3D bioprinting of human liver models with biliary branching morphogenesis for hepatotoxicity, regeneration and NASH

The study of liver disease and drug discovery has been constrained by a lack of liver tissue with intrahepatic bile ducts (IHBD) network. Here we created functional bioprinting human liver with IHBD through precise temporal-spatial regulation via biochemical and mechanical cues. To investigate the regulatory mechanisms of the cell microenvironment of the human liver on the biliary branching morphogenesis, biomimetic liver tissue formation, and physiological function, transcriptome analysis of model A and model B was performed and compared with 2DD and PHH cells. Overall design: For 2D differentiated HepaRG cell (2DD) culture, HepaRG cells were maintained for 2 weeks in the growth medium and then shifted to the growth medium supplemented with 2% DMSO for the next 2 weeks. To obtain bioprinting liver models A and B, uniformly sized hepatobiliary aggregates (HBAs) were used as building blocks. HBAs were readily produced and non-destructively harvested after 7-day culture within the 3D printing alginate/gelatin scaffolds with HepaRG growth medium. The alginate, gelatin solution, and the HBAs were gently and uniformly mixed. Lego-livers were cultured with low concentration medium A or high concentration medium B for 14 days to obtain model A or B, respectively. Primary human hepatocyte (PHH) was bought from the company and cultured for 3 days as instructed. The samples for RNA-seq were collected in TriZol.

肝脏疾病研究与药物开发长期受限于缺乏带有肝内胆管（intrahepatic bile ducts, IHBD）网络的肝脏组织。本研究通过生化与机械信号的精准时空调控，构建了带有IHBD网络的功能性生物打印人肝脏。为探究人肝脏细胞微环境对胆管分支形态发生、仿生肝组织形成及生理功能的调控机制，本研究对模型A与模型B开展了转录组分析，并与二维分化HepaRG细胞（2DD）及原代人肝细胞（primary human hepatocyte, PHH）进行对照。整体实验设计如下：针对二维分化HepaRG细胞（2DD）的培养，先将HepaRG细胞在生长培养基中培养2周，随后更换为添加2%二甲基亚砜（dimethyl sulfoxide, DMSO）的生长培养基，继续培养2周。为获取生物打印肝脏模型A与模型B，本研究采用尺寸均一的肝胆聚集体（hepatobiliary aggregates, HBAs）作为打印结构单元。将HepaRG生长培养基添加至3D打印海藻酸钠/明胶支架中，经7天培养后可无损收获易于制备的HBAs。将海藻酸钠、明胶溶液与HBAs轻柔混匀。将所得乐高式肝脏分别使用低浓度培养基A与高浓度培养基B培养14天，即可分别获得模型A与模型B。原代人肝细胞（PHH）购自商业公司，按照产品说明书培养3天。RNA测序（RNA-seq）样本采用TriZol试剂进行收集。