Characterisation of a functional rat hepatocyte spheroid model

Area of Science:

• Hepatology
• In vitro toxicology
• Biotechnology

Background:

• Traditional 2D liver cell models exhibit limitations in maintaining physiological phenotype and metabolic activity for xenobiotic testing.
• There is a growing need for more representative 3D in vitro models to accurately assess drug-induced liver injury.
• 3D liver models offer enhanced cellular architecture and function compared to 2D systems.

Purpose of the Study:

• To develop a novel, size-controllable 3D hepatic spheroid model using primary rat hepatocytes (PRH).
• To assess the viability, structural integrity, and functional characteristics of the PRH spheroids over time.
• To evaluate the potential of this 3D model for preclinical and non-clinical toxicity studies.

Main Methods:

• Utilized the liquid-overlay technique for spontaneous self-assembly of PRH into 3D microtissues (spheroids).
• Cultured spheroids for up to 21 days, monitoring viability and albumin production.
• Assessed F-actin distribution, P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) transporter expression and function (CMFDA assay).

Main Results:

• Viable PRH spheroids with compact, in vivo-like structures were maintained for 21 days.
• Sustained albumin production was observed throughout the culture period.
• Polarized expression and functional transport activity of P-gp and MRP2 transporters were confirmed in the spheroids.

Conclusions:

• The developed 3D hepatic spheroid model effectively recapitulates key in vivo characteristics of hepatocytes, including cell-cell contacts, polarity, and functional transporters.
• This well-characterized 3D model shows significant potential for preclinical and non-clinical repeat-dose investigations.
• The model offers a valuable tool to replace, reduce, and refine animal use in applied research for drug safety assessment.