2D细胞培养与体内培养之间的生理差异缺乏使得更多的器官型模型如器官样体（organoids）得以发展。针对多种组织，包括肝脏，已经开发出器官样体模型。目前的器官样体方案特点是依赖胞外基质（ECMs），在2D培养中建立模式，成本昂贵的生长因子以及缺乏细胞多样性、结构和组织。目前的肝脏器官样体模型总体上较简单，由肝细胞或胆管细胞组成，相较于原始组织相对生理相关性较低。我们开发了一种方法，不需要2D模式，独立于ECM，利用小分子来模拟胚胎肝脏发育，从而生产大量类似肝脏的器官样体。通过单细胞RNA测序和免疫荧光，我们展示了类肝细胞库、更高级别的细胞复杂性，显示出血管腔结构以及Kupffer细胞一种群体。器官样体表现出关键的肝脏功能，包括药物代谢、血清蛋白产生、尿素合成和凝血因子产生，同时保留有N-糖基化和功能性等翻译后修饰。这些器官样体可以移植并在小鼠体内长期维持，产生人类白蛋白。器官样体展现了复杂的细胞库，反映了肝脏的特点，并具有新生血管化和肝脏样功能。这些特点是许多应用的先决条件，从细胞疗法、组织工程、药物毒性评估、疾病模拟到基本发育生物学等。© 2023作者。

The lack of physiological parity between 2D cell culture and in vivo culture has led to the development of more organotypic models, such as organoids. Organoid models have been developed for a number of tissues, including the liver. Current organoid protocols are characterized by a reliance on extracellular matrices (ECMs), patterning in 2D culture, costly growth factors and a lack of cellular diversity, structure, and organization. Current hepatic organoid models are generally simplistic and composed of hepatocytes or cholangiocytes, rendering them less physiologically relevant compared to native tissue. We have developed an approach that does not require 2D patterning, is ECM independent, and employs small molecules to mimic embryonic liver development that produces large quantities of liver-like organoids. Using single-cell RNA sequencing and immunofluorescence, we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, and a population of resident macrophages: Kupffer cells. The organoids exhibit key liver functions, including drug metabolism, serum protein production, urea synthesis and coagulation factor production, with preserved post-translational modifications such as N-glycosylation and functionality. The organoids can be transplanted and maintained long term in mice producing human albumin. The organoids exhibit a complex cellular repertoire reflective of the organ and have de novo vascularization and liver-like function. These characteristics are a prerequisite for many applications from cellular therapy, tissue engineering, drug toxicity assessment, and disease modeling to basic developmental biology.© 2023. The Author(s).