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A kind of hemizygous capri cell preparation method

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 achieve collection, etc. Effects of proliferation and activation ability

Inactive Publication Date: 2015-12-30
王盛 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 to bear the process of collecting mononuclear cells by the blood cell separator, ⑤ other conditions that do not meet the requirements of the blood cell separator to collect mononuclear cells, the collection of PBMC cannot be realized, which seriously hinders the implementation of the CAPRI cell therapy plan

Method used

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  • A kind of hemizygous capri cell preparation method
  • A kind of hemizygous capri cell preparation method
  • A kind of hemizygous capri cell preparation method

Examples

Experimental program
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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 100ml of peripheral blood from the patient and 150ml 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 for 20- After 30 minutes, the supernatant was discarded, and 25ml of normal saline was added to the centrifuge tubes, carefully mixed, and slowly distributed along the tube wall into 15ml centrifuge tubes pre-added with 5ml o...

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 120ml of peripheral blood from the patient and 150ml of peripheral blood 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 1800rpm for 20- After 30 minutes, the supernatant was discarded, and 25ml of normal saline was added to the centrifuge tubes, carefully mixed, and slowly distributed along the tube wall into 15ml centrifuge tubes pre-added with 5ml of Ficoll-Hypaque lymphocyte separation medium. After cent...

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 in vitro cytotoxicity of the CAPRI cells (CD3mAb-CAPRI) stimulated by CD3mAb alone and the CAPRI cells (CD3 / CD133mAb-CAPRI) co-stimulated by CD3mAb and CD133mAb For comparison, the specific experimental steps are as follows: CAPRI cells harvested from 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 stained with 0.4% trypan blue and counted As target cells, use serum-free 1640 medium to adjust the cell concentration of effector cells and target cells to 1×106 / ml with different effector-target ratios of 2.5:1, 5:1, 10:1, and 20:1, and add them to 96-well plates , 100 μl / well, in addition, set blank control wells, sample control wells, and sample maximum enzyme activity control wells, set 3 duplicate wells for e...

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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-induced 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 both non-specificity of CIK and DC Specific killing ability, 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, and NK cells And dendritic cells (DC), this part of effector cells accounts 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 ha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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