title: A good manufacturing practice method to ex vivo expand natural killer cells for clinical use.
abstract: BACKGROUND
  Great interest has been raised recently by the design of new adoptive immunotherapeutic strategies based on the in vivo infusion of ex vivo-expanded and activated natural killer (NK) cells. The development of good manufacturing practice (GMP) methods for the efficient production of fully functional NK cells is mandatory for clinical application.
  
  
  METHODS
  Peripheral blood mononuclear cells were obtained by leukapheresis and processed in the GMP facility. For NK-cell enrichment, a two-step immunomagnetic procedure consisting of CD3(+) T-cell depletion followed by CD56(+) cell positive selection was used. Isolated NK cells were suspended in serum-free medium containing autologous plasma, interleukin (IL)-2 and IL-15 in the presence of irradiated autologous feeder cells and cultured for 14 days at 37 °C. IL-2 and IL-15 were also added during the last 24 hours of culture. Expanded cells underwent full quality control testing for cytogenetic characteristics, viability, sterility, phenotype and endotoxin status; functional tests, such as degranulation assays and cytotoxicity, were performed on expanded NK cells before cryopreservation and after thawing.
  
  
  RESULTS
  NK-cell populations expanded on average 15.7±4.7 fold by day 14, with a viability of 96% ±0.5. At the end of the incubation period, 97% ±1.1 of the expanded population was CD56(+) NK cells; these effector cells showed significant up-regulation of the activating receptors NKG2D and DNAM-1. Functional tests demonstrated that expanded NK cells are fully functional with no difference whether tested before cryopreservation or after thawing.
  
  
  CONCLUSIONS
  These data provide the basis for developing new NK-cell-based immunotherapeutic strategies for the treatment of patients with cancer.
authors.affiliation: Haematology, Department of Cellular Biotechnologies and Haematology, Sapienza University, Rome, Italy.
authors.forename: Giovanni F
authors.initials: GF
authors.lastname: Torelli
authors.affiliation: Department of Haematology, Oncology and Molecular Medicine, National Institute of Health, Rome, Italy.
authors.forename: Carmela
authors.initials: C
authors.lastname: Rozera
authors.affiliation: Department of Haematology, Oncology and Molecular Medicine, National Institute of Health, Rome, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Santodonato
authors.affiliation: Haematology, Department of Cellular Biotechnologies and Haematology, Sapienza University, Rome, Italy.
authors.forename: Nadia
authors.initials: N
authors.lastname: Peragine
authors.affiliation: Department of Haematology, Oncology and Molecular Medicine, National Institute of Health, Rome, Italy.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: D'agostino
authors.affiliation: Department of Haematology, Oncology and Molecular Medicine, National Institute of Health, Rome, Italy.
authors.forename: Enrica
authors.initials: E
authors.lastname: Montefiore
authors.affiliation: Department of Haematology, Oncology and Molecular Medicine, National Institute of Health, Rome, Italy.
authors.forename: Maria R
authors.initials: MR
authors.lastname: Napolitano
authors.affiliation: Department of Haematology, Oncology and Molecular Medicine, National Institute of Health, Rome, Italy.
authors.forename: Domenica M
authors.initials: DM
authors.lastname: Monque
authors.affiliation: Department of Haematology, Oncology and Molecular Medicine, National Institute of Health, Rome, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Carlei
authors.affiliation: Haematology, Department of Cellular Biotechnologies and Haematology, Sapienza University, Rome, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Mariglia
authors.affiliation: Haematology, Department of Cellular Biotechnologies and Haematology, Sapienza University, Rome, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Pauselli
authors.affiliation: Transfusion Centre, Department of Molecular Medicine, Sapienza University, Rome, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Gozzer
authors.affiliation: Transfusion Centre, Department of Molecular Medicine, Sapienza University, Rome, Italy.
authors.forename: Mahnaz Shafii
authors.initials: MS
authors.lastname: Bafti
authors.affiliation: Transfusion Centre, Department of Molecular Medicine, Sapienza University, Rome, Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Girelli
authors.affiliation: Haematology, Department of Cellular Biotechnologies and Haematology, Sapienza University, Rome, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Guarini
authors.affiliation: Department of Haematology, Oncology and Molecular Medicine, National Institute of Health, Rome, Italy.
authors.forename: Filippo
authors.initials: F
authors.lastname: Belardelli
authors.affiliation: Haematology, Department of Cellular Biotechnologies and Haematology, Sapienza University, Rome, Italy.
authors.forename: Robin
authors.initials: R
authors.lastname: Foà
mesh_terms: D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D015925:Cryopreservation / Q000379:methods; D005260:Female; D006801:Humans; D007694:Killer Cells, Natural / Q000166:cytology* / Q000637:transplantation; D007937:Leukapheresis / Q000379:methods; D008297:Male