A Reproducible MRI‐Based Pipeline for Longitudinal Tracking of Laser‐Assisted Bioprinted Cells in Three‐Dimensional Constructs

ABSTRACT Laser‐assisted bioprinting (LAB) enables precise spatial patterning of living cells within three‐dimensional constructs; however, the lack of non‐destructive, standardised methods for longitudinal evaluation remains a major limitation to construct optimisation and translation. Magnetic resonance imaging (MRI) offers unique advantages for volumetric and repeated imaging due to its non‐invasiveness, yet its application to complex multilayer bioprinted constructs has not been systematically assessed using quantitative workflows. In this study, we developed and evaluated an MRI‐based pipeline for the longitudinal tracking and quantitative analysis of laser‐assisted bioprinted cells within three‐dimensional constructs of increasing architectural complexity. Iron oxide–labelled endothelial cells were bioprinted onto gelatin‐based biopapers in predefined patterns and assembled into constructs composed of three, six or nine stacked layers. Constructs were monitored at 4.7 T using a high‐resolution three‐dimensional steady‐state free precession sequence for up to 35 days, and quantitative image analysis was independently performed by three operators using the open‐source 3D Slicer image computing platform. MRI enabled clear visualisation of printed patterns and multilayer architectures over time due to the signal drop induced by iron oxide particles. Quantitative analysis demonstrated high inter‐operator reproducibility and revealed construct thickness–dependent differences in volume and signal‐to‐noise ratio. As a proof of concept, stacked bioprinted constructs were additionally visualised post‐mortem in a murine calvarial defect model, enabling discrimination of individual layers and printed patterns. Together, these results establish MRI as a robust, non‐destructive tool for the longitudinal evaluation of complex laser‐assisted bioprinted constructs.