Experimental Study Of The In Vitro Induces Of Dendritic Cell With Serum-contain Medium Or With Serum-free Medium

Objectiv: The major cause of relapse of acute myeloid leukemiais minimal residual disease(MRD).People are trying to treat the MRD.Deriving dendritic cell (DC) from the myeloid magligancies in thepresent of combinations of cytokines in vitro maybe can solve theproblem. DC from the myeloid magligancies can be used as antigenpresenting cells and be used to generate a specific antileukemicimmune response. The medium frequently used contain fetal calfserum(FCS),human blood group AB serum or autologousserum.DCs.DCs are not widely used in clinic,because human serummay actually contain components that retard the generation of DCand the allogenous serum may cause the exogenous antigen andinfection.We use serum-free medium X-VIVO 20, which can avoidthe shortage of all above and be allowed to be used in clinic praticeby FDA. Serum-free medium X-VIVO 20 was used in our study,inorder to explore the basis condition of biotherapy and enhanceserum-free medium and make theDC available to apply to clinic. Method: 1.Human monocytes(MNC) were prepared fromhealthy volunteers by centrifugation of peripheral blood over aFicoll-Paque density gradient.2.The MNCs were cultured in differentmedia, including RPMI1640 supplemented with 10%human bloodgroup AB serum, RPMI1640 supplemented with FCS, serum-freemedium X-VIVO 20 with or without human serum albumin.GM-CSFand IL-4 were added at 0 day.All cultures were maintained at 37℃in 5%CO2 and a humidified atmosphere.samples were cultured for7 days .Calcium ionophore A23187 were added on 1 day before theend of the culture period.3.The cells were observed by invertmicroscope ,cell count was measured at 3 days and 7 days .At the endof culture ,the cells were stained with Wright-Giemsa.4.Method offlow cytometry was used to determine cell surface antigens beforeand after culture by using monoclonal antibodids.The monoclonalantibodies included CD83,CDW123,CD14,HLA-DR. 5.Theproliferation of T cells was performed by methyl thiazolytetrazolium(MTT).6.Preparetion of K562 cell and HL-60 cellfrozen-thawed antigen (FTA):Put K562 cell and HL-60 cell in frozennitrogen , put in 37℃ water,frozen and thaw three times,then keepthem in 4 ℃ 7. The specific cytotoxicity of T cells primed with DCwas examined by MTT assay. Result: 1.Cells were shifted along the DC pathway in allconditions.cells showed characteristic morphological features of DCin all conditions,including irregular cellular surface,variable sizeincreases, small condensed nuclei, typical cytoplasmic motileprocesses.As the time went on, number of DC cultured in all contionsincreased.2.Uncultured cells had high-level expression of CD14,butlow-level expression of CDw123,HLA-DR,and CD83.At the end ofthe cultured period,the proportions of CD14 positive cellsdecreased,while the proportions of CDw123,HLA-DR,and CD83were increased.The change were similar both in serum-containconditions and in serum-free conditions. human serum albumin hadno effection on the change.3.DCs cultured with serum or withoutserum were capable of stimulating the proliferation of allogenetic Tlymphocytes.There were no significant RPMI164ong thosegroups.4.T lymphocytotic primed with the culture-derived DCexhibited more killing activity to K562 and HL-60 than thosecultured in RPMI164 alone.There were significant difference betweenexperimental groups and control groups. T lymphocytotic primedwith the culture-derived DC exhibited the similar killing activity toK562 and HL-60,there were no significant difference. Conclusion: 1.There is no significance in DC number,morphology and epitype between DC induced by serum-free X-VIVO20 medium and DC induced by serum-contained medium.2.There isno significance in proliferation of T cell between DC induced byserum- free X-VIVO 20 medium and DC induced byserum-contained medium.3. There is no significance in antitumor ofCTL intrigued by DC between serum-free X-VIVO 20 medium andserum-contained medium.4. Serum-free X-VIVO 20 medium has adefinite amponent, and it is stable safe, no-MHC-restriction and norisk of exogenous antigen and virus infeetion.