Cellular Composite Device for Combination Therapy of Acute Liver Failure

描述(申请人提供)：肝功能衰竭是第七大死因，在美国每年造成5万人死亡。原位肝移植是治疗急性肝功能衰竭(ALF)的唯一有效方法，但由于器官供体短缺、相关费用高以及需要终生免疫抑制，其应用受到限制。受肝功能衰竭影响的人口目前和预期的增长正在不断上升，需要一种拯救生命的替代移植来支持患者。生物人工肝装置是支持ALF患者作为移植桥梁的合理方法。五种基于细胞的设备已经在人类和猪身上进行了测试，似乎是安全的，但没有一种设备显示出对生存的好处。迄今为止，设备的故障表明了一种无效的行动机制。终末期肝功能衰竭导致全身功能障碍，与炎症反应同时发生。我们假设，一种提供肝细胞支持、细胞保护、抗炎和营养支持的联合治疗方法将覆盖能够稳定患者的广泛的病理过程。在验证性治疗试验中，我们已经证明了人类间充质干细胞(MSCs)自然地分泌具有免疫调节特性的生物活性分子。我们已经开发了基于MSC的设备，这种设备在体外运行并连接到受试者的循环以提供长期支持，并且已经表明，当连接到其中一个设备10小时时，接受ALF的大鼠的存活率从不到15%增加到70%以上，增加了5倍。这个第一阶段项目的总体目标是开发一种用于治疗ALF的复合细胞生物反应器，它将肝细胞和MSC的代谢和分泌整合在一个单元中，并评估这种两细胞设备的额外好处，而不是MSC设备的有效性。该项目的具体目标是：(1)优化MSCs和肝细胞的体外共培养，并模拟肝衰竭血清对共培养功能的影响；(2)将MSCs和肝细胞整合到平板设备中，并在啮齿动物模型中启动生物反应器治疗的治疗测试。在这个项目成功完成后，交付的产品将是一个原型细胞透析盒，可以很容易地扩大规模，并在大型动物身上进行测试。 公共卫生相关性：我们建议开发一种体外生物人工肝设备，为肝功能衰竭患者提供无与伦比的支持。该装置将包含肝细胞和间充质干细胞(MSCs)。MSCs的加入是我们技术中独一无二的，旨在增强暴露在血浆中的肝细胞的代谢功能，并通过活跃的MSC分泌、抗炎和营养分子恢复肝功能衰竭患者功能失调的免疫系统的调节。这种双管齐下的方法将这款设备与目前的原型区分开来。我们的目标是利用共培养的代谢工程进行体外共培养的优化，并在两个肝功能衰竭的大鼠模型上进行微制共培养装置的体内测试。这些研究将确定联合治疗方法是否适用于广泛的肝病病因，并将推动在肝功能衰竭的大型动物模型中进行测试，最终在人类患者中进行测试。

DESCRIPTION (provided by applicant): Liver failure is the 7th leading cause of death and is responsible for 50,000 deaths per year in the United States. Orthotopic liver transplantation is the only proven effective treatment of acute liver failure (ALF), but its use is limited due to organ donor shortage, associated high costs, and the requirement of lifelong immunosuppression. The present and expected growth of the population that is affected by liver failure is ever rising and a life-saving alternative to transplantation is needed to support patients. Bioartificial liver devices are a rational approach to support ALF patients as a bridge to transplantation. Five cell- based devices have been tested in humans and pigs and appear safe, but none have shown a survival benefit. The failure of devices to-date suggests an ineffective mechanism of action. End-stage liver failure leads to systemic dysfunction that is occurring simultaneously with an inflammatory response. We hypothesize that a combination approach to therapy that provides hepatocellular support along with cytoprotection, anti-inflammatory, and trophic support will cover the broad spectrum of pathological processes that can stabilize a patient. In proof-of-principle therapeutic trials, we have demonstrated that human mesenchymal stem cell (MSCs) naturally secrete bioactive molecules that have immunomodulatory properties. We have developed MSC-based devices that are operated outside the body and connect to a subject's circulation to provide long-term support, and have shown that when connected to one of these devices for 10 hours, rats undergoing ALF have a 5-fold increase in survival from less than 15% to over 70%. The overall goal of this Phase I project is to develop a composite cellular bioreactor for the treatment of ALF that integrates both hepatocyte and MSC metabolism and secretion in a single unit, and evaluate the added benefit of this two-cell device over and above the effectiveness of the MSC devices. The project specific aims are: (1) To optimize the in vitro coculture of MSCs and hepatocytes and simulate the effect of liver failure serum on the function of the coculture; and (2) To incorporate MSCs and hepatocytes into flat-plate devices and initiate therapeutic testing of bioreactor treatments in rodent models. Upon successful completion of this project, the deliverable will be a prototype cell-laden dialysis cartridge that can be readily scaled up and tested in large animals. PUBLIC HEALTH RELEVANCE: We propose to develop an extracorporeal bioartificial liver device that offers unparalleled support to patients undergoing liver failure. The device will contain hepatocytes and mesenchymal stem cells (MSCs). The addition of MSCs is unique to our technology and is designed to enhance the metabolic functions of hepatocytes exposed to plasma and restore the regulation of the dysfunctional immune system in patients undergoing liver failure by active MSC secretion anti-inflammatory and trophic molecules. This two-pronged approach distinguishes this device from current prototypes. Our objectives are to perform in vitro optimization of the coculture using metabolic engineering of the coculture and in vivo testing of a microfabricated coculture device in two rat models of liver failure. These studies will determine if the combination therapeutic approach can be indicated for a broad range of liver disease etiologies and will motivate testing in large animal models of liver failure, and ultimately in human patients.