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Development of a new protocol for 2-day generation of mature dendritic cells from human monocytes

Biological Procedures Online volume 5, pages 197–203 (2003)Cite this article

Abstract

We developed a new 2-day protocol for the generation of dendritic cells (DCs) from human monocytesin vitro. First, we demonstrated that 24 hours of culture with GM-CSF and IL-4 are sufficient to generate immature DCs capable of antigen uptake. We then compared two different strategies for DC maturation: proinflammatory mediators were either added together with GM-CSF and IL-4 from the beginning of cell culture or added after 24 hours of differentiation with GM-CSF and IL-4. After 48 hours of total culture period, expression of activation markers was more pronounced in cells generated by the 2-step differentiation and activation method. Our new protocol for 2-day DC differentiation reduces labor, cost and time and also reliably renders high numbers of mature and viable DCs.

Abbreviations

DC:

dendritic cell

moDC:

monocyte-derived DC

PBMC:

peripheral blood mononuclear cells

CD40L:

soluble CD40 ligand-trimer

References

  1. Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, Pulendran B, Palucka K. Immunobiology of dendritic cells.Annu Rev Immunol 2000; 18:767–811.

    Article  PubMed  CAS  Google Scholar 

  2. Cella M, Engering A, Pinet V, Pieters J, Lanzavecchia A. Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells.Nature 1997; 388:782–787.

    Article  PubMed  CAS  Google Scholar 

  3. Schnurr M, Then F, Galambos P, Scholz C, Siegmund B, Endres S, Eigler A. Extracellular ATP and TNF-alpha synergize in the activation and maturation of human dendritic cells.J Immunol 2000; 165:4704–4709.

    PubMed  CAS  Google Scholar 

  4. Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha.J Exp Med 1994; 179:1109–1118.

    Article  PubMed  CAS  Google Scholar 

  5. Romani N, Gruner S, Brang D, Kampgen E, Lenz A, Trockenbacher B, Konwalinka G, Fritsch PO, Steinman RM, Schuler G. Proliferating dendritic cell progenitors in human blood.J Exp Med 1994; 180:83–93.

    Article  PubMed  CAS  Google Scholar 

  6. Randolph GJ, Beaulieu S, Lebecque S, Steinman RM, Muller WA. Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking.Science 1998; 282:480–483.

    Article  PubMed  CAS  Google Scholar 

  7. Czerniecki BJ, Carter C, Rivoltini L, Koski GK, Kim HI, Weng DE, Roros GJ, Hijazi YM, Shuwen X, Rosenberg SA, Cohen PA. Calcium ionophore-treated peripheral blood monocytes and dendritic cells rapidly display characteristics of activated dendritic cells.J Immunol 1997; 159:3823–3837.

    PubMed  CAS  Google Scholar 

  8. Faries MB, Bedrosian I, Xu S, Koski G, Roros JG, Moise MA, Nguyen HQ, Engels FH, Cohen PA, Czerniecki B. Calcium signaling inhibits interleukin-12 production and activates CD83(+) dendritic cells that induce Th2 cell development.Blood 2001; 98:2489–2497.

    Article  PubMed  CAS  Google Scholar 

  9. Schnurr M, Galambos P, Scholz C, Then F, Dauer M, Endres S, Eigler A. Tumor cell lysate-pulsed human dendritic cells induce a T-cell response against pancreatic carcinoma cells: an in vitro model for the assessment of tumor vaccines.Cancer Res 2001; 61:6445–6450.

    PubMed  CAS  Google Scholar 

  10. Palucka KA, Taquet N, Sanchez-Chapuis F, Gluckman JC. Dendritic cells as the terminal stage of monocyte differentiation.J Immunol 1998; 160:4587–4595.

    PubMed  CAS  Google Scholar 

  11. Jonuleit H, Kuhn U, Muller G, Steinbrink K, Paragnik L, Schmitt E, Knop J, Enk EH. Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serumfree conditions.Eur J Immunol 1997; 27:3135–3142.

    Article  PubMed  CAS  Google Scholar 

  12. Feuerstein B, Berger TG, Maczek C, Roder C, Schreiner D, Hirsch U, Haendle I, Leisgang W, Glaser A, Kuss O, Diepgen TL, Schuler G, Schuler-Thurner B. A method for the production of cryopreserved aliquots of antigen-preloaded, mature dendritic cells ready for clinical use.J Immunol Methods 2000; 245:15–29.

    Article  PubMed  CAS  Google Scholar 

  13. Snijders A, Kalinski P, Hilkens CM, Kapsenberg ML. High-level IL-12 production by human dendritic cells requires two signals.Int Immunol 1998; 10:1593–1598.

    Article  PubMed  CAS  Google Scholar 

  14. Dauer M, Obermaier B, Herten J, Haerle C, Pohl K, Rothenfusser S, Schnurr M, Endres S, Eigler A. Mature dendritic cells derived from human monocytes within 48 hours: a novel strategy for dendritic cell differentiation from blood precursors.J Immunol 2003; 170:4069–4076.

    PubMed  CAS  Google Scholar 

  15. Santini SM, Lapenta C, Logozzi M, Parlato S, Spada M, Di Pucchio T, Belardelli F. Type I inteferon as a powerful adjuvant for monocyte derived dendritic cell development and activity in vitro and Hu-PBL-SCID mice.J Exp Med 2000; 191:1777–1788.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Division of Clinical Pharmacology, Medizinische Klinik Innenstadt, University of Munich, Ziemssenstr. 1, 80336, Munich, Germany

    Bianca Obermaier, Jan Herten, Katharina Schad & Stefan Endres

  2. Division of Clinical Pharmacology and Section of Gastroenterology, Medizinische Klinik Innenstadt, University of Munich, Ziemssenstr. 1, 80336, Munich, Germany

    Marc Dauer

  3. Abteilung fuer Klinische Pharmakologie, Medizinische Klinik Innenstadt, University of Munich, Ziemssenstr.1, 80336, Munich, Germany

    Andreas Eigler

Authors
  1. Bianca Obermaier
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  2. Marc Dauer
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  3. Jan Herten
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  4. Katharina Schad
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  5. Stefan Endres
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  6. Andreas Eigler
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Corresponding author

Correspondence to Andreas Eigler.

Additional information

B.O. and M.D. contributed equally to this manuscript.

Published: October 24, 2003

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Obermaier, B., Dauer, M., Herten, J. et al. Development of a new protocol for 2-day generation of mature dendritic cells from human monocytes. Biol. Proced. Online 5, 197–203 (2003). https://doi.org/10.1251/bpo62

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  • DOI: https://doi.org/10.1251/bpo62

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Biological Procedures Online

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