Source
Immune and Gene Therapy Laboratory, Department of Oncology & Pathology, Cancer Centrum Karolinska Karolinska Hospital Stockholm Sweden.
Abstract
BACKGROUND:
Clinical studies require protocols where a sufficient number of well-characterized highly immunogenic DC are produced according to good manufacturing practice (GMP) guidelines.
METHODS:
In the present study, using leukapheresis products from 10 cancer patients, we validated an elutriation technology for large-scale clinical grade production of monocyte-derived DC.
RESULTS:
The elutriation method gave a very high purity (mean+/-SD) (86+/-5.3%) and recovery (66+/-10.4%) of monocytes. Specifically for the two monocyte-rich fractions (3 and 4,) the recovery was 42+/-13% of viable cells that could be further differentiated into immature DC in hydrophobic culture bags using GM-CSF and IL-4. The immature DC exhibited<1% CD83+ expression and >98% phagocytic activity. Maturation with TNF-alpha or poly I:C resulted in DC with expression of CD80+, CD86+ and HLA-DR+ (>99%) and CD83+ (80+/-11.9%), as well as producing IL-12p70 and lacking phagocytic activity (<5%). This cell product can be cryopreserved with cell viability >85% and cell recovery >80% after thawing.
DISCUSSION:
The elutriation procedure, when optimized and if the monocyte content of the starting material exceeds 5%, does not require further selection or depletion using affinity approaches.