title: Development of a clinical-scale method for generation of dendritic cells from PBMC for use in cancer immunotherapy.
abstract: BACKGROUND
  There is growing interest in the use of dendritic cells (DCs) for treatment of malignancy and infectious disease. Our goal was to develop a clinical scale method to prepare autologous DCs for cancer clinical trials.
  
  
  METHODS
  PBMC were collected from normal donors or cancer patients by automated leukapheresis, purified by counterflow centrifugal elutriation and placed into culture in polystyrene flasks at 1 x 10(6) cells/mL for 5-7 days at 37 degrees C, with 5% CO(2), with IL-4 and GM-CSF. Conditions investigated included media formulation, supplementation with heat in activated allogeneic AB serum or autologous plasma and time to harvest (Day 5 or Day 7). DCs were evaluated for morphology, quantitative yield, viability, phenotype and function, including mixed leukocyte response and recall response to tetanus toxoid and influenza virus.
  
  
  RESULTS
  DCs with a typical immature phenotype (CD14-negative, CD1a-positive, mannose receptor-positive, CD80-positive, CD83-negative) were generated most consistently in RPMI 1640 supplemented with 10% allogeneic AB serum or 10% autologous plasma. Cell yield was higher at Day 5 than Day 7, without detectable differences in phenotype or function. In pediatric sarcoma patients, autologous DCs had enhanced function compared with monocytes from which they were generated. In this patient group, starting with 8.0 +/- 3.7 x 10(8) fresh or cryopreserved autologous monocytes, DC yield was 2.1 +/- 1.0 x 10(8) cells, or 29% of the starting monocyte number.
  
  
  CONCLUSIONS
  In the optimized clinical-scale method, purified peripheral monocytes are cultured for 5 days in flasks at 1 x 10(6) cells/mL in RPMI 1640, 10% allogeneic AB serum or autologous plasma, IL-4 and GM-CSF. This method avoids the use of FBS and results in immature DCs suitable for clinical trials.
authors.affiliation: Department of Transfusion Medicine, Warren G. Magnuson Clinical Center, Bethesda, Maryland 20892, USA.
authors.forename: E C
authors.initials: EC
authors.lastname: Wong
authors.forename: V E
authors.initials: VE
authors.lastname: Maher
authors.forename: K
authors.initials: K
authors.lastname: Hines
authors.forename: J
authors.initials: J
authors.lastname: Lee
authors.forename: C S
authors.initials: CS
authors.lastname: Carter
authors.forename: T
authors.initials: T
authors.lastname: Goletz
authors.forename: W
authors.initials: W
authors.lastname: Kopp
authors.forename: C L
authors.initials: CL
authors.lastname: Mackall
authors.forename: J
authors.initials: J
authors.lastname: Berzofsky
authors.forename: E J
authors.initials: EJ
authors.lastname: Read
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D048429:Cell Size; D002470:Cell Survival / Q000187:drug effects; D064987:Cell- and Tissue-Based Therapy / Q000379:methods; D002986:Clinical Trials as Topic; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D003713:Dendritic Cells / Q000166:cytology* / Q000187:drug effects / Q000637:transplantation*; D005434:Flow Cytometry; D005947:Glucose / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007167:Immunotherapy / Q000379:methods*; D015847:Interleukin-4 / Q000494:pharmacology; D019344:Lactic Acid / Q000378:metabolism; D007937:Leukapheresis; D009000:Monocytes / Q000166:cytology* / Q000187:drug effects; D012509:Sarcoma / Q000276:immunology* / Q000628:therapy*; D013997:Time Factors; D014182:Transplantation, Autologous
chemical_list: D003470:Culture Media; D015847:Interleukin-4; D019344:Lactic Acid; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D005947:Glucose