The call for a paradigm change in toxicology from the United States National Research Council in 2007 initiates awareness for the invention and use of human‐relevant alternative methods for toxicological hazard assessment. Simple 2D in vitro systems may serve as first screening tools, however, recent developments infer the need for more complex, multicellular organotypic models, which are superior in mimicking the complexity of human organs. In this review article most critical organs for toxicity assessment, i.e., skin, brain, thyroid system, lung, heart, liver, kidney, and intestine are discussed with regards to their functions in health and disease. Embracing the manifold modes‐of‐action how xenobiotic compounds can interfere with physiological organ functions and cause toxicity, the need for translation of such multifaceted organ features into the dish seems obvious. Currently used in vitro methods for toxicological applications and ongoing developments not yet arrived in toxicity testing are discussed, especially highlighting the potential of models based on embryonic stem cells and induced pluripotent stem cells of human origin. Finally, the application of innovative technologies like organs‐on‐a‐chip and genome editing point toward a toxicological paradigm change moves into action.

entist's or even their own experiments, respectively (Baker and Penny, 2016;Miyakawa, 2020). Among the factors contributing to this reproducibility crisis are selective reporting, low statistical power, or poor analysis and experimental design (Baker and Penny, 2016). In addition, poor starting material -especially for hiPSC research -can be a severe source of irreproducibility (Stacey et al., 2013;Pamies et al., 2017). Therefore, already in 2013 "an urgent need" to establish routine screening methods for the characterization of quality-controlled stem cells was identified (Stacey et al., 2013;Crook et al., 2017).While there is guidance available for Good In Vitro Methods Practices in general (OECD, 2018) or stem cell-based Good Cell Culture Practice specifically (Pamies et al., 2017(Pamies et al., , 2018(Pamies et al., , 2020, giving detailed insights into the broad subject of quality assurance (QA) and quality control (QC) of in vitro (stem cell-based) methods, these leave the average academic researcher with a plethora of QC assays, discussing pros and cons that might or

Biodegradable hydrogels that promote stem cell differentiation into neurons in three dimensions (3D) are highly desired in biomedical research to study drug neurotoxicity or to yield cell-containing biomaterials for neuronal tissue repair. Here, we demonstrate that oxidized alginate-gelatin-laminin (ADA-GEL-LAM) hydrogels facilitate neuronal differentiation and growth of embedded human induced pluripotent stem cell (hiPSC) derived neurospheres. ADA-GEL and ADA-GEL-LAM hydrogels exhibiting a stiffness close to ~5 kPa at initial cell culture conditions of 37 °C were prepared. Laminin supplemented ADA-GEL promoted an increase in neuronal differentiation in comparison to pristine ADA-GEL, with enhanced neuron migration from the neurospheres to the bulk 3D hydrogel matrix. The presence of laminin in ADA-GEL led to a more than two-fold increase in the number of neurospheres with migrated neurons. Our findings suggest that laminin addition to oxidized alginate—gelatin hydrogel matrices plays a crucial role to tailor oxidized alginate-gelatin hydrogels suitable for 3D neuronal cell culture applications.

Stable and predictive neural cell culture models are a necessary premise for many research fields. However, conventional 2D models lack 3D cell‐material/‐cell interactions and hence do not reflect the complexity of the in vivo situation properly. Here two alginate/gellan gum/laminin (ALG/GG/LAM) hydrogel blends are presented for the fabrication of human induced pluripotent stem cell (hiPSC)‐based 3D neural models. For hydrogel embedding, hiPSC‐derived neural progenitor cells (hiNPCs) are used either directly or after 3D neural pre‐differentiation. It is shown that stiffness and stress relaxation of the gel blends, as well as the cell differentiation strategy influence 3D model development. The embedded hiNPCs differentiate into neurons and astrocytes within the gel blends and display spontaneous intracellular calcium signals. Two fit‐for‐purpose models valuable for i) applications requiring a high degree of complexity, but less throughput, such as disease modeling and long‐term exposure studies and ii) higher throughput applications, such as acute exposures or substance screenings are proposed. Due to their wide range of applications, adjustability, and printing capabilities, the ALG/GG/LAM based 3D neural models are of great potential for 3D neural modeling in the future.

There is a call for a paradigm shift in developmental neurotoxicity (DNT) evaluation, which demands the implementation of faster, more cost-efficient, and human-relevant test systems than current in vivo guideline studies. Under the umbrella of the Organisation for Economic Co-operation and Development (OECD), a guidance document is currently being prepared that instructs on the regulatory use of a DNT in vitro battery (DNT IVB) for fit-for-purpose applications. One crucial issue for OECD application of methods is validation, which for new approach methods (NAMs) requires novel approaches. Here, mechanistic information previously identified in vivo, as well as reported neurodevelopmental adversities in response to disturbances on the cellular and tissue level, are of central importance. In this study, we scientifically validate the Neurosphere Assay, which is based on human primary neural progenitor cells (hNPCs) and an integral part of the DNT IVB. It assesses neurodevelopmental key events (KEs) like NPC proliferation (NPC1ab), radial glia cell migration (NPC2a), neuronal differentiation (NPC3), neurite outgrowth (NPC4), oligodendrocyte differentiation (NPC5), and thyroid hormone-dependent oligodendrocyte maturation (NPC6). In addition, we extend our work from the hNPCs to human induced pluripotent stem cell-derived NPCs (hiNPCs) for the NPC proliferation (iNPC1ab) and radial glia assays (iNPC2a). The validation process we report for the endpoints studied with the Neurosphere Assays is based on 1) describing the relevance of the respective endpoints for brain development, 2) the confirmation of the cell type-specific morphologies observed in vitro, 3) expressions of cell type-specific markers consistent with those morphologies, 4) appropriate anticipated responses to physiological pertinent signaling stimuli and 5) alterations in specific in vitro endpoints upon challenges with confirmed DNT compounds. With these strong mechanistic underpinnings, we posit that the Neurosphere Assay as an integral part of the DNT in vitro screening battery is well poised for DNT evaluation for regulatory purposes.