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NK cell transfection efficiency improving method

A technology of NK cells and transfection efficiency, applied in the fields of botanical equipment and methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problem of low effective cell number, low CAR-NK effective cell ratio, and low virus titer and other problems to achieve the effect of improving the transfection efficiency

Active Publication Date: 2018-11-27
国健呈诺生物科技(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently, the CAR-NK gene modification mainly uses the method of lentiviral vector infection. Generally, the untreated lentiviral vector virus titer is low, and NK cells are difficult to infect. Therefore, the CAR-NK prepared by the general method is effective. Low cell ratio, low total effective cell number

Method used

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  • NK cell transfection efficiency improving method
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  • NK cell transfection efficiency improving method

Examples

Experimental program
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Effect test

Embodiment 1

[0052] A method for improving NK cell transfection efficiency, the steps are as follows:

[0053] On day 5 of NK cell expansion in vitro figure 1 , by 5×10 5 / ml·well (12-well plate, the culture medium in each well is 0.8ml) for lentiviral vector infection, add 1 lentiviral vector containing CAR for the first infection, and add 1 μl of co-infective agent 1 at the same time;

[0054] For the second infection after 5 hours, add 1 lentiviral vector containing CAR, add 1 μl of co-infection agent 2, 1 μl of co-infection agent 3, and 1 μl of co-infection agent 4, mix well by gently pipetting, and carry out normal culture;

[0055]The preparation of each raw material involved is as follows:

[0056] 1. Lentiviral vector concentration: use 10cm plate to package lentivirus, lentiviral packaging includes three plasmids, consisting of: psPAX2, pMD2.0G and Plenti-CAR-NK, prepared according to the ratio of 3.2μg, 1.8μg, 5μg, the first day Transfect 293T, collect 30ml lentivirus-containi...

Embodiment 2

[0064] A method for improving NK cell transfection efficiency, the steps are as follows:

[0065] On the 6th day of NK in vitro expansion culture, press 5×10 5 / ml well (12-well plate, 1ml of culture medium in each well) for lentiviral vector infection, add 1 lentiviral vector containing CAR for the first infection, and add 1 μl of co-infective agent 1 at the same time;

[0066] For the second infection after 6 hours, add 1 lentiviral vector containing CAR, add 1 μl of co-infection agent 2, 1 μl of co-infection agent 3, and 1 μl of co-infection agent 4, mix well by gently pipetting, and carry out normal culture;

[0067] The preparation of each raw material involved is as follows:

[0068] 1. Lentiviral vector concentration: use 10cm plate to package lentivirus, lentiviral packaging includes three plasmids, consisting of: psPAX2, pMD2.0G and Plenti-CAR-NK, prepared according to the ratio of 3.2μg, 1.8μg, 5μg, the first day Transfect 293T, collect 30ml lentivirus-containing s...

Embodiment 3

[0076] A method for improving NK cell transfection efficiency, the steps are as follows:

[0077] On the 7th day of NK in vitro expansion culture, press 8×10 5 / ml well (12-well plate, 1.5ml of culture medium per well) for lentiviral vector infection, add 1 lentiviral vector containing CAR for the first infection, and add 1 μl of co-infective agent 1 at the same time;

[0078] For the second infection after 7 hours, add 1 lentiviral vector containing CAR, add 1 μl of co-infection agent 2, 1 μl of co-infection agent 3, and 1 μl of co-infection agent 4, mix well by gently pipetting, and carry out normal culture;

[0079] The preparation of each raw material involved is as follows:

[0080] 1. Lentivirus concentration: Use a 10cm plate to package lentivirus. The lentivirus package includes three plasmids, consisting of psPAX2, pMD2.0G and Plenti-CAR-NK, prepared in proportions of 3.5μg, 2.0μg, and 5.5μg, on the first day Transfect 293T, collect 30ml lentivirus-containing supern...

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Abstract

The invention relates to the technical field of NK cell in-vitro amplification, in particular to a NK cell transfection efficiency improving method. In lentivirus infection of NK cells, an auxiliary infection agent 1 is added while a CAR-containing lentivirus carrier is added in primary infection; an auxiliary infection agent 2, an auxiliary infection agent 3 and an auxiliary infection agent 4 areadded while a CAR-containing lentivirus carrier is added in secondary infection; the auxiliary infection agent 1 refers to polybrene solution, the auxiliary infection agent 2 refers to IL2 solution,the auxiliary infection agent 3 refers to IL12 solution, and the auxiliary infection agent 4 refers to PHA solution. According to a Car-NK transfection method, different auxiliary infection agents areadded in a transfection process, NK cell transfection efficiency is imiproved, and NK cell lentivirus transfection efficiency can be improved from less than 10% to 50%-60%.

Description

technical field [0001] The invention relates to the technical field of NK cell expansion in vitro, in particular to a method for improving the transfection efficiency of NK cells. Background technique [0002] Car-NK is mainly used in the cellular immunotherapy of tumors. At present, the cellular immunotherapy of tumors is mainly based on Car-T technology, and Car-NK is rarely used, mainly because NK cells are difficult to transfect. As far as in vitro experiments are concerned, NK cells have higher killing activity on tumors than T cells, and T cells cannot be used for allogeneic reinfusion due to their high immunogenicity, while NK cells have low immunogenicity and can be used for allogeneic reinfusion. The difficult transfection characteristics of cells limit the application of Car-NK. Therefore, it is very important to improve the transfection efficiency of NK cells. [0003] Currently, the CAR-NK gene modification mainly uses the method of lentiviral vector infection. ...

Claims

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Application Information

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IPC IPC(8): C12N15/867
CPCC12N15/86C12N2740/15043
Inventor 顾雨春尹乐谭声江
Owner 国健呈诺生物科技(北京)有限公司
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