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Culture method of SNK cells

A culture method and cell technology, which is applied in the fields of medicine, immunology, cell biology and molecular biology, and can solve problems such as cumbersome experimental operations, unfavorable economic production and use, and long cell culture cycles

Inactive Publication Date: 2020-03-24
BEIJING DCTY BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing NK cell culture media OKM-100 and OKM-200 are not conducive to production and use due to their high cost and long cell culture cycle
Although the price of general-purpose RPMI-1640 medium is low, since it does not contain the cytokine IL-2, it is necessary to add an appropriate amount of IL-2 to the culture system every day, resulting in cumbersome and inconvenient experimental operations. Therefore, we are looking for a simple and economical medium. New cell culture systems are becoming more important

Method used

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  • Culture method of SNK cells
  • Culture method of SNK cells
  • Culture method of SNK cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] 1. Experimental materials and methods:

[0065] 1. The MICA gene (SEQ ID No.3) is connected to the 41BBL gene (SEQ ID No.2) through the first connecting gene (SEQ ID No.4).

[0066] 2. The IL-2 gene (SEQ ID No.1) is connected to the GFP gene (SEQ ID No.6) through the second linker gene (SEQ ID No.5).

[0067] 3. Lentiviral packaging

[0068] The IL-2-GFP fusion gene and the MICA+41BBL fusion gene were synthesized for the whole gene, and the virus supernatant was harvested through lentiviral packaging, concentrated and then transfected into PBMC. The specific operation process is as follows:

[0069] 1) Use gene synthesis technology to synthesize the fusion gene of IL-2-GFP and the fusion gene of MICA+41BBL (Jinweizhi Biotechnology Co., Ltd.), clone them into pCDH vector respectively, and extract the plasmid by transformation amplification to obtain enough target gene plasmid , the specific operation is as follows:

[0070] a) Take out the competent cells from the ref...

Embodiment 2

[0138] Experimental results:

[0139] Example 1: Autologous PBMC constructs trophoblasts, denoted as PBMC-A

[0140] Comparative Example 1: Allogeneic PBMC (HLA-matched) to construct trophoblast cells, denoted as PBMC-B

[0141] Comparative example 2: Allogeneic PBMC (HLA mismatch) constructs trophoblast cells, which is denoted as PBMC-C

[0142] 1. Cell growth curves of SNK cultured in different culture systems

[0143] Three kinds of trophoblasts from different sources were co-cultured with the same PBMC, and it was found by sampling and counting every day ( Figure 1~3 ), the PBMCs infected with IL-2 decreased cell viability and the total number of cells two days after infection with the virus. From the third day after the virus infection (ie D5), the cells gradually began to grow in a curve, and the growth curve was almost the same as that of The PBMC group supplemented with IL-2 every day was the same, until the 17th day of cell culture, the cell viability remained abo...

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Abstract

The invention provides an SNK cell culture method, which belongs to the technical field of medicine, immunology, cytobiology and molecular biology, and the method comprises the step of co-culturing PBMC carrying an IL-2 gene and PBMC carrying a 41BBL gene and an MICA gene to obtain SNK cells. The 41BBL gene and the MICA gene are carried, so that the proliferation of CD56, CD16, and NK cells can bestimulated; the obtained SNK cells have a good killing effect on various solid tumors, the PBMC carrying the IL-2 gene continuously expresses IL-2 under the condition that the PBMC growth condition is met, and IL-2 does not need to be added into a culture system.

Description

technical field [0001] The invention belongs to the technical fields of medicine, immunology, cell biology and molecular biology, and in particular relates to a method for cultivating SNK cells. Background technique [0002] Peripheral blood mononuclear cells isolated from peripheral blood (hereinafter referred to as PBMC) need to be induced by an appropriate amount of cytokine IL-2 to differentiate into NK cells when cultured in vitro. Therefore, the role of IL-2 in the cell culture medium of PBMC is quite critical. The existing NK cell culture media OKM-100 and OKM-200 are economically unfavorable for production and use due to their high cost and long cell culture cycle. Although the price of general-purpose RPMI-1640 medium is low, since it does not contain the cytokine IL-2, it is necessary to add an appropriate amount of IL-2 to the culture system every day, resulting in cumbersome and inconvenient experimental operations. Therefore, we are looking for a simple and econ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0783
CPCC12N5/0646C12N2501/2302C12N2501/599C12N2502/11
Inventor 焦顺昌张嵘李营营
Owner BEIJING DCTY BIOTECH CO LTD
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