Abstract: Objective:?To develop a novel strategy for the construction and cultivation of three-dimensional cell spheroids from dental pulp stem cells (DPSCs) with coaxial-electrospray method. Methods:?DPSCs were isolated from clinically collected teeth and then identified through flow cytometry. Core-shell microcapsules were constructed. By adjusting the concentration and flow rates of internal and external phase solutions and cell density, the morphology and size of the microcapsules were observed to get the best preparation condition. Cell viability was detected through live/dead staining and CCK-8 assay, and the spheroids was observed using cytoskeletal staining. Results:?DPSCs were successfully isolated and cultured. Cell-laden microcapsule with alginate layer and carboxymethylcellulose core structure was fabricated. When the cell density within microcapsules reached 1×108 cells/mL, the DPSCs within a single microcapsule formed a spheroid spontaneously with a diameter of (101.93±10.11) μm within 24 hours. Live/dead staining showed the cell survival rate did not decrease within 7 days(P>0.05). CCK-8 assay revealed an increase in cell proliferation rate to (114.91%±7.70%) after 4 days of culture (P<0.05), which further grew to (169.05%±5.29%) at 7 days (P<0.001). Cytoskeletal staining revealed that the cultured cells formed dense spheroids. Conclusions:? These core-shell microcapsules could promote the formation of DPSCs cell spheroids and maintain their long-term survival.