A Microfluidic-platform Mini-Liver System for Human Liver Biology Studies

描述（由申请人提供）：肝脏是人体最大的器官。它参与了身体内环境稳定所需的无数代谢过程，也可以消除有害化学物质的毒性。肝脏接受来自肝门静脉和肝动脉的血液。肝门静脉携带来自肠、胰腺、脾脏和胆囊的血液，并使肝脏暴露于来自这些来源的营养物质和毒素。肝脏暴露于摄入物质的一个不幸的结果是，其中一些物质，如酒精，对肝脏有害。肝脏也是持续性病毒感染的靶点，例如B型肝炎病毒（HBV）的慢性感染。流行病学研究已经将某些类型的传染性病原体和酒精消费与肝脏疾病和肝癌联系起来;然而，由于缺乏实验上易于处理的人类肝脏模型系统，试图充分表征人类中的许多疾病受到阻碍。限制人类肝脏模型系统开发的障碍包括难以在传统细胞培养环境中维持分化的肝细胞以及难以获得大量正常人类肝细胞。肝细胞是肝脏中的主要细胞类型，负责肝脏的许多活动;正常的肝细胞功能通过与其他肝细胞如肝窦内皮细胞（LSEC）的交流和接触来维持。整个肝脏代谢过程的异质性引起了关于什么定义功能性肝脏单位的争论。然而，即使考虑到这种异质性，肝窦和周围细胞也可以被视为肝脏最基本的结构和功能单位。本建议旨在：1.）使用模拟肝窦功能单元的微流体平台开发新的“迷你肝”培养系统，以及2.）通过两项重点研究来测试该系统的实用性，这些研究分析了B肝炎病毒（HBV）复制和酒精暴露对特定肝细胞信号传导途径的影响。微流体平台将在模拟肝窦功能单元的微通道中具有肝细胞和LSEC的分层共培养物。我们将首先开发一个简单的肝细胞/胶原蛋白/LSEC夹层结构，然后将其扩展为包括胆汁清除通道，如有必要，还包括额外的血窦驻留细胞，如枯否细胞或星状细胞。流体的交换将通过微通道和外部流量控制单元进行调节。我们假设这个系统将产生一个真实的，简单的肝窦功能单位。目前的建议重点是建立这个系统，并研究HBV感染和酒精暴露对肝细胞生理的影响。我们的长期目标是开发一种新的人类肝脏模型系统，可用于研究正常人类肝脏生物学。该系统的未来应用可能包括了解其他感染因子对肝脏的影响、肝脏暴露于毒素的后果以及各种氧浓度对已知肝细胞代谢异质性的影响的研究。 公共卫生相关性：我们对各种人类肝脏疾病如何发展的理解，例如与持续性病毒感染或暴露于毒素或酒精相关的疾病，一直受到难以获得大量人类肝细胞并在传统细胞培养系统中生长时维持其肝脏特异性功能的阻碍。我们建议利用生物反应器和微流控平台技术的最新进展来创建一种新型的人类肝脏模型系统，其中少量的人类肝脏细胞将在环境控制的微通道中培养，以创建功能性肝脏单元和用于研究人类肝脏功能和疾病的新型模型。

DESCRIPTION (provided by applicant): The liver is the largest organ in the body. It is involved in a myriad of metabolic processes required for body homeostasis and can also detoxify harmful chemicals. The liver receives blood from the hepatic portal vein and the hepatic artery. The hepatic portal vein carries blood from the intestines, pancreas, spleen and gallbladder and exposes the liver to nutrients and toxins derived from these sources. An unfortunate result of the liver's exposure to ingested substances is that some of these substances, such as alcohol, are harmful to the liver. The liver is also a target for persistent viral infections, such as chronic infections with the hepatitis B virus (HBV). Epidemiological studies have linked some types of infectious agents and alcohol consumption with liver diseases and liver cancer; however, attempts to fully characterize many of these diseases in humans have been hampered by the absence of experimentally tractable human liver model systems. Impediments that have limited development of human liver model systems include the difficulty in maintaining differentiated hepatocytes in traditional cell-culture environments and the difficulty in obtaining large numbers of normal human hepatocytes. Hepatocytes are the predominant cell type in the liver and are responsible for many activities that are attributed to the liver; normal hepatocyte function is maintained by communication and contact with other liver cells such as liver sinusoidal endothelial cells (LSEC). The heterogeneity in metabolic processes throughout the liver has generated debate over what defines a functional liver unit. However, even considering this heterogeneity, the liver sinusoid and surrounding cells can be viewed as the most basic structural and functional unit of the liver. This proposal is directed towards: 1.) Developing a novel "mini-liver" culture system using a microfluidic platform that simulates a liver sinusoid functional unit, and 2.) Testing the utility of this system with two focused studies that analyze the effect of hepatitis B virus (HBV) replication and alcohol exposure on specific hepatocyte signaling pathways. The microfluidic platform will have a layered co-culture of hepatocytes and LSECs in a microchannel that mimics a liver sinusoid functional unit. We will initially develop a simple hepatocyte/collagen/LSEC sandwiched configuration and then expand this to include a channel for bile removal and, if necessary, additional sinusoid resident cells such as Kupffer or stellate cells. Exchange of fluid will be regulated through microchannels and external flow control units. We hypothesize that this system will generate an authentic, simple liver sinusoid functional unit. The current proposal focuses on establishing this system and studying the impact of HBV infections and alcohol exposure on hepatocyte physiology. Our long-term goal is to develop a novel human liver model system that can be used to study normal human liver biology. Future use of this system could include studies to understand the impact of other infectious agents on the liver, the consequence of liver exposure to toxins and the effects of various oxygen concentrations on the known heterogeneity of hepatocyte metabolism. PUBLIC HEALTH RELEVANCE: Our understanding of how various human liver diseases develop, such as those associated with persistent viral infections or exposure to toxins or alcohol, has been hampered by the difficulty of acquiring large numbers of human hepatocytes and maintaining their liver-specific functions when grown in traditional cell-culture systems. We propose to use recent advances in bioreactor and microfluidic platform technology to create a novel human liver model system in which small number of human liver cells will be cultured in environmentally controlled microchannels to create a functional liver unit and a novel model for studying human liver functions and diseases.