Comparative Evaluation of HepG2 Spheroid Generation Methods for Toxicology.

Area of Science:

• * Hepatology and Drug Discovery
• * 3D Cell Culture and Tissue Engineering

Background:

• * Three-dimensional liver spheroids are vital for studying hepatocyte function and drug-induced liver injury.
• * Inconsistent spheroid formation methods limit reproducibility in research.

Purpose of the Study:

• * To systematically compare different HepG2 spheroid culture methods.
• * To identify optimal methods for reproducible and functional in vitro liver models.

Main Methods:

• * Comparison of HepG2 spheroid culture using 35/96-microwell agarose gels, ultra-low attachment (ULA) plates, and hanging drops.
• * Evaluation across various seeding densities and culture durations.

Main Results:

• * Spheroid geometry significantly impacts hepatocyte function, influencing nutrient and oxygen diffusion.
• * 35-microwell agarose gels produced the most circular spheroids with higher albumin secretion and ATP production.
• * No single method offered optimal repeatability, throughput, and functional robustness.

Conclusions:

• * Optimal spheroid culture method selection depends on application-specific needs (e.g., throughput, cost, function).
• * Hybrid approaches combining plate-based scalability and agarose-based functional performance are recommended.
• * Standardized, high-content platforms are crucial for liver metabolism, toxicity, and drug discovery research.