Cell-Type-Specific High Throughput Toxicity Testing in Human Midbrain Organoids

Abstract

Toxicity testing is a crucial step in the development and approval of chemical compounds for human contact and consumption. However, existing model systems often fall short in their prediction of human toxicity in vivo because they may not sufficiently recapitulate human physiology. The complexity of three-dimensional (3D) human organ-like cell culture systems (“organoids”) can generate potentially more relevant models of human physiology and disease, including toxicity predictions. However, so far, the inherent biological heterogeneity and cumbersome generation and analysis of organoids has rendered efficient, unbiased, high throughput evaluation of toxic effects in these systems challenging. Recent advances in both standardization and quantitative fluorescent imaging enabled us to dissect the toxicities of compound exposure to separate cellular subpopulations within human organoids at the single-cell level in a framework that is compatible with high throughput approaches. Screening a library of 84 compounds in standardized human automated midbrain organoids (AMOs) generated from two independent cell lines correctly recognized known nigrostriatal toxicants. This approach further identified the flame retardant 3,3′,5,5′-tetrabromobisphenol A (TBBPA) as a selective toxicant for dopaminergic neurons in the context of human midbrain-like tissues for the first time. Results were verified with high reproducibility in more detailed dose-response experiments. Further, we demonstrate higher sensitivity in 3D AMOs than in 2D cultures to the known neurotoxic effects of the pesticide lindane. Overall, the automated nature of our workflow is freely scalable and demonstrates the feasibility of quantitatively assessing cell-type-specific toxicity in human organoids in vitro.

Induced pluripotent stem cell-derived brain organoids as potential human model system for chemotherapy induced CNS toxicity

“…For analysis of acute effects of paclitaxel on protein expression, mature brain organoids (day 58 in culture) were treated for 14 h, transferred to fresh medium and collected either 10 h or 18 h later corresponding to 24 and 32 h time points respectively. To investigate long-term effects on cell type composition, mature brain organoids (day 58 in culture) were incubated with paclitaxel for 14 h and kept in culture for 6 days after the start of the exposure to assure clearance of dead cells as described by others before ( Renner et al, 2021 ). Samples were cryo-grinded with EPPI-pestles (Schuett Biotec, Göttingen, Germany) and resuspended in RIPA-Buffer (50 mM Tris-HCl, 140 mM NaCl, 1% Triton-X-100, 1% Sodium deoxycholate, 0.1% sodium dodecyl sulphate supplemented with cOmplete™ Mini Protease Inhibitor Cocktail [Roche, France]) using 15 µl per organoid or 100 µl per vial NPCs.…”

“…While we could previously show that iPSC-derived neuronal precursor cells (NPC) are similarly susceptible to paclitaxel as murine neural stem cells ( Huehnchen et al, 2017 ), which might contribute to PCCI by influencing hippocampal neurogenesis, modelling PCCI “in a dish” is far more complex. This is due to the three-dimensional structure of the brain and the underlying interplay of different cell types (neuronal precursors, mature neurons, astrocytes, oligodendrocytes, microglia, endothelial cells), which could all be differently affected by cytotoxic drugs and thereby contribute individually or together to PCCI ( Renner et al, 2021 ). The recent establishment of organoids with their self-organization of progenitor cells into aggregates which form 3D organ-specific tissue ( Lancaster et al, 2013 ) holds great potential to study mechanisms of disease as well as drug effects and/or toxicity.…”

The Application of Brain Organoids in Assessing Neural Toxicity

“…It would be challenging to combine brain organoids with high-throughput methods for toxicity screening because efficient testing of a large number of compounds at various concentrations requires automated high-throughput workflows, which challenges the capacity and practicality of a model. Renner et al (2021) developed a highly homogeneous and reproducible 3D model system of the human midbrain for high-throughput screening applications. They verified the capability of high-throughput screening by applying this system to assess the general neurotoxic and dopaminergic neuron-specific toxic effects of a library of 84 compounds, and they demonstrated the feasibility of quantitatively assessing cell-type-specific toxicity in human organoids.…”

Human Brain Organoid: A Versatile Tool for Modeling Neurodegeneration Diseases and for Drug Screening

“…By automating the entire workflow from generation to analysis, the intra- and inter-batch reproducibility was enhanced as demonstrated by RNA-seq and HCI. It turned out that this platform could be automated to generate the reproducible prediction of the drug effects on neurological disorders such as PD at the single-cell level albeit within a complex organoid environment [ 72 ].…”