ObjectivesUmbilical cord mesenchymal stromal cells (UCMSCs) can be considered to become a new gold standard for MSC-based therapies. A serum and xeno-free, chemically defined and no-plate-coating-based culture system will greatly facilitate development of robust, clinically acceptable bioprocesses for reproducibly generating quality-assured UCMSCs. Materials and methodsIn this study, we report for the first time, such a serum-free, xeno-free, completely chemically defined and no-plate-coating-based culture system for the isolation and expansion of UCMSCs, whose biological characteristics were evaluated and compared with serum-containing medium (SCM) methods. ResultsThis culture system not only supported UCMSC primary cultures but also allowed for their expansion at low seeding density. Compared to SCM, UCMSCs in SFM exhibited (i) higher proliferative and colony-forming capacities; (ii) distinctly different morphologies; (iii) similar phenotype; (iv) similar pluripotency-associated marker expression; (v) superior osteogenic, but reduced adipogenic differentiation capacitities. In addition, UCMSCs cultured in SFM retained similar immunomodulatory properties to those in SCM. ConclusionsOur findings demonstrate the feasibility of isolating and expanding UCMSCs in a completely serum-free, xeno-free, chemically defined and no-plate-coating-based culture system and represent an important step forward for development of robust, clinically acceptable bioprocesses for UCMSCs. Further, this provides a superior study platform for UCMSCs biology in a controlled environment.