Development of a closed-system process for clinical-scale generation of DCs: evaluation of two monocyte-enrichment methods and two culture containers.

BACKGROUND Clinical immunotherapy trials using DCs depend on large-scale methods for DC generation that fulfil current good manufacturing practice requirements. Our goal was to develop data on two variables, monocyte-enrichment method and culture container, which could be used to design a closed-system process for ex vivo generation of immature DCs. METHODS Mononuclear cells were collected by leukapheresis and enriched for monocytes by either counterflow centrifugal elutriation, or immunomagnetic selection using Isolex, an automated closed-system device. Monocytes were cultured for 7 days in serum-free medium with GM-CSF and IL-4, using either plastic flasks or gas-permeable Stericell bags. Monocytes and cultured DCs were evaluated for yield, flow cytometric phenotype, and in vitro function in MLR, and autologous recall responses to tetanus toxoid and influenza virus. RESULTS Enriched monocyte products from elutriation and immunomagnetic selection were equivalent in yield and purity, and were capable of generating immature DCs in either flasks or bags. DCs from all four culture conditions were equivalent in yield, phenotype, and in vitro function. Mean DC yield was 67-80% per seeding monocyte, and 11-13% per starting mononuclear cell (MNC). A leukapheresis product containing 5 x 10(9) MNCs processed by this method could therefore yield approximately 5 x 10(8) immature DCs. DISCUSSION In this manufacturing process, the Isolex system was equivalent to elutriation, and Stericell bags were equivalent to flasks. Together, the Isolex system and Stericell bags can be incorporated into a closed-system process to generate immature DCs.

[1]  A. Donnenberg,et al.  Implementation of a semiclosed large scale counterflow centrifugal elutriation system , 1987, Journal of clinical apheresis.

[2]  Dirk Schadendorf,et al.  Vaccination of melanoma patients with peptide- or tumorlysate-pulsed dendritic cells , 1998, Nature Medicine.

[3]  F. Sallusto,et al.  Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha , 1994, The Journal of experimental medicine.

[4]  Edgar G. Engleman,et al.  Vaccination of patients with B–cell lymphoma using autologous antigen–pulsed dendritic cells , 1996, Nature Medicine.

[5]  G. Schuler,et al.  Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application. , 1999, Journal of immunological methods.

[6]  R. Steinman,et al.  Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells. , 1999, The Journal of clinical investigation.

[7]  R. Steinman,et al.  Dendritic cells and the control of immunity , 1998, Nature.

[8]  P. Gane,et al.  IL-13 induces CD34+ cells isolated from G-CSF mobilized blood to differentiate in vitro into potent antigen presenting cells. , 1997, Journal of immunological methods.

[9]  A. Anichini,et al.  Massive ex vivo generation of functional dendritic cells from mobilized CD34+ blood progenitors for anticancer therapy. , 1995, Experimental hematology.

[10]  J. Timmerman,et al.  Dendritic cell vaccines for cancer immunotherapy. , 1999, Annual review of medicine.

[11]  G. Potron,et al.  Adherent-free generation of functional dendritic cells from purified blood monocytes in view of potential clinical use. , 1998, Hematology and cell therapy.

[12]  B. Czerniecki,et al.  Calcium ionophore-treated peripheral blood monocytes and dendritic cells rapidly display characteristics of activated dendritic cells. , 1997, Journal of immunology.

[13]  D. Niederwieser,et al.  Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability. , 1996, Journal of immunological methods.

[14]  G. Schuler,et al.  Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability. , 1997, Advances in experimental medicine and biology.

[15]  K. Yoneda,et al.  Distinct subsets of dendritic cells resembling dermal DCs can be generated in vitro from monocytes, in the presence of different serum supplements. , 2000, Journal of immunological methods.

[16]  B. Mcrae,et al.  Interferon-α and -β inhibit the in vitro differentiation of immunocompetent human dendritic cells from CD14+ precursors , 2000 .

[17]  R. Herberman,et al.  A system for obtaining large numbers of cryopreserved human monocytes purified by leukapheresis and counter-current centrifugation elutriation (CCE). , 1983, Journal of immunological methods.

[18]  A. N. Park,et al.  Interferon‐α and granulocyte‐macrophage colony‐stimulating factor differentiate peripheral blood monocytes into potent antigen‐presenting cells , 1998, Journal of leukocyte biology.

[19]  M. Toungouz,et al.  Generation of immature autologous clinical grade dendritic cells for vaccination of cancer patients. , 1999, Cytotherapy.

[20]  N. Bhardwaj,et al.  A monocyte conditioned medium is more effective than defined cytokines in mediating the terminal maturation of human dendritic cells. , 1997, Blood.

[21]  J. Berzofsky,et al.  Development of a clinical-scale method for generation of dendritic cells from PBMC for use in cancer immunotherapy. , 2001, Cytotherapy.