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• W1965432868 abstract "Background We evaluated a clinical-grade protocol for the manufacture of mature DC from CD14+ precursors derived from normal donors and patients suffering from CML and stage IV malignant melanoma. We manufactured six products for CML patients and five for melanoma patients and administered them as vaccines in phase I clinical trials. Methods We isolated CD14+ cells from apheresis products by immunomagnetic separation and incubated them in X-VIVO 15™ medium supplemented with human AB serum, GM-CSF and IL-4 for 7 days, and with additional tumor necrosis factor (TNF)-α, IL-1β, IL-6 and prostaglandin E2 for 3 days. Some cells were electroporated and transfected with mRNA isolated from melanoma tissue. DC were characterized by flow cytometry for the expression ofCD83, CD86 and CD14. Results CD14+ cells constituted 14.4 ± 6.2% (mean ± SD) of nucleated cells in apheresis products and 98.3 ± 3.6% of isolated cells. Normal DC and CML DC were 77.4 ± 7.3% CD83+ and 93.5 ± 7.0% CD86+. Corresponding values for electroporated DC from melanoma patients were 66.1 ± 7.2% and 94.1 ± 7.8%. The yield of CD83+ DC from isolated CD14+ cells was 18.1 ± 7.2% for normal and CML patients and 9.8 ± 3.7% for melanoma patients. DC viability was 92.7 ± 5.8%; after cryopreservation and thawing it was 77 ± 13.5%. Discussion Our method yielded viable and mature DC free of bacteria and mycoplasma. This robust and reproducible method provides cells of consistent phenotype and viability. Cryopreservation in single-dose aliquots allows multiple DC vaccine doses to be manufactured from a single apheresis product. We evaluated a clinical-grade protocol for the manufacture of mature DC from CD14+ precursors derived from normal donors and patients suffering from CML and stage IV malignant melanoma. We manufactured six products for CML patients and five for melanoma patients and administered them as vaccines in phase I clinical trials. We isolated CD14+ cells from apheresis products by immunomagnetic separation and incubated them in X-VIVO 15™ medium supplemented with human AB serum, GM-CSF and IL-4 for 7 days, and with additional tumor necrosis factor (TNF)-α, IL-1β, IL-6 and prostaglandin E2 for 3 days. Some cells were electroporated and transfected with mRNA isolated from melanoma tissue. DC were characterized by flow cytometry for the expression ofCD83, CD86 and CD14. CD14+ cells constituted 14.4 ± 6.2% (mean ± SD) of nucleated cells in apheresis products and 98.3 ± 3.6% of isolated cells. Normal DC and CML DC were 77.4 ± 7.3% CD83+ and 93.5 ± 7.0% CD86+. Corresponding values for electroporated DC from melanoma patients were 66.1 ± 7.2% and 94.1 ± 7.8%. The yield of CD83+ DC from isolated CD14+ cells was 18.1 ± 7.2% for normal and CML patients and 9.8 ± 3.7% for melanoma patients. DC viability was 92.7 ± 5.8%; after cryopreservation and thawing it was 77 ± 13.5%. Our method yielded viable and mature DC free of bacteria and mycoplasma. This robust and reproducible method provides cells of consistent phenotype and viability. Cryopreservation in single-dose aliquots allows multiple DC vaccine doses to be manufactured from a single apheresis product." @default.
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• W1965432868 date "2004-12-01" @default.
• W1965432868 modified "2023-10-18" @default.
• W1965432868 title "Clinical-grade manufacturing of DC from CD14+ precursors: experience from phase I clinical trials in CML and malignant melanoma" @default.
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• W1965432868 doi "https://doi.org/10.1080/14653240410005357-1" @default.
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