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.

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.

The development of industrial bioprocesses starts with creating a producer cell line in research labs. High-throughput screening identifies the most productive cell, which is then passed to process engineering. This selection occurs under lab conditions, while cells must perform under different large-scale bioprocess conditions with fluctuating pH, oxygen, and nutrients. BiProMicro bridges this gap, linking cell line development with bioprocess development through predictive screening. Using microfluidic single-cell cultivation, BiProMicro studies cell lines under precisely controlled, adjustable conditions. Microfluidic cultivation with live cell imaging enables detailed observation of cellular behavior. Rapid changes in conditions simulate the environmental gradients of large-scale bioreactors. This approach assesses cell line robustness and suitability for industrial-scale bioproduction early, ensuring a smooth transition from lab to large-scale.

Antleron developed a custom bioreactor setup with a mineralized tissue model mimetic of the human bone, to enhance in-vitro implant assessment through human mesenchymal stem cells’ differentiation towards the osteo-lineage. Cell viability and differentiation are measured via lactate production, alkaline phosphatase activity, and gene expression. Implants, e.g. from titanium or polymer, were tested for interaction with the model environment. Successful cell ingrowth was shown for the different evaluated implant materials, indicating their potential for in vivo applications as well as validating the performance of the bone-mimetic in-vitro testing environment. This bespoke bioreactor serves as a scalable, biologically relevant model for bone implant testing, potentially reducing animal testing and accelerating personalized implant development.

The TRACERx programme investigates tumor evolution in early-stage resectable non-small cell lung cancer (NSCLC) patients. My role at the Swanton laboratory involves generating bronchial and alveolar organoids from various normal and tumour lung regions. The samples are collected from surgical resection at UCLH immediately after surgery and processed at the Francis Crick Institute. The first step is dissociating the sample into single cells. After this, we undertake further processing immediately or store it in the liquid nitrogen until further processing. Fully processed samples at single cell stage are suspended in GELTREX in either complete or minimal medium, to prevent contamination with normal airway organoids.Organoid formation takes three weeks on average, with a success rate of 2.5%-5% for cancer and 75% for normal organoids. We perform rigorous checks prior to biobanking samples at passage 3. A standard protocol includes H&E staining, immunohistochemistry with markers (CK5, TTF1, p63), Short Tandem Repeat analysis, and checks for mycoplasma and common human viruses. In select cases, we also perform DNA sequencing to compare the genomic landscape of organoids with the original tumour. We now have organoids from 9 patients with lung cancer and 22 tumour adjacent normal lung organoids. In order to investigate clonal heterogeneity, we have multi-regional cancer organoids for 6 patients. The available organoid biobank is being utilised to investigate various mechanistic questions in lung cancer progression such as the mechanistic changes responsible for cancer initiation, how T-cell interaction modulates clonal selection, and how co-culture with various other microenvironmental cells modulate treatment response. Ongoing efforts focus on generating alveolar organoids from primary lung tissue and developing organoids from patients with specific driver mutation backgrounds. We also hope to develop organoid lines representing ethnic diversity. The ultimate aim of this project is to significantly contribute to understanding tumor evolution and effective therapeutic strategies through the systematic derivation of patient-derived multicellular models.

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.

Mammalian cells, these wild living creatures, are sometimes difficult to tame and control in culture. Different medium and serum charges, culture conditions, operator handling, and not to forget the cell age, its population doublings, have a significant impact on the cell quality. Either you live with it and say, “this is biology”, or you try to standardize your cell culture. We will present ways to establish a “Good Cell Culture Practice”, convert cultured cells into a precise reagent, and give guidance on the qualification of cell banks.