Spontaneous generation and survival of blood dendritic cells in mononuclear cell culture without exogenous cytokines (original) (raw)
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Vaccine, 2003
Dendritic cells (DCs) are antigen-presenting cells that play a critical role in the induction of cytotoxic T-lymphocytes. An optimal method for the generation of DC for clinical use remains to be established. The aim of our study was to find an optimal cytokine combination for DC generation from peripheral blood stem cells (PBSC) and peripheral blood mononuclear cells (PBMC) in serum-free conditions. Serial immunophenotyping enabled us to observe changes in DC content during the culture as well as the development of maturation and activation markers. As a source for DC culture, we used PBSC from patients with multiple myeloma after stem cell mobilization using cyclophosphamide and G-CSF, or PBMC from healthy donors without mobilization. The cells were cultured in a serum-free medium with different cytokine combinations including GM-CSF, TNF-alpha, Flt-3, CD40L, IFN-gamma, IL-1alpha, IL-6, PGE1, and IL-4. The cell cultures were evaluated by immunophenotyping. For PBMC, interleukin-12 assay was performed. For PBSC, the yield of DC as determined by CD83+ cell count ranged from 0. 6 x 10(5) to 30.1 x 10(4) (mean: 9.4 x 10(4)) of DC generated per 1 x 10(6) of initially plated nucleated cells from apheresis. This yield corresponded to (0.3-19.1) x 10(5) (mean: 4.3 x 10(5)) per 1 x 10(6) of CD34+ cells in the apheresis products. For PBMC, the yield was (0.4-24.8) x 10(4) (mean: 2.4 x 10(4)) of DC generated per 1 x 10(6) of initially plated mononuclear cells from venous blood. The cultured cells expressed the mature immunophenotype. No significant differences in cell yield or immunophenotype were detected when comparing different cytokine combinations.
Transfusion, 2009
Background-To generate clinical-grade dendritic cells (DCs) ex vivo for immunotherapy trials, peripheral blood monocytes are typically cultured in GM-CSF and IL-4, and then matured using one or more agents. Duration of the initial DC culture is one important variable that has not been systematically evaluated for its effect on the characteristics of the final mature DC product. Study Design-DCs were generated from elutriated peripheral blood monocytes by incubation in medium containing 2000 units/ml each of GM-CSF and IL-4 for 3 to 7 days, followed by maturation with LPS and IFNγ. DC yield, viability, flow cytometric phenotype, and cytokine production were evaluated. Results-The % yield and viability of mature DCs were similar after initial culture durations of 3 or 7 days. Mature DCs expressed abundant CD80, CD86, CD83, and CCR7 regardless of initial culture duration, but 3-day DCs expressed these antigens in a more consistent and homogenous manner. Mature DCs produced much more IL12 and less IL10 in response to restimulation with CD40L when the initial culture duration was 3 days, rather than 7 days. Analogous changes were observed in mature DCs prepared using lower concentrations of GM-CSF/IL4 or when the alternative maturation cocktails poly-(I:C)/IFNγ and CD40L/IFNγ were used. Conclusion-Extended initial culture of DCs in GM-CSF/IL4 does not affect yield or viability of subsequently matured DCs, but can adversely affect their ability to homogeneously express high levels of costimulatory molecules and to produce IL12.
Folia histochemica et cytobiologica / Polish Academy of Sciences, Polish Histochemical and Cytochemical Society, 2005
Culture medium or medium supplement is one of the factors responsible for dendritic cell (DC) generation, but little is known about the influence of various media on DC culture. In our study we generated DC from adherent monocytes of human peripheral blood in the presence of GM-CSF, IL-4 and TNF-alpha. The following culture media were used: RPMI 1640 supplemented with 2% human serum albumin; RPMI 1640 supplemented with 2% TCH serum replacement; X-VIVO 15 and Panserin 501. Flow cytometry analysis revealed that in all media cells were CD83+ and lost CD14. Interestingly, the use of Panserin and RPMI with albumin preferentially gave rise to CD1a+ DC, whereas in X-VIVO and RPMI with TCH we observed both CD1a+ and CD1a-. Our results showed that RPMI with TCH yielded the highest percentage of cells expressing both CD80 and CD86 molecules and, in contrast to other media, the higher percentage of CD86+ cells in comparison to CD80+ cells.
Journal of Immunological Methods, 1996
We have investigated an improved method for generating sizable numbers of mature dendritic cells from nonproliferating progenitors in human blood. The procedure uses 1% human plasma in the place of 10% fetal calf serum and involves two steps. The first step or 'priming' phase is a 6-7 day culture of T cell depleted mononuclear cells in medium supplemented with GM-CSF and IL-4. The second step or 'differentiation' phase requires the exposure to macrophage conditioned medium. This medium cannot be replaced by several known cytokines such as TNF-(u, IL-l, IL-6, IL-12 and IL-15, and cannot be inhibited with neutralizing antibodies to IL-1, TNF-o, IL-6 or IL-12 alone, or in combination. Using this two-step approach, we obtain substantial yields. About 1-3 X lo6 mature dendritic cells are generated from 40 ml of blood vs. < 0.1 X 1 O6 from noncytokine treated blood. The dendritic cells derive from progenitors found primarily in a radioresistant population of CD14+ and adherent blood mononuclear cells and have all the features of mature cells. They include a stellate cell shape, nonadherence to plastic, and very strong T cell stimulatory activity. Strong APC function was evident for both the proliferation of allogeneic T cells in the MLR, and the generation by syngeneic T cells of class I restricted, CTL responses to influenza virus. A panel of dendritic cell restricted markers is also expressed, including CD83, ~55, and perinuclear CD68. All of these dendritic cell properties are retained for at least 3 days when the cytokines are removed, suggesting that these populations are stable and terminally differentiated. We suggest that these cells will be effective in vivo as adjuvants for active immunotherapy.
