Effects of leukapheresis protocol, cell processing and cryopreservation on the generation of monocyte-derived DC for immune therapy.
BACKGROUND
Many clinical trials of DC-based immunotherapy involve administration of monocyte-derived DCs (Mo-DC) on multiple occasion. We aimed to determine tbe optimal cell processing procedures and timing (leukapheresis, RBC depletion and cryopreservation) for generation of Mo-DC for clinical purposes.


METHODS
Leukapheresis was undertaken using a COBE Spectra. Two instrument settings were compared - the standard semi-automated software (Version 4.7) (n = 10) and the fully automated software (Version 6.0) (N = 40). Density gradient centrifugation using Ficoll, Percoll, a combination of these methods or neither for RBC depletion were compared. Outcomes (including cell yield and purity) were compared for cryopreserved unmanipulated monocytes and cryopreserved Mo-DC.


RESULTS
Software Version 6.0 provided significantly better enrichment for monocytes (P < 0.05) but 25% fewer total monocytes. Final Mo-DC purity was not influenced by leukapheresis or RBC depletion method, but was critically dependent on monocyte adherence. Version 6.0 produced significantly lower RBC and platelet contamination (P < 0.0005) but in vitro RBC depletion could not routinely be omitted. Only 5-6% of monocytes harvested resulted in Mo-DC (95% lost in cell processing or failing to differentiate).


CONCLUSIONS
Cell losses remained significant despite attempts to minimise processing steps during Mo-DC generation. Reduction in RBC and platelets achieved with software version 6.0 was insufficient to offset the disadvantage of the lower monocyte yield. Substantial savings in materials and other costs can be achieved if Mo-DC for multiple treatments are generated from cryopreserved monocytes rather than from fresh monocytes.
Department of Medicine University of Queensland Brisbane, Brisbane, Australia.
A
A
Tazbirkova
M
M
Okai
D C
DC
Horley
T M
TM
Crough
A
A
Maksoud
M
M
Nieda
A J
AJ
Nicol
D002469:Cell Separation; D015925:Cryopreservation / Q000379:methods*; D003713:Dendritic Cells / Q000378:metabolism*; D006801:Humans; D007167:Immunotherapy; D007937:Leukapheresis / Q000379:methods*