Multicellular Organotypic Mouse Model of Alcoholic Liver Disease

酒精性肝病的多细胞器官型小鼠模型

• 批准号: 10667672
• 负责人: Salman R Khetani
• 金额: $ 24.29万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667672
• 关键词: 3-Dimensional; AODR mortality; Affect; Alcoholic Liver Diseases; Alcohols; Benchmarking; Biological Models; Cell Communication; Cell Separation; Cell physiology; Cells; Cholesterol; Closure by clamp; Coculture Techniques; Collagen; Complex; Coupled; Cytochrome P450; Data; Development; Diet; Dimensions; Disease; Embryo; Encapsulated; Endothelial Cells; Engineering; Ethanol; Exclusion; Extracellular Matrix; Fibroblasts; Fibrosis; Fructose; Future; Gel; Gene Expression; Grant; Hepatic; Hepatic Stellate Cell; Hepatocyte; Hepatotoxicity; Human; In Vitro; Individual; Inflammatory; Inflammatory Response; Intestines; Kupffer Cells; Liver; Liver diseases; Malignant Neoplasms; Mediating; Microfluidics; Modeling; Molecular; Morbidity - disease rate; Mus; Organ; Outcome; Pathogenesis; Pathology; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Physiological; Progressive Disease; Property; Recombinants; Research; Research Personnel; Resources; Role; Sampling; Steatohepatitis; System; Testing; Tissues; Trans Fats; Vertebral column; alcohol effect; alcohol exposure; cell assembly; cell type; chronic alcohol ingestion; comparison control; disease phenotype; fibrogenesis; hepatocellular injury; in vitro Model; in vivo; in vivo Model; liver function; mortality; mouse model; nonalcoholic steatohepatitis; novel; self assembly; single-cell RNA sequencing; transcriptome sequencing; western diet

ABSTRACT Alcohol-associated liver disease (ALD) is the major cause of alcohol-related mortality and encompasses steatosis, steatohepatitis with or without progressive fibrosis, hepatocellular injury, and loss of liver function. Despite considerable research, the specific mechanisms underlying ALD development and progression have not been fully elucidated, partly due to the lack of in vitro and in vivo model systems that recapitulate human ALD. While live mice are used to model features of human ALD, current models are labor-intensive, induce significant mortality (unlike humans), include diets that induce confounding non-alcoholic steatohepatitis, and/or cannot reproduce all features of human ALD. In contrast, we developed a simple-to-administer 16-week western diet alcohol (WDA) model that recapitulates the inflammatory, fibrotic, and gene expression aspects of human alcohol-associated steatohepatitis (ASH). However, it is difficult to investigate the direct effects of alcohol on multiple liver cell types and elucidate the cell-cell interactions important for ALD pathogenesis in vivo. In contrast, while primary liver cells can be isolated with high purity from livers to build in vitro models, they rapidly lose phenotypic functions in 2D monocultures. To mitigate this limitation, we utilized high-throughput droplet microfluidics to fabricate highly monodisperse extracellular matrix (ECM)-based engineered 3D liver microtissues containing hepatocytes and liver non-parenchymal cells (NPCs) that functionally outperform conventional self- assembled cell spheroids and cells embedded within bulk gels. Here, we will leverage the above advances to test the novel hypothesis that microtissues containing multiple primary mouse liver cells can recapitulate the critical features of ASH as in the WDA mouse. In aim 1, we will fabricate and optimize long-term (4+ weeks) 3D liver microtissues containing primary mouse hepatocytes, liver endothelial cells, hepatic stellate cells, and Kupffer cells; the role of ECM and tissue size will be investigated towards inducing high (physiologic) and stable cell functions. We will further assess the effects of in vitro ethanol exposure on each cell type within microtissues. In aim 2, we will investigate cell-cell interactions in liver microtissues derived from cells isolated from the WDA mice. Microtissues will be cultured with or without ethanol and cellular functions as well as single cell RNA sequencing data will be compared to control microtissues and existing RNA sequencing data from freshly isolated cells from WDA mice. We will further examine the role of each of the alcohol-specific NPC type in maintaining hepatic function and regulating inflammatory responses and fibrogenesis in microtissues. Our efforts will yield a first-of-its-kind in vitro organotypic mouse liver model with long-term functions, which will be utilized to elucidate the direct effects of alcohol on multiple liver cell types, the extent to which the ALD disease phenotype can be maintained in culture, and the effects of liver NPCs on hepatocellular functions in ALD. Our platform could serve as a resource to other investigators, and could be coupled with other tissue types (e.g., intestine) within microfluidic systems to elucidate the role of organ-organ crosstalk in progressive ALD.

摘要 酒精相关性肝病（ALD）是酒精相关死亡的主要原因， 脂肪变性、伴有或不伴有进行性纤维化的脂肪性肝炎、肝细胞损伤和肝功能丧失。 尽管有大量的研究，ALD发展和进展的具体机制还没有 已经完全阐明，部分原因是缺乏体外和体内模型系统，概括人类ALD。 虽然使用活小鼠来模拟人ALD的特征，但目前的模型是劳动密集型的，诱导显著的细胞毒性。 死亡率（与人类不同），包括诱导混杂的非酒精性脂肪性肝炎的饮食，和/或不能 复制人类ALD的所有特征。相比之下，我们开发了一个简单的管理16周的西方饮食 酒精（WDA）模型，该模型概括了人类的炎症、纤维化和基因表达方面， 酒精相关性脂肪性肝炎（ASH）。然而，很难调查酒精对人体的直接影响。 多种肝细胞类型和阐明细胞间相互作用的重要ALD发病机制在体内。与此相反， 虽然原代肝细胞可以从肝脏中高纯度地分离出来，以建立体外模型，但它们很快就会丧失， 2D单一栽培中的表型功能。为了减轻这种限制，我们利用高通量液滴 微流体技术用于制造基于高度单分散细胞外基质（ECM）的3D工程化肝脏微组织 含有肝细胞和肝脏非实质细胞（NPC），其功能优于常规的自体肝细胞， 组装的细胞球状体和包埋在大块凝胶内的细胞。在这里，我们将利用上述进展， 测试新的假设，即含有多个原代小鼠肝细胞的微组织可以重现 ASH的关键特征，如在WDA小鼠中。在目标1中，我们将制作和优化长期（4周以上）3D 肝微组织，包含原代小鼠肝细胞、肝内皮细胞、肝星状细胞，和 库普弗细胞;将研究ECM和组织大小对诱导高（生理）和稳定的 细胞功能。我们将进一步评估体外乙醇暴露对微组织内每种细胞类型的影响。 在目标2中，我们将研究从WDA分离的细胞衍生的肝微组织中的细胞-细胞相互作用 小鼠微组织将在有或没有乙醇和细胞功能以及单细胞RNA的情况下培养 测序数据将与对照微组织和来自新鲜组织的现有RNA测序数据进行比较。 从WDA小鼠分离的细胞。我们将进一步研究每种酒精特异性NPC类型在以下方面的作用： 维持肝功能并调节炎症反应和微组织中的纤维化。我们的努力 将产生第一个具有长期功能的体外器官型小鼠肝脏模型， 为了阐明酒精对多种肝细胞类型的直接影响，ALD疾病 表型可以在培养中维持，以及肝脏NPC对ALD中肝细胞功能的影响。我们 平台可以作为其他研究者的资源，并且可以与其他组织类型（例如， 肠），以阐明器官-器官串扰在进行性ALD中的作用。