On the reproducibility of extrusion-based bioprinting: round robin study on standardization in the field.

The outcome of three-dimensional (3D) bioprinting heavily depends, amongst others, on the interaction between the developed bioink, the printing process, and the printing equipment. However, if this interplay is ensured, bioprinting promises unmatched possibilities in the health care area. To pave the way for comparing newly developed biomaterials, clinical studies, and medical applications (i.e. printed organs, patient-specific tissues), there is a great need for standardization of manufacturing methods in order to enable technology transfers. Despite the importance of such standardization, there is currently a tremendous lack of empirical data that examines the reproducibility and robustness of production in more than one location at a time. In this work, we present data derived from a round robin test for extrusion-based 3D printing performance comprising 12 different academic laboratories throughout Germany and analyze the respective prints using automated image analysis (IA) in three independent academic groups. The fabrication of objects from polymer solutions was standardized as much as currently possible to allow studying the comparability of results from different laboratories. This study has led to the conclusion that current standardization conditions still leave room for the intervention of operators due to missing automation of the equipment. This affects significantly the reproducibility and comparability of bioprinting experiments in multiple laboratories. Nevertheless, automated IA proved to be a suitable methodology for quality assurance as three independently developed workflows achieved similar results. Moreover, the extracted data describing geometric features showed how the function of printers affects the quality of the printed object. A significant step toward standardization of the process was made as an infrastructure for distribution of material and methods, as well as for data transfer and storage was successfully established.

Virtual Reality as Tool for Bioprinting Quality Inspection: A Proof of Principle.

As virtual reality (VR) has drastically evolved over the past few years, the field of applications of VR flourished way beyond the gaming industry. While commercial VR solutions might be available, there is a need to develop a workflow for specific applications. Bioprinting represents such an example. Here, complex 3D data is generated and needs to be visualized in the context of quality control. We demonstrate that the transfer to a commercially available VR software is possible by introducing an optimized workflow. In the present work, we developed a workflow for the visualization of the critical quality attribute (cQA) cell distribution in bioprinted (extrusion-based) samples in VR. The cQA cell distribution is directly influenced by the pre-processing step mixing of cell material in the bioink. Magnetic Resonance Imaging (MRI) was used as an analytical tool to generate spatially resolved 2.5 and 3D data of the bioprinted objects. A sample with poor quality in respect of the cQA cell distribution was identified as its inhomogeneous cell distribution could be displayed spatially resolved in VR. The described workflow facilitates the usage of VR as a tool for quality inspection in the field of bioprinting and represents a powerful tool for visualization of complex 3D MRI data.

Magnetic resonance imaging as a tool for quality control in extrusion-based bioprinting.

Bioprinting is gaining importance for the manufacturing of tailor-made hydrogel scaffolds in tissue engineering, pharmaceutical research and cell therapy. However, structure fidelity and geometric deviations of printed objects heavily influence mass transport and process reproducibility. Fast, three-dimensional and nondestructive quality control methods will be decisive for the approval in larger studies or industry. Magnetic resonance imaging (MRI) meets these requirements for characterizing heterogeneous soft materials with different properties. Complementary to the idea of decentralized 3D printing, magnetic resonance tomography is common in medicine, and image data processing tools can be transferred system-independently. In this study, a MRI measurement and image analysis protocol was evaluated to jointly assess the reproducibility of three different hydrogels and a reference material. Critical parameters for object quality, namely porosity, hole areas and deviations along the height of the scaffolds are discussed. Geometric deviations could be correlated to specific process parameters, anomalies of the ink or changes of ambient conditions. This strategy allows the systematic investigation of complex 3D objects as well as an implementation as a process control tool. Combined with the monitoring of metadata this approach might pave the way for future industrial applications of 3D printing in the field of biopharmaceutics.

Automated image processing as an analytical tool in cell cryopreservation for bioprocess development.

The continuous availability of cells with defined cell characteristics represents a crucial issue in the biopharmaceutical and cell therapy industry. Here, development of cell banks with a long-term stability is essential and ensured by a cryopreservation strategy. The strategy needs to be optimized for each cell application individually and usually comprises controlled freezing, storage at ultra-low temperature, and fast thawing of cells. This approach is implemented by the development of master and working cell banks. Currently, empirical cryopreservation strategy development is standard, but a knowledge-based approach would be highly advantageous. In this article, we report the development of a video-based tool for the characterisation of freezing and thawing behaviour in cryopreservation process to enable a more knowledge-based cryopreservation process development. A successful tool validation was performed with a model cryopreservation process for the ß-cell line INS-1E. Performance was evaluated for two working volumes (1.0 mL and 2.0 mL), based on freezing-thawing rates (20 °C to - 80 °C) and cell recovery and increase of biomass, to determine tool flexibility and practicality. Evaluation confirmed flexibility by correctly identifying a delay in freezing and thawing for the larger working volume. Further more, a decrease in cell recovery from 0.94 (± 0.14) % using 1.0 mL working volume to 0.61 (± 0.05) % using a 2.0 mL working volume displays tool practicality. The video-based tool proposed in this study presents a powerful tool for cell-specific optimisation of cryopreservation protocols. This can facilitate faster and more knowledge-based cryopreservation process development In this study, a video-based analytical tool was developed for the characterisation of freezing and thawing behaviour in cryopreservation process development. Evaluation of the practicality and flexibility of the developed tool was done based on a scale-up case study with the cell line INS-1E. Here, the influence of sample working volume on process performance was investigated. Increasing the volume from 1to 2 mL led to a delay in freezing and thawing behaviour which caused cell recovery loss. We believe that the developed tool will facilitate more directed and systematic cryopreservation process development.

Cell Separation in Aqueous Two-Phase Systems - Influence of Polymer Molecular Weight and Tie-Line Length on the Resolution of Five Model Cell Lines.

The availability of clinical-scale downstream processing strategies for cell-based products presents a critical juncture between basic research and clinical development. Aqueous two-phase systems (ATPS) facilitate the label-free, scalable, and cost-effective separation of cells, and are a versatile tool for downstream processing of cell-based therapeutics. Here, we report the application of a previously developed robotic screening platform, here extended to enable a multiplexed high-throughput cell partitioning analysis in ATPS. We investigated the influence of polymer molecular weight and tie-line length on the resolution of five model cell lines in "charge-sensitive" polyethylene-glycol (PEG)-dextran ATPS. We show, how these factors influence cell partitioning, and that the combination of low molecular weight PEGs and high molecular weight dextrans enable the highest resolution of the five cell lines. Furthermore, we demonstrate that the separability of each cell line from the mixture is highly dependent on the polymer molecular weight composition and tie-line length. Using a countercurrent distribution model we demonstrate that our screenings yielded conditions that theoretically enable the isolation of four of the five cell lines with high purity (>99.9%) and yield.

High-throughput downstream process development for cell-based products using aqueous two-phase systems.

As the clinical development of cell-based therapeutics has evolved immensely within the past years, downstream processing strategies become more relevant than ever. Aqueous two-phase systems (ATPS) enable the label-free, scalable, and cost-effective separation of cells, making them a promising tool for downstream processing of cell-based therapeutics. Here, we report the development of an automated robotic screening that enables high-throughput cell partitioning analysis in ATPS. We demonstrate that this setup enables fast and systematic investigation of factors influencing cell partitioning. Moreover, we examined and optimized separation conditions for the differentiable promyelocytic cell line HL-60 and used a counter-current distribution-model to investigate optimal separation conditions for a multi-stage purification process. Finally, we show that the separation of CD11b-positive and CD11b-negative HL-60 cells is possible after partial DMSO-mediated differentiation towards the granulocytic lineage. The modeling data indicate that complete peak separation is possible with 30 transfers, and >93% of CD11b-positive HL-60 cells can be recovered with >99% purity. The here described screening platform facilitates faster, cheaper, and more directed downstream process development for cell-based therapeutics and presents a powerful tool for translational research.

High-throughput cell quantification assays for use in cell purification development - enabling technologies for cell production.

High-throughput screening (HTS) technology is gaining increasing importance in downstream process development of cell-based products. The development of such HTS-technologies, however, is highly dependent on the availability of robust, accurate, and sensitive high-throughput cell quantification methods. In this article, we compare state-of-the-art cell quantification methods with focus on their applicability in HTS-platforms for downstream processing of cell-based products. Sensitivity, dynamic range, and precision were evaluated for four methods that differ in their respective mechanism. In addition, we evaluated the performance of these methods over a range of buffer compositions, medium densities, and viscosities, representing conditions found in many downstream processing methods. We found that CellTiter-Glo™ and flow cytometry are excellent tools for high-throughput cell quantification. Both methods have broad working ranges (3-4 log) and performed well over a wide range of buffer compositions. In comparison, CyQuant® Direct and CellTracker™ had smaller working ranges and were more sensitive to changes in buffer composition. For fast and sensitive quantification of a single cell type, CellTiter-Glo™ performed best, while for more complex cell mixtures flow cytometry is the method of choice. Our analysis will facilitate the selection of the most suitable method for a specific application and provides a benchmark for future HTS development in downstream processing of cell-based products.