[Optimization of tolerogenic dendritic cell preparation technique in rats with collagen-induced arthritis]

Objective To yield high-quality tolerogenic dendritic cells (TolDCs) for therapeutic intervention by a refined technique of TolDC preparation from the spleen of modified collagen-induced arthritis (CIA) rats. Methods The refinements took place amid monocellular cell isolation process, including both the preparation of single cell suspension and the adjustment of incubation time after plate seeding of mononuclear cells. TolDCs were induced by administration of NF-κB oligonucleotide (ODN) decoy at the initiation of cell culture. Cell morphology was examined under a microscope and cell viability was revealed by trypan blue staining. Expression of classical cell identity and activation makers, CD103 (OX62), CD80 and CD86 was determined by flow cytometry; meanwhile, DC-stimulated lymphocyte proliferation was measured by MTT assay following mixed lymphocyte reaction (MLR). Results With the established approach, the viability ratio of resulting cells reached over 90% and the proportion of OX62 positive ones was above 87.4%, which altogether confirmed their ideal DC phenotype. Functionally, the tolerogenic nature of the NF-κB ODN-treated DCs was further unveiled by the low expression of costimulatory molecules (CD80 and CD86) and the abrogated capacity of lymphocyte stimulation effect. Conclusion Compared with TolDCs generated by conventional protocol, CIA rat spleen-derived TolDCs isolated by the optimized splenic mononuclear cell preparation procedure and induced by NF-κB ODN decoy show the higher regulatory activity, higher phenotypic stability, higher purity and tolerogenic properties.