Preparation method of hemizygote CAPRI cells
A hemizygous and cell technology, applied in the field of preparation of hemizygous CAPRI cells, can solve the problems of poor constitution, hinder the implementation of CAPRI cell treatment plan, and cannot realize collection, etc. Effects of proliferation and activation ability
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Embodiment 1
[0027] Example 1: The patient, 60 years old, female, after the modified radical mastectomy for stage I breast cancer, decreased the number of white blood cells due to chemotherapy, and was unable to withstand the process of collecting mononuclear cells by a blood cell separator because of poor physical condition, chose the hemizygous CAPRI cell preparation method of the present invention. The present invention will be described in detail below through examples.
[0028] Take 100 ml of peripheral blood from the patient and 150 ml from the hemizygous donor, add heparin sodium with a final concentration of 1.5×105U / L for anticoagulant treatment, and divide into 50ml centrifuge tubes according to the volume of peripheral blood obtained, and centrifuge at 1600rpm 20-30min, discard the supernatant, and add 25ml normal saline to the centrifuge tubes, mix carefully, and slowly distribute along the tube wall into 15ml centrifuge tubes pre-added with 5ml Ficoll-Hypaque lymphocyte separat...
Embodiment 2
[0033] Example 2: Patient, male, 54 years old, lung squamous cell carcinoma with bone metastases, white blood cells < 4000 / ml after radiotherapy + chemotherapy, not suitable for collecting mononuclear cells with a blood cell separator, choose the hemizygous CAPRI of the present invention Cell preparation method. The present invention will be described in detail below through examples.
[0034] Take 120 ml of peripheral blood from the patient and 150 ml from the hemizygous donor, add heparin sodium at a final concentration of 1.5×105U / L for anticoagulant treatment, and divide into 50ml centrifuge tubes according to the volume of peripheral blood obtained, and centrifuge at 1800rpm 20-30min, discard the supernatant, and add 25ml normal saline to the centrifuge tubes, mix carefully, and slowly distribute along the tube wall into 15ml centrifuge tubes pre-added with 5ml Ficoll-Hypaque lymphocyte separation medium, centrifuge at 2000rpm for 20min Finally, the cells in the centrifu...
Embodiment 3
[0039] Example 3: CAPRI cell toxicity test in vitro.
[0040]The present invention also detects the in vitro cytotoxicity of the obtained hemizygous CAPRI, the CAPRI cells (CD3 mAb-CAPRI) stimulated by CD3 mAb alone and the CAPRI cells (CD3 / CD133mAb-CAPRI) stimulated by CD3 mAb and CD133 mAb co-stimulated The specific experimental steps are as follows: the harvested CAPRI cells produced by two different stimulation methods were used as effector cells, and the human lung squamous cell carcinoma NCI-H520 cell line in the logarithmic growth phase was treated with 0.4% trypan Blue staining and counting were used as target cells, and the effector cells and target cells were adjusted to 1×106 / ml in serum-free 1640 medium with different effect-target ratios of 2.5:1, 5:1, 10:1, and 20:1. Add 100 μl / well to a 96-well plate, and set up blank control wells, sample control wells, and sample maximum enzyme activity control wells, and set 3 replicate wells in each well, and place them in a...
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