Biological Standardization of Human ASC-Based Biofabrication for Reproducible Macroscale Tissue Constructs

The lack of standardized protocols across laboratories poses a significant barrier to the comparability of experimental outcomes and the efficient transfer of biofabrication technologies. Rigorous biological standardization not only enhances reproducibility but also facilitates alignment with national and international regulatory frameworks, which are essential for the clinical translation of tissue engineering strategies. This study introduces a standardized biofabrication workflow comprising monolayer culture of hADSCs, spheroid formation, 3D printing of Gelatin Methacryloyl (GelMA) scaffolds, and spheroid bioassembly. Quality control metrics were integrated to ensure reproducibility. The 3D-printed GelMA scaffold was designed with two central pores, each capable of housing hundreds of spheroids. Printing fidelity was confirmed by comparing scaffold dimensions to the CAD model, with minor non-significant deviations. hADSCs formed uniform spheroids within 24 h in agarose micromolds. When seeded into scaffold pores, spheroids underwent continuous fusion over 72 h, forming macroscale constructs confirmed by confocal imaging. Scaffold design and printing accuracy ensured structural support, while spheroid uniformity enabled predictable formation of the construct. The fusion of spheroids within the scaffold pores yielded tissue constructs suitable for transplantation, with or without the scaffold, highlighting the versatility of this bioassembly approach. The findings underscore the pivotal role of biological standardization in advancing reproducible methodologies for the biofabrication of living tissue constructs, with promising implications for future clinical applications in regenerative medicine.