NK cell in-vitro amplification culture method

Abstract

The invention discloses an NK (Natural Killer) cell in-vitro amplification culture method which comprises the following steps: on the 0th day, inoculating mononuclear cells into a culture bottle containing an NK cell culture solution, autologous plasma and an exosome of HEK293 cells, and standing and culturing in an incubator for 14 days; during the culture period, supplementing the NK cell culture solution and the autologous plasma once every two days, and controlling the cell density to be 1.0 * 10 < 6 > cells / mL; wherein the exosome of the HEK293 cell also needs to be supplemented on the seventh day; and after 14 days of culture, collecting centrifugal cells to obtain the NK cells. According to the culture method, the exosome of the HEK293 is used for replacing trophoblast cells added in the process of culturing the NK cells by a feeder method, so that the safety risk possibly caused by directly using the trophoblast cells is reduced; meanwhile, the HEK293 exosome is added and is combined with the NK cell surface receptor, so that synergistic stimulation is provided, the NK cells are activated and amplified at a high speed, and the multiplication capacity and purity of the NK cells are greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological cell culture, and in particular to a method for in vitro proliferation and culture of NK cells. Background Art

[0002] NK cells, also known as natural killer cells, are a type of granular lymphocytes and an indispensable part of the human immune system. They play an important role in anti-tumor, anti-viral and immune regulation. NK cells can not only identify and destroy abnormal cells (such as tumor cells and virus-infected cells), but in some cases, they are also involved in the development of allergic reactions and autoimmune diseases.

[0003] At present, the in vitro proliferation of NK cells is mainly achieved by two methods: one is feeder method trophoblast cell culture, and the other is pure factor stimulation. Trophoblast cell culture technology usually uses genetically modified trophoblast cells, which express stimulatory molecules and membrane-bound cytokines on their surface. After these cells are inactivated by irradiation, they are co-cultured with NK cells to activate and promote the proliferation of NK cells. The pure factor rule is to add a variety of biological factors to stimulate NK cells during NK culture to achieve the purpose of NK cell expansion. However, even after the trophoblast cells are inactivated, there are still potential risks. Compared with trophoblast cell culture, pure factor cultured NK cells usually perform poorly in terms of activity and purity.

[0004] Therefore, developing a method that can effectively promote NK cell proliferation while maintaining high cell purity is an important issue that needs to be urgently addressed in the field of immunology research. Summary of the invention

[0005] Based on the above problems, the problem to be solved by the present invention is to provide a high-activity, high-purity, safe and efficient in vitro expansion and culture method of NK cells.

[0006] The technical solution of the present invention is as follows:

[0007] A method for in vitro expansion and culture of NK cells comprises the following steps:

[0008] On day 0, first, 1.0×10 7 ~8.0×10 7 Mononuclear cells were inoculated into a coated culture flask containing 27 mL of NK cell induction medium; then 3 mL of autologous plasma and 1.0×10 8 ~5.0×10 8 The exosomes of HEK293 cells were collected by 1000 particles / mL. Finally, the culture flask was placed in an incubator at 37°C and 5% CO2.

[0009] On the third day, NK cell induction culture medium and autologous plasma were added to the culture flask at a volume ratio of 9:1;

[0010] On the fifth day, the culture flask was supplemented with NK cell induction culture medium and autologous plasma at a volume ratio of 19:1;

[0011] On the 7th day, the cells and culture medium were transferred to the culture bag, and the NK cell induction culture medium and autologous plasma with a volume ratio of 99:1 and 0.5×10 8 ~2.0×10 8 Particles / mL exosomes from HEK293 cells;

[0012] From the 9th day to the 13th day, the medium was changed every two days, and fresh NK cell expansion medium was added to maintain the cell density at 1.0×10 6 Pieces / mL;

[0013] On the 14th day, the culture was stopped, the cells were collected, centrifuged at 500 g for 10 min, the supernatant was removed, and the cell pellet was washed several times with PBS to obtain NK cells.

[0014] In one embodiment, in the above-mentioned NK cell in vitro expansion culture method, the NK cell induction culture medium includes KBM581 culture medium, which contains IL-15 with a final concentration of 100 to 2000 IU / mL and IL-2 with a final concentration of 500 to 2000 IU / mL; the NK cell expansion culture medium includes KBM581 culture medium, which contains IL-2 with a final concentration of 500 to 2000 IU / mL.

[0015] In one embodiment, in the above-mentioned NK cell in vitro expansion and culture method, the exosomes of HEK293 cells are obtained by the following steps:

[0016] HEK293 cells were overexpressed with mIL-21 and 4-1BBL to obtain HEK293 cells modified with mIL-21 and 4-1BBL genes;

[0017] The gene-modified HEK293 cells were expanded and cultured in HEK293 serum-free medium for 2 to 3 days to obtain expanded cells;

[0018] The expanded cells were inoculated into a culture flask containing HEK293 serum-free medium. After culturing for 2 days, the cells were centrifuged at 500 g for 10 min, and the supernatant was collected to obtain the first exosome stock solution of HEK293 cells. The cell precipitate was also collected.

