Development of a complex 3D in vitro alternative model to evaluate the safety of advanced materials

Drug-induced liver injury (DILI) i.e. , liver damage caused by pharmaceutical and other chemical substances, represents a significant clinical challenge in both its diagnosis and treatment, with DILI remaining a major factor in drug attrition and market withdrawal, accounting for approximately 50 % of reported cases of acute liver failure. For decades, conventional in vitro two-dimensional (2D) monolayer cultures and in vivo animal models have been the gold standard in preclinical studies of DILI. However, these models exhibit critical limitations in the accurate replication of metabolic functionality and complexity of human hepatic tissue, a particular disadvantage in the context of drug testing. Rooted in the 3Rs principle of Replacement, Reduction and Refinement, and driven by regulatory frameworks such as REACH (Regulation (EC) No 1907/2006) and Directive 2010/63/EU, which promote ethical alternatives in toxicological studies, one strategy for overcoming issues prevalent when assessing DILI has been the development and adoption of NAMs, or New Approach Methodologies. The use of these innovative and alternative advanced non-animal in vitro systems, which can include 3D cell spheroids, organoids and organ-on-chip (OoC) technology, have shown promising results in recapitulating tissue-mimetic and biologically relevant aspects of target tissue and organs, specifically regarding the liver. Supported by European Union (EU) initiatives like Horizon Europe, and the Joint Research Centres EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM), a core aim of NAMs is to bridge the pre-clinical knowledge gap when evaluating the safety of both drugs and advanced materials (AMs) by developing more predictive and mechanistic safety assessments. Challenges remain however regarding regulatory acceptance, model standardisation and integration into the existing risk assessment frameworks. In spite of these hurdles, NAMs have the potential to transform toxicology, by providing more human-relevant, efficient and ethical alternatives to conventional testing methods. With this in mind, this work deals with the development of a state-of-the-art complex 3D hepatic spheroid model, cultured from both parenchymal (human hepatocellular carcinoma; HepG2) and non-parenchymal (a mixed population of Kupffer cells (KCs), hepatic stellate cells (HSCs) and liver sinusoidal endothelial (LSEC)) cells. This promising NAM not only reconstructs the intrinsic architecture of the human liver, but also facilitates chronic and repeated viability testing over 30 days. The models ability to assess both safety and efficacy to a panel of common hepatotoxins and two representative advanced materials was assessed by ATP quantification, with this study demonstrating the reliability of using NAMs-based systems for in vitro DILI assessment and their revolutionary potential for pre-clinical safety screening as a novel human-relevant liver model. • Drug-induced liver toxicity (DILI) represents a clinically relevant problem in terms of diagnosis and treatment of the disease. • To date, DILI is also a key contributor to the attrition and withdrawal of drugs from the market, with 50 % instances reported of acute liver failure. • Traditional in vitro and in vivo systems such as 2D monolayer and animal studies have been used to recapitulate the main tissue/organ-related functions of the human liver. • Both models present shortcomings in reproducing the main metabolic functionality of the human hepatic tissue, especially when drug testing is performed. • New Approach Methodologies (NAMs) bridges the pre-clinical knowledge gap and evaluate the safety of both drugs and advanced materials. • In this work, 3D complex hepatic spheroids were cultured from a mix of parenchymal (Human Hepatocellular Carcinoma cell line; HepG2) cells and nonparenchymal (a mixed population of KupQer, stellate and liver sinusoidal endothelial cells (LSECs)). • This NAM model was used to assess the safety of a panel of common hepatotoxins and advanced materials. • This model is proving to be a reliable approach not only to reconstruct the human liver architecture but also for testing cytotoxicity and viability for a prolonged period of 30 days.