Comparative flow cytometry immunophenotyping of ASCs expanded in conventional flasks versus automated bioreactors.

Introduction: Adipose-derived stem cells (ASCs) hold significant promises for various regenerative approaches, necessitating the production of a substantial quantity of in vitro expanded ASCs for clinical applications. While ASC expansion is traditionally performed in tissue culture polystyrene (TCP) flasks, the Quantum Cell Expansion System, a hollow fiber bioreactor (HFB), offers an automated and closed system for cell expansion, presenting advantages over manual culture methods. In this study, we compared ASC cultures from a HFB system with traditional TCP flasks, focusing on immunophenotypes. Methods: ASCs from three donors were cultured and underwent equivalent population doublings in both systems. The cell number was counted to compare the growth rate. Furthermore, the individual expressions of 15 surface markers and their co-expression of 5 (CD73, CD90, CD105, CD166, and CD201) and 8 epitopes (CD34, CD36, CD146, CD248, CD271, CD274, and Stro-1) were analyzed by using multicolor flow cytometry. Results: ASCs expanded in the HFB and TCP system showed a comparable growth rate. Except for a significant downregulation of CD201 in HFB (p = 0.008), other surface marker expression profiles were largely comparable between HFB and TCP, with no statistically significant differences observed. While both systems met ISCT criteria for ASC identity, the HFB supported a broader diversity of clonal lineages (greater immunophenotypical heterogeneity), particularly by preserving both CD274-positive and -negative subpopulations. In contrast, TCP culture selectively favored CD201+ and CD274+ clones, indicating environment-driven shifts in subpopulation dynamics. Conclusion: The expansion method significantly influences the phenotypic composition of ASCs. HFB systems offer a promising alternative for large-scale ASC manufacturing by better maintaining subpopulation diversity. These findings emphasize the need for functional validation of ASC subtypes and careful consideration of expansion platforms in clinical-grade cell production.