Optimizing parameters for clinical-scale production of high IL-12 secreting dendritic cells pulsed with oxidized whole tumor cell lysate

J Transl Med. 2011 Nov 14;9:198. doi: 10.1186/1479-5876-9-198.


Background: Dendritic cells (DCs) are the most potent antigen-presenting cell population for activating tumor-specific T cells. Due to the wide range of methods for generating DCs, there is no common protocol or defined set of criteria to validate the immunogenicity and function of DC vaccines.

Methods: Monocyte-derived DCs were generated during 4 days of culture with recombinant granulocyte-macrophage colony stimulating factor and interleukin-4, and pulsed with tumor lysate produced by hypochlorous acid oxidation of tumor cells. Different culture parameters for clinical-scale DC preparation were investigated, including: 1) culture media; 2) culture surface; 3) duration of activating DCs with lipopolysaccharide (LPS) and interferon (IFN)-gamma; 4) method of DC harvest; and 5) cryomedia and final DC product formulation.

Results: DCs cultured in CellGenix DC media containing 2% human AB serum expressed higher levels of maturation markers following lysate-loading and maturation compared to culturing with serum-free CellGenix DC media or AIM-V media, or 2% AB serum supplemented AIM-V media. Nunclon™Δ surface, but not Corning(®) tissue-culture treated surface and Corning(®) ultra-low attachment surface, were suitable for generating an optimal DC phenotype. Recombinant trypsin resulted in reduced major histocompatibility complex (MHC) Class I and II expression on mature lysate-loaded DCs, however presentation of MHC Class I peptides by DCs was not impaired and cell viability was higher compared to cell scraping. Preservation of DCs with an infusible cryomedia containing Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin, and DMSO yielded higher cell viability compared to using human AB serum containing 10% DMSO. Finally, activating DCs for 16 hours with LPS and IFN-γ stimulated robust mixed leukocyte reactions (MLRs), and high IL-12p70 production in vitro that continued for 24 hours after the cryopreserved DCs were thawed and replated in fresh media.

Conclusions: This study examined criteria including DC phenotype, viability, IL-12p70 production and the ability to stimulate MLR as metrics of whole oxidized tumor lysate-pulsed DC immunogenicity and functionality. Development and optimization of this unique method is now being tested in a clinical trial of autologous oxidized tumor lysate-pulsed DC in clinical-scale in recurrent ovarian, primary peritoneal or fallopian tube cancer (NCT01132014).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Culture Techniques / methods*
  • Cell Differentiation / drug effects
  • Cell Extracts / pharmacology*
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Cryopreservation
  • Culture Media / pharmacology
  • Dendritic Cells / cytology*
  • Dendritic Cells / drug effects
  • Dendritic Cells / metabolism*
  • Humans
  • Hypochlorous Acid / pharmacology
  • Interleukin-12 / biosynthesis
  • Interleukin-12 / metabolism*
  • Lymphocyte Culture Test, Mixed
  • Oxidation-Reduction / drug effects
  • Phenotype
  • Time Factors
  • Trypsin / metabolism


  • Cell Extracts
  • Culture Media
  • Interleukin-12
  • Hypochlorous Acid
  • Trypsin

Associated data

  • ClinicalTrials.gov/NCT01132014