的改进。 与公共健康相关：全国范围内的肥胖率继续以惊人的速度攀升。控制肥胖流行的不同方法包括扩大健康生活教育项目，减少不健康、高脂肪食品的营销，以及提供低成本的锻炼计划。作为科学家，我们可以通过增加对脂肪细胞功能机制的理解和高通量筛选新药来为减少肥胖做出贡献。因此，这项建议描述了一种创新的工程方法，利用一种新型的3D中空纤维生物反应器，在本地3D架构中研究脂肪组织，该反应器允许脂肪细胞的长期3D培养和药物的快速筛选。

DESCRIPTION (provided by applicant): We have developed a novel, 3D bioreactor technology that permits the long-term culture of adipocytes, which is not possible using traditional 2D cell culture methods. In this study, we will utilize our technology to rapidly and effectively screen the effects of drugs on human adipose tissue function. We will examine the function of adipocytes in both obese and non-obese patients. One of the parameters we will study is cytokine/adipokine expression. With the bioreactor technology, we are able to rapidly and easily analyze daily expression of cytokines in the media. New drugs may target cytokine expression of adipocytes. It has been shown that involved cytokine behavior in obesity includes the increased expression of, but not limited to: MCP-1 (monocyte chemotactic protein-1, which can recruit macrophages to adipose tissue), TNF-1 (tumor necrosis factor-1, a pro-inflammatory mediator secreted by macrophages), and IL-8 (interleukin-8, a pro-inflammatory cytokine secreted by macrophages). Also of interest is the expression of anti-inflammatory cytokines, such as IL-10 (interleukin-10). While these mediators have been examined using human and murine adipose tissue in 2D in vitro culture, improved experimental systems are necessary to allow the development of high throughput assays for drug discovery. Therefore, the specific aims of this proposal are to: 1) Isolate and characterize human adipose-derived stem cells from both male and female patients, age 40-60 years, (non-obese, vs. obese patients); 2) Develop a novel, multi-compartment, hollow fiber 3D perfusion bioreactor technology for ASC culture in 3D bioreactor; 3) Utilize the 3D perfusion bioreactor system as a tool to study the effects of drug therapies on adipose function. In summary, cell-cell contact in a 3D culture system mimicking natural adipose tissue represents an improvement over current petri-dish technologies aimed at developing high throughput assays for drug discovery. PUBLIC HEALTH RELEVANCE: Nation-wide obesity rates continue to climb at an alarming rate. Different approaches to controlling the obesity epidemic include expanding education programs about healthy living, reducing the marketing of unhealthy, high-fat foods, and providing low-cost exercise programs. As scientists, we can contribute to a reduction in obesity by increasing our understanding of the mechanisms of adipose cell function and the high throughput screening of new drugs. As such, this proposal describes an innovative, engineering approach to studying adipose tissue in a native, 3D architecture, utilizing a novel 3D hollow fiber bioreactor which permits both the long-term 3D culture of adipocytes and the rapid screening of drugs.