[0019] Adding an equal volume of HEK293 serum-free medium to the HEK293 serum-free medium in the culture flask to the collected cell pellet, culturing the cells for 2 days, centrifuging at 500 g for 10 min, and collecting the supernatant again to obtain a second exosome stock solution of HEK293 cells;

[0020] The mixed solution of the first exosome stock solution and the second exosome stock solution was purified using a PEG6000 solution to obtain exosomes from HEK293 cells.

[0021] In one embodiment, in the above-mentioned NK cell in vitro expansion and culture method, HEK293 cells modified with mIL-21 and 4-1BBL genes are obtained by the following steps:

[0022] The cell density was 0.5 × 10 5 ~2.0×10 5 HEK293 cells at 1 × 10 cells / ml were seeded into each well of a 24-well plate and 1.0 × 10 6 Add 1 mL of HEK293 serum-free medium to each cell and then place in a 37°C, 5% CO2 incubator for 24 h;

[0023] After 24 hours, take out the 24-well plate, add 100 μL to 500 μL of HEK293 serum-free medium to each well, and continue culturing for 1 hour;

[0024] After 1 h, add the lentiviral concentrate to each well of the 24-well plate at an MOI of 1 to 5, and continue incubating for 6 h;

[0025] After 6 h, 700 μl of HEK293 serum-free medium was added to each well of the 24-well plate and culture was continued for 72 h;

[0026] After 72 hours, puromycin with a final concentration of 1 μg / mL was added to each well of the 24-well plate to screen out HEK293 cells stably expressing mIL-21 and 4-1BBL, thereby obtaining HEK293 cells modified with the mIL-21 and 4-1BBL genes.

[0027] In one embodiment, in the above-mentioned NK cell in vitro expansion culture method, after obtaining the HEK293 cells modified with mIL-21 and 4-1BBL genes, the HEK293 cells need to be further expanded and cultured:

[0028] The HEK293 cells modified with 2hmIL-21 and 4-1BBL genes were treated with 50 ng / mL mitomycin, and the HEK293 cells were expanded to obtain the amplified HEK293 cells modified with mIL-21 and 4-1BBL genes for later use.

[0029] In one embodiment, in the above-mentioned NK cell in vitro expansion and culture method, when the PEG6000 solution is used to purify the mixed solution of the first exosome stock solution and the second exosome stock solution, the method comprises the following steps:

[0030] The collected first exosome stock solution and the second exosome stock solution were mixed evenly, and the mixed solution was centrifuged at 3000g for 30 minutes for the first centrifugation treatment; after the centrifugation, a first supernatant was obtained;

[0031] Add PEG6000 precipitate with a concentration of 8 w / w% to the first supernatant, and the volume ratio of the first supernatant to the precipitate is 1:2, shake well and let stand at 2-8°C for 8 hours;

[0032] After standing, the mixture of the first supernatant and the precipitate was centrifuged for a second time at 13000 g for 1 h. After the centrifugation, the second supernatant was removed to obtain a suspension of the exosome stock solution.

[0033] The suspension of the exosome stock solution was filtered through a filter membrane to obtain the exosomes of HEK293 cells.

[0034] In one embodiment, in the above-mentioned NK cell in vitro expansion and culture method, the coated culture flask is obtained by the following treatment:

[0035] 50 ng / mL CD16 monoclonal antibody and 50 ng / mL CD3 monoclonal antibody were added to the culture flask, and the flask was placed in a 4°C refrigerator for 12 hours to obtain a coated culture flask.

[0036] The NK cell in vitro expansion and culture method of the present invention uses HEK293 exosomes to replace the trophoblast cells (i.e., tumor cells, such as K562 cells) added during the NK cell culture process by the feeder method, thereby reducing the safety risks that may be caused by the direct use of trophoblast cells; at the same time, the addition of HEK293 exosomes binds to the NK cell surface receptors, provides synergistic stimulation, activates the NK cells and causes them to expand at a high speed, and greatly improves the proliferation ability and purity of the NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The growth and expansion curves of NK cells in Examples 1 to 5;

[0038] Figure 2The flow cytometry graphs of the NK cell phenotypes in Examples 1 to 5 are as follows; wherein, Figure a represents the NK cell CD3-CD56+ phenotype detection graph in Example 1, Figure b represents the NK cell CD3-CD56+ phenotype detection graph in Example 2, Figure c represents the NK cell CD3-CD56+ phenotype detection graph in Example 3, Figure d represents the NK cell CD3-CD56+ phenotype detection graph in Example 4 (Comparative Example 1), and Figure e represents the NK cell CD3-CD56+ phenotype detection graph in Example 5 (Comparative Example 2). DETAILED DESCRIPTION

[0039] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0040] The NK cell culture method for activating NK cells by exosomes provided by the present invention utilizes genetically modified HEK293 cells, collects and purifies exosomes after culture, and adds them to NK cell culture. The NK cell culture method can avoid the safety risks brought about by the direct use of trophoblast cells (i.e., tumor cells) in the feeder method, and also solves the problems of limited proliferation ability and low cell purity of NK cell growth cultured by the pure factor method.

[0041] The NK cell culture method provided by the present invention has the following process steps.

