Establishment and functional in vitro characteristics of three-dimensional collagen HepaRG microsphere

doi:10.3969/j.issn.2095-4344.3149

Abstract

Abstract: BACKGROUND: Some recent studies have shown that the three-dimensional (3D) model of HepaRG cells can better mimic the in vivo microenvironment and show better liver differentiation and function compared with two-dimensional culture.
OBJECTIVE: HepaRG was selected to prepare 3D collagen microspheres, and the adaptive culture and functional expression of cells in the collagen microspheres were evaluated.
METHODS: Collagen hydrogel was used as the scaffold for 3D HepaRG and HepG2 microspheres. Stable cell spheres were formed. HepaRG microspheres, HepG2 microspheres, HepaRG two-dimensional culture and HepG2 two-dimensional culture were used as controls. At 1, 6, and 12 days of culture, cell survival was detected by the Live/Dead assay staining. After 1, 6, 12, and 16 days of culture, the urea synthesis and CYP3A4 secretion of the supernatant were detected in each group. After 12 days of culture, relative expression of CYP3A4, CYP1A2, UGT1A1, and CPS1 mRNA was detected by qPCR. The expression levels of hepatocyte marker albumin and CYP3A4 protein were determined using western blot assay.
RESULTS AND CONCLUSION: (1) In 12 days of culture, Live/Dead assay staining showed that the cell viability in the 3D collagen microsphere was well-maintained and the amount of central necrotic cells was small, with high cell viability. In the 3D collagen microsphere, especially HepaRG cells, multiple cellular clusters formed and adjacent clusters were connected closely, which created a cross-linked structure. (2) After 1, 6, and 12 days of culture, the urea content of HepaRG 3D collagen microspheres was higher than that of HepaRG two-dimensional culture (P < 0.05). After 1, 6, 12, and 16 days of culture, the urea content of HepG2 3D collagen microspheres was higher than that of HepG2 two-dimensional culture (P < 0.05). After 1, 6, and 12 days of culture, the secretion of CYP3A4 in HepaRG 3D collagen microspheres was higher than that in HepaRG two-dimensional culture (P < 0.05). After 6 and 12 days of culture, the secretion of CYP3A4 in HepG2 3D collagen microspheres was higher than that in HepG2 two-dimensional culture (P < 0.05). (3) The relative expression of CYP3A4, CYP1A2, UGT1A1, and CPS1 mRNA in HepaRG 3D collagen microspheres was higher than that in HepaRG two-dimensional cells (P < 0.05), and the relative expression of CYP1A2 in HepG2 3D collagen microspheres was higher than that in HepG2 two-dimensional culture (P < 0.05). (4) The expression levels of albumin and CYP3A4 protein in HepaRG 3D collagen microspheres were higher than those of HepG2 3D collagen microspheres, ordinary microspheres, and two-dimensional culture (P < 0.05). (5) These results indicated the high-level expression of hepatocyte functions in 3D collagen HepaRG microsphere, which could be taken as a reference in drug metabolism evaluation in vitro and tissue engineering application.

Key words: material, HepaRG cell, three-dimensional culture, collagen microsphere, cellular phenotype, cytochrome P450 system, urea secretion, gene expression

CLC Number:

Li Shao, Liang Yongkang, Gao Yi, Peng Qing. Establishment and functional in vitro characteristics of three-dimensional collagen HepaRG microsphere[J]. Chinese Journal of Tissue Engineering Research, 2021, 25(16): 2541-2547.

Figures/Tables 9

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