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Reagent and reagent kit for in vitro culture of NK cells through union of dual antibodies and thymosin, and culture method

A NK cell, in vitro culture technology, applied in cell culture active agents, biochemical equipment and methods, culture process, etc., can solve the problems of spreading infectious diseases, allergy to xenogeneic proteins, etc., achieving high proliferation, high lethality, lightening painful effect

Inactive Publication Date: 2020-09-22
珠海贝索细胞科学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, the culture medium used mainly uses fetal calf serum or calf serum as nutrients for cell culture. Although there is sufficient supply and low price, there is a risk of potential transmission of infectious diseases, and there will also be ethical impacts in clinical treatment. and foreign proteins may cause allergies

Method used

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  • Reagent and reagent kit for in vitro culture of NK cells through union of dual antibodies and thymosin, and culture method
  • Reagent and reagent kit for in vitro culture of NK cells through union of dual antibodies and thymosin, and culture method
  • Reagent and reagent kit for in vitro culture of NK cells through union of dual antibodies and thymosin, and culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] This example is used to introduce a reagent for culturing NK cells in vitro in combination with thymosin of the present invention and its preparation and quality testing of the kit. The NK cell culture reagent, kit and culture method provided by the invention can improve the purity and quantity of the obtained NK cells, are simple and efficient, not only reduce the cost of cell culture, but also improve the safety of the cell culture.

[0044] A reagent for culturing NK cells in vitro in combination with a double antibody combined with thymosin, including an induction solution whose main components are CD161, CD137, thymosin and IL-2, and whose main components are the activation of IL-2, IL-12, IL-7 and IL-15 Liquid, the main component is the amplification liquid of IL-2 and IL-21. In the induction solution, the effective concentration ranges of CD161, CD137, thymosin and IL-2 are respectively 500ng / ml~1500ng / ml, 100ng / ml~1000ng / ml, 100-500ug / ml and 50IU / ml~500IU / ml . ...

Embodiment example 1

[0078] Implementation case 1 result:

[0079] Test items Inducing solution Activation solution Amplification solution determination skills requirement endotoxin <0.06EU / ml

[0080] Table 1

Embodiment 2

[0081] Embodiment 2: Test kit stability

[0082] 1. Preparation of natural killer cells (NK for short)

[0083] (1) Mononuclear cell isolation

[0084] Randomly select 3 healthy volunteers, collect 20ml of peripheral blood from healthy volunteers with sodium heparin anticoagulant tube, and separate cells within two hours. Slowly pour 20ml of fresh blood on 15ml of lymphatic separation fluid, centrifuge at 800xg for 20min, slowly rise and fall. After centrifugation, blood is separated into 4 layers consisting of plasma (upper layer), mononuclear cells between plasma and separation fluid (layer 2), separation fluid (layer 3), and erythrocyte layer (bottom layer). First collect the upper layer of plasma with a sterile pipette and collect it into a centrifuge tube. Do not aspirate the mononuclear cell layer by mistake. The plasma needs to be heated at 56°C for 30 minutes, and centrifuged at 1200xg for 10 minutes at room temperature. Tubes were stored at 4°C until removed before...

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Abstract

The invention relates to a reagent and reagent kit for in vitro culture of NK cells through union of dual antibodies and thymosin, and a culture method. The reagent comprises inducing fluid includingmain components of CD161, CD137 and IL-2, activating fluid including main components of IL-2, IL-12, IL-7 and IL-15, and amplifying fluid including main components of IL-2 and IL-21. Components of theinducing fluid, the activating fluid and the amplifying fluid in the prepared cell reagent can stimulate proliferation of the NK cells, secretion of cytokine, regulation of activation and disintegration of the NK cells, so that the reagent can increase the purity and the quantity of the obtained NK cells. In addition, the reagent uses serum-free cell culture media free of animal-derived components, and ectogenic animal-derived protein is not introduced, so that the possibility of animal-derived pollution is reduced, and the safety of the reagent is improved.

Description

technical field [0001] The invention belongs to the technical field of immune cell therapy, and relates to a double-antibody combined thymosin peptide in vitro culture NK cell reagent, kit and culture method. [0002] technical background [0003] As a special lymphocyte, natural killer cell (NK) contains different types of immunological functions, mainly distributed in peripheral blood, lymph nodes, bone marrow, and spleen, and its immunophenotype is characterized by CD3 - CD56 + CD16 + , accounting for 10%-20% of peripheral blood lymphocytes. NK cells can be activated through a variety of receptors on their surface, mainly including cytokine receptors, membrane integral molecule natural cytotoxic receptors (CD16, NKp46, NKp30 NKp44, NKp80) immunoglobulin-like killer receptors (NKG2) family, DHAM-1 / CD266 family, SLAM family / SLAM, 2B4, NTB-A (CD84) / receptor for non-self antigen recognition (Ly49H) and some other receptors (such as CD18, CD2, TLR-3 / - 9 etc.). [0004] At ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/599C12N2501/2302C12N2501/2307C12N2501/2312C12N2501/2315C12N2501/2321C12N2501/30C12N2500/90
Inventor 孔伟圣王燕蓝欣何娟娟
Owner 珠海贝索细胞科学技术有限公司
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