title: Retroviral transduction of peripheral blood leukocytes in a hollow-fiber bioreactor.
abstract: BACKGROUND
  Peripheral blood white cells (leukocytes) (PBLs) have been used as effective targets for genetic manipulation by transduction with retroviruses in open systems. A semi-closed hollow-fiber bioreactor was tested for culturing and transducing lymphocytes.
  
  
  METHODS
  PBLs were isolated from five healthy donors, and 5 x 10(7) cells were cultured in hollow-fiber bioreactors for 4 days after stimulation with anti-CD3 in medium containing 200 units per mL of recombinant interleukin 2. Transduction with retroviral vector containing the gene for iduronate-2-sulfatase and G 418 resistance, L2SN, was performed daily on Days 4, 5, 6, and 7, and the cells were expanded for an additional 3 days.
  
  
  RESULTS
  PBLs from three donors were harvested from the bioreactor after transduction and expansion, and 4.5 x 10(9), 7.0 x 10(9), and 2.9 x 10(9) cells were recovered, representing 90-, 136-, and 58-fold expansions. The transduction frequency of L2SN was 10, 5, and 1 percent, respectively. For additional expansion of PBLs, in two cases the bioreactor was reinoculated with 5 x 10(7) cells, which were expanded again for 16 and 8 days, respectively, yielding 1.4 x 10(9) and 3.1 x 10(9) cells, which reflected an additional 28- and 62-fold expansion of cells. PBLs from two other donors were transduced and expanded in the bioreactor, and then 0.8 mg per mL of G 418 was added to the medium in an attempt to enrich the transduced population. Although 2.5 and 10 percent of the cells were transduced, cell death and absence of expansion in the presence of G 418 resulted in final cell lots with viabilities of only 4 and 8 percent. In all cases, the harvested cells tested negative in bacterial and fungal cultures.
  
  
  CONCLUSIONS
  Hollow-fiber bioreactors are an efficient and effective system for the retroviral transduction and expansion of PBLs for clinical gene therapy.
authors.affiliation: Department of Laboratory Medicine, University of Minnesota Medical School, Minneapolis, USA.
authors.forename: R
authors.initials: R
authors.lastname: Shankar
authors.forename: C B
authors.initials: CB
authors.lastname: Whitley
authors.forename: D
authors.initials: D
authors.lastname: Pan
authors.forename: S
authors.initials: S
authors.lastname: Burger
authors.forename: J
authors.initials: J
authors.lastname: McCullough
authors.forename: D
authors.initials: D
authors.lastname: Stroncek
mesh_terms: D019149:Bioreactors; D001782:Blood Donors; D015316:Genetic Therapy / Q000295:instrumentation*; D006128:Growth; D006801:Humans; D007962:Leukocytes / Q000502:physiology; D016532:Mucopolysaccharidosis II / Q000628:therapy; D014161:Transduction, Genetic