title: Differential effects of autologous serum on CD34(+) or monocyte-derived dendritic cells.
abstract: Dendritic cells (DC) with potentially important clinical applications have been generated from human peripheral blood monocytes and CD34(+) cells in the presence of recombinant cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) + interleukin-4 (IL-4) and GM-CSF + tumor necrosis factor-alpha (TNF-alpha), respectively. Many of the studies generating DC have included fetal calf serum, which is not desirable due to the risk of immune reactions and infectious disease transmission. Additionally, low DC yields have been reported using serum-free media. In this study, we investigate supplementing serum-free media with autologous serum and plasma for DC generation from monocytes and CD34(+) cells. Our results show that functional DC can be reproducibly obtained in the presence of autologous serum using monocytes and CD34(+) cells as the starting populations. However, with the addition of autologous serum, a differential effect is observed in the phenotypic characterization of these culture-derived DC. Monocytes cultured for 7 days in X-VIVO 15 serum-free media in the presence of GM-CSF + IL-4 showed down-regulation of CD14 with increased expression of HLA-DR, mannose receptor, CD80, and CD86, along with highly up-regulated CD1a(+) expression. The addition of autologous serum to serum-free media in monocyte cultures resulted in a dose-dependent decrease in the CD1a(+) expression generating a distinct subset of CD1a(+/-) cells expressing HLA-DR, mannose receptor, CD80, and CD86. Upon stimulation with CD40L cells, both monocyte-derived DC subsets CD1a(+/-) and CD1a(++) were capable of maturation measured by CD83 and CD86 up-regulation. Data suggest the differences in the monocyte-derived DC in serum-free (CD1a(++)) or autologous serum (CD1a(+/-)) supplemented cultures is of a qualitative nature, rather than quantitative. CD1a(+) and CD14(+) cells expressing HLA-DR, mannose receptor, CD80, and CD86 were generated in 7 days from CD34(+) cells in serum-free media. A quantitative effect was obtained when cultures were supplemented with autologous serum, resulting in a significant enhancement of CD34-derived DC generated. These results demonstrate generation of DC from two different starting populations using serum-free media that can be enhanced with the addition of autologous serum. Interestingly, a differential effect was observed in the phenotypic characterization of these culture-derived DC.
authors.affiliation: Nexell Therapeutics, Inc., Cell Biology, Irvine, CA 92618, USA. mloudovaris@nexell.com
authors.forename: M
authors.initials: M
authors.lastname: Loudovaris
authors.forename: M
authors.initials: M
authors.lastname: Hansen
authors.forename: Y
authors.initials: Y
authors.lastname: Suen
authors.forename: S M
authors.initials: SM
authors.lastname: Lee
authors.forename: P
authors.initials: P
authors.lastname: Casing
authors.forename: J G
authors.initials: JG
authors.lastname: Bender
mesh_terms: D015703:Antigens, CD / Q000032:analysis / Q000187:drug effects; D018952:Antigens, CD34; D001769:Blood; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D016207:Cytokines / Q000494:pharmacology; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D006684:HLA-DR Antigens / Q000032:analysis / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D037181:Lectins, C-Type; D007959:Lymphocyte Culture Test, Mixed; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D009000:Monocytes / Q000166:cytology / Q000187:drug effects*; D010949:Plasma; D011956:Receptors, Cell Surface / Q000032:analysis / Q000187:drug effects
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D003470:Culture Media; D016207:Cytokines; D006684:HLA-DR Antigens; D037181:Lectins, C-Type; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D011956:Receptors, Cell Surface