Article
Serum-free generation of dendritic cells for clinical application in malignant glioma patients
Serumfreie Generierung dendritischer Zellen bei Patienten mit malignen Gliomen für die klinische Applikation
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Published: | May 8, 2006 |
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Objective: Vaccination with dendritic cells (DC) has emerged recently as a promising active immunotherapeutic approach for various tumors. DC are generated ex-vivo, loaded with tumor antigens and administered to patients. This strategy requires generation of functionally competent dendritic cells in sufficient numbers und purity under conditions suitable for clinical application. Here, a serum-free protocol has been established and adapted to GMP large-scale DC production for malignant glioma patients
Methods: Monocytes were enriched immunomagnetically to over 95% CD14+ purity and cultured in GMP-quality plasma-supplemented X-Vivo 15 standard medium (XV15) or serum-free CellGroDC medium (SF) in 24-well plates with GM-CSF and IL-4 for the first 6 days, followed by an additional 3-day culture period in the presence of GM-CSF, IL-4 and TNFα, to induce DC maturation. For clinical samples, large-scale immunomagnetic CD14+ enrichment was performed on a CliniMACS, cells were grown in a closed Teflon culture bag system and the maturation cocktail of cytokines included GM-CSF, IL-4, TNFα, IL-1β, IL-6 and PGE2. Resulting DC were characterized by flow cytometry and functionally.
Results: DC generation under SF culture conditions outperformed the standard plasma-supplemented medium. There was a lower frequency of residual CD14+ monocytes (1.7±0.7% vs. 15.1±4.4%) and a higher frequency of cells expressing the mature DC marker CD83 (69.7±5.5% vs. 32.9±7.8%). Expression of co-stimulatory molecules was slightly elevated and substantial expression of CD1a was observed only in SF cultures. For both culture systems, DC showed the morphology, HLA-class II translocation and antigen-uptake activity of bona fide DC. Mature DC potently induced naïve T-cell proliferative and TH1-cytokine (IL-2, IFNγ) responses. Levels were higher for cells grown in SF medium. For clinical samples (n=3), monocytes were enriched from leukapheresis products of patients to 98.0±0.05% CD14+ purity, yielding 2.6±0.3x109 monocytes. After 9 days of culture, 22.6±2.7% of cells were recovered sufficient for at least 5 vaccines, with a purity of CD83+ mature DC of 84.6±2.9%.
Conclusions: We have established a procedure for serum-free ex-vivo generation of functionally competent DC in sufficient numbers and purity for clinical application.