Cartilage tissue consists of chondrocyte cells and surrounding extracellular matrix. Chondrocytes pass through different stages from progenitor cell (origin form mesenchymal stem cells) to proliferating cells that secrete proteins forming the extracellular matrix to hypertrophic chondrocytes that form bloated cell bodies. Formation of spheroid cultures of musculoskeletal cell types like chondrocytes will help to investigate the differentiation and interaction of chondrocytes with other cells. We tested and compared primary cells isolated from newborn mice (knee) with chondrocyte cell line ATDC5 with regard to formation of spheroid, proliferation, differentiation and formation of extracellular matrix. Both reveal to be a good basis for development of co-cultures with other cell types like endothelial cells and osteoprogenitors.

The use of non-covalent self-assembly has become a prominent strategy for the construction of increasingly functional biomaterials for a range of applications. A variety of molecular building blocks can be used for this purpose, one such block that has attracted considerable attention in the last 20 years is de-novo designed peptides. Our group work focusses on the development of a technological platform for the design of novel biofunctional 3D hydrogels scaffolds exploiting the self-assembly of so-called -sheet forming peptides. These scaffolds can be easily functionalised using specific biological. Through the fundamental understanding of the self-assembly and gelation processes of these peptides across length scales we have been able to design hydrogels with tailored properties.

For over two decades, Oxford Optronix has been dedicated to ensuring reproducibility in cellular research. Here we discuss two widely utilized systems that contribute to this mission in both cell manufacturing and organoid research. Firstly, the GelCount, an automated imaging and analysis platform for colony, spheroid, and organoid counting/sizing is presented. This automation not only saves time and eradicates human bias, but also greatly improves the reproducibility of all counts across a study. Secondly, the HypoxyLab incubator and workstation is introduced with its unique approach for setting a precise oxygen concentration environment independent of altitude or atmospheric pressure changes - a critical factor affecting reproducibility of every hypoxia/physoxia based study.

We developed a novel lensfree imaging method exploiting the optical properties of the cell itself for imaging inside the incubator, which allows non-invasive, super compact, lable-free, live-cell monitoring. By applying AI to determine key cell culture parameters such as confluence, proliferation, and cell motility, high-quality, automated, objective, and real-time data can be collected. Applying our lensfree microscopy (LM) method, we find that memory effects from heterogeneous cell culture conditions lead to an increase of variance during subsequent assays like e.g. omics-readouts or other cell based assays, like wound healing assays, motility and proliferation assays significantly. Furthermore, our LM is also suitable for 3D applications and will enable quantification of organoid growth dynamics and interactions.

Hollow fiber bioreactors provide a fundamentally more in vivo like way of culturing cells. They represent the only way to culture cells at in vivo like cell densities permitting the cells to generate their own specific microenvironment. More physiologic conditions result in uniform and complete post-translational modifications, reduced apoptosis and host cell protein contamination, and for complex cell-to-cell interactions to develop. Some cells will spontaneously form spheroids and others cells behave as they do in vivo. Specific examples include blood brain barrier, placenta co-culture, cryptosporidium culture and malaria sporozoite production for vaccines. Many techniques not possible by any other means can be performed in a 3-D hollow fiber bioreactor.

Transfection is the process to bring nucleic acids into eukaryotic cells. The application of nanoparticles is a standard way to achieve this goal. However, once they reach the cellular membrane, they are endocytosed and trapped in early endosomes, which are acidified to become lysosomes. This process leads to degradation, and it is established that only one percent of the genetic material is able to escape the endosome into the cytosol, making this step the major current obstacle for efficient delivery of the genetic cargo. Agrostemmoside E, a plant secondary metabolite, triggers endosomal escape and application of this molecule in cellular assays can reduce genetic material needed for any transfection assay, since more genetic material reaches the cytosol.

To identify potential intestinal carcinogens, we developed a cell transformation assay using mouse intestinal organoids (mASC-IOs) and assessed the transformation potential on 14 representative chemicals. We optimized the experiment protocol by determining the cytotoxicity, amplification and colony formation of chemical-treated mASC-IOs, and assessed its accuracy and in vivo relevance through characterizing tumorigenicity, pathology of subcutaneous tumor, and CRC-related molecular signatures. Remarkably, the carcinogenicity of 14 chemicals showed strong concordance with epidemiological findings and in vivo mouse studies. The quantitative analysis of anchorage-independent growth was significantly correlated with the tumorigenicity of tested chemicals in a dose-dependent manner. Importantly, the activity of chemical-transformed mASC-IOs was correlated with the differentiation status of subcutaneous tumors and the changes in oncogenic pathways associated with CRC development.

Generation of a highly expressing, regulatory acceptable clonal cell line is the first key step in the development of a biopharmaceutical. To provide the key characteristics (Productivity, Stability, Quality, Monoclonality, and Scalability) from a cell line is challenging especially when using traditional techniques. Significant bottlenecks develop when screening for these rare cells and lengthen the cell line development workflows. Similarly, cell engineering strategies for biotherapeutics discovery frequently add costs and time. Our Cyto-Mine® platform, powered by picodroplet-based microfluidic technology, streamlines the cell line development workflow. It offers a powerful solution to the challenges of screening large numbers of cells allowing isolation of the highest value clones while ensuring monoclonality and enables a viable high-throughput cell engineering workflow.

