PMID- 12670372
OWN - NLM
STAT- MEDLINE
DCOM- 20031223
LR  - 20190819
IS  - 0042-9007 (Print)
IS  - 0042-9007 (Linking)
VI  - 84
IP  - 3
DP  - 2003 Apr
TI  - Optimized culture conditions for the generation of dendritic cells from 
      peripheral blood monocytes.
PG  - 228-36
AB  - BACKGROUND AND OBJECTIVES: Dendritic cells (DCs) are promising adjuvants for 
      clinical immunotherapy, but they are scantily distributed. Therefore, numerous in 
      vitro methods have been developed to expand these cells while maintaining their 
      normal functions. Current culture systems generally require the use of fetal 
      bovine serum (FBS)-supplemented media in order to attain DCs with high 
      immunostimulatory activity. However, the presence of exogenous animal proteins 
      sets limits for their use in clinical trials. The purpose of this study was to 
      establish a simple, efficient and FBS-free method for the generation of human DCs 
      for clinical application. MATERIALS AND METHODS: We compared monocyte-derived DCs 
      generated in a standard FBS-supplemented medium vs. DCs generated in an 
      autologous plasma (AutoPl)-supplemented medium, with regard to their yield, 
      function and longevity. Peripheral blood monocytes were isolated from buffy coats 
      by two consecutive 2-h adherence steps in tissue culture flasks. The adherent 
      cells were differentiated into DCs within 2 weeks by adding 
      granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), 
      c-kit ligand and tumour necrosis factor-alpha (TNF-alpha). Every 2-3 days, the 
      cells in suspension were analysed for their immunophenotype and apoptosis rate by 
      flow cytometry. Their function was demonstrated by their allostimulatory and 
      migratory capacity, as well as by their proteolytic activity. RESULTS: We show 
      that more than 30 x 10(6) DCs can be achieved per unit of buffy coat using either 
      AutoPl- or FBS-supplemented media. The purity of the DCs was 53.4% and 65% (P > 
      0.05) in AutoPl- and FBS-based medium, respectively. DCs grown in AutoPl media 
      showed a CD80high CD83+ CD86high CD14neg HLA-DR+ CD1aneg phenotype, while 
      FBS-generated DCs exhibited a CD80high CD83+ CD86high CD14neg HLA-DR+ CD1ahigh 
      phenotype. The apoptosis rate in both culture conditions increased from 10% to 
      25% over 1 week. AutoPl-generated DCs were shown to be equally strong stimulators 
      for proliferation of allogeneic T lymphocytes as FBS-generated DCs. In addition, 
      the capacity to migrate in response to macrophage inflammatory protein-1alpha 
      (MIP-1alpha) and stromal-cell-derived factor 1alpha (SDF-1alpha) was similar in 
      both groups, whereas the response to MIP-3beta was reduced in AutoPl-derived 
      cells. Zymography analysis of supernatants from 5-day-old cultures demonstrated 
      that AutoPl-generated DCs produced higher amounts of matrix metalloproteinases, 
      suggesting that they have an enhanced capability to traffic through peripheral 
      tissues. CONCLUSIONS: Our findings indicate that plastic-adherent peripheral 
      blood cells, when cultured with GM-CSF, IL-4, c-kit-ligand and TNF-alpha in 
      autologous human plasma-supplemented media, are a potent source of functional DCS 
      that may be of value for human therapy.
FAU - Moldenhauer, A
AU  - Moldenhauer A
AD  - Institut fur Transfusionsmedizin, Charite, Humboldt-Universitat, Berlin, Germany. 
      anja.moldenhauer@charite.de
FAU - Nociari, M M
AU  - Nociari MM
FAU - Dias, S
AU  - Dias S
FAU - Lalezari, P
AU  - Lalezari P
FAU - Moore, M A S
AU  - Moore MA
LA  - eng
GR  - CA-08748/CA/NCI NIH HHS/United States
GR  - CA-75192/CA/NCI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - England
TA  - Vox Sang
JT  - Vox sanguinis
JID - 0413606
RN  - 0 (Culture Media)
RN  - 0 (Stem Cell Factor)
RN  - 0 (Tumor Necrosis Factor-alpha)
RN  - EC 3.4.24.- (Metalloendopeptidases)
SB  - IM
MH  - Blood Cells/cytology
MH  - Cell Culture Techniques/*methods
MH  - Culture Media/pharmacology
MH  - Dendritic Cells/*cytology/metabolism/physiology
MH  - Humans
MH  - Immunophenotyping
MH  - Metalloendopeptidases/metabolism
MH  - Monocytes/*cytology
MH  - Stem Cell Factor/pharmacology
MH  - Tumor Necrosis Factor-alpha/pharmacology
EDAT- 2003/04/03 05:00
MHDA- 2003/12/24 05:00
CRDT- 2003/04/03 05:00
PHST- 2003/04/03 05:00 [pubmed]
PHST- 2003/12/24 05:00 [medline]
PHST- 2003/04/03 05:00 [entrez]
AID - 283 [pii]
AID - 10.1046/j.1423-0410.2003.00283.x [doi]
PST - ppublish
SO  - Vox Sang. 2003 Apr;84(3):228-36. doi: 10.1046/j.1423-0410.2003.00283.x.