[0042] 1. Obtaining Mononuclear Cells

[0043] Mononuclear cells can be obtained from umbilical cord blood or peripheral blood (PBMC). In the present invention, peripheral blood is selected, that is, mononuclear cells need to be separated from peripheral blood, and then cultured into NK cells from the mononuclear cells.

[0044] The specific process of obtaining mononuclear cells is as follows:

[0045] S11. Collect peripheral blood from volunteers, draw 80 ml of blood, place in a sterile sodium heparin blood collection tube, transfer the blood into a 50 ml centrifuge tube, and centrifuge at 700 g for 10 min;

[0046] S12. After the centrifugation is completed, the upper plasma is transferred into another centrifuge tube with a pipette, and the mouth of the centrifuge tube is sealed with a sealing film to avoid contamination, and then inactivated;

[0047] S13, centrifuge at 1500g for 30min again, remove the supernatant to obtain the plasma cell pellet, take an equal volume of PBS to the cell pellet and resuspend and mix, slowly add the mixed blood cell suspension to the lymphocyte separation solution (Fico ll layer) to ensure clear stratification, and then centrifuge at 700g for 30min;

[0048] S14. After the centrifugation stops, aspirate the middle layer (white film layer), and try to aspirate the cell layer at the junction of the two liquid surfaces, put the white film layer into a new centrifuge tube and resuspend it with DPBS; centrifuge the cell suspension again at 700g for 30 minutes;

[0049] S15. After centrifugation, discard the supernatant, wash the cell pellet twice with DPBS, and then resuspend the cell pellet to 100 mL with DPBS. The suspension at this time is the mononuclear cells of peripheral blood (PBMC); set aside.

[0050] 2. Culture of Genetically Modified HEK293 Cells

[0051] S21. HEK293 cells (purchased from Youkang Biotechnology (Beijing) Co., Ltd.) were cultured at 0.5×10 5 ~2.0×10 5 The cells were inoculated into each well of a 24-well plate at a density of 1.0 × 10 cells / mL. 6 1 mL of HEK293 serum-free medium was added to each cell, and then placed in a 37°C, 5% CO2 incubator for 24 h; the cell density can be 0.8×10 5 ~1.5×10 5 / mL, 0.5×10 5 / mL, 1×10 5 / mL, 2×10 5 / M l; the preferred cell density is 1×10 5 Pieces / mL;

[0052] S22. After 24 hours, the 24-well plate was taken out, and 100 μL to 500 μL of HEK293 serum-free medium (commercially purchased from Youkang Biotechnology (Beijing) Co., Ltd.) was added to each well, and the culture was continued for 1 hour; wherein, the amount of HEK293 serum-free medium added can be 200 μL to 400 μL, 100 μL, 300 μL, or 500 μL; preferably, the amount of HEK293 serum-free medium added is 300 μL;

[0053] S23. After 1 hour, add the lentiviral concentrate containing 293T cells to each well of the 24-well plate according to MOI=1-5, and perform cell transfection for 6 hours; wherein the MOI can be: MOI=2-4, MOI=1, MOI=3, MOI=5; preferably MOI=3;

[0054] After S24 and 6h, 500 μL to 1000 μL of HEK293 serum-free medium was added to each well of the 24-well plate for culturing, and the culture was continued for 72h; wherein, the amount of HEK293 serum-free medium added can be 600 μL to 800 μL, 500 μL, 700 μL, or 1000 μL; preferably, the amount of HEK293 serum-free medium added is 700 μL;

[0055] After S25 and 72h, 0.5-2μg / mL puromycin was added to each well of the 24-well plate to screen out cells stably expressing mIL-21 and 4-1BBL, i.e., HEK293 cells modified with mIL-21 and 4-1BBL genes; wherein the amount of puromycin added can be 0.8-1.8μg / mL, 0.5μg / mL, 1μg / mL, 1.5μg / mL, 2μg / mL; preferably, the amount of puromycin added is 2μg / mL;

[0056] S26. Treat the cells stably expressing mIL-21 and 4-1BBL with 50 ng / mL mitomycin for 2 h, expand the cell culture, and obtain the required number of mIL-21 and 4-1BBL gene-modified HEK293 cells for later use.

[0057] 3. Obtaining exosomes from HEK293 cells

[0058] The steps for preparing exosomes are as follows:

[0059] S31, take the 0.5×10 5 ~3.0×10 6 The genetically modified HEK293 cells were added into HEK293 serum-free medium and cultured for 2 to 3 days. After one passage, the amplified HEK293 cells were obtained. The number of HEK293 cells seeded was 0.8×10 5 ~2.8×10 6 pcs, 0.5×10 6 pcs, 1×10 6 pcs, 1.5×10 6 pcs, 2×10 6 5.5×10 6 pcs, 3.0×10 6 The preferred number of HEK293 cells to be seeded is 2.0×10 6 The volume of HEK293 serum-free medium can be 20-50 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, etc.