This presentation delves into the application of engineering involving the integration of robotics, automation, and AI to automate life sciences experiments. We propose the utilization of AI methods within a robot-assisted platform for generating three-dimensional tumor tissue models. Recent technological advancements and innovative engineering solutions have the potential of standardization of the automatic production of the models. Offering flexibility in adjusting various experimental parameters. Manually analyzing such data is time-consuming and subject to inaccuracies stemming from individual subjectivity. Hence, there is a compelling need for a robust and efficient solution to evaluate the morphology of these models. Particularly for quality assessment and assessing therapeutic effects for individualized patient therapy.

Inclined settling technology has been successfully demonstrated in biopharmaceutical manufacturing for achieving high cell densities and viabilities for extended culture durations. We have developed a novel compact scalable device with smaller footprint and higher inclined settling surface area and fabricated it as a single-use disposable plastic BioSettler for recycling of live and productive CHO cells back to the bioreactor, and continuous removal of dead cells and debris. We have now demonstrated it as an inclined settling bioreactor combining the features of a gentle air-lift cell culture bioreactor with the selective cell retention features of the inclined settler, making it useful for growing suspension cells like CHO, IPSC clusters, and attachment dependent cells like MSCs on microcarrier beads and HEK cells.

Our group focuses on modeling autologous cancer models. Cells from patients' primary tumors, metastases, and healthy tissue are isolated. The resulting cell lines are partially immortalized, characterized in detail, and included after extensive quality control in a master cell bank. This allows the establishment of autologous tumor models that reliably mimic the in vivo situation. To date, we have developed sarcoma, chordoma, glioblastoma, pancreas, breast, penis and melanoma models for drug screening, metabolic pathway analysis and tumor development. The communication of tumor cells with cells in the tumor environment is elucidated by characterizing released extracellular vesicles. The importance of patient-derived cell models in combination with advanced 3D cell culture experiments is a must for translational research.

Current organoid culture protocols are laborious and time-consuming, since organoids are cultured manually in droplets of gel. Orgonex offers the RPMotion for rapid and cost-effective production of organoids in four individually controlled 50ml bioreactors. The RPMotion consists of IP-protected technology specifically tailored to organoid culture and is compatible with standard laboratory infrastructure. It contains an intuitive user interface in the control unit and has a modular setup, enabling the culture of multiple organoid conditions in parallel. The RPMotion creates a dynamic environment that enables an average 5-fold faster organoid expansion compared to current culture methods, which results in up to 60% cost reduction of culture ingredients, as well as 75% reduction of labor.

The most common in vitro model for testing pharmaceuticals and cosmetic products is the 2D cell monolayer. However, it has a significant drawback as it oversimplifies the interactions within the model (1). In contrast, 3D in vitro models, including spheres and equivalents, provide a more accurate representation of cell-cell interactions. However, sphere creation methods may lack repeatability or often require additional equipment. Moreover, many existing protocols for cell viability assays are tailored to 2D models, hence the optimization step before conducting experiments on the 3D in vitro models is needed. Our study indicates potential for further optimization of 3D cell culture protocols.

In vitro cell culture systems play a crucial role in biomedical research. In this context, 3D in vitro model systems are becoming increasingly relevant due to their capacity to resemble in vivo microenvironment. Our chemically defined, animal-free coating solution can be easily applied to cell culture consumables creating cell and protein repellent surfaces that are highly suitable for the generation of perfectly round shaped 3D spheroids in a reproducible and rapid way. In addition, our polymeric platform can be functionalized with biologically relevant molecules that can specifically interact with cultivated cells thereby triggering crucial signaling pathways as well as functions.

The rapid advancement of 3D bioprinting opens new opportunities and creates ever-complex challenges. One of them is the capability to 3D print biostructures with high throughput and small feature sizes. This is necessary for such applications as lab-on-chip or printing organs with embedded functioning vasculature. In this talk we present a 3D bioprinter ‘’Vital Light 3D’’ designed to meet this challenge. Based on the femtosecond 2-photon polymerization technique paired with spatial beam shaping it is capable of printing cm-sized objects in a few hours while maintaining µm level precision. We discuss these capabilities in detail explaining how they are achieved, showing already printed sample structures, and highlighting how this printing technology can transform the 3D bioprinting field in the future.

The trend to use complex human-like assay systems for drug discovery and drug profiling continues to increase. Also, therapeutic modalities like cells, tissues and mini organs are increasingly applied to patients. All these probes must not be frozen but need to be shipped to collaborators or patients at distinct conditions. The Cellbox offers a temperature, gas and pH controlled mobile incubator to ship such probes around the world. We will present the Cellbox in various application of such demand.