CN111286488A - In vitro culture method of natural killer cells - Google Patents

Abstract

The invention relates to the technical field of biological pharmacy, in particular to a natural killer cell in-vitro culture method, which comprises the steps of dividing the whole culture process into two stages of induction culture and proliferation culture, wherein hyaluronic acid and a CD16 antibody are used for pre-coating a culture bottle in the induction culture, vitamin E and quercetin are added in the culture process, sesamin and RetroNectin are used for pre-coating the culture bottle in the proliferation culture, glutathione and porphyrin- α are added in the culture process, the coating components and the added auxiliary components of the culture bottle are mutually matched with effective components in culture media in all stages, the activation and proliferation of NK cells are synergistically promoted, the amplification efficiency of the NK cells is improved, and the activity of the NK cells is improved.

Description

A kind of natural killer cell culture method in vitro

technical field

The invention relates to the technical field of biopharmaceuticals, in particular to a method for culturing natural killer cells in vitro.

Background technique

The human immune system is regulated by a complex mechanism. When the immune system is abnormal, it may cause an imbalance of the immune system, and various difficult-to-treat diseases such as cancer may occur. Therefore, the development of immune cell therapy, a method of treating immune-related diseases by alleviating the imbalance of the immune system and restoring it to a normal state, has come into the spotlight.

Natural killer cells (NK) are a type of human immune cells derived from bone marrow lymphocytes. In the human body, NK cells are mainly distributed in peripheral blood and spleen, accounting for approximately 5% to 15% of peripheral blood lymphocytes; at the same time, they also exist in small amounts in other tissues such as lymph nodes. NK cells have their own unique properties relative to other types of lymphocytes. Its most important property is that NK cells do not express specific antigen-recognition receptors. Therefore, in the process of killing target cells, NK cells do not need to recognize target cells through a specific mechanism. They are the first line of defense for the human body to quickly resist the invasion of foreign substances, and can kill various pathogens broadly. At the same time, the killing effect of NK cells is not limited by the major histocompatibility complex, and can rapidly kill and dissolve a variety of tumor cells. In recent years, a large number of clinical studies have shown that NK cells play a very important role in resisting tumors and viral infections.

Since the content of NK cells in peripheral blood lymphocytes is only about 10%, NK cells used in clinical applications usually need to be expanded by in vitro culture to meet the clinical dosage. The conventional method of in vitro culture is to use high doses of IL-2 to expand NK cells, which can only be expanded several times, and a large amount of peripheral blood needs to be drawn from patients to achieve the number of cells required for treatment. Therefore, in recent years, people have carried out a lot of research to improve the efficiency of NK cell culture in vitro, including adding IL-5, IL-21, GM-CSF and other inducing factors and proliferating factors to the culture medium. Although these methods of improving the medium have improved the expansion efficiency of NK cells to a certain extent, the injection of NK cells cultured in vitro to exert their immune function in vivo also needs to ensure that the NK cells have high activity. Therefore, how to improve the expansion efficiency of NK cells, prevent cell death and aging, and maintain cell activity has become an urgent technical problem to be solved.

SUMMARY OF THE INVENTION

In order to overcome the defects of the prior art, the purpose of the present invention is to provide a method for culturing natural killer cells in vitro, which has high amplification efficiency and maintains cell activity.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows:

A method for culturing natural killer cells in vitro, comprising the following steps:

1) Prepare a mononuclear cell suspension:

Resuspend the isolated peripheral blood mononuclear cells in the induction medium to obtain a mononuclear cell suspension;

2) Induction culture:

Add hyaluronic acid and CD16 antibody to the culture flask to coat the culture flask and use it as a culture flask for induction culture; inoculate the mononuclear cell suspension of step 1) into the culture flask for induction culture, and cultivate for 24 hours. Supplement the induction medium in time, and add vitamin E and quercetin to the culture flask every 6 hours to obtain the induction culture suspension;

3) Proliferation culture:

Add sesamin and RetroNectin to a new culture flask to coat the culture flask as a culture flask for proliferation culture; inoculate the induced culture suspension obtained in step 2) into a culture flask for proliferation culture, under the environment of proliferation culture medium Cultivate for 14 days, supplement the proliferation medium in time during the cultivation process, and add glutathione and porphyrin-α to the culture flask every 2 to 3 days, that is, complete;

The composition of the induction medium includes basal medium, IL-2, IL-5, autologous serum, and amino acids; the composition of the proliferation medium includes basal medium, IL-15, GM-CSF, autologous serum, and amino acids.

