Culture of myeloid dendritic cells from bone marrow precursors

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Summary

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Cite this Article

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Boudreau, J., Koshy, S., Cummings, D., Wan, Y. Culture of myeloid dendritic cells from bone marrow precursors. J. Vis. Exp. (17), e769, doi:10.3791/769 (2008).

Abstract

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors.

Protocol

This protocol has been adapted from Lutz et al.1

Harvest and processing of bone marrow

  1. Isolation of the tibia and femur: Euthanize the donor mouse and spray the legs with 70% ethanol. Grasp the first ankle firmly with blunt forceps and begin to cut away the skin and underlying musculature to expose the tibia. To avoid damaging the bone, cut slowly and parallel to the tibia, leaving the knee joint intact.

    To clean the femur, immobilize the knee joint by grasping with blunt forceps. Clean away the musculature with a pair of sharp scissors and curved forceps. Continue upward until the hip joint is exposed and scissors can be placed between the head of the femur and the hip joint. Remove the bones by cutting between femur and hip joint and place into phosphate buffered saline (PBS) on ice.
  2. Removal of bone marrow: Clean remaining musculature from the bones using scissors and curved forceps. Cut off the epiphyses of each bone and locate the center cavity. Using a 10mL syringe loaded with PBS and a 25G0.5” needle, flush the bone marrow into a non-tissue coated petri dish.
  3. Processing of bone marrow: Use the rubber end of the plunger from a 1mL syringe to dissociate the bone marrow into a single-cell suspension (use an up and down, not scraping motion). Collect bone marrow in a conical falcon tube, and rinse residual bone marrow with PBS.

Culture of dendritic cells

  1. Resuspend cells in DC media and count DC precursors on a hemocytometer (Figure 1). You will see cells of varying sizes; the DC precursors are the largest and brightest. Differentially count only these cells. From two femurs and two tibias of a wild-type C57Bl/6 mouse (6-8 weeks old), you should expect between 25-40 x 106 total DC precursors.



    Figure 1

  2. Culture cells at a density of 2 x 105 DC precursors/mL in DC media supplemented with 40 ng/mL recombinant murine GM-CSF. Plate cells in non-tissue-coated polystyrene petri plates.

Add media (day 3)

To refresh the media, add half of the total volume of fresh media supplemented with 40 ng/mL GM-CSF.

Replace one third of the media (day 6) and maturation

To refresh the media, carefully remove one third of the total volume of media and replace this volume with fresh DC media supplemented with 40 ng/mL GM-CSF on day 6 of culture.

If desired, dendritic cells can be stimulated for maturation using cytokines or toll-like receptor ligands. In the video, DCs were matured by overnight stimulation with 5 ng/mL CpG.

Harvest of dendritic cells

DC culture is complete (Figure 2). Cells will be both in suspension and loosely adhered to the plate. Adhered cells can be removed by scraping the dish with a tissue culture scraper and rinsing with PBS. The total number of cells will increase 5-8 fold during the week-long culture and differentiation period, therefore, expect to harvest 1-1.6 x 106 cells/mL.

Figure 2

Figure 2

Reagents

  1. DC media
    1. RPMI-1640 media, supplemented with:
    2. 10% Fetal bovine serum
    3. 100 IU/mL penicillin
    4. 100 μg/mL streptomycin
    5. 1%L-glutamine
    6. 0.1% 2-mercaptoethanol
    7. 1X non-essential amino acids
    8. 1X sodium pyruvate
  2. Recombinant murine GM-CSF
    1. rmGM-CSF (Peprotech inc, New Jersey, catalogue no. 315-03)

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Discussion

DCs are useful for studies of innate and adaptive immune interactions, and can be employed as a vaccine vector. In this video, we have demonstrated the steps to isolate bone marrow and differentiate myeloid dendritic cells in vitro. Following the culture period, these cells can be visualized microscopically both as adherent cells, which often possess dendrites, and non-adherent round cells. The DCs can be further manipulated for antigen presentation by pulsing with antigen or stimulated for cytokine production and costimulatory molecule upregulation using cytokines and/or toll-like receptor ligands (for review, see Gilboa, 2008(2)).

