For three-dimensional bio-printed cell-laden hydrogel tissue constructs, the well-designed internal porous geometry is tailored to obtain the desired structural and cellular properties. However, significant differences often exist between the designed and as-printed scaffolds because of the inherent characteristics of hydrogels and cells. In this study, an iterative feedback bio-printing (IFBP) approach based on optical coherence tomography (OCT) for the fabrication of cell-laden hydrogel scaffolds with optimal geometrical fidelity and cellular controllability was proposed. A custom-made swept-source OCT (SS-OCT) system was applied to characterize the printed scaffolds quantitatively. Based on the obtained empirical linear formula from the first experimental feedback loop, we defined the most appropriate design constraints and optimized the printing process to improve the geometrical fidelity. The effectiveness of IFBP was verified from the second run using gelatin/alginate hydrogel scaffolds laden with C3A cells. The mismatch of the morphological parameters greatly decreased from 40% to within 7%, which significantly optimized the cell viability, proliferation, and morphology, as well as the representative expression of hepatocyte markers, including CYP3A4 and albumin, of the printed cell-laden hydrogel scaffolds. The demonstrated protocol paves the way for the mass fabrication of cell-laden hydrogel scaffolds, engineered tissues, and scaled-up applications of the 3D bio-printing technique.

The expansion and harvest of stem cells at clinically relevant scales is critical for cell-based therapies. These approaches need to be robust and cost-effective, support the functional maintenance of desired cell behaviors, and allow for simple harvest. Here, we introduce a real-time monitoring 3D printing approach to fabricate scaffolds with quadruple hierarchical structure that meet these design goals for stem cell expansion. Specifically, a versatile strategy was developed to produce scaffolds from alginate and gelatin with approximately 102 μm interconnected macropores, 300 μm microfilaments, 1.3 mm hollow channels, and centimeter-scale overall dimensions. The scaffolds exhibited good pattern fidelity and stable mechanical properties (compressive modulus value was 22-fold that of hydrogels from the same materials), facilitating uniform and efficient cell seeding with high viability (98.9%). The utility of the scaffold was shown with the 3D culture of HepaRG cells and embryonic stem cells (ESCs) with aggregated morphology, and significantly enhanced cell proliferation was observed compared to those of cultures on flat surfaces, obtaining approximately 2 × 108 cells within a single culture. Interestingly, the functional behavior of the cells was dependent on the cell type, as ESCs maintained their pluripotency, while HepaRG cells improved their hepatic differentiation. Cells were harmlessly harvested through chelating the calcium ions in the cross-linked alginate and de-cross-linking the scaffolds, indicating the potential of this study for scalable stem cell culture for numerous downstream applications.