Preservation of transporter function in 3D primary human hepatocyte cultures allows liver toxicity studies

Introduction: Primary human hepatocytes (hHeps) in 2D culture are the “gold standard” for in vitro toxicity studies. Since these conditions do not reflect tissue in vivo properties, hepatocytes rapidly lose their biotransformation capacity. To mimic in vivo conditions, we developed a 3D culture system maintaining hepatocytes polarity and function.

Materials and methods: hHeps were isolated with two-step collagenase perfusion (according to ethical guidelines) and seeded in collagen I gels in 96-well-plates. As quality criteria for our system we measured cell viability, production of glucose and urea, functionality of hepatocyte specific transporters, namely multidrug resistance-associated protein-1 (MRP1) and permeability glycoprotein (P-gp) transporter, Cytochrome P450 (CYP) expression and performed toxicity assays (Acetaminophen and Diclophenac).

Results: The survival of hHeps embedded into collagen I gels was demonstrated by resazurin conversion and life-dead staining. In addition we detected increased glucose and urea production in 3 D versus 2D, as well as higher transporter activity for MRP1 and P-pg. In line with these findings, hHeps showed intoxication in 3D with Acetaminophen and Diclophenac, while under 2D conditions the effect was less pronounced.

Conclusion: Our data reveal an improvement of hHeps biotransformation capacity in our system. The observed intoxication of hHeps in 3D is most likely due to the preservation of functional drug transporter, which may explain Acetaminophen and Diclophenac intoxication observed in some patients. In summary our data highlight the specific role of in vitro 3D cultivation, reflecting tissue in vivo properties and which is suitable for detailed in vitro toxicity assays.