Comparison Of Different Methods For Dendritic Cell Generation From Mouse Bone Marrow

Document Type : Original Article




Objective: Dendritic cells (DCs) are the most effective antigen-presenting cells with many applications. However, DCs can be generated in vitro by various methods from bone marrow cells, each with varying pros and cons. Thus, we evaluated three different methods to generate DCs from mice bone marrow cells.
Materials and Methods: BM cells from C57BL/6 mice were cultured in the presence of 1000 U/ml of GM-CSF either with or without 500 U/ml of IL-4 for 6 days as first and second method respectively. In the third method, BM cells were cultured in bacterial plates in the presence of 200 U/ml GM-CSF for 10 days. For further maturation, the cultures were extended for two more days using 50ng/ml TNF-α. The purity of the obtained DCs, their subtypes and maturation states were determined using flow cytometric analysis and the capacity to induce the allogenic T cell proliferation was determined using 3H-thymidine incorporation.
Results: The purity of generated DCs (CD11c+) in both methods of GM-CSF plus IL-4 and culturing in bacterial plates was more than that of GM-CSF method. Culturing in bacterial plates resulted in the generation of higher number of DCs. However, DCs were resistant to maturation induction by maturation factors. Expression of maturation markers (CD86 and MHCII) on DCs was up-regulated in the presence of IL-4 compared to the other two methods and the cells acted more efficiently in the induction of allogenic T cells proliferation. All the three methods resulted in the successful generation of myeloid (CD11b+) DCs.
Conclusion: A combination of GM-CSF and IL-4 results in the generation of more pure, more mature, and functionally more effective DCs.