OM-197-MP-AC adjuvant properties: the in vitro maturation of normal and leukemic dendritic cells in a serum-free culture model

The effect of hydrocortisone on the in vitro maturation of human foetal kidney was investigated. Following legal therapeutic abortions, explants of renal cortex from foetuses aged 13–18 weeks were cultured for 5 days in serum-free Leibovitz's L-15 medium at 37 °C in a mixture of 95% air – 5% CO2, without hormone (controls) or with hydrocortisone at concentrations of 12.5, 25, or 50 ng/mL, which are the levels representative of different gestational periods. During the studied period of culture, the overall architecture of the renal structures was preserved without any evident signs of nephrogenesis induced by hydrocortisone. DNA synthesis was measured by incorporation of [3H]thymidine and was stimulated on day 5 by 80% with the addition of hydrocortisone at 12.5 ng/mL, and by 131% with 50 ng/mL. In autoradiograms, the sites of [3H]thymidine incorporation were the same after hydrocortisone addition, but the number of labelled nuclei was higher in 5-day explants supplemented with hydrocortisone at 50 ng/mL. The activities of some brush border enzymes (leucylnaphthylamidase, maltase, and alkaline phosphatase) were not influenced by hydrocortisone when compared with controls. Trehalase activity was decreased on day 5 with 12.5 and 50 ng/mL. A concentration of 12.5 ng/mL diminished γ-glutamyltransferase activity by 29% on day 5. The incorporation of [3H]leucine into proteins was not influenced by any concentration of the glucocorticoid hormone. This study indicates that hydrocortisone directly influences cell proliferation and certain brush border enzymic activities in human developing kidney maintained in organ culture.Key words: hydrocortisone, organ culture, human foetus, kidney, development.

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Experimental attempt to produce mRNA transfected dendritic cells derived from enriched CD34+ blood progenitor cells

Open Medicine ◽

10.2478/s11536-007-0072-9 ◽

2008 ◽

Vol 3 (1) ◽

pp. 21-28

Author(s):

Paula Lazarova ◽

Gunnar Kvalheim ◽

Liana Gercheva ◽

Krassimir Metodiev

Keyword(s):

Prostate Cancer ◽

Dendritic Cells ◽

Progenitor Cells ◽

Cultured Cells ◽

Ex Vivo ◽

High Dose ◽

Serum Free ◽

Gm Csf ◽

Cd34 Cells

AbstractIt Peripheral blood progenitor enriched CD34+ cells (PBPC) are rather often used as stem cell background in cancer patients following high dose therapy. Keeping in mind that precursor dendritic cells (DCs) originate from haematopoietic progenitor cells, purified CD34+ cells might also serve as starting cells for ex-vivo production of DC. The aim of the present study is to develop a clinical grade procedure for ex-vivo production of DC derived from enriched CD34+ cells. Various concentrations of CD34+ cells were grown in gas-permeable Teflon bags with different serum-free and serum-containing media supplemented with GM-CSF, IL-4, TNF-a, SCF, Flt-3L and INF-a. Serum-free CellGroSCGM medium for 7 days followed by CellGroDC medium in 7 days gave equal results as serum-containing medium. Following incubation, the cultured cells containing immature DCs were concentrated and transfected with tumour mRNA from human prostate cancer cell lines employing a highly efficient electroporation procedure. Thawed transfected DCs were able to elicit primary T-cell responses in vitro against antigens encoded by the prostate cancer mRNA as shown by ELISPOT assay using mock-transfected DCs as control. The results of our study show that frozen enriched CD34+ cells can be an alternative and efficient source for production of DCs for therapeutic purpose.

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Poor In Vitro Maturation and Pro-Inflammatory Cytokine Response of Dendritic Cells in Children at Genetic Risk of Type 1 Diabetes

Scandinavian Journal of Immunology ◽

10.1111/j.0300-9475.2004.01521.x ◽

2004 ◽

Vol 60 (6) ◽

pp. 647-652 ◽

Cited By ~ 22

Author(s):

S. Skarsvik ◽

M. Tiittanen ◽

A. Lindstrom ◽

R. Casas ◽

J. Ludvigsson ◽

...

Keyword(s):

Type 1 Diabetes ◽

Dendritic Cells ◽

Genetic Risk ◽

In Vitro Maturation ◽

Inflammatory Cytokine ◽

Cytokine Response ◽

Inflammatory Cytokine Response

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flt3 Ligand in Cooperation With Transforming Growth Factor-β1 Potentiates In Vitro Development of Langerhans-Type Dendritic Cells and Allows Single-Cell Dendritic Cell Cluster Formation Under Serum-Free Conditions

Blood ◽

10.1182/blood.v90.4.1425.1425_1425_1434 ◽

1997 ◽

Vol 90 (4) ◽

pp. 1425-1434 ◽

Cited By ~ 11

Author(s):

Herbert Strobl ◽

Concha Bello-Fernandez ◽

Elisabeth Riedl ◽

Winfried F. Pickl ◽

Otto Majdic ◽

...

