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Through the biofabrication technology using cellular aggregates (spheroids), an artificial blood vessel derived from autologous cells of human dermal fibroblast was successfully produced and is expected to be applied clinically. However, the production period remains a major problem, and the organizational construction must be optimized. It is necessary to focus on the formation and fusion of multicellular spheroids (MCS) derived from human dermal fibroblast (HDFB) and consider what affects MCS formation and fusion because these are the basis of tissue construction. In this study, we evaluated HDFB-derived MCS formation and fusion as the basis of the manufacturing process, focusing on the PI3K/AKT cascade in biofabrication technology using MCS. HDFB were used to assess the degree of MCS formation and fusion. TGF-β1 was used as an activator of the PI3K/AKT pathway, whereas LY294002 and COA-Cl were used as its inhibitors. Compared with the control group, the suppression of the PI3K/AKT pathway significantly inhibited MCS formation and fusion, whereas the activation of the PI3K/AKT pathway significantly promoted MCS formation and fusion. We identified six genes (ITGB1, PTK2, MAP2K1, PDK1, GJA1, and PI3KR1) related to cell adhesion in the PI3K/AKT pathways and confirmed a significant difference in gene expression. This study showed that the formation and fusion of HDFB-derived MCS were inhibited by suppressing the PI3K/AKT cascade, whereas it was promoted by activating the cascade with TGF-β1. This research may help elucidate the mechanism of MCS formation and fusion and optimize MCS and tissue construction, which are the basis for tissue engineering technology, and contribute to the development of regenerative medicine. [ABSTRACT FROM AUTHOR]