[0060] S32, inoculating the amplified HEK293 cells into a T75 culture flask containing HEK293 serum-free medium, culturing for 2 days, centrifuging the cells at 500g for 10min, collecting the supernatant, obtaining the first exosome stock solution of HEK293 cells, and collecting the cell precipitate at the same time; wherein, the subsequent treatment of the first exosome stock solution is transferred to step S34, and the treatment of the cell precipitate is transferred to step S33; the volume of HEK293 serum-free medium can be 20-50mL, 20mL, 25mL, 30mL, 35mL, 40mL, 45mL, 50mL, etc.; preferably, the volume of HEK293 serum-free medium is 35mL;

[0061] S33, adding an equal volume of HEK293 serum-free medium to the collected cell pellet as that in the culture flask, culturing for 2 days, centrifuging the cells at 500g for 10 minutes, collecting the supernatant again, and obtaining a second exosome stock solution; wherein the volume of the HEK293 serum-free medium can be 20-50mL, 20mL, 25mL, 30mL, 35mL, 40mL, 45mL, 50mL, etc.; preferably, the volume of the HEK293 serum-free medium is 35mL;

[0062] S34, combining and mixing the collected first exosome stock solution and the second exosome stock solution, and centrifuging the obtained mixed solution at 3000 g for 30 min for the first centrifugation; after the centrifugation, obtaining a first supernatant;

[0063] S35, adding PEG6000 precipitate with a concentration of 8 w / w% to the first supernatant, and the volume ratio of the first supernatant to the precipitate is 1:2, shaking well and letting stand at 2-8° C. for 8 h;

[0064] S36, after standing, centrifuging at 13000 g for 1 h, and subjecting the mixture of the first supernatant and the precipitate to a second centrifugation; after the centrifugation, removing the second supernatant to obtain a suspension of the exosome stock solution;

[0065] S37. Filter the suspension of the exosome stock solution with a 0.22 μm filter membrane to obtain the exosomes of HEK293 cells; set aside.

[0066] 4. Preparation of NK cell culture medium

[0067] In the present invention, the culture medium that needs to be added during the NK cell culture process includes an induction culture medium for inducing the transformation of mononuclear cells into NK cells, and an expansion culture medium for promoting the rapid expansion and growth of NK cells.

[0068] S41, preparing 1000 mL of NK cell induction culture medium: using KBM581 medium as the basal medium, adding cytokines such as IL-15 and IL-2 to the basal medium, and making the final concentrations of IL-15 and IL-2 in the KB581 medium be 100-2000 IU / mL and 500-2000 IU / mL, respectively;

[0069] S42. Prepare 1000 mL of NK cell expansion culture medium: use KBM581 medium as the basal medium, add IL-2 cytokine to the basal medium, and make the final concentration of IL-2 in KB581 medium be 500-2000 IU / mL.

[0070] Preferably, the NK cell induction culture medium contains IL-15 at a final concentration of 500 to 1500 IU / mL and IL-2 at a final concentration of 800 to 1500 IU / mL, or contains IL-15 at a final concentration of 1000 IU / mL and IL-2 at a final concentration of 1000 IU / mL.

[0071] Preferably, the NK cell expansion medium contains IL-2 at a final concentration of 800 to 1500 IU / mL, or contains IL-2 at a final concentration of 1000 IU / mL.

[0072] 5. NK cell culture

[0073] S51, adding 50 ng / mL CD16 monoclonal antibody and 50 ng / mL CD3 monoclonal antibody to the T75 culture flask, and placing it in a 4°C refrigerator for 12 hours to obtain a pretreated coated culture flask;

[0074] S52, day 0, first, take 1.0×10 7 ~8.0×10 7 The mononuclear cells obtained in step S15 were inoculated into a coated culture bottle containing 27 mL of the NK cell induction culture medium prepared in step S41; then 3 mL of autologous plasma and 1.0×10 8 ~5.0×10 8 The exosomes of HEK293 cells obtained in step S37 were granules / mL; finally, the T75 culture flask was placed in an incubator at 37°C and 5v / v% CO2 for static culture; wherein the number of mononuclear cells inoculated can be 2.0×10 7 ~7.0×10 7 pcs, 1.0×10 7 pcs, 2.0×10 7 pcs, 2.5×10 7 pcs, 3.0×10 7 pcs, 4.0×10 7pcs, 5.0×10 7 pcs, 6.0×10 7 8.0×10 7 Preferably, the number of mononuclear cells seeded is 6.0×10 7 The amount of exosomes from HEK293 cells can be 2.0×10 8 ~4.0×10 8 particles / mL, 3.0×10 8 particles / mL, 4.0×10 8 Particles / mL, etc.

