Hi Elin,

to my knowledge 5-6 days were sufficient for DC differentiation (some people in my lab were also changing half of the medium at day 3 , and as you said, without doubling the quantity of the cytokines). After 7 days of differentiation, i don't think you need to add further cytokines, but GMCSF seems important for their viability. Also, I agree with John's comments, I think you might adapt your differentiation protocol depending on what gives you the better differentiation rate, but also depending on what is your readout (ie you want to study DC fonctions or just want to use them as APC to prime T cells). In my opinion a good thing to do is to look at CD14 expression that should disappear with DC differentiation; this should give you an idea of the differentiation rate. Also, as CD83 is a DC-specific maturation marker, you might verify that your matured DC espress it (by the use of LPS as a positive control for exemple). A good thing to keep in mind is that the yield of DC differentiation will greatly vary between different FCS lots.

Concerning the adherent cells, to my knowledge it might be monocytes. Using the cells that are not adherent should be fine. But in the mouse, after the differentiation process, most of the immature DC are adherent, while the mature DC are in the supernatant. So again, you should look at CD14 expression and perhaps CD83 on the 2 fractions (adherent vs non-adherent). In principle, the more you manipulate DCs, the more they will get matured, but it is up to you, depending on what you expect.

Hope it helps!

romain

Hi Elin,

regarding the concentration of IL4 and GM-CSF you should not double the concentration of IL-4 and GM-CSF when changing half the medium. Just use the normal supplemented medium used at d1. According to my experience adhering cells have not fully differentiated into moDCs so I would focus on the loosely detached cells. It might be worth, though, to compare the firm adhering cells to the loose fraction but they should rather have a moacrophage-like phenotype.

Good luck with your project. Henning

Hi Elin,

As you probably noticed from the answers, there is no definitive answer to your question! DCs are extremely sensitive to the culture conditions and though you will get cells that you can characterize as DCs from many different protocols, each one of them will be slightly different. You will need to decide what is your "target cell" and check your protocol accordingly. In general, in our cultures, the addition of cytokines along the culture period do not affect significantly the phenotype of the cells - but the culture medium, the anticoagulant during blood collection and very small changes in pH do!

Good luck with your project,

Alex

Hi all,

Thank you so much for your advices. I understand that there is not ONE DC differentiation protocol but rather it is a matter of finding the one that works for you. My readout is CD83, HLA DR and CD86 and eventually mixed lymphocyte reaction. I will have a look at the different fractions (adhered cells vs non adherent).

Thanks again!

Hi Elin,

There are DC differentiation and DC maturation.

There are some parameters which are important during these culture and activation processes. I would refer to the paper that we published in 2006 in Scandinavian Journal in Immunology, PJ Royer as the first author. I added this full paper in my ResearchGate personnal windows. You have access to this paper.

Good luck for your research, and contact me if you need more details.

Hi Elin,

there are a lot of publications about various DC differentiation and maturation protocols. While culture time for differentiation has traditionally been 5 to 6 days, as you seem to be trying to do, it has been shown that 24 to 48 hours with IL-4 and GM-CSF are sufficient (Dauer et al., J Immunol 2003). What matters the most with respect to DC characteristics, certainly is the "DC maturation" during the last 24 to 48 hours of the culture. The gold standard has long been the combination of TNF, IL-1beta, IL-6 and PGE2 (Jonuleit et al., Eur J Immunol 1997), but depending on what you want to achieve with your DCs, other protocols might be far better. Within the next days to weeks, a publication of mine comparing the above mentioned Jonuleit-DCs with TLR-matured DCs will appear in PLoS One (Lichtenegger FS et al.). This kind of TLR-DCs has been developed in our lab (Spranger et al., J Immunol 2010) and will be used in a clinical study soon.

Hope this helps and good luck with your research!