Further, in step 2), the specific method of adding hyaluronic acid and CD16 antibody to the culture flask to coat the culture flask includes taking physiological saline, adding hyaluronic acid and CD16 antibody, and configuring it to contain 3-6 mg/ml hyaluronic acid. Acid and 5~8μg/ml CD16 antibody coating solution, add 5~10ml PBS and 1~2ml coating solution to the culture flask, after overnight at 4°C, discard the coating solution, wash with PBS, and you are done .

Further, in step 3), adding sesamin and RetroNectin to the culture medium, the specific method for coating the culture flask includes taking physiological saline, adding sesamin and RetroNectin, and being configured to contain 50-60 μg/ml sesamin and 5-8 μg /ml RetroNectin coating solution, add 5~10ml of PBS and 1~2ml of coating solution to the culture flask. After overnight at 4°C, discard the coating solution and wash with PBS.

Further, the specific composition of induction medium is: DMEM medium, 5% autologous serum, 1% glutamine, 1% non-essential amino acid, 500～750IU/ml IL-2, 35～60mg/ml IL-5;

The specific composition of the proliferation medium is: Cellgro-SCGM medium, 5% autologous serum, 1% glutamine, 1% non-essential amino acids, 25~50ng/ml IL-15, 0.5~1μg/ml GM-CSF.

Further, in step 2), according to the volume of the culture medium in the culture bottle, the dosage of vitamin E added to the culture bottle is 10-20 mg/ml, and the dosage of quercetin is 15-20 μg/ml.

Further, in step 3), according to the volume of the culture medium in the culture flask, the amount of glutathione added to the culture flask is 5-10 μg/ml, and the amount of porphyrin-α added is 0.6-1 μg/ml.

Further, the specific method for preparing the mononuclear cell suspension in step 1) includes aseptically collecting peripheral blood, diluting the peripheral blood with buffer, adding Ficoll separation medium, centrifuging and washing to obtain peripheral blood mononuclear cells, and separating the peripheral blood mononuclear cells. The nucleated cells are resuspended in induction medium according to a certain concentration, that is, complete.

The natural killer cell in vitro culture method of the invention divides the whole culture process into two stages: induction culture and proliferation culture. The culture flask is pre-coated with hyaluronic acid and CD16 antibody in the induction culture, and vitamin E and quercetin are added in the culture process. ; Proliferation culture uses sesamin and RetroNectin to pre-coat the culture flask, and adds glutathione and porphyrin-α during the culture process; the coating components of the culture flask, the added auxiliary components and the active ingredients in the medium at each stage They cooperate with each other and synergistically promote the activation and proliferation of NK cells. While improving the expansion efficiency of NK cells, the activity of NK cells is also improved.

Detailed ways

The technical solutions of the present invention will be described in detail below through specific embodiments.

Example 1

A method for culturing natural killer cells in vitro, comprising the following steps:

1) Configure the culture medium:

A: Induction medium: take DMEM medium, add 5% autologous serum, 1% glutamine, 1% non-essential amino acids, 600IU/ml IL-2, 45mg/ml IL-5;

B: Proliferation medium: take Cellgro-SCGM medium, add 5% autologous serum, 1% glutamine, 1% non-essential amino acids, 35ng/ml IL-15, 0.7μg/ml GM-CSF;

The non-essential amino acids are serine and alanine;

2) Prepare the mononuclear cell suspension:

Aseptically collect peripheral blood, and dilute the peripheral blood with PBS buffer at a ratio of 1:2; take a centrifuge tube and add Ficoll separation medium, then add the diluted peripheral blood to the centrifuge tube, centrifuge and wash to obtain peripheral blood mononuclear cells , the peripheral blood mononuclear cells were resuspended in induction medium at a concentration of 5×10 ⁵ /ml;