[0075] S53. On the third day, the culture flask was supplemented with NK cell induction medium and autologous plasma at a volume ratio of 9:1;

[0076] S54, on the 5th day, the culture flask was supplemented with NK cell induction culture medium and autologous plasma at a volume ratio of 19:1;

[0077] S55. On the 7th day, the cells and culture medium in the culture flask were transferred to the culture bag, and NK cell induction culture medium and autologous plasma with a volume ratio of 99:1 and 0.5×10 8 ~2.0×10 8 Particles / mL exosomes from HEK293 cells;

[0078] In steps S52 to S55 of the present invention, the total volume of NK cell culture fluid and autologous plasma is preferably controlled to be 500 mL; and the volumes of NK cell induction culture fluid and autologous plasma in step S52 are 27 mL and 3 mL, respectively; therefore, in step S53, the volumes of NK cell induction culture fluid and autologous plasma are 27 mL and 3 mL, respectively; in step S54, the volumes of NK cell induction culture fluid and autologous plasma are 133 mL and 7 mL, respectively; in step S55, the volumes of NK cell induction culture fluid and autologous plasma are 297 mL and 3 mL, respectively;

[0079] S56. From the 9th day to the 13th day, the medium was changed every two days, the waste culture medium was discarded, and the NK cell expansion culture medium prepared in step S42 was added, and the cell density in the culture bag was maintained at 1.0×10 6 Pieces / mL;

[0080] S57. On the 14th day, the culture was stopped, the cells were collected, centrifuged at 500 g for 10 min, the supernatant was removed, and the cell pellet was washed several times with PBS to obtain in vitro expanded NK cells.

[0081] The technical aspects of the present invention are further described in detail below through some specific embodiments and comparative examples.

[0082] In the following embodiments, mononuclear cells are derived from the above step S15 of the present invention, the genetically modified HEK293 cell culture is derived from the above step S26 of the present invention, and the exosomes of NK cells are derived from the above step S37 of the present invention.

[0083] 1. NK cell culture

[0084] Example 1

[0085] 1.1. Preparation of NK cell culture medium

[0086] Prepare 1000 mL of NK cell induction culture medium: use KBM581 medium as the basal medium, add cytokines such as IL-15 and IL-2 to the basal medium, and make the final concentrations of IL-15 and IL-2 in KB581 medium be 1000 IU / mL and 1000 IU / mL respectively.

[0087] 1000 mL of NK cell expansion culture medium was prepared: KBM581 medium was used as the basal medium, IL-2 cytokine was added to the basal medium, and the final concentration of IL-2 in the KB581 medium was set to 1000 IU / mL.

[0088] 1.2 NK cell culture

[0089] On day 0, first, take the 6.0×10 7 Mononuclear cells were inoculated into a coated T75 culture flask containing 27 mL of NK cell induction medium; then 3 mL of autologous plasma and 3.0×10 8 particles / mL exosomes; finally, the T75 culture flask was placed in an incubator at 37°C and 5v / v% CO2 for static culture;

[0090] On day 3, 27 mL of NK cell induction medium and 3 mL of autologous plasma were added to the culture flask;

[0091] On day 5, the culture flask was supplemented with 133 mL of NK cell induction medium and 7 mL of autologous plasma;

[0092] On the 7th day, the cells in the culture flask were transferred to the culture bag, and 297 mL of NK cell induction culture medium, 3 mL of autologous plasma and 1.0 × 10 8 The total volume of NK cell culture medium and autologous plasma was controlled to be 500 mL from day 0 to day 7.

[0093] From the 9th day to the 13th day, the medium was changed every two days. The waste culture medium was discarded with a pipette and a certain amount of fresh NK cell expansion medium was added. The cell density was controlled at 1.0×10 6 Pieces / mL;

[0094] On the 14th day, the culture was stopped, the cells were collected into a centrifuge tube, centrifuged at 500 g for 10 min, the supernatant was removed, the cell pellet was collected and washed several times with PBS to obtain high-purity and high-activity NK cells.

[0095] Example 2

[0096] 2.1. Preparation of NK cell culture medium

[0097] Prepare 1000 mL of NK cell induction culture medium: use KBM581 medium as the basal medium, add cytokines such as IL-15 and IL-2 to the basal medium, and make the final concentrations of IL-15 and IL-2 in KB581 medium be 100 IU / mL and 2000 IU / mL, respectively.

[0098] 1000 mL of NK cell expansion culture medium was prepared: KBM581 medium was used as the basal medium, IL-2 cytokine was added to the basal medium, and the final concentration of IL-2 in the KB581 medium was set to 2000 IU / mL.

[0099] 2.2 NK cell culture

[0100] On day 0, first, take the 1.0×10 7 Mononuclear cells were inoculated into a coated T75 culture flask containing 27 mL of NK cell induction medium; then 3 mL of autologous plasma and 5.0×10 8 particles / mL exosomes; finally, the T75 culture flask was placed in an incubator at 37°C and 5v / v% CO2 for static culture;

[0101] On day 3, 27 mL of NK cell induction medium and 3 mL of autologous plasma were added to the culture flask;

[0102] On day 5, the culture flask was supplemented with 133 mL of NK cell induction medium and 7 mL of autologous plasma;

[0103] On the 7th day, the cells in the culture flask were transferred to a culture bag, and 297 mL of NK cell induction culture medium, 3 mL of autologous plasma, and 2.0 × 10 8 The total volume of NK cell culture medium and autologous plasma was controlled to be 500 mL from day 0 to day 7.

[0104] From the 9th day to the 13th day, the medium was changed every two days. The waste culture medium was discarded with a pipette and a certain amount of fresh NK cell expansion medium was added. The cell density was controlled at 1.0×10 6 Pieces / mL;

[0105] On the 14th day, the culture was stopped, the cells were collected into a centrifuge tube, centrifuged at 500 g for 10 min, the supernatant was removed, the cell pellet was collected and washed several times with PBS to obtain high-purity and high-activity NK cells.

