A CTL preparation method for efficient proliferation and targeted killing of tumors
A high-efficiency proliferation and targeting technology, applied in the direction of blood/immune system cells, vertebrate cells, animal cells, etc., can solve the problems of high proportion of Treg cells, reduce cell killing activity, etc., achieve high-efficiency proliferation, inhibit transformation, inhibit differentiation effect
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Embodiment 1
[0044] Example 1: Preparation of CTL cells
[0045] 1. Preparation of PBMCs
[0046] Collect 50ml of peripheral blood from tumor patients, centrifuge at 2500rpm (rev / min) for 10min, and suck out the upper layer of plasma for later use. Add 0.9% physiological saline for injection to the blood cells, mix well, and dilute the blood cells. Slowly add the diluted peripheral blood to the human lymphocyte separation liquid layer with a specific gravity of 1.077, the volume ratio of the two is 1:1. Centrifuge at 600g for 30min, gently suck out the mononuclear cell layer in the form of white mist, and transfer it to another in a centrifuge tube. Diluted with 0.9% normal saline for injection, 400g, centrifuged for 5min, washed 3 times and centrifuged at low speed to obtain PBMC.
[0047] 2. MiniMACS immunomagnetic bead negative sorting method to remove CD4 + CD25 + Treg cells
[0048] The cells were resuspended in MACS (Magnetic-activated cell sorting, magnetic bead separation sys...
Embodiment 2
[0068] 1. Preparation of PBMCs
[0069] Collect 50ml of peripheral blood from tumor patients, centrifuge at 2500rpm for 10min, and suck out the upper layer of plasma for later use. Add 0.9% physiological saline for injection to the blood cells, mix well, and dilute the blood cells. Slowly add the diluted peripheral blood to the human lymphocyte separation liquid layer with a specific gravity of 1.077, the volume ratio of the two is 1:1. Centrifuge at 600g for 30min, gently suck out the mononuclear cell layer in the form of white mist, and transfer it to another in a centrifuge tube. Diluted with 0.9% normal saline for injection, 400g, centrifuged for 5min, washed 3 times and centrifuged at low speed to obtain PBMC.
[0070] 2. MiniMACS immunomagnetic bead negative sorting method to remove CD4 + CD25 + Treg cells
[0071] Resuspend the cells with MACS buffer, pass through a 100 μm filter to obtain suspended single cells, and adjust the cell density to 5×10 8 / ml. Add bio...
Embodiment 3
[0088] 1. Preparation of PBMC and 2. MiniMACS immunomagnetic bead negative sorting method to remove CD4 + CD25 + Treg cells adopt the same method as in Example 1;
[0089] 3. Separation of DC cells and T lymphocytes:
[0090] will result in CD3 + , CD4 + , CD8 + , CD14 + Mix the cells, aliquot into 30ml of serum-free AIM-Ⅴ medium, at 37°C, 5% CO by volume 2 After static incubation in the incubator for 2 hours, the suspension cells (ie, T lymphocytes) and adherent cells (ie, DCs) were obtained, and the suspension cells were transferred to a new culture flask.
[0091] 4. Preparation of mature DC cells:
[0092] Add 20 ml of serum-free AIM-Ⅴ medium containing 50 ng / ml GM-CSF and 25 ng / ml IL-4 to the adherent DC cells, place at 37 ° C, and the volume percentage is 5% CO 2 cultured in an incubator for 5 days. On the 6th day, 50 μg / ml of the whole tumor cell antigen of the tumor patient was added, and the DC cells loaded with the whole tumor cell antigen were obtained on t...
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