Methylcellulose in culture medium regulates size, cell viability, cytokine production, and exosome secretion in adipose-derived stem cell spheroids cultured using SphereRing® system

Knee osteoarthritis (OA) is a degenerative disease that impairs joint function. Adipose-derived stem cells (ADSCs) are promising for therapy due to their regenerative potential, particularly when cultured as spheroids to enhance paracrine activity. However, conventional plate-based spheroid culture faces challenges such as scalability limits, retrieval difficulties, excessive enlargement, and necrotic core formation, all of which may compromise therapeutic efficacy. An optimized, scalable method is therefore required for clinical application. This study aimed to establish a spheroid culture protocol using the SphereRing® device with methylcellulose (MC) to regulate spheroid size, improve viability, and enhance secretion of therapeutic factors including exosomes and interleukin-10 (IL-10). ADSCs were cultured in SphereRing® with 0.5–1 % MC for 3 days, and spheroid size, morphology, viability, and secretory profiles were assessed. Spheroids cultured with 0.75 % MC exhibited the most uniform size distribution, higher circularity, reduced necrotic core formation, and significantly greater viability compared to untreated controls. Moreover, IL-10 and exosome secretion were markedly increased in the 0.75 % MC group. Patient-derived ADSCs showed comparable improvements. MC-treated spheroids also maintained superior viability after exposure to synovial fluid, simulating intra-articular conditions. These findings suggest that incorporating MC into the SphereRing® system enables scalable and uniform spheroid production, establishing a clinically relevant platform for ADSC-based therapy in knee OA with potential to improve treatment consistency and outcomes. • A novel spheroid culture method using SphereRing® and methylcellulose (MC) was established. • MC at 0.75 % improved spheroid size uniformity and minimized necrotic core formation. • MC-treated spheroids exhibited enhanced cell viability and reduced dead cell ratio. • Exosome and IL-10 secretion were significantly increased in 0.75 % MC-treated spheroids. • MC-treated ADSC spheroids maintained viability in synovial fluid, supporting clinical use.