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Serum-free cell culture solution for in-vitro amplification of nature killer cells and nature killer T cells

A technology of natural killer cells and cell culture, which is applied in the direction of cell culture active agents, tissue culture, animal cells, etc., and can solve the problems of cells being limited to the use of hosts and cell recipient rejection

Active Publication Date: 2019-12-31
PRECISION BIOTECH TAIWAN CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005]However, known natural killer cell in vitro expansion technology still needs to rely on host plasma or serum, or serum from other animal sources, to provide sufficient nutrients for cell growth , the addition of other animal-derived serum substances during the preparation process may cause rejection by the recipient of the cells, and the addition of host plasma or serum during the preparation process will limit the use of the amplified cells to the host, which has become a clinical development. A major bottleneck in immune cell therapy, accordingly, the present invention proposes to solve the above-mentioned known problems

Method used

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  • Serum-free cell culture solution for in-vitro amplification of nature killer cells and nature killer T cells
  • Serum-free cell culture solution for in-vitro amplification of nature killer cells and nature killer T cells
  • Serum-free cell culture solution for in-vitro amplification of nature killer cells and nature killer T cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, the test of serum-free culture fluid

[0051] The purpose of this example is to establish a serum-free cell culture medium that can be used to expand natural killer cells (NK cells) / natural killer T cells (NKT cells) in vitro, and this cell culture medium does not need to add additional autologous plasma or peripheral blood samples. Serum, or other animal-derived serum; peripheral blood mononuclear cells were obtained as described above, and the peripheral blood samples were obtained from autologous serum, SILAC Advanced DMEM / F-12, UltraGRO TM Or RPMI-1640 medium four kinds of cell culture medium for culture, wherein, the autologous serum of the source of the peripheral blood sample contains 500IU / ml rhIL-2 and 50ng / ml Anti-CD3 monoclonal antibody, SILAC Advanced DMEM / F -12. UltraGRO TM and RPMI-1640 medium respectively contained 500IU / ml of rhIL-2, 50ng / ml of Anti-CD3 monoclonal antibody and 2U / ml of heparin. After culturing, the cell density was evaluat...

Embodiment 2

[0055] Embodiment 2, optimization of initial cell number

[0056] Continuing from the above, this example continues to discuss the optimal initial cell density when expanding NK / NKT cells with serum-free cell culture medium, which obtains peripheral blood mononuclear cells as described above, and uses UltraGRO TM As a basal medium, add 500IU / ml rhIL-2, 50ng / ml Anti-CD3 monoclonal antibody and 2U / ml heparin for culture, and then evaluate the cell density by the aforementioned cell counting method.

[0057] As shown in Table 2, the initial cell density was 1.00x10 6 , 2.00x10 6 , 3.00x10 6 and 5.00x10 6 cells / ml was cultured, and the cells were taken out from the culture plate on the 5th, 7th, 9th, 12th, 14th, 16th, 18th and 20th day after culture for counting, and the cell density was readjusted to 1.00x106cells / ml, the results can be It is known that when NK / NKT cells are expanded in vitro with these initial cell densities, the cell survival rate can be maintained above 90...

Embodiment 3

[0060] Embodiment 3, optimization of heparin (Heparin) concentration in serum-free cell culture medium

[0061]In one embodiment of the present invention, heparin is used as part of the role of replacing serum. Therefore, this embodiment continues to discuss. Under the culture conditions of serum-free cell culture medium and initial cell density established in Examples 1 and 2, Can further enhance the heparin concentration of cell growth efficiency; It obtains the aforementioned peripheral blood mononuclear cells, with a preferred embodiment 2.00×10 6 cells / mL is the initial cell density, using UltraGRO TM The cell culture medium (containing 500 IU / ml of rhIL-2 and 50 ng / ml of Anti-CD3 monoclonal antibody) was cultured, and the test groups were respectively containing 2, 5 or 10 U / ml of heparin.

[0062] As shown in Table 3, the group cultured with 2U / ml heparin had the most stable cell expansion trend, and the total number of cells obtained on the 20th day of culture was 1.5...

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Abstract

The invention relates to a serum-free cell culture solution for in-vitro amplification of nature killer cells and nature killer T cells. The serum-free cell culture solution comprises a basal culturemedium, a human interleukin-2 recombinant protein (rhIL-2), heparin and an anti-CD3 monoclonal antibody (anti-CD3 monoclonal antibody); when the cell culture solution is used for amplifying nature killer cells (NK cells) and nature killer T cells (NKT cells) in vitro, the NK / NKT cells can be effectively proliferated without adding serum, and the obtained cells have high cell toxicity and can be used for preparing a pharmaceutical composition.

Description

technical field [0001] The present invention relates to a cell culture medium for expanding natural killer cells (NK cells) and natural killer T cells (NKT cells) in vitro, especially a kind of cell culture medium that can be expanded by three to seven thousand times in vitro without adding serum The cell culture medium of NK / NKT cells, and the NK / NKT cells amplified by the cell culture medium, the cell survival rate is higher than 85%, and the active NK / NKT cells reach more than 50%, which can kill cancer cells The effect is more than 76%. Background technique [0002] Immunotherapy (Immunotherapy) generally refers to the way of preventing and treating diseases by strengthening the organism's own immune system, or externally endowing the organism with immunity, which has the characteristics of high specificity, high efficiency and persistence; Over the past decade, immune cell therapy derived from the concept of immunotherapy has gradually been widely discussed. [0003] ...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/2302C12N2501/91C12N2501/515
Inventor 叶明功
Owner PRECISION BIOTECH TAIWAN CORP
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