Call for Speakers 2024

In recent years organoids have advanced to prominent and versatile tools in 3R research, representing valuable alternatives for animal testing and physiologically relevant disease models at the same time. As the federal Public Health Institute, the RKI aims to establish an in-house multi-species, multi-organ organoid platform. The objective is a broad methodology in order to strengthen the Institute`s pandemic preparedness plans for novel and re-emerging pathogens, which often have to be handled in a BSL4 laboratory. So far, bronchial and nasal lung organoids have been generated from adult stem cells derived from various primary and commercial cells. Characterisation on mRNA and protein level showed that, with small variances, all organoids consist of a physiologically applicable cell composition.

One property characterizing solid tumors, is the mechanical stress. This restricts the cell proliferation, migration and invasion of weakly malignant cells, while stimulating the selection of the most aggressive clones found in a heterogeneous population. Based on this fact, we designed a new kind of artificial tumor- known as Tumor-like microcapsules- which mimic the biomechanical properties sensed by confined cancer cells in vivo. Cells cultured in these matrices show a better, faster, and stronger upregulation of most hallmarks described in neoplastic pathologies, compared to traditional cancer spheroids. Our results have been validated in vitro and in vivo (i.e. zebrafish model, mice), showing that the biomechanical stress is extremely relevant to design new strategies to culture cancer cells in 3D.

In this presentation, I will discuss the acoustofluidic trap, a tool for precise 3D cell proliferation and function analysis in levitation. Our prototype integrates with any microscope, enabling continuous perfusion experiments with temperature and flow control under optical inspection. Furthermore, I will present a mathematical model and an FEM-based COMSOL simulation to define the acoustic mode and nodal positions in a spherical cavity, aligned with the microscope’s field of view and depth of field. In sterile conditions, we conducted 55-hour continuous perfusion experiments with the K562 cell line, allowing deterministic monitoring. This acoustofluidic platform facilitates in vitro cell testing, imitating in vivo conditions for cell function tests and cell–cell interactions.

The capacity of primary stem cells to maintain their in vivo functionalities during in vitro cultivation is compromised by standard culture conditions because they do not recapitulate the in vivo situation. In contrast, it is crucial to mimic the physiologic situation to maintain cellular functionalities for successful applications in cell based therapies (CBT). To gain results from cell-based assays that resemble the in vivo condition and therefore can be considered as relevant for translation onto the human organism, not only the cellular environment has to mimic the in vivo situation but also the cells have to be isolated, expanded, differentiated and cultured under conditions that do not alter their original behavior nor characteristics due to an artificial environment or treatment. A physiologic cell culture environment can be achieved by establishing 3D cultivation, physiologic (hypoxic) oxygen concentrations, dynamic culture conditions and by using cell culture media which closely resemble the in vivo fluids. Therefore, we strive for optimizing 3D conditions already from isolation and expansion to facilitate the generation of „healthy“ cells and effective cell based products. To guide cellular differentiation processes we utilize functional 3D tissue-like structures under defined and controlled dynamic conditions, using specialized incubator and bioreactor systems and feeding strategies to enable a more physiological cellular development. For a safe translation to clinical application all CBT products should be produced in a physiological setting, including dynamic 3D culture conditions, hypoxic O2 concentration, and antibiotic and xeno-free culture media.

The majority of the current cancer research is based on 2D cell cultures and animal models. These methods have limitations, including different expression of key factors involved in carcinogenesis and metastasis, depending on culture conditions. Addressing these differences is crucial in obtaining physiologically relevant results. Stemness and epithelial-mesenchymal transition (EMT) is linked to the increased invasive potential and metastasis, thus exploring the expression of this markers in a different growth conditions is essential. We report plasticity of expression of selected stem cell and EMT markers in different culture conditions, pointing to the importance of spatial parameters. The most significant difference is the expression of adherent cell junction protein E-cadherin, which changes dramatically between standard 2D culture, floating spheroid culture and matrigel scaffolded culture. As a step towards understanding the reasons causing these discrepancies, we have created a mathematical model of tensions within the 3D bioprinted culture.

Automated systems enhance throughput, accuracy, and efficiency in life science labs but are usually limited to specific, labor-intensive steps. The Robotic-enabled Biological Automation platform (ReBia) automates the entire tissue culture workflow, including microscopic imaging, for creating three-dimensional human tissue models like epidermal skin, airway epithelium, and spheroid-based tumors. This automation supports the 3R initiatives—replacement, reduction, and refinement of animal testing—by improving availability and standardization. AI integration in the ReBia platform monitors Tissue Engineering processes to increase robustness. Machine learning will further aid data analysis, detecting morphological changes in response to drug treatments, thus enhancing drug screening and efficacy assessment. A comparative drug screening studies will highlight the full potential of automation and AI in life sciences.