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A cryopreservation method and immune cell technology, applied in the field of cell biology, can solve the problems of easy aging of cells, low cell viability, and damage of cells by ice crystals
Active Publication Date: 2017-12-05
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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Problems solved by technology
However, the existing immune cell cryopreservation method is easy to form ice crystals to damage the cells during the cryopreservation process. After recovery, the cell viability is low, the number of cell proliferation is insufficient, and the cells are prone to aging.
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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 mononuclear cells with 2-3mL X-VIVO15 serum-free medium, slowly add the same volume of freezing solution 1 to make 4-6mL cell freezing suspension, divide into 4-6 tubes for freezing, each tube 1mL, frozen storage density: 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.
[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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Abstract
The invention belongs to the field of cytobiology and particularly discloses an immunological cell freeze-saving method. According to the immunological cell freeze-saving method, mononuclear cells are resuspended in an X-VIVO15 serum-free medium, same volume of freeze-saving solution is slowly added so as to prepare a cell freeze-saving suspension, the cell freeze-saving suspension is cooled to -80 DEG C, and then the cell freeze-saving suspension is freeze-saved at an ultralow temperature of -196 DEG C. Experiments indicate that the recovered cells freeze-saved by the immunological cell freeze-saving method have cell viability and cell multiplication quantity obviously superior to those of cells obtained by recovery of cells freeze-saved by a conventional freeze-saving scheme, have K562 cell killing ability equal to that of the cells which are not freeze-saved, and preserve the killing activity of the cells.
Description
technical field [0001] The invention belongs to the field of cell biology, and in particular relates to a cryopreservation method of immune cells, in particular to a method for cryopreservation of immune cells and recovery after cryopreservation. Background technique [0002] Immune cells refer to cells involved in or related to immune responses, including lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells, etc. Immune cells can be divided into many types, and various immune cells play important roles in the human body. Immune cells include phagocytes and lymphocytes, and T cells and B cells in lymphocytes are the main cell groups that exert specific immunity. Mononuclear cells isolated from blood include: T cells, B cells, NK cells and DC cells, etc., which are the initial cell source in immune cell therapy. After induction and expansion of various cytokines, a group of cells with high killing activity can be obtained. immune cell population. ...
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