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Acknowledgements

JB is supported by studentships from the Natural Sciences and Engineering Research Council (NSERC). Support for this project has been provided by the Canadian Institutes of Health Research, Grant MOP-67066.

Materials

Name Type Company Catalog Number Comments
recombinant murine GM-CSF Reagent Peptrotech 315-03

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References

  1. Lutz, M. B., Kukutsch, N., Ogilvie, A. L., Rossner, S., Koch, F., Romani, N., Schuler, G. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods. 223, 77-92 (1999).
  2. Gilboa, E. DC-based cancer vaccines. J. Clin. Invest. 117, 1195-1203 (2007).

Comments

14 Comments

  1. Adherent cells are macrophages not DCs. They are >95% F4/80 positive macrophages.

    Reply
    Posted by: Anonymous
    December 12, 2008 - 1:21 PM
  2. I confirm that!

    Reply
    Posted by: Imane A.
    October 10, 2012 - 6:12 AM
  3. The expression level of Class II molecules is strong? Have you tested the level of this marker in your cells?

    Reply
    Posted by: Anonymous
    January 30, 2009 - 10:27 AM
  4. These cells are generally about ²5% MHC II+ and expression is increased >65% after stimulation (ie with LPS)

    Reply
    Posted by: Jeanette B.
    May 6, 2009 - 6:39 PM
  5. How long can dendritic cells be cultured for before use in an experiment? How often should media be changed if you are not using them immediately?

    Reply
    Posted by: Sarah L.
    May 5, 2009 - 3:48 PM
  6. I have never tried to culture them longer than 10 days, but they are healthy at that point. Maybe someone else has? If you do culture them longer, they should be split 1/² or 1/3 (keep the media that they're in and top up with fresh media) and more frequently if the media begins to yellow. They grow quickly at this stage!    

    Reply
    Posted by: Jeanette B.
    May 6, 2009 - 6:43 PM
  7. Do you know if splitting the cells will activate them? I am afraid I'll lose a lot of cells... (not sure if I should expect all of them to re-attach or not...)

    Reply
    Posted by: Anonymous
    May 12, 2010 - 3:43 PM
  8. 14 days.

    Reply
    Posted by: Imane A.
    October 10, 2012 - 6:14 AM
  9. when i culture the cells i often see a few small (maybe 1 or ²mm) of cells. It is visible to the eye and just looks like a clump of cells under the microscope. I use GM-CSF to mature the cells but nothing to stimulate them as i have to do that as part of my experiment at a later date. I was just wondering is it normal for the cells to clump like that if they are not activated?

    Reply
    Posted by: Anonymous
    June 5, 2009 - 9:48 AM
  10. It is normal to have these small clusters early in the culture period. They should be very rare by day 7 of culture, even after stimulation.

    Reply
    Posted by: Jeanette B.
    June 6, 2009 - 9:30 AM
  11. Hi, just like to ask...Should I just collect the non-adherent cells after GM-CSF differentiation for subsequent culturing if the adherent cells are macrophages?
    I intend to follow the protocol of Lutz and the whole generation of DCs will take a period of 1² days. I hope the cells can survive for a long period.

    Reply
    Posted by: Anonymous
    June 18, 2009 - 5:30 AM
  12. why do you not use IL-4 + rmGM-CSF?, and why do you use 40 ng/mL of GM-CSF, if Luzt use ²0ng/ml? Where do you mature the DC, in the same plate?

    Reply
    Posted by: paula c.
    August 15, 2009 - 10:38 AM
  13. I work with Sprague Dawley Rats and my experience is that when I use the combination of GMCSF and IL4 the capacity of the cells to stimulate T cell activation/diferrentiation decrease.....in comparisson withthe ones culture just with GMCSF

    Reply
    Posted by: Anonymous
    August 21, 2009 - 9:10 AM
  14. When you replace/change the media...Are you lossed a lot of cells????

    Reply
    Posted by: Anonymous
    August 21, 2009 - 9:13 AM

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