Effects of leukapheresis protocol, cell processing and cryopreservation on the generation of monocyte-derived DC for immune therapy

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Abstract
Many clinical trials of DC-based immunotherapy involve administration of monocyte-derived DCs (Mo-DC) on multiple occasions. We aimed to determine the optimal cell processing procedures and timing (leukapheresis, RBC depletion and cryopreservation) for generation of Mo-DC for clinical purposes. 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, Percoli, 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. 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 leultapheresis 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 T’OIItineiy be omitted. Only 5–6% of monocytes harvested resulted in Mo-DC (95% lost in cell processing or foiling to differentiate). Cell losses remained significant despite attempts to minimise processing steps during Mo-DC generation. Reduction in RBC and platekts 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.