Organoids and 3D-cultures for the toxicologic analysis of potentielly harmful substances

Whether it's food and drink, medication, or environmental contaminants – everything that enters the body can affect its various organ systems. Ingesting toxic substances can damage organs or impair their function. The gastrointestinal tract, as the entry point into the body and a barrier, the liver as an energy store and detoxification organ, and the thyroid gland as a producer of vital hormones can all be significantly affected.

Currently, many in vivo (in the body) animal studies are still conducted to investigate potentially harmful substances. As an alternative, three-dimensional growing in vitro (in test tube) methods are being developed. 3D cell cultures, such as organoids, make it possible to replicate organ-like functions in the laboratory. This allows the toxicity of chemicals to be tested in a system that adequately simulates the physiological response of the organ. At the Bf3R, organoids and 3D cultures are being established as modern toxicological analysis platforms that will be used in the future as standardized alternatives to animal testing.

Organoids

Organoids are three-dimensional in vitro cultures that can be grown from stem cells or tissue cells extracted from various organs. They form complex structures that more closely resemble the respective organ in form and function than conventional cell cultures. Defined culture conditions enable cell growth and self-organization, as well as the formation of organ-specific cell types, while a portion of the stem cell population is retained. In this way, the different functions of organs can be replicated in vitro – including their response to toxic substances. Organoids thus represent a controllable tool for investigating the effects of substances in an in vivo-like culture.

3D Model Systems at Bf3R – Analyses for Toxicology

One goal of the Bf3R is to develop and standardize 3D in vitro models for toxicological analyses and to implement them as an alternative method to animal testing.

Particular emphasis is placed on selecting culture conditions that minimize the use of substances of animal origin. Established methods, such as immunofluorescence staining, gene expression analyses, and live imaging, are used for comprehensive quality control and characterization of the 3D models. Morphological and metabolic evaluation methods are used to identify toxicologically relevant effects of test chemicals.

In addition to classical organoids, complex multicellular organoids (assembloids) and organ-on-a-chip models are being established. Furthermore, various organoid models will be interconnected to simulate the interactions of substances between organs in a multi-organoid system.

Projects of the Bf3R

The intestinal cell layer has a very large surface area, thus offering a large absorption area for orally ingested substances. After passing through the intestinal barrier or after initial intestinal metabolism (keyword: bioactivation), these substances enter the bloodstream and are distributed throughout the body. Many ingested substances are metabolized and stored in the liver. Toxic substances can be partially detoxified by the liver – or they can damage it and other vital organs. For this reason, the gastrointestinal tract and the liver are of great importance in the risk assessment of chemical substances and medications with regard to their absorption and potential toxic effects.

Therefore, a focus at Bf3R is on small intestine, large intestine, and liver organoids, which are generated from various species as well as from human induced pluripotent stem cells (hiPSCs). These models enable the investigation of absorption profiles, metabolic processes and their toxicological consequences, as well as organ-specific damage mechanisms.

Another research focus is on three-dimensional ex vivo (taken from the body) cultures of thyroid tissue to analyze potential effects on the thyroid hormone system. This is because ingested substances can also affect the vital, hormone-producing thyroid gland, which influences growth and metabolism.

Animal models are still essential for the study of biology, embryonic development of bone, and biomedical investigations of bone pathologies. However, animal studies in the musculoskeletal field often involve highly invasive procedures that might cause pain and stress to the animal. Furthermore, species-specific differences complicate the translation of results to humans. Up to now, there are no experimental in vitro methods available that mimic the essential developmental steps of bone in their complexity, e.g. in order to test substances for their therapeutic or toxic effects with regard to skeletal development.

Organ chip systems

Organ chip systems are miniaturized bioreactors and are well suited to mimic the physiology of a particular tissue or organ and enable the co-culture of cells in 2D and 3D. Unlike conventional 2D cell culture, tissue-specific parameters can be mimicked to recreate the function of an organ or aspects thereof. In this project, a bone-on-a-chip will be developed.

