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Preparation method of Car-NK cell for small cell lung cancer

A technology of small cell lung cancer and NK cells, applied in the field of biomedicine, can solve the problem of inability to carry out horizontal analogy, and achieve the effect of sufficient and convenient material sources

Inactive Publication Date: 2018-01-09
ANHUI HUIEN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First of all, small cell lung cancer itself is also a systematic concept, and no completely similar cases have appeared, so horizontal analogy cannot be made

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] S1. Collect 100mL of peripheral blood from the patient, put it into a blood collection tube covered with anticoagulant, and add 100mL of normal saline to dilute;

[0021] S2. Slowly add the diluted peripheral blood to the Ficoll lymphocyte separation medium, the volume ratio is 1:2, shake evenly and then centrifuge at 1500r / min for 20min, and the separated cells are treated with 5mmol / L PME at room temperature for 40min for later use;

[0022] S3. Human peripheral blood mononuclear cells treated in S2 are placed in NK medium, wherein, cytokines AIMV, 1000U / mL IL-2, 10ng / mL IL-12, 10% human AB serum are added to the NK medium , adjust the cell concentration to 3x10 5 , placed at 37°C, 5% CO 2 Cultivate continuously in the incubator for 48 hours, and collect the adherent cells;

[0023] S4. Add the cells collected in S3 to the NK medium, and adjust the cell concentration to 1×10 5 , continue to cultivate for 12 days, and supplement the culture medium every 3 days;

[...

Embodiment 2

[0027] S1. Collect 100mL of peripheral blood from the patient, put it into a blood collection tube covered with anticoagulant, and add 100mL of normal saline to dilute;

[0028] S2. Slowly add the diluted peripheral blood to the Ficoll lymphocyte separation medium, the volume ratio is 1:2, shake evenly and then centrifuge at 2000r / min for 20min, and the separated cells are treated with 5mmol / L PME at room temperature for 40min for later use;

[0029] S3. Human peripheral blood mononuclear cells treated in S2 are placed in NK medium, wherein, cytokines AIMV, 1000U / mL IL-2, 10ng / mL IL-12, 10% human AB serum are added to the NK medium , adjust the cell concentration to 5x10 5 , placed at 37°C, 5% CO 2 Cultivate continuously in the incubator for 72 hours, and collect the adherent cells;

[0030] S4. Add the cells collected in S3 to the NK medium, and adjust the cell concentration to 2×10 5 , continue culturing for 15 days, and supplement the culture medium every 3 days;

[003...

Embodiment 3

[0034] S1. Collect 100mL of peripheral blood from the patient, put it into a blood collection tube covered with anticoagulant, and add 100mL of normal saline to dilute;

[0035] S2. Slowly add the diluted peripheral blood to the Ficoll lymphocyte separation medium, the volume ratio is 1:2, shake evenly, centrifuge at 1800r / min for 20min, and treat the separated cells with 5mmol / L PME at room temperature for 40min for later use;

[0036] S3. Human peripheral blood mononuclear cells treated in S2 are placed in NK medium, wherein, cytokines AIMV, 1000U / mL IL-2, 10ng / mL IL-12, 10% human AB serum are added to the NK medium , adjust the cell concentration to 4x10 5 , placed at 37°C, 5% CO 2 Cultivate continuously in the incubator for 60 hours, and collect the adherent cells;

[0037] S4. Add the cells collected in S3 to the NK medium, and adjust the cell concentration to 1.5×10 5 , continue culturing for 14 days, and supplement the culture medium every 3 days;

[0038] S5, trans...

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Abstract

The invention discloses a preparation method of a Car-NK cell for small cell lung cancer. The method comprises the following steps: collecting peripheral blood of a patient and isolating an NK cell with an Ficoll method; placing a peripheral blood mononuclear cell in an NK medium, adjusting the cell concentration, placing the cell in an incubator for continuous culture for 48-72 h, and collectingan adherent cell; adding the cell to the NK medium for continuous culture for 12-15 days, and supplementing a culture solution once every three days; transfecting the NK cell with lentivirus and performing multiplication culture to obtain the Car-NK cell; preparing a small cell lung cancer cell preparation.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a preparation method of Car-NK cells for small cell lung cancer. Background technique [0002] Small cell lung cancer is one of the basic types of lung cancer, which belongs to undifferentiated carcinoma, and its pathological types include oat cell type, intermediate cell type and compound oat cell type. One-third of lung cancer patients are of this type. Small cell lung cancer is a highly malignant tumor with bad biological behavior and dire prognosis. Comparing the same spreading range, small cell lung cancer has a shorter symptom period before diagnosis and a shorter survival period after diagnosis than other types of lung cancer. [0003] For the survival cycle of patients with small cell lung cancer, the medical community has not been able to have a definite conclusion. First of all, small cell lung cancer itself is also a systematic concept, and no completely similar cases hav...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867A61K35/17A61P35/00
Inventor 张正亮
Owner ANHUI HUIEN BIOTECH
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