Figure 1.

Three-dimensional floating culture in HA gel. (A): First step (left). HA gel is prepared by mixing sterilized HA powder and culture medium (DMEM) overnight. Middle: Second step. Human ASCs (1.0 × 10⁴ cells per cm²) are disseminated on the HA gel. Right: Final step. After incubation for 48 hours (37°C, 5% CO₂), ASCs formed spheroids in the gel. For retrieval, culture medium is added to dilute HA gel and incubated for 3 hours. The diluted supernatant containing ASC spheroids is transferred into a silicon tube, and ASC spheroids are collected as a pellet by centrifugation (760g, 5 minutes). (B): Three-dimensional culture in HA gels (0%, 2%, 3%, 4%, 5%, or 10%) or on a nonadhesive dish. Human ASCs were seeded in the HA gels of various (0%–10%) concentrations. In 2%–3% HA gel ASC formed spheroids, but some of them sank and proliferated on the bottom of the dish like an explant culture. In contrast, in 4%–5% HA gel formation of floating spheroids was completed at 48 hours without further substantial change in size afterward. Spheroids were not formed in 10% HA gel. On a nonadhesive dish ASC spheroids continued growing until day 7. Scale bars = 100 μm. Abbreviations: 3D, three-dimensional; ASC, adipose-derived stem/stromal cell; d, day(s); DMEM, Dulbecco’s modified Eagle’s medium; h or hr, hour(s); HA, hyaluronic acid; hASCs, human adipose-derived stem/stromal cells; O/N, overnight.