In-vitro large-scale amplification method of natural killer cells

Abstract

The invention discloses an in-vitro large-scale amplification method of natural killer cells. The method comprises the following steps: collecting and separating peripheral blood mononuclear cells; sorting natural killer cells; culturing the natural killer cells; and collecting the natural killer cells. The method utilizing immunomagnetic beads to sort the natural killer cells has the advantages of simplicity in establishing and operation, and high comprehensive efficiency; and the effect of the method adopting in-vitro amplification of the immunomagnetic bead shorted NK cells is 2-3 times the effect of present amplification methods, so the method disclosed in the invention has great application values in the field of in-vitro large-scale amplification.

Description

technical field

[0001] The invention relates to the technical field of immune cell therapy, in particular to a method for culturing human cells. Background technique

[0002] The natural killer cell (NK cell) in vitro large-scale expansion method is a process for in vitro expansion of NK cells that meet clinical needs in the field of autoimmune cell therapy. This method mainly collects donor peripheral blood to separate mononuclear cells. In the culture environment, serum-free medium is used for culture, and a variety of required cytokines are added to achieve the purpose of large-scale expansion of NK cells.

[0003] The large-scale expansion method of NK cells has been extensively studied and applied in the field of immune cell therapy, because the expansion efficiency, purity and efficacy of NK cells directly affect the effect of NK cell therapy, so the expansion as high as possible Increased efficiency and purity are important indicators pursued by the industry. Becaus...

Examples

Embodiment 1

[0060] The in vitro large-scale expansion method of natural killer cells in this embodiment is carried out according to the following steps:

[0061] 1 Collect peripheral blood mononuclear cells (PBMC) from the donor.

[0062] Directly draw 80-100mL of venous blood from the donor, and add anticoagulant—heparin;

[0063] 2. Isolation of PBMCs

[0064] 2.1 Centrifuge at 2000rpm for ten minutes at room temperature, carefully absorb the upper plasma layer, inactivate in a 56-degree water bath for 30 minutes, centrifuge at 4000rpm for 10 minutes, carefully absorb the supernatant and store it at 4°C for later use.

[0065] 2.2 Dilute the patient's blood sample with sterile saline at a ratio of 1:1 to reduce the loss of PBMC. After mixing well, slowly add the blood sample into the 50mL centrifuge tube filled with lymphocyte separation medium at a ratio of 1:1.

[0066] 2.3 Horizontal centrifugation: speed 2000rpm, 30min.

[0067] 2.4 After the centrifugation is completed, the abs...

Embodiment 2

[0095] The large-scale in vitro expansion method of natural killer cells in this example differs from Example 1 in that: the anticoagulant added to the venous blood collected in step 1 is sodium citrate; the centrifuge used in step 2.4 Tubes are 250 mL capacity; step 4.4 is performed on day 4; step 4.5 is performed on day 6; step 4.6 is performed on day 9; step 4.8 is performed on day 12; step 4.9 is performed on day 15.

technical field

[0001] The invention relates to the technical field of immune cell therapy, in particular to a method for culturing human cells. Background technique

[0002] The natural killer cell (NK cell) in vitro large-scale expansion method is a process for in vitro expansion of NK cells that meet clinical needs in the field of autoimmune cell therapy. This method mainly collects donor peripheral blood to separate mononuclear cells. In the culture environment, serum-free medium is used for culture, and a variety of required cytokines are added to achieve the purpose of large-scale expansion of NK cells.

[0003] The large-scale expansion method of NK cells has been extensively studied and applied in the field of immune cell therapy, because the expansion efficiency, purity and efficacy of NK cells directly affect the effect of NK cell therapy, so the expansion as high as possible Increased efficiency and purity are important indicators pursued by the industry. Becaus...

Examples

Embodiment 1

[0060] The in vitro large-scale expansion method of natural killer cells in this embodiment is carried out according to the following steps:

[0061] 1 Collect peripheral blood mononuclear cells (PBMC) from the donor.

[0062] Directly draw 80-100mL of venous blood from the donor, and add anticoagulant—heparin;

[0063] 2. Isolation of PBMCs

[0064] 2.1 Centrifuge at 2000rpm for ten minutes at room temperature, carefully absorb the upper plasma layer, inactivate in a 56-degree water bath for 30 minutes, centrifuge at 4000rpm for 10 minutes, carefully absorb the supernatant and store it at 4°C for later use.

[0065] 2.2 Dilute the patient's blood sample with sterile saline at a ratio of 1:1 to reduce the loss of PBMC. After mixing well, slowly add the blood sample into the 50mL centrifuge tube filled with lymphocyte separation medium at a ratio of 1:1.

[0066] 2.3 Horizontal centrifugation: speed 2000rpm, 30min.

[0067] 2.4 After the centrifugation is completed, the abs...

Embodiment 2

[0095] The large-scale in vitro expansion method of natural killer cells in this example differs from Example 1 in that: the anticoagulant added to the venous blood collected in step 1 is sodium citrate; the centrifuge used in step 2.4 Tubes are 250 mL capacity; step 4.4 is performed on day 4; step 4.5 is performed on day 6; step 4.6 is performed on day 9; step 4.8 is performed on day 12; step 4.9 is performed on day 15.

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