Development of a technology platform for large-scale clinical grade production of DC.
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.


CONCLUSIONS
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.
Adamson
L
L
Immune and Gene Therapy Laboratory, Department of Oncology & Pathology, Cancer Centrum Karolinska Karolinska Hospital Stockholm Sweden.
Palmborg
A
A
Svensson
A
A
Lundqvist
A
A
Hansson
M
M
Kiessling
R
R
Masucci
G
G
Mellstedt
H
H
Pisa
P
P
D015703:Antigens, CD / Q000378:metabolism; D002478:Cells, Cultured; D015925:Cryopreservation; D046508:Culture Techniques; D003713:Dendritic Cells / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D006684:HLA-DR Antigens / Q000378:metabolism; D006801:Humans; D007136:Immunoglobulins / Q000378:metabolism; D018189:Immunomagnetic Separation; D007937:Leukapheresis; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism; D009000:Monocytes / Q000166:cytology* / Q000378:metabolism; D000097802:CD83 Antigen
D015703:Antigens, CD; D006684:HLA-DR Antigens; D007136:Immunoglobulins; D008562:Membrane Glycoproteins; D000097802:CD83 Antigen