3) Induction culture:

A: Coating of culture flasks: take physiological saline, add hyaluronic acid and CD16 antibody, prepare a coating solution containing 5 mg/ml hyaluronic acid and 7 μg/ml CD16 antibody, add 7 ml of PBS, 1.5 ml of coating solution, after overnight at 4°C, discard the coating solution and wash with PBS;

B: Inoculate the mononuclear cell suspension prepared in step 2) into the coated culture flask, supplement the induction medium in time during the culture process, and add vitamin E to the culture flask every 6 hours to a concentration of 15 mg/ ml, quercetin to a concentration of 18 μg/ml, and cultured for 24 hours to obtain an induced culture suspension;

4) Proliferation culture:

A: Coating of culture flasks: take physiological saline, add sesamin and RetroNectin, prepare a coating solution containing 55μg/ml sesamin and 7μg/ml RetroNectin, add 8ml of PBS and 1.5ml of coating to the culture flask After overnight at 4°C, discard the coating solution and wash with PBS;

B: Inoculate the induced culture suspension prepared in step 3) into the coated culture flask, replenish the proliferation medium in time during the culture process, and add glutathione to the medium every 3 days to a concentration of 7 μg /ml, porphyrin-α to a concentration of 0.8 μg/ml, cultured for 14 days and harvested.

Example 2

A method for culturing natural killer cells in vitro, comprising the following steps:

1) Configure the culture medium:

A: Induction medium: take DMEM medium, add 5% autologous serum, 1% glutamine, 1% non-essential amino acids, 500IU/ml IL-2, 60mg/ml IL-5;

B: Proliferation medium: take Cellgro-SCGM medium, add 5% autologous serum, 1% glutamine, 1% non-essential amino acids, 25ng/ml IL-15, 1μg/ml GM-CSF;

The non-essential amino acids are serine and alanine;

2) Prepare the mononuclear cell suspension:

Aseptically collect peripheral blood, and dilute the peripheral blood with PBS buffer at a ratio of 1:2; take a centrifuge tube and add Ficoll separation medium, then add the diluted peripheral blood to the centrifuge tube, centrifuge and wash to obtain peripheral blood mononuclear cells , the peripheral blood mononuclear cells were resuspended in induction medium at a concentration of 5×10 ⁵ /ml;

3) Induction culture:

A: Coating of culture flasks: take physiological saline, add hyaluronic acid and CD16 antibody, and prepare a coating solution containing 3 mg/ml hyaluronic acid and 8 μg/ml CD16 antibody, add 10 ml of PBS, 2 ml of PBS to the culture flask After overnight at 4°C, the coating solution was discarded and washed with PBS;

B: Inoculate the mononuclear cell suspension prepared in step 2) into the coated culture flask, supplement the induction medium in time during the culture process, and add vitamin E to the culture flask every 6 hours to a concentration of 10 mg/ ml, quercetin to a concentration of 20 μg/ml, and cultured for 24 hours to obtain an induced culture suspension;

4) Proliferation culture:

A: Coating of culture flasks: take physiological saline, add sesamin and RetroNectin, prepare a coating solution containing 50μg/ml sesamin and 8μg/ml RetroNectin, add 5ml of PBS and 1ml of coating solution to the culture flask , after overnight at 4°C, discard the coating solution and wash with PBS;

B: Inoculate the induced culture suspension prepared in step 3) into the coated culture flask, replenish the proliferation medium in time during the culture process, and add glutathione to the medium every 2 days to a concentration of 5 μg /ml, porphyrin-α to a concentration of 1 μg/ml, cultured for 14 days and harvested.