[0106] Example 3

[0107] 3.1. Preparation of NK cell culture medium

[0108] Prepare 1000 mL of NK cell induction culture medium: use KBM581 medium as the basal medium, add cytokines such as IL-15 and IL-2 to the basal medium, and make the final concentrations of IL-15 and IL-2 in KB581 medium be 2000 IU / mL and 500 IU / mL, respectively.

[0109] 1000 mL of NK cell expansion culture medium was prepared: KBM581 medium was used as the basal medium, IL-2 cytokine was added to the basal medium, and the final concentration of IL-2 in the KB581 medium was 500 IU / mL.

[0110] 3.2 NK cell culture

[0111] On day 0, first, take the 8.0×10 7 Mononuclear cells were inoculated into a coated T75 culture flask containing 27 mL of NK cell induction medium; then 3 mL of autologous plasma and 1.0×10 8 The exosomes of HEK293 cells were counted into particles / mL. Finally, the T75 culture flask was placed in an incubator at 37°C and 5v / v% CO2 for static culture.

[0112] On day 3, 27 mL of NK cell induction medium and 3 mL of autologous plasma were added to the culture flask;

[0113] On day 5, the culture flask was supplemented with 133 mL of NK cell induction medium and 7 mL of autologous plasma;

[0114] On the 7th day, the cells in the culture flask were transferred to the culture bag, and 297 mL of NK cell induction culture medium, 3 mL of autologous plasma and 1.0 × 10 8The exosomes of HEK293 cells were granules / mL; wherein, from day 0 to day 7, the total volume of NK cell culture medium and autologous plasma was controlled to be 500 mL;

[0115] From the 9th day to the 13th day, the medium was changed every two days. The waste culture medium was discarded with a pipette and a certain amount of fresh NK cell expansion medium was added. The cell density was controlled at 1.0×10 6 Pieces / mL;

[0116] On the 14th day, the culture was stopped, the cells were collected into a centrifuge tube, centrifuged at 500 g for 10 min, the supernatant was removed, the cell pellet was collected and washed several times with PBS to obtain high-purity and high-activity NK cells.

[0117] Example 4 (Comparative Example 1, no exosomes were added during NK culture)

[0118] 4.1. Preparation of NK cell culture medium

[0119] Prepare 1000 mL of NK cell induction culture medium: use KBM581 medium as the basal medium, add cytokines such as IL-15 and IL-2 to the basal medium, and make the final concentrations of IL-15 and IL-2 in KB581 medium be 1000 IU / mL and 1000 IU / mL respectively.

[0120] 1000 mL of NK cell expansion culture medium was prepared: KBM581 medium was used as the basal medium, IL-2 cytokine was added to the basal medium, and the final concentration of IL-2 in the KB581 medium was 1000 IU / mL.

[0121] 4.2 NK cell culture

[0122] On day 0, first, take the 6.0×10 7 The mononuclear cells were inoculated into a coated T75 culture flask containing 27 mL of NK cell induction culture medium; then 3 mL of autologous plasma was added; finally, the T75 culture flask was placed in an incubator at 37°C and 5 v / v% CO2 for static culture;

[0123] On day 3, 27 mL of NK cell induction medium and 3 mL of autologous plasma were added to the culture flask;

[0124] On day 5, the culture flask was supplemented with 133 mL of NK cell induction medium and 7 mL of autologous plasma;

[0125] On day 7, the cells in the culture flask were transferred to a culture bag, and a volume ratio of 297 mL of NK cell induction culture medium and 3 mL of autologous plasma was added;

[0126] From the 9th day to the 15th day, the medium was changed every two days, the waste culture medium was discarded with a pipette, and a certain amount of fresh NK cell expansion culture medium was added, and the cell density was controlled at 1.0×10 6 Pieces / mL;

[0127] On the 16th day, the culture was stopped, the cells were collected into a centrifuge tube, centrifuged at 500 g for 10 min, the supernatant was removed, the cell pellet was collected and washed several times with PBS to obtain NK cells.

[0128] Example 5 (Comparative Example 2, other process NK cell culture process)

[0129] 1) Prepare NK cell induction medium and expansion medium

[0130] Prepare 500 mL of NK cell induction medium: use KBM581 medium as the basal medium, and the basal medium contains 1000 U / ml I L-2, 50 ng / ml I L-15, and 10 μM StemRegen in 1 at a final concentration;

[0131] Prepare 2500 mL of NK cell expansion medium: use KBM581 medium as the basal medium, and the basal medium contains 1000 U / ml I L-2 and 50 ng / ml I L-21 at a final concentration;

[0132] 2) NK cell culture

[0133] Inoculation on day 0: Take 6.0×10 6 The mononuclear cells / mL were inoculated into a coated T75 culture flask containing 27 ml of induction medium and 3 ml of serum, and placed in a 37°C, 5% CO2 incubator for 5 days; the cells were replenished once on the 3rd day with 27 ml of induction medium and 3 ml of serum;

[0134] On day 5, cells were collected, supernatant was removed by centrifugation, cells were resuspended in 133 ml expansion medium and 7 ml serum, and the cell suspension was transferred to a culture bag with a total volume of 200 ml;

[0135] Rehydration on day 7: add 200 ml expansion medium to a total volume of 400 ml;

[0136] On day 9, the cells were replenished with 400 ml of expansion medium, making the total volume 800 ml;

[0137] On the 11th day, the cells were replenished with 800 ml of expansion medium, and the total volume was 1600 ml;

[0138] On the 13th day, the cells were replenished with 800 ml of expansion medium, making the total volume 2400 ml;

[0139] On the 16th day, the culture was stopped, and the NK cells and culture medium in the culture bag were transferred to a centrifuge tube. The tube was centrifuged and the supernatant was removed. The cell precipitate was collected and washed with PBS for 2 to 3 times to obtain NK cells.

