Immunological cell freeze-saving method
A cryopreservation method and technology of immune cells, applied in the field of cell biology, can solve the problems of low cell viability, ice crystal damage to cells, easy aging of cells, etc.
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Embodiment 1
[0024] Example 1: Isolation of mononuclear cells
[0025] 1. Draw 20mL of peripheral blood, add it to a 50mL centrifuge tube, and centrifuge at 700g for 10min;
[0026] 2. Draw 8-9mL of the upper layer of plasma to a 15mL centrifuge tube for later use, and add 2 times the volume of normal saline to dilute the lower layer of blood;
[0027] 3. Take a new 50mL centrifuge tube, add 15mL lymphocyte separation medium, and slowly add diluted blood to ensure clear stratification.
[0028] 4. Centrifuge at 700g for 20-30min, and the centrifuge speed drops to zero.
[0029] 5. Extract the middle buffy coat cells to obtain mononuclear cells, add normal saline to wash twice, count, and the cell volume is 2×10 7 -3×10 7 .
Embodiment 2
[0030] Embodiment 2, the immune cell cryopreservation method of the present invention
[0031] 1. Autologous plasma was inactivated at 56°C for 30 minutes, filtered through a 0.22 μm filter membrane, and set aside.
[0032] 2. Prepare cryopreservation solution 1 according to the ratio of DMSO: autologous plasma: X-VIVO15 medium volume ratio of 1:2:2, and pre-cool in a refrigerator at 2-8°C.
[0033] 3. Resuspend the mononuclear cells with 2-3mL X-VIVO15 serum-free medium, slowly add the same volume of cryopreservation solution 1 to make 4-6mL cell cryopreservation suspension, divide into 4-6 tubes for cryopreservation, each tube 1mL , the freezing density is: 4×10 6 cell / mL-6×10 6 cell / mL.
[0034] 4. Cool down to -80°C with a programmed cooling device, and transfer to a liquid nitrogen tank for freezing.
Embodiment 3
[0035] Embodiment 3, routine cryopreservation method
[0036] The cryopreservation solution was prepared according to the ratio of DMSO:FBS:X-VIVO15 serum-free medium volume ratio of 1:2:7, and the mononuclear cells were resuspended in the cryopreservation solution and then frozen in liquid nitrogen.
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