CN113493766B

CN113493766B - NK cell in-vitro culture method for improving cell amplification multiple

Info

Publication number: CN113493766B
Application number: CN202111039919.8A
Authority: CN
Inventors: 马士棋; 陈旭; 陈刚
Original assignee: Excell Biology Taicang Co ltd
Current assignee: Suzhou Ecosai Biotechnology Co.,Ltd.
Priority date: 2021-09-07
Filing date: 2021-09-07
Publication date: 2021-12-21
Anticipated expiration: 2041-09-07
Also published as: CN113493766A

Abstract

The invention discloses an NK cell in-vitro culture method for improving cell expansion multiple, wherein dextran has the effects of activating natural killer cells and macrophages, promoting immune cell expansion and the like, and is used as a raw material of products such as health nutrition supplements, functional oral liquids and the like in recent years. Dextran with different molecular weights has different auxiliary effects on the growth of immune cells, low molecular dextran can increase the osmotic pressure of a culture medium to simulate the plasma effect so as to assist in activating NK cells, and small molecular dextran can promote the expansion of the NK cells by increasing the content of free calcium ions and cAMP in the cells and reducing cell clustering. The invention creatively introduces the dextran, discloses a dextran adding method, limits the dosage of the dextran, can improve the cell expansion multiple by more than 60 percent in the in-vitro culture process of NK cells, improves the proportion of the NK cells by more than 10 percent, and has higher practicability.

Description

NK cell in-vitro culture method for improving cell amplification multiple

Technical Field

The invention relates to the technical field of NK cell culture, in particular to an NK cell in-vitro culture method for improving cell amplification multiple.

Background

Natural Killer cells, NK cells, are important immune cells in the body, not only related to anti-tumor and anti-viral infection, but also have a certain regulatory effect on T lymphocytes and B lymphocytes in the human body. NK cells are a class of immune cells not associated with a specific immune response, and their mediated lysis is not affected by the Major Histocompatibility Complex (MHC), and are therefore also referred to as the "heart" of the body's natural immune system. NK cells can not only remove parasitic bacteria, viruses and aging variant cells in human bodies, but also have extremely strong removal effect on cancer cells, and the adoptive immunotherapy using the NK cells is an important means for the immunotherapy of tumor cells clinically at present. The number of the NK cells is related to the killing effect of the NK cells on tumor cells, so how to optimize the in vitro culture process of the NK cells, improve the proportion of the NK cells and the amplification multiple of the NK cells is the key point of the research on adoptive immunotherapy of the NK cells.

Dextran (dextran), also called dextran and dextrane, is a polymer of glucose and can be produced by fermentation of sucrose with Leuconostoc mesenteroides. Dextran can be classified into high molecular dextran (average molecular weight Mw = 100000-. Dextran is often used as a medicinal injection-grade raw material, has small side effect, is safe to human bodies, and is a raw material very suitable for in vitro culture of immune cells.

Based on the situation, the application discloses an NK cell in-vitro culture method for improving cell expansion multiple, so that the NK cell proportion is improved in the NK cell in-vitro culture process.

Disclosure of Invention

The invention aims to provide an NK cell in-vitro culture method for improving cell expansion multiple, so as to solve the problems in the background technology.

In order to solve the technical problems, the invention provides the following technical scheme:

an in vitro culture method of NK cells for improving cell expansion multiple comprises the following steps:

(1) isolating the PBMC cells;

(2) transferring the separated PBMC cell suspension to a culture flask coated with a CD16 antibody at D0 days, adding a culture medium, and performing cell activation culture at 37 ℃ under the 5% carbon dioxide culture condition for 3D;

(3) transferring the cells to a new culture flask at D3 days, adding culture medium, and supplementing liquid to maintain cell density of 1-1.5 × 10⁶And culturing for 4 days, reducing the proportion of the heat-inactivated autologous plasma to 1% at D7 days, continuing culturing for 10 days, and centrifuging at D17 days to harvest the cells.

In a more optimized scheme, 50-150 μ g/mL of dextran is added at D0 day, and the average molecular weight of dextran is Mw = 20000.

In an optimized protocol, 100. mu.g/mL dextran was added at D0.

In a more optimized scheme, from D3 days, the solution is supplemented with dextran with the concentration of 50-200 μ g/mL, and the average molecular weight of the dextran is Mw = 9000.