[0140] 2. Experimental testing and result representation

[0141] 1. NK cell proliferation and growth count detection

[0142] For the NK cell culture in Examples 1 to 5, samples were taken on day 0, day 3, day 5, day 7, day 11, day 14, and day 16, respectively, and the number of cells was detected using a cell counter. The test results are shown in Table 1. Figure 1 The NK cell growth curve was drawn based on the data in Table 1.

[0143] Table 1 NK cell growth count

[0144]

[0145] Figure 1 As shown in Table 1: 1) in Examples 1 to 3, the number of NK cells expanded after 13 days of culture has exceeded the number of NK cells expanded after 16 days of culture in Examples 4 and 5; 2) since the sampling and cell number counting began on the 3rd day, the number of expanded NK cells in Examples 1 to 3 was higher than that in Examples 4 and 5, especially after the 7th day, the NK cells in Examples 1 to 3 showed rapid expansion, and the number of expanded NK cells was much higher than that in Examples 4 and 5.

[0146] Figure 1 The cell counting statistical results in Table 1 illustrate that in Examples 1 to 3, the HEK293 cell exosomes added on day 0 bind to the NK cell surface receptors, activate the NK cells to rapidly proliferate, and can further induce mononuclear cells to grow into NK cells. The HEK293 cell exosomes are supplemented again on day 7 to continuously stimulate the NK cells, which can further stimulate the rapid proliferation and growth of NK cells.

[0147] 2. NK cell flow cytometry phenotype detection

[0148] Figure 2 It is a CD3-CD56+ phenotype detection diagram of NK cells in Examples 1 to 5; wherein, the sampling time for NK cell phenotype detection is: the 14th day of NK cell culture in Examples 1 to 3, and the 16th day of NK cell culture in Examples 4 and 5.

[0149] Take the NK cells cultured in Examples 1 to 5 respectively, prepare a cell suspension and adjust the cell concentration to 1×105 / ml, add labeled CD3-CD56+ monoclonal antibody, incubate at room temperature in the dark for 20 minutes, wash away excess antibody, and detect by flow cytometry. The test results are as follows Figure 2 As shown; where:

[0150] Figure a shows the NK cell CD3-CD56+ phenotype detection diagram in Example 1;

[0151] Figure b shows the phenotype detection diagram of NK cells CD3-CD56+ in Example 2;

[0152] Figure c shows the phenotype detection diagram of NK cells CD3-CD56+ in Example 3;

[0153] Figure d shows the phenotype detection diagram of NK cells CD3-CD56+ in Example 4 (Comparative Example 1);

[0154] Figure e shows the NK cell CD3-CD56+ phenotype detection diagram in Example 5 (Comparative Example 2).

[0155] Depend on Figure 2 It can be seen that the CD3-CD56+ contents of the NK cells cultured in Examples 1 to 5 were 92.66%, 82.80%, 87.42%, 67.20% and 65.61%, respectively.

[0156] Depend on Figure 2 It can be seen from the cell flow cytometry that in the NK cell culture process of Examples 1 to 3, exosomes of NK cells are added. At this time, the proportion of NK effector cells can be as high as 82-93%. This is because after the addition of exosomes, the surface receptors of NK cells bind to the exosomes, activating NK cells to make them proliferate at a high speed; while in the NK cell culture process of Examples 4 and 5, exosomes of HEK293 cells are not added, and the proportion of NK effector cells is less than 70%. This shows that the exosomes of HEK293 cells provided by the present invention activate the culture of NK cells and can improve the purity of NK cells.

[0157] It should be understood that the above description of the preferred embodiments of the present invention is relatively detailed and cannot be regarded as limiting the scope of patent protection of the present invention. The scope of patent protection of the present invention shall be based on the attached claims.

Claims

1. A method for in vitro expansion and cultivation of NK cells, characterized in that: The steps include: On day 0, first, 1.0×10 7 ~8.0×10 7 Mononuclear cells were inoculated into a coated culture flask containing 27 mL of NK cell induction medium; then 3 mL of autologous plasma and 1.0×10 8 ~5.0×10 8 The exosomes of HEK293 cells were collected in 10000 particles / mL. Finally, the culture flask was placed in an incubator at 37°C and 5% CO2. On the third day, NK cell induction culture medium and autologous plasma were added to the culture flask at a volume ratio of 9:1; On the fifth day, the culture flask was supplemented with NK cell induction culture medium and autologous plasma at a volume ratio of 19:1; On the 7th day, the cells and culture medium were transferred to the culture bag, and the NK cell induction culture medium and autologous plasma with a volume ratio of 99:1 and 0.5×10 8 ~2.0×10 8 Particles / mL exosomes from HEK293 cells; From the 9th day to the 13th day, the medium was changed every two days, and fresh NK cell expansion medium was added to maintain the cell density at 1.0×10 6 Pieces / mL; On the 14th day, the culture was stopped, the cells were collected, centrifuged at 500 g for 10 min, the supernatant was removed, and the cell pellet was washed several times with PBS to obtain NK cells.

