PhD Scientific Days 2026

Molecular Medicine 1.

Validation of HepG2, HepaRG and Primary Human Hepatocyte Spheroids for Long-term Applications

Name of the presenter

Pacsuta, Johanna

Institute/workplace of the presenter

Semmelweis University Doctoral School/ Charles River Laboratories Hungary

Authors

Pacsuta Johanna¹, Mártonné-Tóth Beáta², Bujdosó-Székely Virág², Jemnitz Katalin¹, Gáborik Zsuzsanna²
1: Semmelweis University Doctoral School/ Charles River Laboratories Hungary
2: Charles River Laboratories Hungary

Text of the abstract

Spheroid cultures are actively investigated to improve the predictability of in vitro assays and to evaluate ADME-Tox properties of drug candidates. Primary human hepatocyte (PHH) spheroids were previously described to be phenotypically and functionally stable over several weeks in culture. Due to significant interindividual varieties and higher associated costs other hepatocyte cell types, such as HepG2 or HepaRG, have been applied as models. HepG2 cells retain some liver-like characteristics and serve as an easy-to-handle system. In terms of hepatic function, HepaRG cells represent an intermediate model between HepG2 and PHH cells, although their maturation into hepatocyte-like cells requires a long differentiation process.
We aimed to compare the properties of these hepatocyte cultures to determine their predictivity for ADME-Tox studies. First, we investigated the effect of spheroid size on cell viability. Next, we measured the presence of the necrotic core and the generation of biliary ducts in the spheroids by High Content Imaging. The functionality of hepatocytes was measured by their albumin and urea secretion and cytochrome P450 (CYP) enzyme expression with fluorescence-based kits and real-time PCR, respectively.
The 500 cells/spheroid setup was prone to flotation, while 250- and 1000 cells/spheroid provided a more stable culturing system. HepG2 spheroids developed a strong necrotic core over time, while HepaRG and PHH cells remained alive even in the center of the spheres. Bile ducts could be detected only in PHH spheroids. All cell types secreted albumin, while urea was secreted by only PHH and HepG2. The CYP enzyme expression also differs between the three models: HepG2 cells do not express CYP enzymes; HepaRG cells expressed CYP1A2, 2B6, 2C8, 2C9, 2C19, and 3A4 but not CYP2D6; PHH expressed all investigated CYP enzymes.
We conclude that spheroids from PHH and HepaRG at 250 and 1000 cells/ spheroid have the most physiological relevance and provide the most straightforward culturing possibilities. We intend to apply these validated models for metabolic stability assessment of low-clearance compounds and for long-term toxicity predictions.
This research was supported by the 2024-2.1.2-EKÖP-KDP new National Excellence Program of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund.