Background & Aims Biofabricated tissue models are promising tools for in vitro drug testing and regenerative medicine. Yet, most remain small (<1 cm3) compared to physiological tissues (up to 200 cm3) and are typically matured under static plate culture. Dynamic culture with precise physicochemical control could enable scale-up and improve reproducibility but is not yet standard. The setup of the latter strategy is presented herein. Methodology A perfusion strategy was developed to cultivate large bioprinted tissues under controlled conditions. Custom components were designed, 3D-printed, and integrated with an Ambr250 bioreactor. These parts were validated as autoclavable and leak-proof. The Ambr250 acted as a regulated medium vessel (oxygen concentration, temperature, pH). An 8 cm3 macroporous bioprinted tissue, housed in a custom chamber, was perfused using peristaltic pumping (1.5 mL/min). Tissue morphology and flow rates going through the chamber were assessed non-destructively using 7 T MRI velocimetry. Final tissue analysis was performed on day 21. Results The system maintained stable temperature, dissolved oxygen, and pH over 21 days across a wide oxygen range (20–80 % Air sat. DO). A setpoint-dependent dissolved oxygen concentration offset was identified between the tissue culture chamber and the regulation vessel due to oxygenation of medium through flexible tubing. MRI sequences allowed to reconstruct 3D morphology of the tissue and the liquid phase. Velocimetry sequence revealed flow paths and velocity magnitude distribution of fluid flowing through the tissue, highlighting heterogeneous flow paths that diverged from CAD-based simulations. Three large mesenchymal stem cell-based tissues were successfully perfused in minimal medium and were characterised (live cell count & distribution, differentiation). Conclusion We present a novel perfusion platform enabling controlled culture of large macroporous bioprinted tissues. Future work will refine quantitative flow velocimetry and develop a CFD-based digital model to map local microenvironments, advancing reproducible large-scale tissue maturation.
Introduction Three‐dimensional (3D) bioprinting offers new possibilities to recreate in vivo‐like microenvironments for mammalian cells, with potential applications in therapeutic bioproduction. Methods In this study, we evaluate the growth, central carbon metabolism, and IgG1 monoclonal antibody (mAb) bioproduction of Chinese hamster ovary (CHO) DG44 cells embedded in 3D‐bioprinted hydrogel constructs under different cultivation strategies: batch, semicontinuous, and both modes preceded by a 3D amplification step in an amplification medium. Results and Discussion Across all 3D conditions, CHO DG44 cells exhibited markedly reduced and limited growth over 14 days, most likely due to restricted nutrient diffusion and limited available space imposed by the intrinsic porosity of the bioink. Glucose was not depleted under any condition. In semicontinuous cultures, glucose consumption and lactate production remained approximately constant over the full culture duration, whereas both processes stopped midrun in batch cultures. The relatively low overall glucose consumption indicates that higher cell‐to‐medium ratios could be implemented to improve nutrient utilisation. mAb production closely followed lactate metabolism, with sustained production in semicontinuous cultures and an early plateau in batch mode, resulting in 2‐ to 3‐fold lower final mAb amounts in batch compared with semicontinuous cultures. Introducing a 3D amplification phase more than doubled cumulative mAb production after 14 days. Overall, low growth, low titres, low space–time yields, and residual nutrients in the supernatant show that the current 3D‐bioprinting strategy is not yet optimal for mAb manufacturing. However, despite this apparent weakness of the 3D‐bioprinted environment for production, the 3D specific productivity of 3D‐amplified semicontinuous conditions was found to be higher than the global suspension specific productivity. Consequently, our data suggest that increasing the cell‐to‐ medium ratio and reducing the construct size to mitigate diffusion limitations enhance cell‐specific productivity and process efficiency in 3D‐bioprinted CHO‐ based bioproduction systems.
The rubredoxin-rubredoxin reductase complex is an ancient redox system that protects anaerobic bacteria from the detrimental oxygen, promotes the utilization of liquid hydrocarbons as carbon source in aerobic proteobacteria, is a component of the photosystem II of phototrophic organisms and has evolved into a defensive weapon of pathogenic bacteria against killing by the mammalian host. The highly virulent Pseudomonas aeruginosa strain TBCF10839 utilizes its rubredoxin – rubredoxin reductase complex to grow on alkanes in the aquatic environment and to degrade reactive oxygen species in order to survive in the phagolysosome of human neutrophils.
Background & Aims Adoptive T-cell therapies continue to demonstrate transformative clinical benefit, yet their durability and safety remain limited in part by the differentiation state and subset composition of the infused cells. There is growing evidence that shows that stem-like T-cell subsets (Tscm/Tnaïve) correlate with improved persistence, lower exhaustion, and enhanced therapeutic responses. Additionally, CD8+ T cells serve as the primary cytotoxic effectors that mediate tumor clearance, and a higher percentage of CD8+ T cells in the final product has been associated with more effective clinical outcomes. This underscores the need for activation systems capable of generating T-cell products that are both stem-like and CD8-enriched. Methodology n/a Results Here we show that activation with NanoSpark STEM-T T Cell Activator consistently promotes enrichment of stem-like phenotypes in both CD4+ and CD8+ populations in xeno-free and animal-component free media. STEM-T also yields higher proportions of CD8+ T cells following activation and expansion, a shift aligned with phenotypic profiles associated with improved effector durability and in vivo persistence. Further data show that STEM-T induces a more moderate activation signature, with lower CD25 and CD69 expression, compared with other soluble activators. This gentle activation correlates with reduced PD-1 and LAG-3 levels over time, indicating decreased exhaustion and preservation of functional potential. RUO and GMP formats show consistent performance, underscoring translational relevance. Conclusion Together, these findings suggest that STEM-T drives a differentiation trajectory marked by early memory preservation, CD8+ enrichment, and reduced exhaustion, which are increasingly associated with more effective and persistent adoptive T-cell products.
