Development of a clinical-scale method for generation of dendritic cells from PBMC for use in cancer immunotherapy

Background There is growing interest bt the use of dendritic cells (DCs) fo r treatment of malignancy and infectious disease Our goal was to develop a clinical-scale method to prepare autologous DCs for cancer clinical trials Methods PBMC were collected from normal donors or cancer patients by automa ted leukapheresis, purified by counterflow centrifugal elutriation and plac ed into culture in polystyrene flasks at 1 X 10(6) cells/mL for 5-7 days at 37 degreesC, with 5% CO2, with IL-4 and GM-CSF. Conditions investigated in cluded media formulation, supplementation with heat-inactivated allogeneic AB serum or autologous plasma and time to harvest (Day 5 or Day 7). DCs wer e evaluated for morphology, quantitative yield viability phenotype and func tion, including mixed leukocyte response and recall response to tetanus tor oid and influenza virus. Results DCs with a typical immature phenotype (CD14-negative, CD1a-positive , mannose receptor-positive, CD80-positive, CD83-negntive) were generated m ost consistently in RPMI 1640 supplemented with 10% allogeneic AB serum or 10% autologous plasma. Cell yield was higher at Day 5 than Day 7, without d etectable differences in phenotype or function. In pediatric sarcoma patien ts autologous DCs bad enhanced function compared with monocytes from which they were generated. In this patient group, starting with 8.0 +/- 3.7 x 10( 8 fr)esh or cryopreserved autologous monocytes, DC yield was 2.1 +/- 1.0 x 10(8) cells, or 29% of the starting monocyte number: Discussion In the optimized clinical-scale method, purified peripheral mono cytes are cultured for 5 days in flasks at 1 X 10(6) cells/mL in RPMI 1640, 10% allogeneic AB serum or autologous plasma, IL-4 and CM-CSF: This method avoids the use of FBS and results in in immature DCs suitable for clinical trials.