Generation of large numbers of highly purified dendritic cells from bone marrow progenitor cells after co-culture with syngeneic murine splenocytes

Exp Mol Pathol. 2013 Apr;94(2):336-42. doi: 10.1016/j.yexmp.2012.12.001. Epub 2012 Dec 23.

Abstract

Dendritic cells (DCs) are called the sentinels of the human immune system because of their function as antigen presenting cells (APCs) that elicit a protective immune response. Given that DCs have been used for many years as target cells in a great number of experiments, it became essential to devise a new method for producing DCs in higher quantities and of greater purity. Here we report a novel technique for obtaining more dendritic cells, and with higher purity, from in-vitro co-culture of bone marrow (BM) cells with splenocytes. From a total of 20 × 10(6) BM cells and 120 × 10(6)splenocytes, 3 × 10(6) BM cells along with 20 × 10(6)splenocytes were co-cultured in petri dishes for DC generation; 120 × 10(6) splenocytes from one C57BL/6 mouse were also co-cultured in petri dishes for DC generation. BM cells were the control group cultured in the same conditions except for the presence of splenocytes. Purity and maturation state of DCs were checked by lineage surface markers (CD11c, CD11b, CD8α, and F4/80) and the expression levels of MHCII as well as co-stimulatory molecules (CD86, CD80, and CD40). Endocytosis and thymidine uptake capacity were also used to test the functionality of DCs. The levels of IL-12p70, IL-23, and IL-10 were also checked in the supernatant of cultured cells by ELISA. The number of DCs derived from co-culture of BM and splenocytes (DCs(TME)) was at least twice that of BM-derived DCs in the absence of splenocytes. In addition, the purity of DCs after co-culture of BM and splenocytes was greater than that of DCs in the control culture (90.2% and 77.2%, respectively; p<0.05). While functional assays showed no differences between co-culture and control groups, IL-10 levels were significantly lower in DCs(TME) compared to BM-derived DCs in the absence of splenocytes (193 pg/ml and 630 pg/ml, respectively; p<0.05). The results of the present study show that the generation of DCs from BM progenitors is accelerated in the presence of syngeneic splenocytes. Given the larger number of generated DCs, and with higher purity, in this technique, DCs(TME) could be more advantageous for DC-based immunotherapy and vaccination techniques.

MeSH terms

  • Animals
  • Antigens, Surface / metabolism
  • Biomarkers / analysis
  • Bone Marrow Cells / cytology*
  • Cell Culture Techniques*
  • Coculture Techniques
  • Cytokines / metabolism
  • Dendritic Cells*
  • Endocytosis
  • Female
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Spleen
  • Stem Cells / cytology*
  • Thymidine / metabolism

Substances

  • Antigens, Surface
  • Biomarkers
  • Cytokines
  • Thymidine