Optimized culture conditions for the generation of dendritic cells from peripheral blood monocytes

Moldenhauer, Anja; Nociari, Marcelo; Dias, Sérgio; Lalezari, Parviz; Moore, Malcolm A.S.

doi:10.1046/j.1423-0410.2003.00283.x

Cited by 27 publications

(22 citation statements)

References 41 publications

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“…Phase-contrast microscopy of liquid cultures and light microscopy of cytospin preparations were carried out as previously described to document the morphology and maturation features of the investigated cells [29]. For cocultures studied by electron microscopy, EC were grown in 24-well plates on inserted coverslips (Thermanox; Nunc; Wiesbaden, Germany; http://www.nuncbrand.com) to confluence prior to the addition of progenitor cells with or without TNF-α.…”

Section: Morphology and Electron And Confocal Microscopymentioning

confidence: 99%

“…The apoptosis rate was lower in cocultures with direct contact to TNF-α-stimulated EC than in those without direct contact to TNF-α-stimulated EC or in absence of EC. Under in vitro conditions, fully differentiated DC usually undergo rapid apoptosis [29]. This can be delayed by treating them with TNF family members like TNF-related activation-induced cytokine (TRANCE) [54].…”

Section: Intercellular Dialoguementioning

confidence: 99%

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Tumor Necrosis Factor Alpha‐Stimulated Endothelium: An Inducer of Dendritic Cell Development from Hematopoietic Progenitors and Myeloid Leukemic Cells

et al. 2004

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Especially when exposed to inflammatory stimuli, endothelial cells (EC) have been shown to promote the maturation of monocytes into dendritic cells (DC) and the long-term proliferation of CD34 + cells by constitutive cytokine production and direct cellular contact. We therefore hypothesized that cytokine-stimulated EC would induce hematopoietic progenitor cells to develop into mature dendritic cells. To test this theory, human CD34 + cells derived from cord blood or leukapheresis products were cultured with a monolayer of either interleukin (IL)-1β β, IL-4, or tumor necrosis factor (TNF)-α α-stimulated human umbilical cord EC. The cells in suspension were analyzed weekly over a period of 6 weeks. IL-1β β supported cell expansion, whereas IL-4 had no effect on cell expansion or DC differentiation. Only TNF-α α-stimulated EC induced the development of mature, allostimulatory DC with a high expression of CD83, HLA-DR, CD1a, and costimulatory molecules like CD80 and CD86. Acute myeloid leukemia cells from the cell line Kasumi-1 also developed DC-like features when cocultured with TNF-α α-stimulated EC. Direct contact between endothelial and progenitor cells increased the number of developing DC. Cell cycle analysis and apoptosis studies demonstrated a reduced G 2 M fraction, an increased S fraction, and a decrease in TNF-α α-dependent apoptosis of DC developing in the presence of endothelial cells. As shown by electron and confocal microscopic studies, intimate interactions between EC and DC occurred, resulting in the internalization of the developing DC within the EC monolayer and a bidirectional exchange of proteins. We conclude that, via the action of TNF-α α, inflamed human endothelium can induce CD34 + and leukemic cells to differentiate into dendritic cells.

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Section: Morphology and Electron And Confocal Microscopymentioning

confidence: 99%

Section: Intercellular Dialoguementioning

confidence: 99%

Tumor Necrosis Factor Alpha‐Stimulated Endothelium: An Inducer of Dendritic Cell Development from Hematopoietic Progenitors and Myeloid Leukemic Cells

et al. 2004

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“…The FBS commonly used as a source of serum proteins in cell culture precludes the clinical application of cells thus produced, owing to the risk of xenosensitization against bovine proteins, the possible transfer of bovine diseases, and the effects of immune regulation of DCs by other substances [18,20,21]. To avoid these limitations, investigators have reported the generation of DCs under serum-free conditions or when supplemented with several human-derived proteins, such as autologous serum, autologous plasma, or human serum albumin.…”