In the optimized scheme, from D3 days, the solution is supplemented with dextran with the concentration of 100 mug/mL.

In an optimized scheme, the culture medium is NK culture medium containing IL-2, IL-15, IL-12, IL-21 and heat-inactivated autologous plasma.

In an optimized scheme, the final concentration of the IL-2 is 1000IU/mL, the final concentration of the IL-15 is 50ng/mL, the final concentration of the IL-12 is 10ng/mL, the final concentration of the IL-21 is 10ng/mL, and the adding proportion of the heat inactivated autologous plasma is 10%.

In an optimized scheme, the NK culture medium is any one of SCGM NK culture medium, Corning NK culture medium and XVIVO15 culture medium.

In the optimized scheme, in the step (2), the cell density in the culture flask is 2 multiplied by 10⁶one/mL.

In an optimized scheme, in the step (1), PBMC cells are freshly separated from human whole blood by adopting a density centrifugation method.

Compared with the prior art, the invention has the following beneficial effects:

dextran has the effects of activating natural killer cells and macrophages, promoting immune cell expansion and the like, and is recently used as a raw material of products such as health nutrition supplements, functional oral liquids and the like. Dextran with different molecular weights has different auxiliary effects on the growth of immune cells, low molecular dextran can increase the osmotic pressure of a culture medium to simulate the plasma effect so as to assist in activating NK cells, and small molecular dextran can promote the expansion of the NK cells by increasing the content of free calcium ions and cAMP in the cells and reducing cell clustering. The invention provides an NK cell in-vitro culture method for improving cell expansion multiple, which can improve the cell expansion multiple by more than 60% and improve the NK cell proportion by more than 10% in the NK cell in-vitro culture process.

The invention relates to an in vitro culture method of NK cells for improving cell expansion fold, wherein the dextran adding method comprises the steps of adding dextran with average molecular weight Mw =20000 during activation of NK cells, adding dextran with average molecular weight Mw =9000 during the culture process after activation of NK cells, wherein the dextran adding concentration with average molecular weight Mw =20000 is 50-150 mu g/mL, the dextran adding concentration with average molecular weight Mw =9000 is 50-200 mu g/mL, and more preferably, the dextran adding concentration with average molecular weight Mw =20000 is 100 mu g/mL, and the dextran adding concentration with average molecular weight Mw =9000 is 100 mu g/mL.

The invention creatively introduces dextran, discloses a dextran adding method, limits the dosage of the dextran, can improve the cell expansion multiple by more than 60 percent in the in-vitro culture process of NK cells, and improves the proportion of the NK cells by more than 10 percent; meanwhile, the dextran adopted by the application has clear source, no animal source component, no toxicity to cells, simple use method and excellent performance, can be widely applied to actual production and has higher practicability.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a graph showing the cell viability of the experimental group-the control group III in example 1 of the present invention;

FIG. 2 is a graph showing the cell viability of the control group four to the control group seven in example 1 of the present invention;

FIG. 3 is a graph showing the cell expansion fold of the experimental group-control group III in example 1 of the present invention;

FIG. 4 is a graph showing the fold expansion of cells in the control group IV-control group VII in example 1 of the present invention;

FIG. 5 is a graph showing the results of flow assay of the experimental group of example 1 of the present invention;

FIG. 6 is a diagram showing the results of the flow assay in control group seven in example 1 of the present invention;

FIG. 7 is a graph showing the cell viability in the experimental group-control group III in example 2 of the present invention;

FIG. 8 is a graph showing the cell viability of the control group four to the control group seven in example 2 of the present invention;

FIG. 9 is a graph showing the fold amplification of cells in the experimental group-control group III in example 2 of the present invention;

FIG. 10 is a graph showing the fold expansion of cells in the control group IV-control group VII in example 2 of the present invention;

FIG. 11 is a graph showing the results of flow assay of the experimental group of example 2 of the present invention;

FIG. 12 is a diagram showing the results of the flow assay in control group VII in example 2 of the present invention;

FIG. 13 is a graph showing the cell viability in the experimental group-control group III according to example 3 of the present invention;

FIG. 14 is a graph showing the cell viability of the control group four-control group seven in example 3 of the present invention;

FIG. 15 is a graph showing the fold expansion of cells in the experimental group-control group III in example 3 of the present invention;