Many questions, some answers:

a) Concentration of cytokines is dependent on the source/supplier of GM-CSF. The “amount” of cytokine is not relevant, the biologic activity is important! If possible, use IU/ml (international units). 200 IU/ml GM-CSF + 200 IU/ml IL-4 are enough. For detail information sees: Dendritic cell immunotherapy: mapping the way. Figdor CG, de Vries IJ, Lesterhuis WJ, Melief CJ. Nat Med. 2004 May;10(5):475-80. Review. A DC protocol used for clinical application of DC assured normally the best quality of DC. I have published a protocol in 1997: Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions. Eur J Immunol. 1997 Dec;27(12):3135-42.

b) The principles of monocyte-derived DC: GM-CSF is added as a general growth factor, essential for all conventional DC. IL-4 inhibits the dominant differentiation pathway into macrophages. Only a part of plastic-adherent monocytes (use 6-well-plates instead of flasks!) differentiate in presence of GM.CSF + IL-4 into immature DC. Therefore, at day 7, the non-adherent cells only should be transferred on new plates.

c) Terminal differentiation of DC needs additional stimulation (cytokine cocktail or TLRL combination)! TNF-a only is not able to induce a stable dendritic phenotype (see N. Romani, original protocols). A simple test: cytokine washout: culture the resulting cells without cytokines for additional 2 days. Only non-adherent, CD80/CD83+ cells with dendritic morphology are real conventional DC.

d) DC are professional APC (see literature of RM Steinman). These cells are very strong T cell stimulators. Therefore, they have to die a few days after arrival in the lymph node. Yes, terminal differentiated (in contrast to tissue-resistant immature DC) die.

I'd just add a point that monocyte-derived DCs are just that, DCs derived in culture that resemble 'Tip DCs' or 'inflammatory DCs'. So much work focuses on these DCs because they are easy to get, but it may be a bit like the guy in the Rumi story who drops and loses his keys in a dark place but looks for them under a lamppost because that 'is where the light is'. Murine splenic DCs come in at least 3 or 4 functional flavors, yet precious little is known about human DC subsets functioning in lymphoid organs, the gut etc. So all this talk about the best way to get monocyte-derived DCs is fine, but how far can you generalize your data to DCs in vivo?

Hi Edward,

You are right, but the question is: What is your aim?

If you want to use or characterize the best DC subset for vaccination of tumor patients or inhibition of autoimmune disease, monocyte-derived DC are the gold standard, homogeneous population clear functional properties, extreme T cell stimulators after terminal differentiation (tumor vaccine), good protocols for tolerogenic moDC also available (see all the publication von Steinman, Schuler, Romani, Steinbrink…) an most important: large amounts of high quality syngenic DC can be easily generated for the patients under GMP conditions.

We have also sorted human DC subsets (CD1a+ DC, CD141+ DC, CD16+ DC) from peripheral blood. All subsets showed significant less capacity to induce T cell proliferation and differentiation. However, all signals (cytokines, TLRL) described to induce terminal differentiation of moDC are also effective in activation of peripheral DC directly ex vivo.

Nevertheless, one of our important research activities is the generation and improvement of humanized mice that hopefully allows a better understanding of human DC subsets in vivo. Since mice are not man.

Hi Elin,

Sorry but I can't give you any answer about your question. My subject of interest is actually "parasitology" and "animal behavior". I didn't no more continue in the immunoparasitology. I've just studied immunology for my master, and I later oriented my research on animal parasitology, specially about lemurs' parasites.

Best regards

Hi Elin,

The adherence of monocytes to plastic is dependent on the culture disk. We have tested different suppliers 10 years ago (in collaboration with a pharmaceutical company). The result: Coster are the best one for DC differentiation.

Additionally, if you want to use the DC for cancer patient treatment, you should use X-VIVO-15 (GMP-media).

