人类肝脏类器官（HLO）是代表人类肝脏生理学的可靠工具。然而，它们的使用受到限制，尤其是在基础科学领域。造成这种情况的原因之一是处理 HLO 的系统方法不足，包括培养系统、功能评估和基因转导。在这里，我们通过慢病毒转导生成并表征了稳定且同时过表达 R-spondin1、肝细胞生长因子、成纤维细胞生长因子 (FGF) 7 和 FGF10 的小鼠 L 细胞。细胞的条件培养基有助于 HLO 生长，作为市售重组蛋白的替代品，从而显着降低其培养成本。通过切换培养基来控制细胞的增殖和成熟阶段，以促进肝细胞功能的评估，包括胰岛素反应性和细胞内脂质积累。基因表达分析表明，HLO 高表达参与脂质代谢的基因。重要的是，HLO 分泌生理成熟的极低密度脂蛋白，这在小鼠和已建立的细胞系中很少观察到。在病毒感染期间通过瞬时二维培养实现了向 HLO 的有效基因转导。这项研究为在分子生物学、药理学、毒理学和再生医学等各个研究领域中利用 HLO 提供了宝贵的平台。本文受版权保护。保留所有权利。本文受版权保护。版权所有。

Human liver organoids (HLOs) are reliable tools to represent physiological human liver biology. However, their use is limited especially in basic sciences. One of the reasons for this would be the insufficient systematic methodology to handle HLOs, including culture system, functional assessment, and gene transduction. Here, we generated and characterized mouse L cells stably and simultaneously overexpressing R-spondin1, hepatocyte growth factor, fibroblast growth factor (FGF) 7, and FGF10 via lentiviral transduction. The conditioned medium of the cells contributed to HLO growth as a replacement of commercially available recombinant proteins, which leads to significant reduction of their culture cost. Proliferative and maturation phases of the cells were controlled by switching the medium to facilitate the evaluation of hepatocyte function, including insulin responsiveness and intracellular lipid accumulation. Gene expression analysis revealed that HLOs highly expressed genes involved in lipid metabolism. Importantly, HLOs secreted physiologically matured very low-density lipoprotein, which is rarely observed in mice and in established cell lines. Efficient gene transduction into HLOs was achieved via a transient 2-dimensional culture during viral infection. This study provides an invaluable platform for utilizing HLOs in various research fields, such as molecular biology, pharmacology, toxicology, and regenerative medicine. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.