Efficient in-vitro culture kit and culture method for killer cells

A technology of killing cells and in vitro culture, applied in the direction of cell culture active agent, cell culture support/coating, animal cells, etc., can solve the problem of limited cell expansion multiple and killing activity, low expansion efficiency, low purity and lack of NK cells. In vitro expansion system and other problems, to achieve the effect of increasing the operating burden, reducing pain and medical costs, and shortening the culture period

Active 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

However, due to the low content of NK cells in peripheral blood, reports from various research institutions at home and abroad have proposed different expansion methods, mainly based on the expansion system of IL-2, IL-12, and IL-15, and it is difficult to induce and culture NK cells in vitro Large size, low expansion efficiency and NK cell purity, limited cell expansion and killing activity, and lack of effective in vitro expansion system
Although the CIK culture technology is relatively mature, it is easily affected by reagent factors and the skills of experimenters, resulting in the purity of CIK (cell phenotype CD3 + CD56 + ) has some instability

Method used

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  • Efficient in-vitro culture kit and culture method for killer cells
  • Efficient in-vitro culture kit and culture method for killer cells
  • Efficient in-vitro culture kit and culture method for killer cells

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Effect test

Embodiment 1

[0046] This embodiment is used to introduce a high-efficiency killing cell in vitro culture kit of the present invention and its preparation and kit quality detection. The high-efficiency killing cell (CNK) in the present invention refers specifically to the combination of NK and CIK cells. The culture kit can conveniently culture and reinfuse two kinds of cells (NK cells and CIK cells) at the same time in clinical practice, which not only reduces the cost of cell culture, but also improves the safety of cell culture, and makes the culture of NK and CIK cells standardized and simplified .

[0047] A high-efficiency killing cell in vitro culture kit, including, the main components are Anti-CD3 antibody and the coating solution of heparin sodium for injection, the main components are anti-human CD28 antibody, IL-2, IL-1α, and the induction of sapelin Liquid, the main component is the amplification liquid of IL-2 and IL-21. In this embodiment, 1 ml of the coating solution, 1 ml ...

Embodiment example 1

[0080] Implementation case 1 result:

[0081]

[0082]

[0083] Table 1

[0084] As another realization, a high-efficiency killing cell in vitro culture kit, including, the main components are Anti-CD3 antibody and heparin sodium injection coating solution, the main components are anti-human CD28 antibody, IL-2, IL-1α , the induction solution of Sapelin, the main component is the amplification solution of IL-2 and IL-21; in the coating solution, the effective concentration of Anti-CD3 antibody is 100μg / ml; the effective concentration of heparin sodium is 500IU / cm 2 In the induction solution, the effective concentrations of Anti-CD28 antibody, IL-2, IL-1α and Sapelin are respectively 10ng / ml, 100IU / ml, 100IU / ml and 20ng / ml; in the expansion solution, The effective concentrations of IL-2 and IL-21 are 1000IU / ml and 200IU / ml respectively.

[0085] As yet another realization, a high-efficiency killing cell in vitro culture kit, including, the main components are anti-CD3 a...

Embodiment 2

[0086] Embodiment two, kit stability detection

[0087] 1. CNK cell preparation

[0088] (1) Antibody coating of culture flask: pipette 1ml of coating solution on T-75cm 2 Add 4ml of normal saline to the culture bottle, shake the culture bottle gently, and allow the solution to diffuse on the surface of the culture bottle. Incubate for 1 hour at room temperature or store at 4°C until removed before use. Remove the coating solution, wash the culture flask once with normal saline, and use the washed culture flask immediately.

[0089] (2) Mononuclear cell isolation

[0090] 3 healthy volunteers were randomly selected, and 30ml of human peripheral blood anticoagulated with heparin sodium solution was collected from healthy volunteers, slowly poured into two tubes filled with 15ml of lymphocyte separation medium, and centrifuged at room temperature with a centrifugal force of 800xg for 20 minutes. After centrifugation, the blood was divided into 4 layers, and the upper layer o...

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Abstract

The invention relates to an efficient in-vitro culture kit for killer cells. The efficient in-vitro culture kit comprises main components including a coating solution of an Anti-CD3 antibody and heparin sodium for injection, an induction solution of an anti-human CD28 antibody, IL-2, IL-1alpha and sapylin, and an amplification solution of IL-2 and IL-21. The invention constructs the kit for in-vitro coculture of natural killer (NK) and cytokine induced killer (CIK) cells, the cell culture cost is reduced, and the cell culture safety is improved, so that the culture of the NK and CIK cells is standardized and simplified.

Description

technical field [0001] The invention belongs to the technical field of immune cell therapy, and relates to a high-efficiency killer cell (CNK) in vitro culture kit and a culture method. [0002] technical background [0003] Since the 1980s and 1990s, the application of adoptive cell therapy technology in the field of tumor immunotherapy research has been a hot question, mainly including lymphokine-activated killer cells (LAK), cytokine-induced killer cells (CIK), natural Killer cells (NK), natural killer T cells (NKT) and cytotoxic T lymphocytes (CTL), etc. [0004] Natural killer cells (Natural Killer Cells; NK) play an important role in immune defenses such as anti-virus, anti-tumor and anti-infection. Because NK cells do not express the antigen recognition receptors (TCR or BCR) possessed by T and B lymphocytes, and other unique markers, they were once called bare cells. CD 3 - , TCR - 、BCR - 、CD 56 + 、CD 16 + Lymphocytes are considered to be NK cells. CD 56 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N5/0638C12N2533/70C12N2501/515C12N2501/51C12N2501/2301C12N2501/2302C12N2501/2321C12N2500/72C12N2500/90
Inventor 孔伟圣蓝欣冉红王燕
Owner 珠海贝索细胞科学技术有限公司
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