Experimental Study On Induction And Expansion Of Dendritic Cells From Human Peripheral Blood By Using Serum-free Medium In Vitro

Object: Dendritic cells (DC) are the most potent professionalantigen-presenting cells (APC) which can take up, process and presentantigen. They can strongly stimulate proliferation of naive T cells andpromote generation of cytolytic T lymphocyte and T help cells, so theyare considered initiating and adjusting agent of immune response. Activespecific immunotherapy based on dendritic cells has already become amain strategy in the field of tumor immunotherapy. It is difficult topurify DCs because of the low concentration in vitro. So the study ofmass-production of DCs in vitro becomes the key points inImmunology. In most of experiences, culturing DCs in vitro uses fetalbovine serum (FBS). FBS may has the problems of safety and quality.FBS may not be suitable for future clinical use.Methods: In this study, DCs were cultured from human peripheralblood mononuclear cells by using serum-free medium X-VIVO20 withrhGM-CSF (100ng/ml) and rhlL-4 (50ng/ml) for 6 days, rhTNF-a(100ng/ml)was added on the 6th day of culture. PBMC were seedingat the concentration of 5x10~6/ml,6x10~6/ml and 7x10~6/ml in 6-wellfiat-bottomed plates. Suspension cells were harvested at days 6,9 and 12and analysed on morphology, phenotype and function observed. In thisstudy, a serum-free and cytokine-containing culture system for DC expansion was systematically developed and optimized using thetwo-level factorial design, which are cell cuture density and cuture time.X-VIVO 20 was also compared with RPMI-1640, supplemented with10% fetal bovine serum or 10% substitude serum.The study was divided into two parts 1. Monocyte-derived dendriticcells(MoDCs) from human peripheral blood are ideally generated inserum-free medium (SFM). 2. Serum-free medium was compared withserum medium or substitude serum medium.Result:1. Morphological observations were performed using phase-contrastmicroscopy. Cells on day 9 of culture displayed DC-like morphologywith irregular cell shape and multiple cytoplasmic processes.2. Surface marker expression on culture cells was assessed usingflow cytometry. Phenotype analysis on DCs acquired showed that thecells of 6x10~6/ml cultured for 9 days contained a higher percentage ofCDla cells. The positive of CDla is 36.57%±0.92%.3. DCs provoked a proliferation of allo—T lymphocytes in MLR.4. There is difference in the yield of DC between serum-free medium,substitude serum medium or serum medium(P＜0.05). The positive ofCDla is 36.57%±0.92%, 47.33%±5.46% and 65.00%±5.27%respectively.Conclusion: o1. cultured cells exhibited typical morphological characteristics ofDC and had immunological phenotype and function of DC. The resultssuggested the culture method was feasible.2. DC differentiation in medium with or without serum indicated DCdifferentiation was supported by serum . Serum may be an importantfactor in determining the yield of DC.