Korean J Pediatr Hematol Oncol.  2001 Oct;8(2):305-313.

Generation of Mature Dendritic Cells from Peripheral Blood

• Yoo KH
• Kim DH
• Kim SY
• Sung KW
• Koo HH

Abstract

PURPOSE: Dendritic cells (DCs) are the most potent antigen presenting cells and should be differentiated to mature form to induce primary T cell response. In this study, we intended to generate mature DCs from peripheral blood mononuclear cells (PBMCs), so that develope the basis for immunotherapy using DCs.
METHODS
PBMCs were isolated from 25 mL of normal adults' peripheral blood and evenly distributed in 5 wells of a 6-well plate. Nonadherent cells were gently aspirated after 2 hour-incubation under humidified 5% CO2 at 37degrees C. Adherent monocytes were cultured in 3 mL of 10% fetal bovine serum plus RPMI-1640 media containing granulocyte/macrophage-colony stimulating factor (GM-CSF) 200 ng/mL and interleukin (IL)-4 20 ng/mL. To assess the effect of tumor necrosis factor (TNF)-alpha and interferon (IFN)-alpha on DC maturation, either or both were added on day 4 of culture. Cells were harvested on day 4 and 7 to calculate the cell counts, CD83 /HLA-DR cells, and CD86 /HLA-DR cells.
RESULTS
On day 4, large amounts of DCs were observed. CD83 /HLA-DR cells and CD86 / HLA-DR cells were 11.6% and 16.6% of total cells counted and yields were 1.3% and 2.0%, respectively. On day 7, DCs were more frequently observed in all instances and purity ranged from 24.0% to 31.0% as a mean value. The final yields of matue DCs were 2.9~3.4% of PBMCs inoculated. Adding TNF-alpha plus IFN-alpha led to the best yield. But, IFN-alpha alone did not increase the mature DCs compared to the control.
CONCLUSION
We successfully cultured large quantities of mature DCs from PBMCs using GM-CSF and IL-4. IFN-alpha seems to have a synergistic effect when added with TNF-alpha, but further studies are required to prove the clinical significance.

Keyword

Mature dendritic cells; Peripheral blood mononuclear cells; Granulocyte/ macrophage-colony stimulating factor; Interleukin-4; Tumor necrosis factor-alpha; Interferon-alpha

MeSH Terms

Antigen-Presenting Cells
Cell Count
Dendritic Cells*
Granulocyte-Macrophage Colony-Stimulating Factor
HLA-DR Antigens
Immunotherapy
Interferon-alpha
Interferons
Interleukin-4
Interleukins
Monocytes
Tumor Necrosis Factor-alpha
Granulocyte-Macrophage Colony-Stimulating Factor
HLA-DR Antigens
Interferon-alpha
Interferons
Interleukin-4
Interleukins
Tumor Necrosis Factor-alpha