I would like to differentiate monocyte-derived dendritic cells to use as efficient APCs in vitro.
What medium would be recommended (X-vivo, RPMI etc) with which serum (human AB, FCS or none) for this? I am aware that there are many different protocols used by different labs, but wondered if anyone had specific experience with measuring APC functionality after different media/serum differentiation.
Many papers are more geared towards generating DCs for therapies and so use autologous plasma or no serum (inc. protocols from Jonuleit and Bhardwaj). Royer et al (2006) suggest that MoDC differentiated in RPMI+FCS show the highest levels of maturation markers (CD83, CD86) compared to MoDCs with human serum or autologous plasma. Are there any disadvantages to using RPMI/FCS when the DCs will only be used in vitro?
Any help would be greatly appreciated!
Thanks.
This is the protocol that we have been using for years, with good results in in vitro assays:
Human peripheral blood mononuclear cells were isolated from buffy coats from normal donors over a Lymphoprep (Nycomed Pharma, Oslo, Norway) gradient. Monocytes (>95% CD14+ cells) were cultured at 0.7 × 106 cells/mL for 5 days in RPMI 1640 supplemented with 10% fetal calf serum (complete medium) at 37°C in a humidified atmosphere with 5% CO2 containing 1000 U/mL granulocyte macrophage colony-stimulating factor (GM-CSF) (ImmunoTools; Friesoythe, Germany) and 1000 U/mL IL-4 (ImmunoTools) to generate immature MDDCs, with cytokine addition every 2 days. For maturation, MDDCs were treated with 10 ng/mL lipopolysaccharide (LPS; E. coli 055:B5; Sigma).
Hi Amy,
If your aim is to use human dendritic cells as eficient APCs , at some point you would need to show how this cells present antigens / activate T-cells. In this regard, using XVIVO medium suplemented with autologus human serum will probably give you cleaner responses than using FCS. There is always the possibility for your human DCs of presenting some bovine Ags and therefore interfering in your antigen specific responses (if that what's your going to read)
Then, using human AB serum (commercially available) might be a good choice if you want to reduce variability between different donor's serum.
I hope this helps and answers your question!
Best
Victor
Thank you both for your answers, they were very helpful. My initial readout will be antigen-specific proliferation. Do either of you know if there is a correlation between the expression of maturation markers such as CD86 (in terms of MFI, rather than simply the % of mature MDDCs) and antigen-specific stimulation of autologous T cells?
J Leukoc Biol. 2003 Apr;73(4):482-92.
Peritoneal dialysis solutions inhibit the differentiation and maturation of human monocyte-derived dendritic cells: effect of lactate and glucose-degradation products.
Puig-Kröger A1, Pello OM, Selgas R, Criado G, Bajo MA, Sánchez-Tomero JA, Alvarez V, del Peso G, Sánchez-Mateos P, Holmes C, Faict D, López-Cabrera M, Madrenas J, Corbí AL.
This a quite old paper, but we studied the effect of differentiation and maturation agents in human dendritic cells and we included antigen-specific stimulation of T-cells. Maybe can be useful.
Good luck
maybe helpful see paper. In case of measuring cytokines I would prefer X-vivo media.
Article In vitro detection of contact allergens: Development of an o...
Dear Amy,
Some technical details are never published. the difficulty to obtain immadure DC depends of several factors. first, you need to have fresh blood samples in heparin litium tube or buffy coat, and you should isolate cells rapidely, after 2 hours, monocytes became activated and decreased DC recovery. then, quality of sera is critical (low endotoxin is needed), thus many calf serum test is needed to find the better. then dc activation may be done using LPS, TNF/IL1 or CD40L, that depends about your subject and question. immature DC express low level of CD83, CD86, after activation you should verify level expression of both CD86 ad HLA-DR expressions, without high level expression of these antigen, mature DC were low efficient to induce LT proliferation.
in summary/
critical sera quality (bovin-low endotoxin, human sera-control quality/pool of AB healthy donors)
monocyte towards immature DC (5 days) (low CD14/hi CD1a/int HLA-DR/low CD86)
immature DC towards activated (2 days) (hi HLA-DR/hi CD86)
DC/LT ratio (4%, 1%, 0.5%) in autologous or allogenic T
best regards
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