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Culturing method of NK (natural killer) cell

A technology of NK cells and culture methods, which is applied in the field of NK cell culture, can solve the problems of poor cell culture effect, high price of Cellgro medium and IL-15, and achieve the goal of ensuring cytotoxicity, reducing culture costs and saving operation time Effect

Inactive Publication Date: 2015-09-23
WUHAN HAMILTON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Generally speaking, the current NK cell expansion effect is still not satisfactory for practical applications, and the prices of Cellgro medium and IL-15 are very expensive, so the medium generally used in China is generally used to cultivate immune cells. base, but the effect of cell culture is extremely poor

Method used

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  • Culturing method of NK (natural killer) cell
  • Culturing method of NK (natural killer) cell
  • Culturing method of NK (natural killer) cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] NK cell culture from peripheral blood

[0039] Materials and Methods

[0040] The RPMI-1640 medium used in the present invention is selected from GIBCO Company, and recombinant human IL-2 is purchased from the market.

[0041] Step a: Preparation of autologous plasma

[0042] In a biosafety cabinet, divide 40mL-60mL of anticoagulated whole blood in the syringe into two 50mL sterile centrifuge tubes, centrifuge with a centrifuge at a speed of 2500rpm for 10min, and absorb the upper layer of plasma after centrifugation, 56 ℃, 30min to inactivate complement, place in a 4℃ water bath for 15 minutes, then centrifuge with a centrifuge at 2500rpm for 20 minutes, and transfer the supernatant to a new centrifuge tube. Mark and store in a -20°C refrigerator;

[0043] Step b: Isolation of mononuclear cells

[0044] Add 25 mL of buffered saline solution to the unabsorbed blood cells after centrifugation described in step a to dilute the blood cells, and rotate the centrifuge tu...

Embodiment 2

[0056] NK cell culture by conventional method

[0057] Materials and Methods

[0058] The Cellgro SCGM serum-free medium medium used in the conventional method was selected from the company (Freiburg, Germany), and IL-15 and recombinant human IL-2 were purchased from the market.

[0059] Step a to step c are the same as embodiment 1

[0060] Step d: Resuspend cells, inoculate

[0061] Discard the supernatant from the cell suspension obtained in the above step c, take 5 mL and resuspend the cells in CellGro medium containing 10 ng / mL CD3 monoclonal antibody, 10 ng / mL IL-15 and 500 U / mL recombinant human IL-2 (Adjust cell concentration to 1-2x 10 6 / ml), add 5% autologous plasma, transfer to a culture bottle with a surface area of ​​75 square centimeters, 1*10 per bottle 6 / mL density at 37°C, 5% CO 2 In a cell culture incubator with saturated humidity, incubate for 2 hours;

[0062] Step e: culture and identification of NK cells

[0063] Observe the cell state every day....

Embodiment 3

[0064] Embodiment 3: the comparison of the cell expansion multiple of this method and conventional method

[0065] Take the cultured cells from the two groups on the 0th, 5th, 10th, 15th and 20th day respectively, count them after staining with trypan blue, divide the total number of cells on the day of counting by the number of cells before culture (that is, day 0), and the value is is the expansion factor of the cells. This method can dynamically compare the expansion of two groups of cells, the results are shown in Table 2 and Figure 4 Shown: on the 5th, 10th, 15th, and 20th days of culture, the NK cell expansion times of this method were significantly higher than those of the conventional group P<0.01.

[0066] Table 2

[0067] Training days (days)

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Abstract

The invention discloses a culturing method of an NK (natural killer) cell. The culturing method comprises the following steps of (1) separating a mononuclear cell from peripheral blood or umbilical cord blood of a human body; (2) inoculating the mononuclear cell into a culture medium suitable for culturing lymphocyte, adding a CD3 monoclonal antibody, recombinant human interleukin 2 and autologous plasma, and culturing for 3 to 5 days; (3) adding the recombinant human interleukin 2 and the autologous plasma, and culturing; (4) harvesting the NK cell. According to the culturing method of the NK cell, the culturing cost is reduced, the amplification multiple and cell toxicity of the NK cell are guaranteed, the operation time is saved, meanwhile the probability of error operation is reduced, the obtaining efficiency of the NK cell is higher, and the safety of the NK cell is better.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a method for culturing NK cells. Background technique [0002] NK cells are important immune regulatory cells for the body to resist malignant tumors and viral infections. They originate from bone marrow-derived CD34+ hematopoietic progenitor cells and can directly kill tumor cells without the presence of antibodies or prior sensitization. They are important effector cells for tumor immunotherapy. With the in-depth study of the molecular mechanism of NK cells recognizing human tumors, NK cell-based anti-tumor immunotherapy has attracted attention and made significant progress. At present, the research on NK cells in tumor immunotherapy mainly focuses on autologous NK cell adoptive immunotherapy, allogeneic NK cell adoptive immunotherapy and gene-modified NK cell immunotherapy, etc., and has achieved remarkable results. However, how to improve the in vitro prol...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 姚惟琦武栋成袁冰
Owner WUHAN HAMILTON BIOTECH
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