Generation and Maturation of Human Monocyte-derived DCs
BIO-PROTOCOL, 2014
Dendritic cells (DC) are antigen-presenting cells, which play a critical role in the regulation of the adaptive immune response. They act as a bridge between the innate and the adaptive immune systems. An approach to study their function and potentiality is to generate DClike cells by culturing CD14 + monocyte-enriched peripheral blood mononuclear cells (PBMC). In the presence of GM-CSF and IL-4, these cultures give rise to large numbers of DC-like cells. Generating human-DC from PBMC is a useful tool to study biological functions of human DC.
Single step enrichment of blood dendritic cells by positive immunoselection
Journal of Immunological Methods, 2003
Dendritic cells (DC) for cancer immunotherapy protocols are generated most commonly by in vitro differentiation of monocytes with exogenous cytokines (Mo-DC). However, Mo-DC differ in their molecular phenotype and function from blood DC (BDC). Clinical isolation of BDC has been limited to the use of density gradients, which result in low yields of variable purity. We have developed a DC enrichment platform, which uses the CMRF-44 (IgM) or CMRF-56 (IgG) monoclonal antibodies (mAb) to select BDC that express these antigens after a short overnight incubation. After culture of peripheral blood mononuclear cells (PBMC) in autologous/AB serum, biotinylated CMRF-44 was used to select DC in a single step immunomagnetic bead procedure; this produced populations containing up to 99% CMRF-44 + cells, including up to 67% CMRF-44 + CD14 À CD19 À DC, from an initial starting population of approximately 0.5%. We observed consistent differences in the purities obtained from individual donors with a mean of 54% CMRF-44 + cells (range 19 -99%). Similar results were obtained using biotinylated CMRF-56 mAb, an antibody identifying a comparable population in cultured PBMC. We recovered an average of 54% and 66% of the available BDC in separations performed with the CMRF-44 and CMRF-56 mAb, respectively. The reproducibility of the procedure and the ability to perform it in a closed sterile system makes it suitable for clinical use. Larger scale preparations starting from apheresis derived PBMC will produce sufficient BDC for immunotherapy protocols. The purified BDC elicited strong allogeneic mixed leukocyte reactions and HLA classes II-and I-restricted antigen-specific primary immune responses. D 2002 Published by Elsevier Science B.V. 0022-1759/02/$ -see front matter D 2002 Published by Elsevier Science B.V. PII: S 0 0 2 2 -1 7 5 9 ( 0 2 ) 0 0 4 2 9 -5
Differentiation of Monocyte Derived Dendritic Cells in Serum Free Conditions
The Medical Journal of The Islamic Republic of Iran, 1988
Human peripheral blood monocytes (HPBM) were cultured in the absence of human serum and were converted into a state exhibiting a high accessory function expressed by their ability of supporting lymphocyte proliferation. After a prolonged culture in serum free media the monocyte derived cells were highly viable, increased in size and developed veils and dendritiform elongatio'l1s. Paralleling the increase in accessory function, the cells decreased in the expression of markers typical of monocytes and macrophages (M 0), approaching the phenotype of lymphoid dendritic cells. We here define conditions for reproducibly generating these monocyte derived dendritic cells (m-DC) in various serum free media. This study also shows that acidic conditions prevent M0 development and facilitate m-DC differentiation even under serum conditions. MJlR1, Vo/,2, No.2, 91-98,1988
Biotechnic & histochemistry : official publication of the Biological Stain Commission, 2015
Dendritic cells (DC) are antigen-presenting cells (APC) that are important for innate and acquired immune responses. Owing to their involvement in autoinflammation, autoimmunity and cancer, DC are useful cellular models for biomedical research. Appropriate DC production in vitro could aid the study of DC in many human diseases. We used fluorochrome-based flow cytometry assays to analyze the effects of culture period and maturation of monocyte-derived DC (MoDC) on their viability and necrosis, purity, CD11c expression and phagocytic capacity. The morphological changes that occur as purified monocytes become DC were assessed at 24 and 72 h, and 6 and 9 days in culture. The dynamics of certain cell surface markers of monocytes and mature MoDC (mMoDC) also were assessed using fluorescence-based assays. We found that day 6 of culture yielded the most functional immature MoDC (iMoDC) with maximal viability, purity, CD11c expression and appropriate phagocytic capacity. Mass production of v...