Keyword(s):

Dendritic Cells ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β1 ◽

Langerhans Cell ◽

Flt3 Ligand ◽

Serum Free ◽

Cd34 Cells ◽

Serum Free Conditions ◽

Tgf Β1

Using a recently described serum-free culture system of purified human CD34+ progenitor cells, we show here a critical cooperation of flt3 ligand (FL) with transforming growth factor-β1 (TGF-β1) in the induction of in vitro dendritic cell/Langerhans cell (DC/LC) development. The addition of FL to serum-free cultures of CD34+ cells supplemented with TGF-β1, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor α, and stem cell factor strongly increases both percentages (mean, 36% ± 5% v 64% ± 4%; P = .001) and total numbers (4.4- ± 0.8-fold) of CD1a+ dendritic cells. These in vitro-generated CD1a+ cells molecularly closely resemble a particular type of DC known as an epidermal Langerhans cell. Generation of DC under serum-free conditions was found to strictly require supplementation of culture medium with TGF-β1. Upon omission of TGF-β1, percentages of CD1a+ DC decreased (to mean, 10% ± 8%; P = .001) and, in turn, percentages of granulomonocytic cells (CD1a− cells that are lysozyme [LZ+]; myeloperoxidase [MPO+]; CD14+) increased approximately threefold (P < .05). Furthermore, in the absence of TGF-β1, FL consistently promotes generation of LZ+, MPO+, and CD14+ cells, but not of CD1a+ cells. Serum-free single-cell cultures set up under identical TGF-β1– and FL-supplemented culture conditions showed that high percentages of CD34+ cells (mean, 18% ± 2%; n = 4) give rise to day-10 DC colony formation. The majority of cells in these DC-containing colonies expressed the Langerhans cell/Birbeck granule specific marker molecule Lag. Without TGF-β1 supplementation, Lag+ colony formation is minimal and formation of monocyte/macrophage-containing colonies predominates. Total cloning efficiency in the absence and presence of TGF-β1 is virtually identical (mean, 41% ± 6% v 41% ± 4%). Thus, FL has the potential to strongly stimulate DC/LC generation, but has a strict requirement for TGF-β1 to show this costimulatory effect.

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In vitro co-culture model of human monocyte-derived dendritic cells and T cells to evaluate the sensitization of dinitrochlorobenzene

Ecotoxicology and Environmental Safety ◽

10.1016/j.ecoenv.2021.112331 ◽

2021 ◽

Vol 220 ◽

pp. 112331

Author(s):

Lei Bao ◽

Changfu Hao ◽

Juan Wang ◽

Feifei Guo ◽

Zihan Geng ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Human Monocyte ◽

Culture Model

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Effect of striatal cells on in vitro maturation of mesencephalic dopaminergic neurones grown in serum-free conditions

Nature ◽

10.1038/288370a0 ◽

1980 ◽

Vol 288 (5789) ◽

pp. 370-373 ◽

Cited By ~ 218

Author(s):

Umberto di Porzio ◽

Marie-Christine Daguet ◽

Jacques Glowinski ◽

Alain Prochiantz

Keyword(s):

In Vitro Maturation ◽

Serum Free ◽

Serum Free Conditions

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The Role of Tumor Necrosis Factor α in Modulating the Quantity of Peripheral Blood-Derived, Cytokine-Driven Human Dendritic Cells and Its Role in Enhancing the Quality of Dendritic Cell Function in Presenting Soluble Antigens to CD4+ T Cells In Vitro

Blood ◽

10.1182/blood.v91.12.4652.412a03_4652_4661 ◽

1998 ◽

Vol 91 (12) ◽

pp. 4652-4661 ◽

Cited By ~ 1

Author(s):

Bing-guan Chen ◽

Yijun Shi ◽

Jeffrey D. Smith ◽

David Choi ◽

James D. Geiger ◽

...

Keyword(s):

Dendritic Cells ◽

Tumor Necrosis ◽

Tumor Necrosis Factor Α ◽

Serum Free ◽

Enhancing Effect ◽

Pulsed Dc ◽

Factor Α ◽

And Function ◽

Necrosis Factor

Because dendritic cells (DC) are critically involved in both initiating primary and boosting secondary host immune responses, attention has focused on the use of DC in vaccine strategies to enhance reactivity to tumor-associated antigens. We have reported previously the induction of major histocompatibility complex class II-specific T-cell responses after stimulation with tumor antigen-pulsed DC in vitro. The identification of in vitro conditions that would generate large numbers of DC with more potent antigen-presenting cell (APC) capacity would be an important step in the further development of clinical cancer vaccine approaches in humans. We have focused attention on identifying certain exogenous cytokines added to DC cultures that would lead to augmented human DC number and function. DC progenitors from peripheral blood mononuclear cells (PBMC) were enriched by adherence to plastic, and the adherent cells were then cultured in serum-free XVIVO-15 medium (SFM) for 7 days with added granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). At day 7, cultures contained cells that displayed the typical phenotypic and morphologic characteristics of DC. Importantly, we have found that the further addition of tumor necrosis factor α (TNFα) at day 7 resulted in a twofold higher yield of DC compared with non–TNFα-containing DC cultures at day 14. Moreover, 14-day cultured DC generated in the presence of TNFα (when added at day 7) demonstrated marked enhancement in their capacity to stimulate a primary allogeneic mixed leukocyte reaction (8-fold increase in stimulation index [SI]) as well as to present soluble tetanus toxoid and candida albicans (10- to 100-fold increases in SI) to purified CD4+ T cells. These defined conditions allowed for significantly fewer DC and lower concentrations of soluble antigen to be used for the pulsing of DC to efficiently trigger specific T-cell proliferative responses in vitro. When compared with non–TNFα-supplemented cultures, these DC also displayed an increased surface expression of CD83 as well as the costimulatory molecules, CD80 and CD86. Removal of TNFα from the DC cultures after 2 or 4 days reduced its enhancing effect on DC yield, phenotype, and function. Thus, the continuous presence of TNFα over a 7-day period was necessary to achieve the maximum enhancing effect observed. Collectively, our findings point out the importance of exogenous TNFα added to cultures of cytokine-driven human DC under serum-free conditions, which resulted in an enhanced number and function of these APC. On the basis of these results, we plan to initiate clinical vaccine trials in patients that use tumor-pulsed DC generated under these defined conditions.

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