Section: Discussionmentioning

confidence: 99%

“…However, the use of FBS introduces the risk of xenosensitization against bovine proteins, as well as the possible transfer of bovine diseases such as bovine spongiform encephalopathy [20]. Alternatively, serum-free media have been used to generate DCs for clinical immunotherapy.…”

Section: Introductionmentioning

confidence: 99%

Optimization of the concentration of autologous serum for generation of leukemic dendritic cells from acute myeloid leukemic cells for clinical immunotherapy

et al. 2006

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Clinical application of immunotherapy for acute myeloid leukemia (AML) requires the efficient induction of dendritic cells (DCs) from AML blast cells using in vitro culture. We examined the effect of autologous serum on the properties of leukemic DCs derived from leukemic cells of AML patients by culture in AIM-V medium with GM-CSF, IL-4, TNF-alpha, and 0, 2, 5, or 10% human autologous serum. The expressions of CD80, CD83, CD86, and HLA-DR were upregulated under all culture conditions; however, 10% autologous serum induced the highest expression levels of several molecules. The capacity of leukemic DCs to stimulate allogeneic T cells increased with increasing serum concentration. Stimulation of autologous CD3(+) T cells with leukemic DCs grown in the presence of various concentrations of autologous serum resulted in induction of more IFN-gamma-secreting cells than was the case for unprimed CD3(+) T cells. Leukemic DCs cultured with 10% autologous serum induced the highest numbers of IFN-gamma-secreting cells and CD8(+)CD56(+) T cells from autologous T cells. These results suggest that culture of AML blast cells in the presence of autologous serum could be used to generate leukemic DCs for immunotherapy against AML. The highest serum concentration appeared optimal for generating the most potent leukemic DCs.

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“…Cytokines and many Toll-like receptor ligands are now available in GMP quality. Fetal calf serum (FCS) was part of initial DC protocols, but DCs are now mostly generated in the absence of FCS using autologous plasma [26] or in serumfree cultures [27] in order to avoid unwanted loading with xenoantigens being part of FCS [28] . A future approach to the safe clinical use of DCs might be closed culture system.…”

Section: Source Type Of Dcs and Their In Vitro Manipulation For Clinmentioning

confidence: 99%

Current State and Perspectives of Dendritic Cell Vaccination in Cancer Immunotherapy

Farkas

Conrad

Tonel

et al. 2006

Skin Pharmacol Physiol

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Recent progress in the approach towards immunotherapy of cancer consists in molecular definition of tumor antigens, new tools for phenotypical and functional characterization of tumor-specific effector cells and clinical use of novel adjuvants for optimal stimulation of a cancer-specific immune response such as dendritic cells. In spite of these advances and immunological as well as clinical responses in selected patients, mechanisms involved in dendritic-cell-based cancer immunotherapy are still poorly understood. Therefore, a standardized study design and small pilot trials are needed to explore open scientific questions in future clinical trials. This review focuses on the different parameters of dendritic cell biology relevant to cancer immunotherapy and on innovative approaches to hopefully enhance the efficacy of dendritic cell vaccination.

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Optimized culture conditions for the generation of dendritic cells from peripheral blood monocytes

Abstract: Our findings indicate that plastic-adherent peripheral blood cells, when cultured with GM-CSF, IL-4, c-kit-ligand and TNF-alpha in autologous human plasma-supplemented media, are a potent source of functional DCS that may be of value for human therapy.

Cited by 27 publications

References 41 publications

Tumor Necrosis Factor Alpha‐Stimulated Endothelium: An Inducer of Dendritic Cell Development from Hematopoietic Progenitors and Myeloid Leukemic Cells

Tumor Necrosis Factor Alpha‐Stimulated Endothelium: An Inducer of Dendritic Cell Development from Hematopoietic Progenitors and Myeloid Leukemic Cells

Optimization of the concentration of autologous serum for generation of leukemic dendritic cells from acute myeloid leukemic cells for clinical immunotherapy

Current State and Perspectives of Dendritic Cell Vaccination in Cancer Immunotherapy

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