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NK culture medium and amplification culture method thereof

A medium and serum-free medium technology, applied in the field of NK medium and its expansion culture, can solve the problems of expensive instruments and low purity of NK cells, and achieve the effect of high expansion efficiency

Pending Publication Date: 2021-04-20
杭州原生生物科技有限公司
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AI Technical Summary

Benefits of technology

The technical effect that this patented method solves by eliminating any risks associated with obtaining human fetus blood from pregnant women without causing harm during laboratory tests. It uses specialized cells called natural killer (NK) cells instead of animal products like cow milk for specific purposes such as killing tumor-cells while still maintaining their ability to fight off other diseases caused by viruses. This allows N K cells to have better performance over time compared to current methods used for producing these cells.

Problems solved by technology

This patented describes different methods for culturing Natural Killer cell populations called CD34+ lymphocytes or neutrocyte precursors with high levels of activity towards cancerous tumor growth. These techniques require isolating specific types from other normal tissues during their cultivation process which may increase costs compared to current procedures such as immunotherapy. Additionally, these processes involve multiple stages like expansion followed by further manipulations on them, making them difficult to perform at large scales due to costly equipment required.

Method used

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  • NK culture medium and amplification culture method thereof
  • NK culture medium and amplification culture method thereof
  • NK culture medium and amplification culture method thereof

Examples

Experimental program
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Embodiment 1

[0026] Embodiment 1: the preparation of culture medium

[0027] A kind of NK medium comprises, serum-free medium, IL-2, IL-21, IL-15, human autologous serum; Described serum-free medium is Corning KBM581 lymphocyte serum-free medium; Described IL-2 The concentration is between 500-1500 U / mL, specifically, in this embodiment, the concentration of IL-2 is preferably 800 U / mL. The concentration of IL-21 is 5-15 ng / mL, and the concentration of IL-15 is 1-10 ng / mL. In this embodiment, the concentration of IL-21 is preferably 10 ng / mL, and the concentration of IL-15 is preferably 5 ng / mL. Wherein the volume of the human autologous serum is 5-15%, in this embodiment, the volume of the human autologous serum is preferably 10%.

[0028] The control medium is: Corning KBM581 lymphocyte serum-free medium, IL-2 (800U / mL), IL-21 (10ng / mL), IL-15 (5ng / mL), 10% autologous plasma, OKT-3 ( 5ng / mL);

Embodiment 2

[0029] Example 2: Obtaining cord blood mononuclear cells

[0030] A. Obtain 50mL of cord blood;

[0031] B. Preparation of serum: centrifuge 800g of cord blood for 10min, absorb the upper layer of plasma and transfer it to a new 50mL centrifuge tube, place it in a water bath at 56°C for 30min, then cool it at -20°C for 30min, then centrifuge at 1200g for 10min. Take 15-20mL of the upper serum, and add it to the culture system at 5% in the later stage for use in culturing cells.

[0032] C. After absorbing the plasma, reduce the sample to 30mL with normal saline and mix well, slowly add it on the surface of the Ficoll liquid of human lymphocyte separation medium, 700g, and centrifuge at room temperature for 20min.

[0033] D. Discard the supernatant after centrifugation, carefully absorb the buffy coat layer of mononuclear cells, transfer to two 50mL centrifuge tubes with 50mL of normal saline in each tube, each separation tube corresponds to a 50mL centrifuge tube, and mix we...

Embodiment 3

[0036] Example 3: The present invention provides a method for expanding and culturing NK cells

[0037] Take the umbilical cord blood mononuclear cells prepared in Example 2 and resuspend them with the culture medium prepared in Example 1. The density of the cells after resuspension is 2×10 6 cells / mL, inoculate the cells in low-adsorption culture flasks, and place them at 37°C, 5% CO 2 The CD3-CD56+ ratio in the initial umbilical cord blood mononuclear cells was about 10% according to flow cytometry. The medium prepared in Example 1 was supplemented every 2 to 3 days to keep the density of the cells at 2×10 6 cells / mL, continuously cultured for 9 days, and recorded the number of cells regularly. In this experiment, the cells grown in the NK medium of the present invention maintained a good growth trend. After culturing until the ninth day, the cells were taken for flow cytometry, and it was found that the ratio of CD3-CD56+ was about 51.62%. After calculation, the total cel...

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Abstract

The invention discloses an NK culture medium and an amplification culture method thereof. The NK culture medium comprises an NK cell culture medium, wherein the culture medium comprises a serum-free culture medium, IL-2, IL-21, IL-15 and human autoserum. According to the culture medium, fetal calf serum is not needed, so that risks caused by use of exogenous serum can be avoided; a flow cytometer or an immunomagnetic bead technology does not need to be used for separation and purification; and the amplification efficiency is high, relatively high-purity NK cells can be obtained in 9 days, the proportion of CD3-CD56+ is larger than 50%, and the amplification multiple is 121 times.

Description

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Claims

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Application Information

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Owner 杭州原生生物科技有限公司
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