The aim of the current study was to evaluate cell viability and osteogenic differentiation potential in cell spheroids composed of varying ratios of gingiva-derived and bone marrow stem cells cultured in concave microwells. Cell spheroids were established from bone marrow and gingiva-derived stem cells in ratios of 6:0 (Group 1), 2:1 (Group 2), 3:3 (Group 3), 1:2 (Group 4), and 0:6 (Group 5). On days 3 and 5, the viability of the cell spheroids was qualitatively analyzed using a calcein acetoxymethyl ester working solution and an ethidium homodimer-1 live/dead assay. On days 1, 3, 5 and 7, a quantitative cell viability analysis was performed using a Cell Counting Kit-8. Alkaline phosphatase activity assays were performed using a commercially available kit on day 7 to assess osteogenic differentiation. In addition, reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to evaluate runt-related transcription factor 2 (Runx2) and osteocalcin expression. The ratio of gingiva-derived to bone marrow stem cells did not affect the stem cell spheroid morphology. No significant changes in cell viability were noted among the different groups following incubation for 7 days. A consistent alkaline phosphatase activity was measured in co-cultured gingiva-derived and bone marrow stem cell spheroids of varying compositions. Runx2 and osteocalcin expression was increased when co-cultured compared with pure gingiva-derived or bone marrow stem cells. In conclusion, stem cell spheroids established by co-culturing maintained morphology, viability and a high osteogenic differentiation potential during the experimental period of 7 days. These spheroids containing human gingiva-derived and bone marrow stem cells may enhance the osteogenic differentiation potential. The use of multicell spheroids may be a simple and effective strategy for improving stem cell therapy.

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