Exploring multiple monitoring modalities for large-scale 3D tissue cultivation in bioreactor

Abstract As the field of tissue engineering advances toward clinically viable, large-scale biofabrication, there is an urgent need for non-invasive, real-time monitoring tools capable of assessing the dynamic maturation of 3D bioprinted tissues. This study presents a modular analytical framework combining physicochemical, metabolic, morphological, and perfusion monitoring strategies tailored to volumetric engineered tissues. A perfused cultivation platform was developed for 10.8 cm³ bioprinted fibroblast tissues, enabling fine regulation of pH, temperature, and oxygen. Enhanced oxygen control was achieved through dual-gas PID regulation, reducing deviation from 128–22%. Metabolic activity was monitored via online Raman spectroscopy, allowing real-time lactic acid quantification with a prediction precision error of 0.103 g.L⁻¹, despite low secretion levels typical of adherent cells. Morphological evolution was tracked using 7 Tesla MRI, revealing high fidelity to initial designs (87.6% within 1 mm deviation) and providing longitudinal insights into tissue remodeling without labeling or sectioning. Perfusion was evaluated through computational fluid dynamics (CFD) simulations and MRI velocimetry, confirming flow heterogeneity and validating internal fluid distribution. These combined approaches demonstrate the feasibility of a closed-loop, feedback-driven biomanufacturing process that aligns with quality-by-design principles and regulatory expectations for advanced therapy medicinal products (ATMPs). The integration of established tools from pharmaceutical and clinical fields into tissue engineering workflows marks a critical step toward scalable, standardized, and adaptive biofabrication processes capable of supporting the next generation of functional tissue substitutes.