Deng Shuai, Zhu Yanlun, Zhao Xiaoyu, Chen Jiansu, Tuan Rocky S, Chan Hon Fai
Institute for Tissue Engineering and Regenerative Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, People's Republic of China.
Key Laboratory for Regenerative Medicine of the Ministry of Education of China, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, People's Republic of China.
Biofabrication. 2021 Oct 18;14(1). doi: 10.1088/1758-5090/ac2b89.
Three-dimensional (3D) culture techniques, such as spheroid and organoid cultures, have gained increasing interest in biomedical research. However, the understanding and control of extracellular matrix (ECM) effect in spheroid and organoid culture has been limited. Here, we report a biofabrication approach to efficiently form uniform-sized 3D hepatocyte spheroids and encapsulate them in a hybrid hydrogel composed of alginate and various ECM molecules. Cells were seeded in a microwell platform to form spheroid before being encapsulated directly in a hybrid hydrogel containing various ECM molecules, including collagen type I (COL1), collagen type IV (COL4), fibronectin (FN), and laminin (LM). A systematic analysis of the effect of ECM molecules on the primary mouse hepatocyte phenotype was then performed. Our results showed that hydrogel encapsulation of hepatocyte spheroid promoted hepatic marker expression and secretory functions. In addition, different ECM molecules elicited distinct effects on hepatic functions in 3D encapsulated hepatocyte spheroids, but not in 2D hepatocyte and 3D non-encapsulated spheroids. When encapsulated in hybrid hydrogel containing LM alone or COL1 alone, hepatocyte spheroids exhibited improved hepatic functions overall. Analysis of gene and protein expression showed an upregulation of integrin1 and integrin6 when LM was introduced in the hybrid hydrogel, suggesting a possible role of integrin signaling involved in the ECM effect. Finally, a combinatorial screening was performed to demonstrate the potential to screen a multitude of 3D microenvironments of varying ECM combinations that exhibited synergistic influence, indicating a strong positive effect of COL1 and a negative interaction effect of COL1·LM on both albumin and urea secretion. These findings illustrate the broad application potential of this biofabrication approach in identifying optimal ECM composition(s) for engineering 3D tissue, and elucidating defined ECM cues for tissue engineering and regenerative medicine.
三维（3D）培养技术，如球体培养和类器官培养，在生物医学研究中越来越受到关注。然而，对球体和类器官培养中细胞外基质（ECM）效应的理解和控制一直有限。在此，我们报告一种生物制造方法，可有效形成大小均匀的3D肝细胞球体，并将其封装在由藻酸盐和各种ECM分子组成的混合水凝胶中。细胞先接种在微孔平台上形成球体，然后直接封装在含有各种ECM分子的混合水凝胶中，这些分子包括I型胶原（COL1）、IV型胶原（COL4）、纤连蛋白（FN）和层粘连蛋白（LM）。随后对ECM分子对原代小鼠肝细胞表型的影响进行了系统分析。我们的结果表明，肝细胞球体的水凝胶封装促进了肝脏标志物表达和分泌功能。此外，不同的ECM分子对3D封装肝细胞球体的肝功能产生不同影响，但对2D肝细胞和3D未封装球体则无此影响。当单独封装在含有LM或COL1的混合水凝胶中时，肝细胞球体总体上表现出改善的肝功能。基因和蛋白质表达分析表明，当在混合水凝胶中引入LM时，整合素1和整合素6上调，提示整合素信号可能参与ECM效应。最后，进行了组合筛选，以证明筛选多种具有协同影响的不同ECM组合的3D微环境的潜力，表明COL1对白蛋白和尿素分泌有强烈的正向作用，而COL1·LM有负向相互作用。这些发现说明了这种生物制造方法在识别用于工程化3D组织的最佳ECM组成以及阐明用于组织工程和再生医学的特定ECM线索方面具有广泛的应用潜力。
