Three-Dimensional Printing of Hydrogel Scaffolds with Hierarchical Structure for Scalable Stem Cell Culture

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.