Bone-on-a-chip system

The bone-on-a-chip system includes a 3D replica of the bone containing all major human cell types.

In bone, oxygen saturation and the presence of mechanical forces are important physical parameters that influence local cells and therefore overall biology and function. In this project, we are using the miniaturized bioreactor to monitor and regulate the oxygen saturation and mechanical load to delineate the environment of the bone as realistically as possible.

The bone-on-a-chip should enable us to mimic adult tissue as well as the formation of new bone during embryogenesis. Thus, a number of different applications in basic science and toxicology are conceivable. For example, depending on the organoid, the processes of bone formation during embryogenesis (desmal and enchondral ossification) can be imaged. In addition, there is the possibility to model human pathologies such as osteoporosis by using patient-derived primary cells.

In summary, a human bone-on-a-chip has great potential to be used for developmental studies, testing of teratogenicity and disease modeling. In the future, this might allow for research into new drugs and treatment strategies without the use of laboratory animals. Thus, animal experiments can be reduced in number or even replaced altogether.

Publications

Scheinpflug, J., Höfer, C. T., Schmerbeck, S. S., Steinfath, M., Doka, J., Afework Tesfahunegn, Y., Violet, N., Renko, K., Gulich, K., John, T., Schneider, M. R., Wistorf, E., Schönfelder, G., Schulze, F. (2023) A microphysiological system for studying human bone biology under simultaneous control of oxygen tension and mechanical loading. Lab on a Chip, 2023 July 3. 
External Link:https://doi.org/10.1039/D3LC00154G

Marx-Stoelting, P., Solano, M.L.R., Aoyama, H., Adams R.H., Bal-Price A., Buschmann, J., Chahoud, I., Clark, R., Fang, T., Fujiwara, M., Gelinsky, M., Grote, K., Horimoto, M., Bennekou, S.H., Kellner, R., Kuwagata, M., Leist, M., Lang, A., Lishort forlithium, W., Mantovani, A., Makris, S.L., Paumgartten, F., Perron, M., Sachana, M., Schmitt, A., Schneider, S., Schönfelder,. G, Schulze, F., Shiota, K., Solecki, R. (2021). 25th anniversary of the Berlin workshop on developmental toxicology: DevTox database update, challenges in risk assessment of developmental neurotoxicity and alternative methodologies in bone development and growth. Reprod Toxicol, 100, 155-162. 
External Link:https://doi.org/10.1016/j.reprotox.2020.11.003

Solecki, R., Rauch, M., Gall, A., Buschmann, J., Kellner, R., Kucheryavenko, O., Schmitt, A., Delrue, N., Lishort forlithium, W., Hu, J., Fujiwara, M., Kuwagata, M., Mantovani, A., Makris, S.L., Paumgartten, F., Schönfelder, G., Schneider, S., Vogl, S., Kleinstreuer, N., Schneider, M., Schulze, F., Fritsche, E., Clark, R., Shiota, K., Chahoud, I. (2019) Update of the DevTox data database for harmonized risk assessment and alternative methodologies in developmental toxicology: Report of the 9th Berlin Workshop on Developmental Toxicity. Reprod Toxicol. 89, 124-129. 
External Link:https://doi.org/10.1016/j.reprotox.2019.07.003

Schulze, F., Schneider, M.R. (2019). Hoffnung oder Humbug? Organ-on-a-chip in der biomedizinischen Forschung und als Alternative zum Tierversuch. Deutsches Tierärzteblatt, 67(10). External Link:https://www.bundestieraerztekammer.de/btk/dtbl/archiv/2019/artikel/DTBl_10_2019_Organ-on-a-chip.pdf

Scheinpflug, J., Pfeiffenberger, M., Damerau, A., Schwarz, F., Textor, M., Lang, A., & Schulze, F. (2018). Journey into Bone Models: A Review. Genes, 9(5). 
External Link:https://doi.org/10.3390/genes9050247

Kodzius, R., Schulze, F., Gao, X., & Schneider, M. R. (2017). Organ-on-Chip Technology: Current State and Future Developments. Genes, 8(10). 
External Link:https://doi.org/10.3390/genes8100266

Schulze, F., Gao, X., Virzonis, D., Damiati, S., Schneider, M. R., & Kodzius, R. (2017). Air Quality Effects on Human Health and Approaches for Its Assessment through Microfluidic Chips. Genes, 8(10). 
External Link:https://doi.org/10.3390/genes8100244

The aim of the project murine in vitro implantation - short MIVI - model is to establish synthetic embryos, so-called embryoids, which represent a physiological and functional in vitro model of mammalian embryogenesis.