FIG. 16 is a graph showing the fold expansion of cells in the control group four-control group seven in example 3 of the present invention;

FIG. 17 is a graph showing the results of flow assay of the experimental group of example 3 of the present invention;

FIG. 18 is a schematic diagram showing the flow detection results of control group VII in example 3 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the examples described below, the media used were as follows:

CellGenix brand SCGM NK medium (SCGM NK medium for short), with the product number of 20802-0500, the basic culture method is a general method for activating and amplifying NK cells by using CD16/IL-2/IL-15/IL-12/IL-21 cytokines;

a Corning brand NK cell activation/amplification culture medium set (called Corning NK culture medium for short), wherein the product number is 88-570-kit, and the basic culture method is a standard operation method attached to the set;

a serum-free culture medium (XVIVO 15 culture medium for short) of immune cells of Lonza brand XVIVO15, a cargo number BE02-060F, and a basic culture method are methods for activating and amplifying cytokines which are used for NK cells and used by CD 16/IL-2/IL-15/IL-12/IL-21.

The materials and consumable sources used in the examples are those commonly used in the art.

Example 1:

(1) freshly separating PBMC (peripheral blood mononuclear cell) from human whole blood by adopting a density centrifugation method for activating and amplifying NK cells;

(2) taking the separated PBMC cell suspension, transferring the PBMC cell suspension to a T75 culture bottle coated with a CD16 antibody at D0 days, adding an NK culture medium containing IL-2, IL-15, IL-12, IL-21 and heat-inactivated autologous plasma, wherein the final concentration of the IL-2 is 1000IU/mL, the final concentration of the IL-15 is 50ng/mL, the final concentration of the IL-12 is 10ng/mL, the final concentration of the IL-21 is 10ng/mL, the adding proportion of the heat-inactivated autologous plasma is 10%, the NK culture medium is SCGM NK culture medium, the cell density in the culture bottle is 2 multiplied by 10, and the cell density in the culture bottle is 2 multiplied by 10⁶Performing cell activation culture at 37 deg.C under 5% carbon dioxide culture condition for 3 d;

(3) transferring the cells to a new T75 culture flask at D3 days, adding NK medium containing IL-2, IL-15, IL-12, IL-21, and heat-inactivated autologous plasma, specifically adding components and selecting NK medium, and performing cell viability and density test every 1 or 2 days as described in step (2), and supplementing liquid to maintain cell density at 1.5 × 10⁶Keeping the maximum capacity of a T75 culture bottle to be 40mL, discarding redundant cells, culturing for 4D, reducing the proportion of heat-inactivated autologous plasma to 1% in D7 days, continuing culturing for 10D, centrifuging and harvesting cells in D17 days, and performing flow cytometry detection on cell surface antigens CD3 and CD56, wherein CD3-CD56+ is a mark of NK cells.

The procedure disclosed in example 1 was used as experimental group (SCGM NK medium group) and the following control experiments were performed:

control group one (SCGM NK medium + Mw20000 dextran minimum): on the basis of the above culture method, dextran with a final concentration of 50 μ g/mL average molecular weight Mw =20000 was added to the culture medium on day 0 (D0);

control group two (SCGM NK medium + Mw20000 dextran optimum): on the basis of the above culture method, dextran with a final concentration of 100 μ g/mL average molecular weight Mw =20000 was added to the culture medium on day 0 (D0);

control group three (SCGM NK medium + Mw20000 dextran top): on the basis of the above culture method, dextran with a final concentration of 150 μ g/mL average molecular weight Mw =20000 was added to the culture medium on day 0 (D0);

control group four (SCGM NK medium + Mw9000 dextran minimum): on the basis of the above culture method, dextran with a final concentration of 50 μ g/mL average molecular weight Mw =9000 was added to the culture medium at every fluid replacement starting on day 3 (D3);

control group five (SCGM NK medium + Mw9000 dextran optimum): on the basis of the above culture method, dextran with a final concentration of 100. mu.g/mL average molecular weight Mw =9000 was added to the culture medium at every fluid replacement starting on day 3 (D3);

control group six (SCGM NK medium + Mw9000 dextran max): on the basis of the above culture method, dextran with a final concentration of 200. mu.g/mL average molecular weight Mw =9000 was added to the culture medium at every fluid replacement starting on day 3 (D3);

control group seven (SCGM NK medium + dextran addition method of the invention): on the basis of the above culture method, dextran with a final concentration of 100. mu.g/mL average molecular weight Mw =20000 was added to the culture medium on day 0 (D0), and dextran with a final concentration of 100. mu.g/mL average molecular weight Mw =9000 was added to the culture medium at every fluid replacement on day 3 (D3).

