Challenges and solutions in transitioning to animal-free standards: a comprehensive analysis of components in human cell-based developmental neurotoxicity assays

The replacement, reduction, and refinement (3Rs) of animal experiments is a central objective in modern toxicology. Human cell-based in vitro assays have become key tools to implement these principles by providing mechanistically driven and human-relevant New Approach Methodologies (NAMs) for toxicity testing. However, even in approaches that avoid the use of living animals, many protocols still rely on animal-derived cell culture components beyond fetal bovine serum (FBS), such as bovine serum albumin (BSA) in supplements, extracellular matrix (ECM) preparations such as Matrigel, and animal-sourced antibodies, which may introduce variability. This review provides a targeted materials analysis of human cell-based assays within the Developmental Neurotoxicity in vitro Battery (DNT-IVB), examining basal media, supplements, ECM, growth factors, and antibodies. While serum-free media are widely implemented, animal-derived components remain in use, particularly in supplements, ECM, and immunodetection workflows. Guidance documents, including OECD Good in vitro Method Practices (GIVIMP), or Good Cell Culture Practice (GCCP), promote replacing undefined components such as FBS, but ECM and BSA are less explicitly addressed. Recommendations from the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) encourage the use of animal-free antibodies where feasible. We propose a two-pronged strategy: (1) New protocols incorporate animal-free design from inception; (2) established DNT-IVB assays evaluate component reduction where feasible, balancing validation requirements with practicality. Manufacturers can contribute through standardized labeling (“serum-free,” “animal-free”, “xeno-free,” “chemically defined”) and expanded animal-free product availability. Updated GCCP/GIVIMP guidance could explicitly address BSA and ECM as sources of variability alongside serum. By addressing persistent animal-derived reagents, this reagents-focused review advances animal-free DNT-IVB implementation and supports broader 3Rs objectives by providing actionable strategies for animal-free cell culture in human-relevant NAMs.