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Background Ulcerative colitis (UC) is an intractable disease; therefore new therapies need to be developed. CD4+ CD25high regulatory T cells (Treg) significantly ameliorate colitis in animal models. In active UC patients, although Treg are functionally preserved, their proportion in peripheral blood decreases. Thus Treg transfer therapy is expected to be efficacious for UC. During leukapheresis for UC, Treg are depleted, as well as colitogenic effector leukocytes. We therefore designed a leukapheresis/Treg transfer therapy in which Treg are isolated from leukapheresis products and transfused to patients, and studied large-scale germ-free methods of Treg preparation. Methods Using the CliniMACS cell selection system, we conducted Treg isolation experiments from leukapheresis products in which B and CD8+ T cells were depleted, followed by positive selection of CD25+ cells. In some experiments, isolated Treg or non-Treg were expanded with interleukin-2 (IL-2) ± transforming growth factor (TGF)-β1. Expression of a Treg-specific marker, FOXP3, and gut-homing receptors, and suppressor activity of isolated or cultured cells, were analyzed. Results CD4+ CD25high T cells were collected and efficiently enriched with a good recovery rate. Isolated cells preferentially expressed FOXP3 and significantly suppressed T-cell proliferation in vitro. In addition, isolated Treg could be efficiently expanded, and Treg could be induced from non-Treg with TGF-β1 in vitro. TGF-β1 significantly up-regulated αEβ7 and α4β7 integrins. Discussion We have established a method of Treg isolation from leukapheresis products that can be used clinically; therefore, Treg transfer therapy is feasible in combination with leukapheresis for UC. Expansion or induction of Treg in vitro may be another approach to Treg-based immunotherapy. [ABSTRACT FROM AUTHOR]