Supplementary file 1_3D-bioprinted microenvironments shape the growth and IgG1 monoclonal antibody production of CHO DG44 cells.docx

Introduction<p>Three‐dimensional (3D) bioprinting offers new possibilities to recreate in vivo‐like microenvironments for mammalian cells, with potential applications in therapeutic bioproduction.</p>Methods<p>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.</p>Results and Discussion<p>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.