Generation of Mature Dendritic Cells from Peripheral Blood.
- Author:
Keon Hee YOO
1
;
Dong Hyun KIM
;
Su Yeun KIM
;
Ki Woong SUNG
;
Hong Hoe KOO
Author Information
1. Department of Pediatrics, Samsung Seoul Hospital, Sungkyunkwan University School of Medicine, Seoul, Korea. hhkoo@smc.samsung.co.kr
- Publication Type:Original Article
- Keywords:
Mature dendritic cells;
Peripheral blood mononuclear cells;
Granulocyte/ macrophage-colony stimulating factor;
Interleukin-4;
Tumor necrosis factor-alpha;
Interferon-alpha
- MeSH:
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
- From:Korean Journal of Pediatric Hematology-Oncology
2001;8(2):305-313
- CountryRepublic of Korea
- Language:Korean
-
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.
