Amplification and identification of regulatory T cells derived from mice in vitro

Objective To establish a method for in vitro expansion of regulatory T cells (Tregs) for clinical study and immunotherapy. Methods CD4+ T cells were isolated from BALB/c spleens by magnetic activated cell sorting (MACS), then cultured in 24-well plates coated with anti-CD3/CD28 microbeads in the presence of 100 U/ml interleukin-2 (IL-2) and 5 ng/ml recombinant human transforming growth factor-β (rhTGF-β). The purity of the expanded Tregs were tested by flow cytometry. In vitro inhibition of CD4+ CD25-T cells response by expanded Tregs was measured by mixed lymphocyte reaction test. The number and the viability of the expanded Tregs were detemined by trypan blue staining. Results Tregs were expanded up to 20 folds after 5 days in culture, and the activity was (93±4)%. The purity of expanded Tregs were (79.1±1.5)%, and maintained suppressive ability. Conclusions We can obtain large numbers of Tregs with highly purity and suppressive ability, thereby providing a solution to the availability of sufficient Tregs in clinical study and immunotherapy. Key words: T-lymphocytes, regulatory; In vitro techniques; Mice