2. The method for in vitro expansion and cultivation of NK cells according to claim 1, characterized in that: The NK cell induction culture fluid includes KBM581 culture medium, which contains IL-15 with a final concentration of 100 to 2000 IU / mL and IL-2 with a final concentration of 500 to 2000 IU / mL.

3. The method for in vitro expansion and cultivation of NK cells according to claim 1, characterized in that: The NK cell expansion culture medium includes KBM581 culture medium, which contains IL-2 with a final concentration of 500 to 2000 IU / mL.

4. The method for in vitro expansion and cultivation of NK cells according to claim 1, characterized in that: The exosomes of HEK293 cells were obtained by the following steps: HEK293 cells were overexpressed with mIL-21 and 4-1BBL to obtain HEK293 cells modified with mIL-21 and 4-1BBL genes; The gene-modified HEK293 cells were expanded and cultured in HEK293 serum-free medium for 2 to 3 days to obtain expanded cells; The expanded cells were inoculated into a culture flask containing HEK293 serum-free medium. After culturing for 2 days, the cells were centrifuged at 500 g for 10 min, and the supernatant was collected to obtain the first exosome stock solution of HEK293 cells; at the same time, the cell precipitate was collected; Adding an equal volume of HEK293 serum-free medium to the HEK293 serum-free medium in the culture flask to the collected cell pellet, culturing for 2 days, centrifuging the cells at 500 g for 10 min, and collecting the supernatant again to obtain a second exosome stock solution of HEK293 cells; The mixed solution of the first exosome stock solution and the second exosome stock solution was purified using a PEG6000 solution to obtain exosomes from HEK293 cells.

5. The method for in vitro expansion and cultivation of NK cells according to claim 4, characterized in that: The mIL-21 and 4-1BBL gene-modified HEK293 cells are obtained by the following steps: The cell density was 0.5 × 10 5 ~2.0×10 5 HEK293 cells were seeded into each well of a 24-well plate at a concentration of 1.0 × 10 cells / ml. 6 Add 1 mL of HEK293 serum-free medium to each cell and then place in a 37°C, 5% CO2 incubator for 24 h; After 24 h, the 24-well plate was removed, and 100 μL to 500 μL of HEK293 serum-free medium was added to each well, and the culture was continued for 1 h; After 1 h, the lentiviral concentrate was added to each well of a 24-well plate at an MOI of 1 to 5, and the cells were transfected for 6 h; After 6 h, add 500 μL to 1000 μL of HEK293 serum-free medium to each well of the 24-well plate and continue culturing for 72 h; After 72 hours, puromycin was added to each well of the 24-well plate at a final concentration of 0.5 to 2 μg / mL to screen out HEK293 cells stably expressing mIL-21 and 4-1BBL, thereby obtaining HEK293 cells modified with the mIL-21 and 4-1BBL genes.

6. The method for in vitro expansion and cultivation of NK cells according to claim 5, characterized in that: After screening and obtaining cells modified with mIL-21 and 4-1BBL genes, the cells need to be further expanded and cultured: The 2hmIL-21 and 4-1BBL gene-modified HEK293 cells were treated with 50 ng / mL mitomycin, and the HEK293 cells were expanded to obtain the required number of mIL-21 and 4-1BBL gene-modified HEK293 cells for later use.

7. The method for in vitro expansion and cultivation of NK cells according to claim 4, characterized in that: When the PEG6000 solution is used to purify the mixed solution of the first exosome stock solution and the second exosome stock solution, the steps include: The collected first exosome stock solution and the second exosome stock solution were mixed evenly, and the mixed solution was centrifuged at 3000g for 30 minutes for the first centrifugation treatment; After centrifugation, the first supernatant was obtained; Add PEG6000 precipitate with a concentration of 8 w / w% to the first supernatant, and the volume ratio of the first supernatant to the precipitate is 1:2, shake well and let stand at 2-8°C for 8 hours; After standing, the mixture of the first supernatant and the precipitate was centrifuged for a second time at 13000 g for 1 h; after the centrifugation, the second supernatant was removed to obtain a suspension of the exosome stock solution; The suspension of the exosome stock solution was filtered through a filter membrane to obtain the exosomes of HEK293 cells.

8. The method for in vitro expansion and cultivation of NK cells according to claim 1, characterized in that: The coated flasks are obtained by the following treatment: 50 ng / mL CD16 monoclonal antibody and 50 ng / mL CD3 monoclonal antibody were added to the culture flask, and the flask was placed in a 4°C refrigerator for 12 hours to obtain a coated culture flask.

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