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

描述（由申请人提供）：我们开发了一种新型的3D生物反应器技术，该技术允许脂肪细胞的长期培养，这是使用传统的2D细胞培养方法不可能实现的。在这项研究中，我们将利用我们的技术，快速有效地筛选药物对人体脂肪组织功能的影响。我们将研究肥胖和非肥胖患者脂肪细胞的功能。我们将研究的参数之一是细胞因子/脂肪因子表达。利用生物反应器技术，我们能够快速、轻松地分析培养基中细胞因子的日常表达。新药可能靶向脂肪细胞的细胞因子表达。已经显示，肥胖症中涉及的细胞因子行为包括但不限于以下的表达增加：MCP-1（单核细胞趋化蛋白-1，其可以将巨噬细胞募集到脂肪组织）、TNF-1（肿瘤坏死因子-1，由巨噬细胞分泌的促炎介质）和IL-8（白细胞介素-8，由巨噬细胞分泌的促炎细胞因子）。还感兴趣的是抗炎细胞因子如IL-10（白细胞介素-10）的表达。虽然这些介质已检查使用人类和小鼠脂肪组织在2D体外培养，改进的实验系统是必要的，以允许开发高通量的药物发现测定。1）从年龄40-60岁的男性和女性患者中分离和表征人脂肪源性干细胞，（非肥胖患者vs.肥胖患者）; 2）开发用于在3D生物反应器中培养ASC的新型多隔室中空纤维3D灌注生物反应器技术; 3）利用三维灌注生物反应器系统作为研究药物治疗对脂肪功能的影响的工具。总之，模拟天然脂肪组织的3D培养系统中的细胞-细胞接触代表了对旨在开发用于药物发现的高通量测定的当前培养皿技术的改进。 公共卫生相关性：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的教育计划，减少不健康的高脂肪食品的营销，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减少肥胖。因此，该提案描述了一种创新的工程方法来研究天然3D结构中的脂肪组织，利用新型3D中空纤维生物反应器，该生物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.