The best assay to test the quality of mature moDC is the washout (published by N. Romani et al.). Transfer of non-adherent cells into fresh medium without any cytokines. After 24 h, only non-adherent cells are really mature and stable DC that allows the stimulation of allogeneic TC in a ration of 1:400 (DC:TC ratio).

Hi Elin,

to obtain undifferentiated monocyte derived dendritic cells (MoDC), I use a CD14 positive magnetic selection kit (Myltenyi). The medum is RMPI+ FBS (10%) supplemenented with 100ng/ml GMCSF and 50ng/ml IL4. This medium is changed (totally changed) every 2-3 days. These cells are very sensitive and could adhere without any stiùmulation. However, I observed a very important aadherence when the cells are activated (or diffrentiated) with LPS or IL-1 beta cytokine. To detach the cells (cultured in 6 well plates), I use a 1000µl micropette. 

Adherent cells form cytoplasmic extensions that are charateristic .

best regards

Olivier

Can I just ask why various people are saying not to double up the cytokine concentrations for a half feed? I was always under the impression you should double up the concentration so the overall concentration at the end would be the same as the original set up. Is it because there will still be a good amount of cytokines present? Thanks in advance.

Hi Elin

I wouldn't double the cytokines on media exchange. The key is not the quantity, but the biological activity on the c.o.a (and this can vary from lot-lot). If your yield is Ok, stick with normal amounts, but consider adding on day 0 instead of waiting a day. This will help drive the CD14+ to CD11+ and increase the DC/monocyte ratio.

Don't add cytokines on maturation with TNFa. You want to drive genes towards activation (CD80/83/86, MHC Class II, etc.), not mitosis and terminal differentiation, if you want a population of DC that can activate CTL. 

Try using TrypLE select (see link), to gently dislodge the mature DC without damaging them. You can then pipette them easily to a new flask or dish.

Hope these are helpful!

Bruce 

https://www.thermofisher.com/order/catalog/product/12563011

I developed threes protocols for culturing DC cells from human blood monocytes. 

 I am in great trouble, so it is kind of you if you could give some instructions.

Protocol A: Human monocytes were treated with 1000 U/ml GM-CSF (R&D) and 500 U/ml IL-4 (R&) for 48h.

Then the following cocktails for added to cells for 24 h:

10000 U/ml IL-1β(R&D); 1000 U/ml TNF-a(R&D); 1000 U/ml INF-γ(R&D); 10 ng/ml LPS (irradiated, Sigma);

2.5µg/ml R848 (Selleckchem); 500 ng/ml CD40L (Peprotech).

With protocol A, I got the most DC cells, and the cell viability is 97%.

Protocol B: Human monocytes were treated with 1000 U/ml GM-CSF (R&D) and 800 U/ml IL-4 (R&) for 48h.

Then the following cocktails for added to cells for 24 h:

1000 U/ml IL-1β(R&D); 500 U/ml TNF-a(R&D); 1000 U/ml INF-γ(R&D); 10 ng/ml LPS (irradiated, Sigma);

2.5µg/ml R848 (Selleckchem); 1000 ng/ml CD40L (Peprotech); 20µg/ml poly(I:C) (non-irradiated, Sigma).

With protocol B, the cell yield was half of that from protocol A, and the cell viability is 67%.

Protocol C: Human monocytes were treated with 1000 U/ml GM-CSF (R&D) and 800 U/ml IL-4 (R&) for 48h.

Then the following cocktails for added to cells for 24 h with R848 added within the last 20h:

1000 U/ml IL-1β(R&D); 500 U/ml TNF-a(R&D); 1000 U/ml INF-γ(R&D); 10 ng/ml LPS (irradiated, Sigma);

2.5µg/ml R848 (Selleckchem); 1000 ng/ml CD40L (Peprotech); 20µg/ml poly(I:C) (non-irradiated, Sigma).

With protocol C, the cell yield was half of that from protocol A, and the cell viability is 57%.

I do not know why so many cells died in both protocol B and C.

Best!

Xinfeng