A Preliminary Investigation of Embedding In Vitro HepaRG Spheroids into Recombinant Human Collagen Type I for the Promotion of Liver Differentiation

Fang-Chun Liao; Yang-Kao Wang; Ming-Yang Cheng; Ting-Yuan Tu

doi:10.3390/polym14091923

A Preliminary Investigation of Embedding In Vitro HepaRG Spheroids into Recombinant Human Collagen Type I for the Promotion of Liver Differentiation

Polymers (Basel). 2022 May 9;14(9):1923. doi: 10.3390/polym14091923.

Authors

Fang-Chun Liao¹, Yang-Kao Wang², Ming-Yang Cheng¹, Ting-Yuan Tu^{1

3

4}

Affiliations

¹ Department of Biomedical Engineering, National Cheng Kung University, Tainan 70101, Taiwan.
² Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan.
³ Medical Device Innovation Center, National Cheng Kung University, Tainan 70101, Taiwan.
⁴ International Center for Wound Repair and Regeneration, National Cheng Kung University, Tainan 70101, Taiwan.

Abstract

Background: In vitro three-dimensional (3D) hepatic spheroid culture has shown great promise in toxicity testing because it better mimics the cell-cell and cell-matrix interactions found in in vivo conditions than that of the traditional two-dimensional (2D) culture. Despite embedding HepaRG spheroids with collagen type I (collagen I) extracellular matrix (ECM) revealed a much better differentiation capability, almost all the collagen utilized in in vitro hepatocytes cultures is animal-derived collagen that may limit its use in human toxicity testing.

Method: Here, a preliminary investigation of HepaRG cells cultured in different dimensionalities and with the addition of ECM was performed. Comparisons of conventional 2D culture with 3D spheroid culture were performed based on their functional or structural differences over 7 days. Rat tail collagen (rtCollagen) I and recombinant human collagen (rhCollagen) I were investigated for their ability in promoting HepaRG spheroid differentiation.

Results: An immunofluorescence analysis of the hepatocyte-specific functional protein albumin suggested that HepaRG spheroids demonstrated better hepatic function than spheroids from 2D culture, and the function of HepaRG spheroids improved in a time-dependent manner. The fluorescence intensities per unit area of spheroids formed by 1000 cells on days 7 and 10 were 25.41 and 45.38, respectively, whereas almost undetectable fluorescence was obtained with 2D cells. In addition, the embedding of HepaRG spheroids into rtCollagen and rhCollagen I showed that HepaRG differentiation can be accelerated relative to the differentiation of spheroids grown in suspension, demonstrating the great promise of HepaRG spheroids.

Conclusions: The culture conditions established in this study provide a potentially novel alternative for promoting the differentiation of HepaRG spheroids into mature hepatocytes through a collagen-embedded in vitro liver spheroid model. This culture method is envisioned to provide insights for future drug toxicology.

Keywords: 3D culture; HepaRG; in vitro liver model; recombinant human collagen; spheroid.

Abstract

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