gms | German Medical Science

57. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie e. V. (DGNC)
Joint Meeting mit der Japanischen Gesellschaft für Neurochirurgie

Deutsche Gesellschaft für Neurochirurgie (DGNC) e. V.

11. bis 14.05.2006, Essen

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

Meeting Abstract

  • T. Beetz - Neurochirurgische Klinik, Universität Düsseldorf
  • corresponding author M. Rapp - Neurochirurgische Klinik, Universität Düsseldorf
  • Z. Özcan - Institut für Transplantationsdiagnostik und Zelltherapeutika
  • S. De Vleeschouwer - Neurochirurgische Klinik, Universität Düsseldorf
  • H.J. Steiger - Neurochirurgische Klinik, Universität Düsseldorf
  • P. Wernet - Institut für Transplantationsdiagnostik und Zelltherapeutika
  • R.V. Sorg - Institut für Transplantationsdiagnostik und Zelltherapeutika
  • M. Sabel - Neurochirurgische Klinik, Universität Düsseldorf

Deutsche Gesellschaft für Neurochirurgie. Japanische Gesellschaft für Neurochirurgie. 57. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie e.V. (DGNC), Joint Meeting mit der Japanischen Gesellschaft für Neurochirurgie. Essen, 11.-14.05.2006. Düsseldorf, Köln: German Medical Science; 2006. DocP 05.71

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Veröffentlicht: 8. Mai 2006

© 2006 Beetz et al.
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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.