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

Active Publication Date: 2014-08-06
王盛 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above method has strict requirements on the patient’s physical condition when obtaining PBMC. ①The patient’s white blood cells are less than 4000/ml, ②less than 6 weeks after chemotherapy, ③less than 2 weeks after stopping the whitening drug, and ④the patient’s physical condition is poor Unable

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  • Preparation method of hemizygote CAPRI cells
  • Preparation method of hemizygote CAPRI cells
  • Preparation method of hemizygote CAPRI cells

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention relates to a preparation method of hemizygote CAPRI cells. The method comprises the following concrete steps: (1) obtaining peripheral blood of a patient and a hemizygote donor, and separating and purifying a peripheral blood mononuclear cell (PBMC); (2) preparing serum from hemizygote donor plasma; (3) activating the PBMC; (4) expanding the CAPRI cells; (5) harvesting the CAPRI cells; (6) cryopreserving the CAPRI cells. In addition, an early cell treatment step and a double-antibody loading method are added to increase the number of CAPRI cells and improve the tumor killing capability. According to the preparation method provided by the invention, the problem that the CAPRI culture cannot be carried out due to factors of relatively poor condition and the like of the patient in the prior art is solved, the generated cells have a broad-spectrum tumor-killing effect, the PBMC has good cell expansion and activation capability due to the early cell treatment step, and the tumor cell killing effect of the CAPRI cells is improved due to the double-antibody loading mode; the advantages of the CAPRI cells are brought into full play, so that cells from the patient fully cooperate with cells from the hemizygote donor, and the blank in the prior art is filled; moreover, the reactivation capability of cryopreserved cells is greatly improved due to cell cryopreserving liquid provided in the method.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing hemizygous CAPRI cells. Background technique [0002] CAPRI (Cascade Primed Immune cells) cells, also known as cascade-triggered immune cells, are a new type of autologous T cells, which are prepared by the chain activation process of antigen-presenting cells combined with CD3 monoclonal antibody and specific cytokines, and have the non-specificity of CIK There are two types of effector cells: CD4+ and CD8+-based T helper cells and T killer cells (cytotoxic cells), accounting for about 80%, CD3+ and CD56+-based NKT cells, As well as NK cells and dendritic cells (DC), these effector cells account for about 20%. In recent years, it has been very active in tumor biotherapy. It has the characteristics of traditional biotherapy, such as safe and effective, broad tumor killing spectrum, and small side effects, and also has the characteristics of a typica...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A01N1/02
Inventor 齐湘杰王盛李俊萍于登伟
Owner 王盛
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