And (4) conclusion: the cell viability data are shown in table one, and the cell fold expansion data are shown in table two:

watch 1

Watch two

The specific cell viability curve is shown in fig. 1 and fig. 2: dextran is added in the scheme, the cell viability is not influenced, the viability of the experimental group on the 17 th day is 91.6%, and the viability of the seventh control group (namely the scheme disclosed by the application) is 92.9%.

The specific cell expansion curve 3, fig. 4 shows: the dextran is added in the scheme, so that the cell amplification times can be obviously improved, the cell amplification times of an experimental group on the 17 th day are 529 times, the cell amplification times of a control group seven (namely the scheme disclosed by the application) are 891 times, and the cell amplification times can be improved by 68.4% by using the dextran adding method. This effect cannot be achieved by adding only dextran with an average molecular weight Mw =20000 or dextran with an average molecular weight Mw = 9000.

The flow detection results of the experimental group are shown in fig. 5, and the flow detection results of the control group seven are shown in fig. 6: the addition of dextran in the scheme can obviously improve the proportion of NK cells during harvesting, the proportion of NK cells in a test group on the 17 th day (CD 3-CD56 +) is 69%, the proportion of NK cells in a control group seven (namely the scheme disclosed by the application) (CD 3-CD56 +) is 82.5%, and the proportion of NK cells can be improved by 19.6% by using the dextran addition method.

Example 2:

(2) taking the separated PBMC cell suspension, transferring the PBMC cell suspension to a T75 culture bottle coated with a CD16 antibody at D0 days, adding an NK culture medium containing IL-2, IL-15, IL-12, IL-21 and heat-inactivated autologous plasma, wherein the final concentration of the IL-2 is 1000IU/mL, the final concentration of the IL-15 is 50ng/mL, the final concentration of the IL-12 is 10ng/mL, the final concentration of the IL-21 is 10ng/mL, the adding proportion of the heat-inactivated autologous plasma is 10%, the NK culture medium is corning NK culture medium, the cell density in the culture bottle is 2 multiplied by 10, and the cell density in the culture bottle is 2 multiplied by 10⁶Performing cell activation culture at 37 deg.C under 5% carbon dioxide culture condition for 3 d;

(3) cells were transferred on day D3Adding NK culture medium containing IL-2, IL-15, IL-12, IL-21, and heat-inactivated autologous plasma into new T75 culture flask, specifically adding components and selecting NK culture medium, performing cell viability and density test every 1 or 2 days, and supplementing liquid to maintain cell density at 1.5 × 10⁶Keeping the maximum capacity of a T75 culture bottle to be 40mL, discarding redundant cells, culturing for 4D, reducing the proportion of heat-inactivated autologous plasma to 1% in D7 days, continuing culturing for 10D, centrifuging and harvesting cells in D17 days, and performing flow cytometry detection on cell surface antigens CD3 and CD56, wherein CD3-CD56+ is a mark of NK cells.

The procedure disclosed in example 2 was used as an experimental group (corning NK medium group) and the following control experiments were carried out:

control group one (corning NK medium + Mw20000 dextran minimum): on the basis of the above culture method, dextran with a final concentration of 50 μ g/mL average molecular weight Mw =20000 was added to the culture medium on day 0 (D0);

control group two (corning NK medium + Mw20000 dextran optimum): on the basis of the above culture method, dextran with a final concentration of 100 μ g/mL average molecular weight Mw =20000 was added to the culture medium on day 0 (D0);

control group three (corning NK medium + Mw20000 dextran top): on the basis of the above culture method, dextran with a final concentration of 150 μ g/mL average molecular weight Mw =20000 was added to the culture medium on day 0 (D0);

control group four (corning NK medium + Mw9000 dextran minimum): on the basis of the above culture method, dextran with a final concentration of 50 μ g/mL average molecular weight Mw =9000 was added to the culture medium at every fluid replacement starting on day 3 (D3);

