3D-bioprinted microenvironments shape the growth and IgG1 monoclonal antibody production of CHO DG44 cells.

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.