Example 3

A method for culturing natural killer cells in vitro, comprising the following steps:

1) Configure the culture medium:

A: Induction medium: take DMEM medium, add 5% autologous serum, 1% glutamine, 1% non-essential amino acids, 750IU/ml IL-2, 35mg/ml IL-5;

B: Proliferation medium: take Cellgro-SCGM medium, add 5% autologous serum, 1% glutamine, 1% non-essential amino acids, 50ng/ml IL-15, 0.5μg/ml GM-CSF;

The non-essential amino acids are serine and alanine;

2) Prepare the mononuclear cell suspension:

Aseptically collect peripheral blood, and dilute the peripheral blood with PBS buffer at a ratio of 1:2; take a centrifuge tube and add Ficoll separation medium, then add the diluted peripheral blood to the centrifuge tube, centrifuge and wash to obtain peripheral blood mononuclear cells , the peripheral blood mononuclear cells were resuspended in induction medium at a concentration of 5×10 ⁵ /ml;

3) Induction culture:

A: Coating of culture flasks: Take physiological saline, add hyaluronic acid and CD16 antibody, and prepare a coating solution containing 6 mg/ml hyaluronic acid and 5 μg/ml CD16 antibody, add 5 ml of PBS, 1.5 ml of PBS to the culture flask ml of coating solution, after overnight at 4°C, discard the coating solution and wash with PBS;

B: Inoculate the mononuclear cell suspension prepared in step 2) into the coated culture flask, supplement the induction medium in time during the culture process, and add vitamin E to the culture flask every 6 hours to a concentration of 20 mg/ ml, quercetin to a concentration of 15μg/ml, and cultured for 24 hours to obtain an induced culture suspension;

4) Proliferation culture:

A: Coating of culture flasks: take physiological saline, add sesamin and RetroNectin, and prepare a coating solution containing 60 μg/ml sesamin and 5 μg/ml RetroNectin, add 10 ml of PBS and 2 ml of coating solution to the culture flask , after overnight at 4°C, discard the coating solution and wash with PBS;

B: Inoculate the induced culture suspension prepared in step 3) into the coated culture flask, replenish the proliferation medium in time during the culture process, and add glutathione to the medium every 3 days to a concentration of 10 μg /ml, porphyrin-α to a concentration of 0.6 μg/ml, cultured for 14 days and harvested.

Comparative Example 1

The difference between this comparative example and Example 1 is that the culture flask for induction culture is coated with sesamin and RetroNectin, and the culture flask for proliferation culture is coated with hyaluronic acid and CD16 antibody, and the others are the same as in Example 1.

Comparative Example 2

The difference between this comparative example and Example 1 is that glutathione and porphyrin-α were added to replace vitamin E and quercetin during the induction culture, and vitamin E and quercetin were added to replace the glutathione during the proliferation culture. Sathione and Porphyrin-alpha.

Comparative Example 3

The difference between this comparative example and Example 1 is that the use of hyaluronic acid is omitted in the coating process of the culture flask for induction culture, and the other is the same as that of Example 1.

Comparative Example 4

The difference between this comparative example and Example 1 is that the use of sesamin was omitted during the coating process of the culture bottle for proliferation culture, and an equal amount of sesamin was added to the proliferation medium, and the others were the same as in Example 1.

Test Example 1 Comparison of the total number of cell proliferation

Configure a trypan blue solution with pH=7.0~7.2 and a mass concentration of 0.4%, respectively take the NK cell suspensions of Examples 1~3, Comparative Examples 1~4, and 14 days of proliferation and culture, and add an equal volume of trypan blue. After mixing, the cells were stained, and the total number of cells was counted on the cytometer. The results showed that Example 1> Example 2 ≈ Example 3> Comparative Example 3> Comparative Example 4 ≈ Comparative Example 1> Comparative Example 2, and Example 14 days of proliferation The total number of cells after culture was 5 times that of Comparative Example 1 and 10 times that of Comparative Example 2.

Test Example 2 Cell Viability Comparison

A 96-well flat-bottom culture plate was used to set up three experimental wells, three pure effector cell wells and three pure target cell wells, and set up blank wells. K562 was added to the experimental wells and pure target cell wells. NK cells proliferated and cultured for 14 days in Example 1 were added to the experimental wells and the simple effector cell wells, the effector-target ratio was set to 40:1, cultured for 24 hours, 20 μl of WST was added, and the culture was continued for 4 hours, and the OD value was detected by a microplate reader. , killing activity (%)=[1-(OD value of experimental well-OD value of effector cell group)×OD value of pure target cell]×100%;

According to the same method as above, the killing activities of NK cells in Comparative Examples 1 to 4 after 14 days of proliferation and culture were calculated respectively to K562.