control group five (corning NK medium + Mw9000 dextran optimum): on the basis of the above culture method, dextran with a final concentration of 100. mu.g/mL average molecular weight Mw =9000 was added to the culture medium at every fluid replacement starting on day 3 (D3);

control group six (corning NK medium + Mw9000 dextran top): on the basis of the above culture method, dextran with a final concentration of 200. mu.g/mL average molecular weight Mw =9000 was added to the culture medium at every fluid replacement starting on day 3 (D3);

control group seven (kangning NK medium + dextran addition method of the invention): on the basis of the above culture method, dextran with a final concentration of 100. mu.g/mL average molecular weight Mw =20000 was added to the culture medium on day 0 (D0), and dextran with a final concentration of 100. mu.g/mL average molecular weight Mw =9000 was added to the culture medium at every fluid replacement on day 3 (D3).

And (4) conclusion: the cell viability data are shown in table three, and the cell fold expansion data are shown in table four:

watch III

Watch four

Specific cell viability curves are shown in fig. 7 and 8: the dextran is added in the scheme, the cell viability is not influenced, the viability of the experimental group on the 17 th day is 92%, and the viability of the control group seven (namely the scheme disclosed by the application) is 95.2%.

The specific cell expansion curve is shown in fig. 9 and 10: the dextran is added in the scheme, so that the cell amplification times can be obviously improved, the cell amplification times of an experimental group on the 17 th day are 549 times, the cell amplification times of a control group seven (namely the scheme disclosed by the application) are 944 times, and the cell amplification times can be improved by 71.9% by using the dextran adding method; this effect cannot be achieved by adding only dextran with an average molecular weight Mw =20000 or dextran with an average molecular weight Mw = 9000.

The flow assay results of the experimental group are shown in fig. 11, and the flow assay results of the control group seven are shown in fig. 12: the addition of dextran in the scheme can obviously improve the proportion of NK cells during harvesting, the proportion of NK cells in a test group on day 17 (CD 3-CD56 +) is 72.3%, the proportion of NK cells in a control group seven (namely the scheme disclosed by the application) (CD 3-CD56 +) is 86.9%, and the proportion of NK cells can be improved by 20.2% by using the dextran addition method.

Example 3:

(2) taking the separated PBMC cell suspension, transferring the PBMC cell suspension to a T75 culture bottle coated with a CD16 antibody at D0 days, adding NK culture medium containing IL-2, IL-15, IL-12, IL-21 and heat-inactivated autologous plasma, wherein the final concentration of the IL-2 is 1000IU/mL, the final concentration of the IL-15 is 50ng/mL, the final concentration of the IL-12 is 10ng/mL, the final concentration of the IL-21 is 10ng/mL, the adding proportion of the heat-inactivated autologous plasma is 10%, the NK culture medium is XVIVO15 culture medium, the cell density in the culture bottle is 2 multiplied by 10, and the NK culture medium is XVIVO15 culture medium⁶Performing cell activation culture at 37 deg.C under 5% carbon dioxide culture condition for 3 d;

The procedure disclosed in example 3 was used as experimental group (XVIVO 15 medium group) and the following control experiments were carried out:

control group one (XVIVO 15 medium + Mw20000 dextran minimum): on the basis of the above culture method, dextran with a final concentration of 50 μ g/mL average molecular weight Mw =20000 was added to the culture medium on day 0 (D0);

control group two (XVIVO 15 medium + Mw20000 dextran optimum): on the basis of the above culture method, dextran with a final concentration of 100 μ g/mL average molecular weight Mw =20000 was added to the culture medium on day 0 (D0);

control group three (XVIVO 15 medium + Mw20000 dextran maximum): on the basis of the above culture method, dextran with a final concentration of 150 μ g/mL average molecular weight Mw =20000 was added to the culture medium on day 0 (D0);

control group iv (XVIVO 15 medium + Mw9000 dextran minimum): on the basis of the above culture method, dextran with a final concentration of 50 μ g/mL average molecular weight Mw =9000 was added to the culture medium at every fluid replacement starting on day 3 (D3);

control group five (XVIVO 15 medium + Mw9000 dextran optimum): on the basis of the above culture method, dextran with a final concentration of 100. mu.g/mL average molecular weight Mw =9000 was added to the culture medium at every fluid replacement starting on day 3 (D3);

control group six (XVIVO 15 medium + Mw9000 dextran top): on the basis of the above culture method, dextran with a final concentration of 200. mu.g/mL average molecular weight Mw =9000 was added to the culture medium at every fluid replacement starting on day 3 (D3);

control group seven (XVIVO 15 medium + dextran addition method of the invention): on the basis of the above culture method, dextran with a final concentration of 100. mu.g/mL average molecular weight Mw =20000 was added to the culture medium on day 0 (D0), and dextran with a final concentration of 100. mu.g/mL average molecular weight Mw =9000 was added to the culture medium at every fluid replacement on day 3 (D3).

