Osteoarthritis (OA) is an irreversible degenerative condition causing bone deformation in the joints and articular cartilage degeneration with chronic pain and impaired movement. Adipose-derived stem cell (ADSC) or crushed adipose tissue injection into the joint cavity reportedly improve knee function and symptoms, including pain. Stem cell spheroids may be promising treatment options due to their anti-inflammatory and enhanced tissue regeneration/repair effects. Herein, to form human ADSC spheroids, we used first SphereRing® (Fukoku Co., Ltd., Ageo, Japan), a newly developed rotating donut-shaped tube and determined their characteristics by DNA microarray of mRNA analysis. The variable gene expression cluster was then identified and validated by RT-PCR. Gene expression fluctuations were observed, such as COL15A1 and ANGPTL2, related to vascular endothelial cells and angiogenesis, and TNC, involved in tissue formation. In addition, multiplex cytokine analysis in the medium revealed significant cytokines and growth factors production increase of IL-6, IL-10, etc. However, ADSC administration into the joint cavity involves their contact with the synovial fluid (SF). Therefore, we examined how SF collected from OA patient joint cavities affect 2D-culture ADSCs and ADSC spheroids and observed SF induced cell death. ADSC spheroids could become promising OA treatment options, although studying the administration methods and consider their interaction with SF is essential.