Efficient fabrication of monodisperse hepatocyte spheroids and encapsulation in hybrid hydrogel with controllable extracellular matrix effect

Biofabrication. 2021 Oct 18;14(1). doi: 10.1088/1758-5090/ac2b89.

Shuai Deng ¹ ², Yanlun Zhu ¹ ², Xiaoyu Zhao ¹ ² ³, Jiansu Chen ⁴ ⁵ ⁶ ⁷ ⁸, Rocky S Tuan ¹ ⁹, Hon Fai Chan ¹ ² ³ ¹⁰

Affiliations

1 Institute for Tissue Engineering and Regenerative Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, People's Republic of China.
2 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.
3 Shun Hing Institute of Advanced Engineering, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, People's Republic of China.
4 Key Laboratory for Regenerative Medicine, Ministry of Education of China, Jinan University, Guangzhou, People's Republic of China.
5 Aier Eye Institute, Changsha, People's Republic of China.
6 Aier School of Ophthalmology, Central South University, Changsha, People's Republic of China.
7 Institute of Ophthalmology, Medical College, Jinan University, Guangzhou, People's Republic of China.
8 Department of Ophthalmology, First Affiliated Hospital of Jinan University, Guangzhou, People's Republic of China.
9 School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, People's Republic of China.
10 Hong Kong Branch of CAS Center for Excellence in Animal Evolution and Genetics, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, People's Republic of China.

PMID: 34587587 DOI: 10.1088/1758-5090/ac2b89

Abstract

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 Integrinα1 and Integrinα6 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.

Keywords

biomaterial; cell-extracellular matrix interaction; combinatorial screening; hepatocyte spheroid; microfabrication.

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HY-K0301

Cell Counting Kit-8

Cell Proliferation and Cytotoxicity

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