Abstract
Mesenchymal stem cells (MSCs) are considered promising candidates for regenerative and immunomodulatory therapies. However, their clinical use requires xeno-free, good manufacturing practice (GMP)-compatible culture systems. Conventional serum-containing media (FCM) present variability and xenogeneic risk; as such, serum-free (SFM) and animal‑origin‑free (AOF) media have been produced to enhance reproducibility and safety. However, comparative analyses of MSC phenotype, function, and transcriptomic integrity across these media are scarce. Herein, we compared the features of MSC cultures in these different media. Human adipose-derived MSCs from three donors were expanded in FCM, FDA-approved SFM, and AOF media at early, mid, and late passages. The growth kinetics, morphology, immunophenotype, and trilineage potential of these MSCs were assessed. Transcriptomic profiling (RNA-seq), pathway analysis, cell cycle inference, qRT-PCR confirmation, and copy number variation (CNV) analyses were further performed. AOF-supported MSCs exhibited the shortest population doubling time and stable morphology. AOF upregulated G2/M-phase regulators, including Cyclin B-CDK1 complexes and the anaphase-promoting complex/cyclosome, while downregulating negative proliferation and differentiation pathways. Cell cycle profiling confirmed G2/M enrichment. MSC identity (ISCT markers) and low inflammatory profiles, including reduced NGF and CSF1 levels, were maintained. CNV analysis showed no significant differences in genomic instability across media. AOF medium promoted MSC proliferation through selective G2/M activation while maintaining MSC identity, immunomodulation-related marker profiles, and genomic stability under the tested conditions.