Hepatic zonation in a microfluidic liver model: application to drug metabolism

描述（由申请人提供）：新药的肝毒性在药物发现的临床前阶段通常是不可预测的，导致50%的药物无法进入I期临床试验，导致2-6年的时间损失和每种药物约1500万美元的资源损失。目前的体外药物筛选平台通常不考虑肝细胞代谢的分区，这对于准确模拟体内功能至关重要，因为生物转化在不同区域发生的程度不同，会影响肝毒性。尽管人们已经研究了许多代谢地带性诱导剂，最著名的是O2，但我们仍然缺乏对化学诱导剂的浓度和浓度梯度导致生理地带性的系统理解。我们的长期目标是创建一个生理相关的高通量系统，用于预测体外药物筛选。我们的目标是系统地了解化学试剂的浓度梯度如何诱导肝脏代谢分区，并将这种理解纳入快速药物毒性筛选的高通量微流控筛选平台。该研究的核心假设是，在微流控肝细胞培养中，可以使用带化诱导剂（O2、激素、胆汁酸和乙醇）的浓度梯度产生肝细胞代谢的动态带化，以指导形态学和代谢上不同区域的发展，从而阐明外源肝毒性。该建议的基本原理是，当前的微加工技术允许使用微流体来创建更多包含肝细胞异质性的生理肝脏模型。此外，这种微流控平台可以复用高通量分析，允许精确控制和有效的药物筛选。目的1：设计并制作肝细胞培养的多路微流控平台，系统地测定带化诱导剂浓度对细胞代谢的影响。目的2：建立微流控平台，评价单、多分区诱导剂浓度梯度对肝细胞动态代谢的影响。目的3：通过细胞消耗，模拟肝脏生理，在流动方向上形成肝细胞代谢区，并在长期培养中测试各种药物的肝毒性。这些目标的成功结果将提供能够复制带状肝细胞形态和代谢的新型体外微流体培养系统。我们希望阐明沿腺泡的区域诱导剂梯度与肝细胞动态代谢反应之间的联系，并将代谢反应与药物代谢和毒性联系起来。这些研究的更广泛的影响将是开发一种预测性的、高通量的体外药物筛选平台，以取代预测性差的动物模型。

DESCRIPTION (provided by applicant): Hepatotoxicity of new drugs, which is often unpredictable in the preclinical phase of drug discovery, causes failure of 50% of pharmaceuticals that make it to Phase I clinical trials, resulting in ~2-6yr loss in time and ~$15 million in resources per drug. Current in vitro drug screening platforms typically do not consider zonation in hepatocyte metabolism, which is critical for accurate modeling of in vivo function as biotransformation occurs to different extents in different zones, affecting hepatotoxicity. Although many metabolic zonal inducers have been studied, most notably O2, we still lack a systematic understanding of what concentration and concentration gradients of the chemical inducers lead to physiological zonation. Our long-term goal is to create a physiologically relevant, high-throughput system for predictive in vitro drug-screening. Our objective is to develop a systematic understanding of how concentration gradients in chemical agents induce hepatic metabolic zonation and incorporate this understanding into a high-throughput microfluidic screening platform for rapid drug toxicity screening. The central hypothesis is that dynamic zonation of hepatocyte metabolism can be generated using concentration gradients in zonation induction agents (O2, hormones, bile acids, and ethanol) to direct the development of morphologically and metabolically distinct zones within a microfluidic hepatocyte cell culture to elucidate xenobiotic hepatotoxicity. The rationale for this proposal is that current microfabrication technology allows the use of microfluidics to create more physiological liver models that incorporate hepatocyte heterogeneity. Furthermore, such microfluidic platforms can be multiplexed for high- throughput analysis, allowing precisely controlled and efficient drug screening. Aim 1: Design and fabricate a multiplexed microfluidic platform for hepatocyte culture to systematically determine the concentration effects of zonation inducers on cell metabolism. Aim 2: Create a microfluidic platform to assess the effects of concentration gradients of single and multiple zonation induction agents on hepatocyte dynamic metabolism. Aim 3: Create hepatocyte metabolic zonation in the flow-direction via cellular consumption, mimicking liver physiology, and test the hepatotoxicity of various drugs in long-term culture. The successful outcome of these aims will provide novel in vitro microfluidic culture systems capable of reproducing zonal hepatocyte morphology and metabolism. We expect to elucidate the connection between gradients of zonal inducers along the acinus and hepatocyte dynamic metabolic responses, and to correlate metabolic response to drug metabolism and toxicity. The broader impact of the studies will be the development of a predictive, high- throughput in vitro drug-screening platform to replace poorly predictive animal models.