Abstract
The development of robust and scalable culture systems is essential for the clinical-scale production of human umbilical cord (UC)-derived mesenchymal stem/stromal cells (MSCs) (UC-MSCs). While various basal and serum-free media are commercially available, systematic comparisons of their efficacy in supporting the expansion and functional properties of UC-MSCs remain limited. In this study, we conducted a comprehensive evaluation of multiple culture systems, including basal media (α-MEM, DMEM, and DMEM/F12) supplemented with human platelet lysate (HPL), and commercial serum-free media (Corning MSC Xeno-Free SFM, NutriStem XF Medium, Prime-XV MSC Expansion XSFM), for their ability to sustain UC-MSCs proliferation, maintain phenotypic properties, and support functional potency. The results demonstrated that all basal media supported cell growth, with α-MEM (Gibco) and DMEM/F12 showing superior performance over DMEM. Among serum-free formulations, Prime-XV with 2% HPL yielded the highest primary culture output and the shortest population doubling (PD) time (PDT) during passaging. Notably, cells expanded in commercial serum-free media exhibited reduced diameter and higher uniformity. Functional analyses revealed that NutriStem XF Medium supplemented with 2% HPL elicited the strongest immunomodulatory effects in mixed lymphocyte reactions (MLRs). Furthermore, all media maintained trilineage differentiation capacity and satisfied International Society for Cellular Therapy (ISCT) phenotypic criteria. Critically, no tumorigenic potential was detected in vitro or in vivo. Large-scale manufacturing using the selected medium (NutriStem XF + 2% HPL) confirmed consistent expansion kinetics, high viability, stable marker expression, and functional potency across seven production batches. This study provides a rigorous and clinically relevant framework for selecting culture media that ensure both scalability and functional integrity of UC-MSCs, highlighting the promise of serum-free systems for therapeutic manufacturing.