Basic fibroblast growth factor preserves rabbit adipose-derived mesenchymal stromal cells in serum free culture

Importance: Fetal bovine serum (FBS) is widely used for mesenchymal stromal cell (MSC) expansion but carries risks of contamination, batch variability, and xenogeneic exposure that can compromise reproducibility and downstream therapeutic use.Objective: To develop and evaluate a rabbit adipose-derived mesenchymal stromal cell (Rab-ADMSC) serum-free medium (SFM) supplemented with growth factors that maintains key MSC characteristics.Methods: Rab-ADMSCs were isolated from 3 male New Zealand White rabbits.Basic fibroblast growth factor (bFGF), transforming growth factor-β1, and insulin-like growth factor-1 were screened for support of cell viability in SFM.Cells were then cultured in α-Minimum Essential Medium with 10% FBS or in SFM supplemented with bFGF and assessed for morphology, proliferation, expression of stemness-and senescence-associated transcripts, surface marker expression, and tri-lineage differentiation potential.Results: SFM supplemented with bFGF preserved fibroblast-like morphology and supported Rab-ADMSC expansion.Transcript levels of stemness-associated markers and senescenceassociated markers were comparable between culture conditions.Rab-ADMSCs retained osteogenic, adipogenic, and chondrogenic differentiation potential.Flow cytometry showed higher expression of CD81, CD49f, and CD29 in the SFM plus bFGF condition, whereas CD34 and CD45 remained low in both conditions.Conclusions and Relevance: A tailored SFM supplemented with bFGF supports Rab-ADMSC growth while maintaining core MSC phenotypic and functional characteristics, supporting its potential use for more standardized MSC manufacturing in veterinary regenerative applications.