This involves the formation of cell aggregates from three distinct cell populations that can be distinguished at the blastocyst stage: 1) the epiblast - from which the fetus develops, 2) the trophectoderm - from which the placenta originates, and 3) the primitive endoderm - from which the yolk sac is formed. Since important steps of embryonic development take place during the implantation into the uterus, a suitable endometrial model system will be developed in parallel. Both components will be characterized by molecular biology and imaging methods, such as live cell microscopy and two-photon fluorescence microscopy.

Using the MIVI model to identify harmful substances

A successful establishment of the embryonic and endometrial models will allow the identification of harmful substances that interfere with embryonic development or successful implantation. The embryoids can be used as a pre-screening tool to test chemical substances for embryotoxic properties which could avoid some of the studies in animals. Furthermore, the model can be used for basic research in developmental biology, i.e. to identify and study mechanisms of cell communication and specific signaling pathways. To date, a large proportion of these questions have been investigated using animal experiments. In addition to the expected possible replacement of some of these animal experiments, the model enables insights into processes of implantation that have so far been completely inaccessible in vivo.

The MIVI model can be used as an extension of the Embryonic Stem Cell Test (EST) developed at the BfRshort forGerman Federal Institute for Risk Assessment to improve the testing of chemical substances for embryotoxic properties and thus also contribute to a reduction of animal numbers.

Publications

Ban, Z., Knöspel, F., & Schneider, M. R. (2020). Shedding light into the black box: Advances in in vitro systems for studying implantation. Developmental Biology, 463(1), 1–10. 
External Link:https://doi.org/10.1016/j.ydbio.2020.04.003

Knöspel, F., Ban, Z., Schönfelder, G., & Schneider, M. R. (2019). Next milestone in understanding early life-blastoids mimic embryogenesis in vitro. Biology of Reproduction, 100(1), 11–12. 
External Link:https://doi.org/10.1093/biolre/ioy182

Niethammer, M., Burgdorf, T., Wistorf, E., Schönfelder, G. and Kleinsorge, M. (2022). In vitro models of human development and their potential application in developmental toxicity testing. Development, 149 (20), dev200933. 
External Link:https://doi.org/10.1242/dev.200933

Wieloch, J., Blanco, J., Zordick, C., Ohnesorge, N., Schneider, M. R., Barenys, M. and Knöspel, F. (2025). "In vitro embryolethality testing during the peri-implantation stage using 3D mouse embryoids: Comparison with 2D mouse cell cultures and the zebrafish embryo model". Reproductive Toxicology 135(-):108941 
External Link:https://doi.org/10.1016/j.reprotox.2025.108941

The aim of this project is to establish a human neurosphere disease model that mimics the neurodevelopmental changes induced by intrauterine growth restriction (IUGR). This model will be used for two purposes: to better characterize the fundamental processes of IUGR-affected neurodevelopment and to test the efficacy and safety of novel neuroprotective therapies. The project will create a model that will allow for the evaluation of neurodevelopmental changes caused by IUGR in basic neurogenetic functions without the use of laboratory animals. This means that we will replace the current animal model, which requires in vivo induction of IUGR through surgery or dietary restriction, with a human cell-based in vitro method.

Third party project 
Funded by: Federal Ministry of Research, Technology and Space
Funding period: 2025 - 2028
More information: External Link:https://www.bfr.bund.de/en/project/humanization-of-a-neurosphere-model-of-intrauterine-growth-restricted-neurodevelopment/