Impact of culture medium on CD4+ CD25highCD127lo/neg Treg expansion for the purpose of clinical application

A recently discovered population of lymphocytes, called T regulatory cells (Tregs), is characterized by expression of transcription factor Forkhead box P3 (FoxP3). These cells have been successfully used as therapeutic treatments and prophylaxis for graft-versus-host disease (GVHD) and diabetes and might become an attractive alternative to traditional immunotherapy. Here we evaluated how the type of culture medium and the type of serum can influence yield and quality of Tregs after in vitro expansion. We compared Treg fold of expansion and their phenotypical characteristics including expression of FoxP3, CD25, CD127, CD62L and CD45RA in three commercially available culture media (RPMI 1640 (Cellgro; Manassas VA, USA), SCGM (Cellgenix; Freiburg, Germany), and X-VIVO 20 (Lonza; Walkersville, MD, USA)) with addition of human serum (HS, 10%) or fetal bovine serum (FBS, 10%). Among the tested media, X-VIVO 20 supplemented with HS produced the highest yield after 17 days of in vitro expansion (a median of 86-fold expansion, range 30–1365) and highest level of FoxP3 expression (a median of 66.8% of positive cells, range 56–84.8%) in CD4+ CD25hiCD127lo/neg FACS sorted polyclonal Tregs. There was no difference in Tregs yield whether HS or FBS serum was used. In conclusion, the yield of the ex vivo expanded Tregs is related to the type of media applied. Supplementation of the culture with FBS or human serum is equally beneficial.

► Type of culture medium can significantly influence Treg expansion.
► X-VIVO 20 enables highest proliferation rates during Treg expansion.
► Treg expansion outcome is not significantly influenced by the type of serum.