Optimized Mouse BMDC Isolation and Culture under Endotoxin-Free Conditions

Abstract

Introduction: Dendritic cells are very important in basic studies and vaccine research, but isolation and culture of these cells face challenges due to their small number in tissues. Since there is no standard method, we addressed some of the factors affecting the efficiency of dendritic cell isolation and culture from BALB/c mouse bone marrow. Materials & Methods: Bone marrow cells isolated from femur and tibia, were seeded in plates and treated with GM-CSF. On day 3, half of the media was transferred to a new plate with fresh media plus GM-CSF. On day 5, cells were induced with CpG oligonucleotide. Cell survival as well as maturation markers of CD11C, MHC-II and CD86 were investigated by flow cytometry on day 5 (immature cells) and day 7 (mature cells). Ethics code: IR.PII.REC.1395.99 Findings: It was found that the presence of endotoxin in the culture of immature cells, significantly reduces the recovery of dendritic cells (p value <0.05). Less than one million cells seeded in non-treated 6-well plates and transfer of cells to a new plate on day 3 increased the efficiency of immature cell retrieval by preventing early maturation and premature death. Further treatment of immature cells with CpG on day 7 was used to induce the Th1 pathway. Significant difference (p value <0.05) with control group showed that CpG induces maturation after treatment with GM-CSF (without IL-4). Discussion & Conclusions: Optimization of dendritic cell culture in terms of different culture conditions highly impacts the efficiency of immature cell formation and maturation.

Keywords

• bone marrow
• dendritic cells
• cell culture optimization
• endotoxin