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

Abstract：

BACKGROUND:There is growing interest in the use of dendritic cells (DCs) for 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 automated leukapheresis, purified by counterflow centrifugal elutriation and placed into culture in polystyrene flasks at 1 x 10(6) cells/mL for 5-7 days at 37 degrees C, with 5% CO(2), with IL-4 and GM-CSF. Conditions investigated included media formulation, supplementation with heat in activated allogeneic AB serum or autologous plasma and time to harvest (Day 5 or Day 7). DCs were evaluated for morphology, quantitative yield, viability, phenotype and function, including mixed leukocyte response and recall response to tetanus toxoid and influenza virus. RESULTS:DCs with a typical immature phenotype (CD14-negative, CD1a-positive, mannose receptor-positive, CD80-positive, CD83-negative) were generated most 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 detectable differences in phenotype or function. In pediatric sarcoma patients, autologous DCs had enhanced function compared with monocytes from which they were generated. In this patient group, starting with 8.0 +/- 3.7 x 10(8) fresh 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 monocytes 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 GM-CSF. This method avoids the use of FBS and results in immature DCs suitable for clinical trials.

journal_name

Cytotherapy

journal_title

Cytotherapy

authors

Wong EC,Maher VE,Hines K,Lee J,Carter CS,Goletz T,Kopp W,Mackall CL,Berzofsky J,Read EJ

doi

10.1080/146532401753156377

keywords:

subject

Has Abstract

pub_date

2001-01-01 00:00:00

pages

19-29

issue

eissn

1465-3249

issn

1477-2566

pii

S1465-3249(01)70960-7

journal_volume

pub_type

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