Abstract: Bone marrow cells of C57BL/6 mice were induced and cultured under low temperature cryopreservation conditions to obtain dendritic cells (DCs). There was no significant difference in survival rate after cryopreservation of DCs. All groups of DCs expressed high CD11c, low expression of CD80, CD86 and major histocompatibility complex-II (MHC-II) molecules; mixed lymphocyte reaction and There was no significant difference in the levels of interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1) in the culture supernatant. It is indicated that DCs cryopreserved at different times can still maintain stable biological characteristics.
Materials and Methods
1. Experimental animals SPF healthy male C57BL/6 mice and BALB/c mice, 6 to 8 weeks old, were about 20 g.
2. Main reagent RPMI1640 whole culture medium, South American fetal bovine serum recombinant mouse granulocyte-macrophage colony-stimulating factor, recombinant mouse interleukin-4; flow fluorescent antibody FITCan-ti-mouseCD11c, PerCP-CyTM5.5anti-mouseCD80 , APCanti-mouseCD86, PEanti-mouseMHC-II and its isotype control antibody; CellCountingKit-8; mouse interleukin-10, transforming growth factor-β1, ELISA kit.
3. Main instruments Constant temperature cell incubator; inverted microscope; -80 °C ultra-low temperature refrigerator; flow cytometer; microplate reader.
4. Culture of mouse bone marrow-derived DCs and C57BL/6 mice were sacrificed by cervical spine debridement, soaked in 75% alcohol for at least 10 min, and the muscle tissues of the femur and tibia of the mice were aseptically removed. After repeated washing with PBS, the backbone was removed. At both ends, the RPMI1640 whole culture solution was washed into the bone marrow cavity until the washing liquid turned white, the tissue debris was removed by filtration, and the filtrate was collected to obtain mouse bone marrow cells, which were divided into a normal culture group and an early cryopreservation group, and the culture group was used for early cryopreservation. After cryopreservation, the normal culture group was centrifuged at 1200 r/min for 5 min, the supernatant was discarded, and a single cell suspension was prepared using RPMI1640 whole medium containing 12% FBS. After counting, the cell concentration was adjusted to 0.5×106/ml. Add rmGM-CSF and rmIL-4 (the final concentration is 20ng/ml), incubate in a 37°C, 5% CO2 constant temperature incubator, and centrifuge at 1200r/min for 5min to remove the supernatant after 48h. The culture medium and cytokines were further cultured, and the liquid was changed halfway every other day. On the fifth day, the half-quantity liquid exchange was continued, and the cultured cryopreserved group was separated from the normal culture group. After the culture, the frozen storage group was subjected to natural sedimentation, and the suspension cells were collected for cryopreservation. The cell suspension was collected every other day.
5. After cryopreservation and resuscitation of the cells, the cells used for cryopreservation were centrifuged and resuspended in a homemade cryopreservation solution (70% RPMI1640 whole medium, 20% FBS, 10% DMSO), and the cell concentration was adjusted to 5×109. /L, stored at -20 ° C for 1 h, transferred to a -80 ° C ultra-low temperature refrigerator, 24 h later transferred to a liquid nitrogen tank. Recovery after 4 weeks. After the early cryopreservation, the culture group was resuscitated and the DCs were continued to be cultured in the normal culture group. After the culture, the cryopreservation group was made into a cell suspension using whole culture solution, and rmGM-CSF and rmIL-4 were added, cultured for 1 day, exchanged and supplemented with cytokines, and cells were collected every other day.
6. Observe cell survival rate Take 0.1 ml of DCs suspension after resuscitation, add 0.8 ml PBS, add 0.1 ml of 0.4% trypan blue staining solution and mix (final concentration 0.04%), stain for 3 min, observe under inverted microscope The unstained viable cells and the dead cells stained blue were counted, and the survival rate of DCs was calculated. Cell viability (%) = total number of viable cells / (total number of viable cells + total number of dead cells) x 100%.
7. Flow cytometry was used to detect the molecular phenotype of mouse bone marrow-derived DCs. The DCs suspension of each group was collected, and the cell concentration of the sample was adjusted to 1×106/ml. The antibody and its isotype control label were labeled according to the flow antibody specification at 4 °C. Incubate for 30 min under light, centrifuge, wash and resuspend in 500 μl PBS solution. Flow cytometry was used to detect the expression of CD11c, CD80, CD86 and major histocompatibility complex-II molecules on each cell surface.
8. The CCK-8 method was used to detect the mixed lymphocyte reaction. The spleen of BALB/c mice was aseptically taken and cut, and the cell suspension was obtained by grinding and filtering. The allogeneic T cells were obtained by nylon hair column method (adjusted concentration was 2). ×106/ml). In each of the DCs obtained in steps 1.4 and 1.5, mitomycin C (final concentration 25 μg/ml) was added, incubated at 37 ° C for 45 min, and washed twice with PBS for 1:5, 1:10, 1: 20, 1:40 ratio DCs and T cells were added to 96-well plates, and each group was set up with 3 duplicate wells. At the same time, DCs cells and T cells were used as negative controls. The culture medium was used as a blank control. After culture for 68 hours, 10 μl of CCK-8 solution was added to each well, and after further incubation for 4 hours, the optical density (OD) value of each well at 450 nm was measured by a microplate reader.
9. Detection of IL-10 and TGF-β1 levels in the culture supernatant by ELISA The supernatants of the above groups of DCs were collected, and the levels of IL-10 and TGF-β1 in the culture supernatant were determined according to the procedure of the ELISA kit.
10. Statistical analysis Five samples were detected in each group. The single factor analysis was performed using SPSS 17.0 statistical software. The quantitative data were expressed by x珋±s. P<0.05 was statistically significant.