And (4) conclusion: cell viability data are shown in table five and cell fold expansion data are shown in table six:

watch five

Watch six

Specific cell viability curves are shown in fig. 13 and 14: dextran is added in the scheme, the cell viability is not influenced, the viability of an experimental group on the 17 th day is 90.1%, and the viability of a control group seven (namely the scheme disclosed by the application) is 94.7%.

The specific cell expansion graph 15, fig. 16 shows: the dextran is added in the scheme, so that the cell amplification multiple can be obviously improved, the cell amplification multiple of an experimental group on the 17 th day is 481 times, the cell amplification multiple of a control group seven (namely the scheme disclosed by the application) is 813 times, and the cell amplification multiple can be improved by 69% by using the dextran adding method. This effect cannot be achieved by adding only dextran with an average molecular weight Mw =20000 or dextran with an average molecular weight Mw = 9000.

The flow assay results of the experimental group are shown in fig. 17, and the flow assay results of the control group seven are shown in fig. 18: the scheme can obviously improve the proportion of NK cells in harvesting by adding dextran, the proportion of NK cells in a test group on the 17 th day (CD 3-CD56 +) is 75.8 percent, the proportion of NK cells in a control group seven (namely the scheme disclosed by the application) (CD 3-CD56 +) is 86.1 percent, and the proportion of NK cells can be improved by 13.6 percent by using the dextran adding method.

From the above data, it can be seen that: the invention creatively introduces dextran, discloses a dextran adding method, limits the dosage of the dextran, can improve the cell expansion multiple by more than 60 percent in the in-vitro culture process of NK cells, and improves the proportion of the NK cells by more than 10 percent; meanwhile, the dextran adopted by the application has clear source, no animal source component, no toxicity to cells, simple use method and excellent performance, can be widely applied to actual production and has higher practicability.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An NK cell in-vitro culture method for improving cell expansion multiple is characterized in that: the method comprises the following steps:

(1) isolating the PBMC cells;

(3) transferring the cells to a new culture flask at D3 days, adding culture medium, and supplementing liquid to maintain cell density of 1-1.5 × 10⁶Culturing for 4 days, reducing the proportion of heat-inactivated autologous plasma to 1% in D7 days, continuing culturing for 10 days, and centrifuging to harvest cells in D17 days;

adding dextran 50-150 μ g/mL in D0 days, wherein the average molecular weight Mw of the dextran is 20000; from D3 days, adding dextran with concentration of 50-200 μ g/mL, wherein the average molecular weight Mw of the dextran is 9000;

the culture medium is an NK culture medium containing IL-2, IL-15, IL-12, IL-21 and heat-inactivated autologous plasma; the final concentration of the IL-2 is 1000IU/mL, the final concentration of the IL-15 is 50ng/mL, the final concentration of the IL-12 is 10ng/mL, the final concentration of the IL-21 is 10ng/mL, and the adding proportion of the heat inactivated autologous plasma is 10 percent; the NK culture medium is any one of SCGM NK culture medium, Corning NK culture medium and XVIVO15 culture medium.

2. The method for culturing NK cells with increased cell expansion fold according to claim 1, wherein the method comprises the following steps: dextran 100. mu.g/mL was added on day D0.

3. The method for culturing NK cells with increased cell expansion fold according to claim 1, wherein the method comprises the following steps: from day D3, the replenisher added dextran at a concentration of 100. mu.g/mL.

4. The method for culturing NK cells with increased cell expansion fold according to claim 1, wherein the method comprises the following steps: in the step (2), the cell density in the culture flask is 2X 10⁶one/mL.

5. The method for culturing NK cells with increased cell expansion fold according to claim 1, wherein the method comprises the following steps: in the step (1), PBMC cells are freshly separated from human whole blood by a density centrifugation method.