Phenotypic characterization of equine monocyte-derived dendritic cells generated ex vivo utilizing commercially...

The impact of culture conditions on equine monocyte-derived dendritic cells (MoDC) generation has not been fully characterized. We hypothesized that 1) MoDC could be cultured in a commercially available serum-free medium (AIM-V); and 2) that differential culture conditions would influence MoDC viability, yield and phenotype. MoDC generated from adult horses were cultured under variable conditions in a series of experiments. Viability was assessed using trypan blue and propidium iodide staining. Yield was determined by manual hemocytometer counting. Phenotype was assessed by flow cytometric analysis of surface markers (MHC class-II, CD86 and CD14). Data were analyzed using paired t-tests and repeated measures ANOVA. Two MoDC populations that differed in size and phenotype were identified: larger MoDC (LgMoDC) and smaller MoDC (SmMoDC). Medium type, plate chemistry, or length of monocyte adhesion time did not impact MoDC viability or yield. LgMoDC generated in serum-free medium expressed more MHC class-II and CD86 (P ≤ 0.03). A prolonged duration in culture reduced MoDC yield (P ≤ 0.04). MoDC can be consistently and reliably generated using AIM-V serum-free medium in standard tissue culture plates with a recommended culture duration of 3-4 days.