title: [Isolation, amplification and identification of human natural CD4⁺CD25⁺ regulatory T cells in vitro].
abstract: OBJECTIVE
  To establish a method for in vitro expansion of human natural CD4⁺CD25⁺ T regulatory cell (Treg) cells for clinical study and immunotherapy.
  
  
  METHODS
  Human natural CD4⁺CD25⁺ Treg were isolated from peripheral blood monocyte cells (PBMCs) by magnetic activated cell sorting (MACS) and expanded by CD3/CD28 expansion beads, IL-2 and rapamycin. The number and the viability of the freshly isolated and expanded Treg were detemined by trypan blue staining. The phenotype and the purity of the freshly isolated and expanded Treg were analyzed by FACS. Treg suppression activity was assessed by mixed lymphocyte reaction (MLR) assay.
  
  
  RESULTS
  Human natural Treg were expanded up to 2 000 folds after 3 weeks in culture, and the activity was more than 97%. The expanded Treg retained Treg phenotype as shown by their freshly isolated counterparts, and the purity of CD4⁺CD25⁺FoxP3⁺ Treg was (94.22 ± 2.12)%. The expanded Treg demonstrated a similar potent suppression of both proliferating auto- and allo- CD4⁺CD25⁻ effector T cells in vitro in a cell number-dependent manner.
  
  
  CONCLUSIONS
  An in vitro expansion of human natural Treg was established to obtain large numbers of human Treg with highly suppressive phenotype and function, thereby providing a solution to the availability of sufficient human natural Treg in clinical study and immunotherapy.
authors.affiliation: Department of Endocrinology, Second Xiangya Hospital, Central South University; Diabetes Center, Central South University; Institute of Metabolism and Endocrinology, Central South University; Key Laboratory of Diabetes Immunology, Ministry of Education, Changsha 410011, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Hu
authors.forename: Xin
authors.initials: X
authors.lastname: Su
authors.forename: Lingjia
authors.initials: L
authors.lastname: Liu
authors.forename: Yufei
authors.initials: Y
authors.lastname: Xiang
authors.forename: Qiqi
authors.initials: Q
authors.lastname: Yu
authors.forename: Shounan
authors.initials: S
authors.lastname: Yi
authors.forename: Zhiguang
authors.initials: Z
authors.lastname: Zhou
mesh_terms: D018929:Cell Culture Techniques; D002469:Cell Separation; D002478:Cells, Cultured; D006801:Humans; D007376:Interleukin-2; D007963:Leukocytes, Mononuclear; D007959:Lymphocyte Culture Test, Mixed; D050378:T-Lymphocytes, Regulatory / Q000166:cytology*
chemical_list: D007376:Interleukin-2