Comparing the killing activities of NK cells obtained by different culture methods, the results show that Example 1>Comparative Example 3>Comparative Example 4>Comparative Example 1>Comparative Example 2.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that it can still be The technical solutions described in the foregoing embodiments are modified, or some technical features thereof are equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (8)

1. a natural killer cell in vitro culture method, is characterized in that, comprises the following operation steps: 1) Prepare a mononuclear cell suspension: Resuspend the isolated peripheral blood mononuclear cells in the induction medium to obtain a mononuclear cell suspension; 2) Induction culture: Add hyaluronic acid and CD16 antibody to the culture flask to coat the culture flask as a culture flask for induction culture; inoculate the mononuclear cell suspension of step 1) into the culture flask for induction culture, and cultivate for 24 hours. Supplement the induction medium in time, and add vitamin E and quercetin to the culture flask every 6 hours to obtain the induction culture suspension; 3) Proliferation culture: Add sesamin and RetroNectin to a new culture flask to coat the culture flask as a culture flask for proliferation culture; inoculate the induced culture suspension obtained in step 2) into a culture flask for proliferation culture, under the environment of proliferation culture medium Cultivate for 14 days, supplement the proliferation medium in time during the cultivation process, and add glutathione and porphyrin-α to the culture flask every 2 to 3 days, that is, complete; The composition of the induction medium includes basal medium, IL-2, IL-5, autologous serum, and amino acids; the composition of the proliferation medium includes basal medium, IL-15, GM-CSF, autologous serum, and amino acids. 2. The method for culturing natural killer cells in vitro as claimed in claim 1, wherein in step 2), adding hyaluronic acid and CD16 antibody in the culture flask to coat the culture flask The concrete method comprises taking physiological saline, adding Hyaluronic acid and CD16 antibody, configure a coating solution containing 3-6 mg/ml hyaluronic acid and 5-8 μg/ml CD16 antibody, add 5-10 ml of PBS and 1-2 ml of coating solution to the culture flask, 4 After overnight at °C, the coating solution was discarded and washed with PBS to complete. 3. natural killer cell in vitro culture method as claimed in claim 1, is characterized in that, in step 3), in substratum, add sesamin and RetroNectin and the concrete method that culture flask is coated comprises getting physiological saline, adding sesamin and RetroNectin, configured into a coating solution containing 50-60 μg/ml sesamin and 5-8 μg/ml RetroNectin, add 5-10 ml of PBS and 1-2 ml of coating solution to the culture flask, and discard it after overnight at 4°C The coating solution, washed with PBS, is completed. 4. The method for culturing natural killer cells in vitro as claimed in claim 1, wherein the specific composition of the induction medium is: DMEM medium, 5% autologous serum, 1% glutamine, 1% non-essential amino acids, 500 ~750IU/ml IL-2, 35-60mg/ml IL-5. 5. The method for culturing natural killer cells in vitro as claimed in claim 4, wherein the specific composition of the proliferation medium is: Cellgro-SCGM medium, 5% autologous serum, 1% glutamine, 1% non-essential amino acids , 25～50ng/ml IL-15, 0.5～1μg/ml GM-CSF. 6. The method for culturing natural killer cells in vitro as claimed in claim 1, wherein in step 2), according to the volume of the culture medium in the culture flask, the consumption of adding vitamin E to the culture flask is 10～20mg/ml, adding The dosage of quercetin is 15-20 μg/ml. 7. The method for culturing natural killer cells in vitro as claimed in claim 1, wherein in step 3), according to the volume of the culture medium in the culture flask, the consumption of adding glutathione to the culture flask is 5～10 μg/ml , and the amount of porphyrin-α added is 0.6 to 1 μg/ml. 8. The method for culturing natural killer cells in vitro as claimed in claim 1, wherein step 1) the specific method for preparing mononuclear cell suspension comprises aseptically collecting peripheral blood, diluting the peripheral blood with buffer, adding Ficoll to separate The solution is centrifuged and washed to obtain peripheral blood mononuclear cells. The peripheral blood mononuclear cells are resuspended in an induction medium according to a certain concentration, and the process is completed.