小鼠胎肝细胞透明质酸3D培养体系的建立

Stem-cell Derived Functional Mouse Hepatic Organoids in Hyaluronan Hydrogels

为了利用透明质酸建立小鼠胎肝细胞3D培养体系,分离获得胚胎12~14d胎肝细胞,利用KM培养基进行初步2D肝干/祖细胞的筛选培养,并利用透明质酸及KM培养基配制水凝胶建立3D细胞培养体系.胎肝细胞在2D体系中呈现克隆状生长.分离培养获得的肝干/祖细胞,克隆在透明质酸建立的3D培养体系保持增殖活性,并进一步获得肝细胞功能特性,表现为3D培养上清中白蛋白合成和尿素水平显著增加.定量PCR结果显示,随着3D培养时间的延长,其肝细胞干性标志如AFP、CK19、Ep CAM、Prox1等表达水平都大幅度降低且接近成年小鼠肝脏表达水平.本研究成功建立基于透明质酸的小鼠胎肝细胞的3D无血清培养体系,并可促进小鼠胎肝细胞的肝细胞功能进一步成熟.

To establish a 3 D organoid culture system of mouse fetal liver cell using hyaluronic acid, the fetal liver cells isolated from 12 to 14 days mouse embryos were preliminary screening of 2 D liver stem/progenitor cells by Kubota＇s Medium（KM） and 3 D organoid culture in hyaluronan hydrogel. Fetal liver cells grew as clones in 2 D cell culture system. Liver stem/progenitor cell colonies maintained proliferation activity. The albumin and urea levels in supernatant increased significantly, which indicated hepatic functional maturation of the cell colonies in3 D hyaluronic hydrogels. The results of Q-PCR showed that the expression levels of liver stem/progenitor markers,such as AFP, CK19, Ep CAM and Prox1, significantly reduced and were close to the expression level in adult mouse liver. In this study, stem-cell derived, functional mouse hepatic organoids in hyaluronan hydrogels was successfully established.