Comparison of 3D culture systems on mesenchymal stem cell longevity, regenerative potential, and secretome production.

BACKGROUND/AIMS: Mesenchymal stem/stromal cells (MSCs) are widely investigated for regenerative therapies, yet current expansion methods often compromise their stem-like properties, limiting large-scale clinical translation. We aimed to evaluate whether a novel hydrogel-based Bio-Block® platform preserved intrinsic adipose-derived MSC (ASCs) phenotype and secretome compared with conventional systems. MATERIALS/METHODS: ASCs were cultured for four weeks in 2D, spheroids, Matrigel, or Bio-Blocks. Cultures were assessed for proliferation, senescence, apoptosis, trilineage differentiation, stem-like gene expression, secretome protein and extracellular vesicle (EV) production, and EV potency on endothelial cells (ECs). RESULTS: Bio-Block ASCs exhibited ~2-fold higher proliferation than spheroid and Matrigel groups, with senescence reduced 30-37% and apoptosis decreased 2-3-fold. Trilineage differentiation and stem-like markers (e.g. LIF, OCT4, IGF1) were significantly higher in Bio-Block ASCs. Secretome protein declined 35%, 47%, and 10% in 2D, spheroid, and Matrigel, respectively, but was preserved in Bio-Blocks. Similarly, EV production increased ~44% in Bio-Blocks, while other systems declined 30-70%. Bio-Block EVs enhanced EC proliferation, migration, and VE-cadherin expression, whereas spheroid EVs induced senescence and apoptosis. CONCLUSION: This study highlights the critical influence of culture systems on scaling robust MSC-based therapies and introduces a biomimetic platform that represents a potential scalable strategy for producing high-potency and robust regenerative therapies.