| Objective:To compare the influence of two different cryopreservation methods on biological characteristics of mouse bone marrow-derived dendritic cells.Methods:1. Culture and grouping mice bone marrow-derived dendritic cells Killing C57BL/6 mice by cervical dislocation to obtain mouse bone marrow cells under sterile conditions, and divided them into 3 groups:group A (routine culture), group B (early cryopreservation) and group C (cultured frozen). Make group A into single cell suspension, then rmGM-CSF and rmIL-4 are added and dispnsed into two ventilation culture flasks, supernatant was removed by centrifugation after 48 hours, and RPMI1640 medium containing 12% FBS and cytokines are added and cultured. Group B for cryopreservation directly, and group C, the first five days of culture with the group A, then collecting the suspended cells for cryopreservation after natural sedimentation on the 5th day.2. Cryopreservation and culture after resuscitation of cells After washing and centrifugation, cells for cryopreservation are resuspended in homemade freezing medium (70% RPMI 1640 medium,20% FBS,10% DMSO), and transferred to a -80℃ ultra-low temperature freezer after stored for one hour at -20℃, then transferred to liquid nitrogen tank after 24 hours, and resuscitated in 4 weeks. After resuscitation, and continue to culture DCs from group B according to the steps of group A. Made group C into single cell and rmGM-CSF and rmIL-4 are added into the suspension, then culture it for a day, adding cytokines again after the medium was changed, and cells suspension are collected every other day.3. The observation of cells survival rateCalculating the survival rate of DCs in B, C groups by trypan blue staining after the recovery.Cells survival rate (%)=Number of live cells/(Total Number of live cells+Total Number of dead cells)×100%。4. The detection of molecular phenotype of mouse bone marrow-derived dendritic cells using flow cytometryDCs suspension of each group were collected and labeled by antibody and isotype according to the specification of flow antibody, and detect the expression of CD11c, CD80, CD86 and MHC-II molecules on cell surface in each group by flow cytometry.5. The detection of mixed lymphocyte reaction (MLR) using CCK-8 method Taking out the spleen of BALB/c mice and cut it into pieces under sterile conditions, and obtaining cell suspension after grinding, and using lymphocyte separation medium to obtain lymphocytes, nylon wool column method to obtain allogeneic T cells. Each group of mature dendritic cells are obtained using the above methods, and the mitomycin C was added and incubated at 37℃ for 45 min, after washing, adding DCs and T cells in 96-well plates respectively according to the ratio of 1:5,1:10,1:20,1:40, and set three holes in each groups, and provided DCs and T cells as negative control while culture medium as blank control, both of them co-cultured for 68h, adding 10μl CCK8 solution to each well, and cultured for 4h, then determining the OD value of each well by using Microplate Reader at 450nm.6. The detection of IL-10 and TGF-β1 level in culture supernatant using Elisa method Collecting the immature DCs supernatant in groups above, and measure the IL-10 and TGF-β1 levels of supernatant according to the steps in the specification of Elisa kit.Results:1. The survival rate after revival of two cryopreserved groups are (85±3.9)% and(83± 6.1)% respectively but it is not statistically significant with P>0.05.2. Three groups are all have high expression on CD11c, low expression on CD80, CD86 and MHC-Ⅱ.3. Mixed lymphocyte reaction indicates that B, C groups’ ability of stimulating allogeneic T cell proliferation has decreased, in comparison to group A. However, the difference is no statistical significance as well (P>0.05).4. Differences on IL-10 and TGF-β1 in culture supernatant fluid are not statistically significant (P>0.05).Conclusion:Cryopreserved DCs and DCs differentiated from cryopreserved mice bone-marrows cells can maintain stable biological characteristics, which means they can be used for cellular immune treatment of atherosclerosis in mice. |
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