Amplification method of high-purity anti-tumor NK (Natural Killer) cells

Abstract

The invention relates to the technical field of cell in-vitro culture. The invention aims to provide an amplification method of high-purity anti-tumor NK cells. The amplification method comprises the following steps: S1, obtaining initial NK cells; s2, performing double-stage culture; a double-stage culture mode is adopted, the first-stage culture period is 8 days, the second-stage culture period is 10 days, and after the first-stage culture is finished, NK cells with full cytoplasm are separated through a density gradient centrifugation method for second-stage culture; s3, harvesting the NK cells. The method has the advantages of simplicity and convenience in operation, controllable cost, suitability for large-scale production and the like, and a more efficient and economical solution can be provided for preparation of the high-purity anti-tumor NK cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell in vitro culture, and particularly relates to a high-purity anti-tumor NK cell expansion method. BACKGROUND

[0002] Natural killer (NK) cells, as important effector cells in the immune system, can directly recognize and kill tumor cells without prior sensitization, showing great potential in the field of tumor immunotherapy. However, the number of NK cells isolated from peripheral blood is limited, and problems such as decreased cell activity and insufficient purity often occur during in vitro culture, which limits its clinical application. Currently, NK cell expansion technology mainly faces two challenges: one is how to maintain the anti-tumor activity of cells while achieving efficient proliferation; the other is how to remove non-functional cells or mixed cells produced during culture to ensure the high purity and safety of the final product.

[0003] Traditional NK cell expansion methods mostly use a single culture system to promote cell proliferation by adding cytokines (such as IL-2, IL-15) and feeder cells. Although this method can increase the number of NK cells, due to the lack of dynamic regulation mechanism, cell overactivation, exhaustion or abnormal phenotype differentiation often occur in the later stage of culture, leading to decreased killing ability. In addition, non-specifically proliferating immune cells (such as T cells, monocytes) are mixed in, further reducing the purity of NK cells and increasing the risk of clinical application.

[0004] In recent years, the two-stage culture strategy has gradually become a hot spot in NK cell expansion research. This strategy simulates the development microenvironment of NK cells in vivo and regulates cell proliferation and functional differentiation in stages. In the first stage, the goal is rapid proliferation, and high-concentration cytokines and suitable culture conditions are used to promote the expansion of NK cells. In the second stage, the culture system is adjusted to optimize cell function. Based on this, the present application innovatively introduces density gradient centrifugation as a key linking step between the first and second stages. This method can efficiently enrich the subpopulation of superior NK cells with high proliferation potential and killing activity based on the difference in cell density, and remove senescent or non-functional cells with abnormal density, thereby significantly improving the purity and uniformity of the cells. Compared with traditional screening techniques, the density gradient centrifugation method has the advantages of simple operation, controllable cost, and suitability for large-scale production, and is expected to provide a more efficient and economical solution for the preparation of high-purity anti-tumor NK cells. SUMMARY

[0005] The present application aims to provide a high-purity anti-tumor NK cell expansion method, which has the advantages of simple operation, controllable cost, and suitability for large-scale production, and can provide a more efficient and economical solution for the preparation of high-purity anti-tumor NK cells.

[0006] To achieve the above-mentioned purposes, the technical scheme adopted by the present application is as follows: a method for expanding high-purity anti-tumor NK cells, comprising the following steps:

[0007] S1, obtaining starting NK cells;

[0008] Single-nucleated cells are separated from peripheral blood obtained from a human body by using a density gradient centrifugation method; T cells, B cells and monocytes are removed from the single-nucleated cells by using an immunomagnetic bead sorting method, so as to enrich and separate NK cells; and the enriched NK cells are purified by using an NK cell specific antibody and a flow cytometry method;

[0009] S2, two-stage culture;

[0010] The two-stage culture method is adopted, the culture period of the first stage is 8 days, the added amount of the cytokines in the culture medium is 1000 U / mL of IL-2, 50 ng / mL of IL-15 and 10 ng / mL of IL-21, the culture period of the second stage is 10 days, the added amount of the cytokines in the culture medium is 800 U / mL of IL-2, 80 ng / mL of IL-15 and 20 ng / mL of IL-21, and after the first-stage culture is completed, the density gradient centrifugation method is used to separate the NK cells with full cytoplasm for the second-stage culture;

[0011] S3, harvesting the NK cells.

[0012] Preferably, the step S2 of the two-stage culture comprises the following steps:

[0013] S2.1, first-stage culture;

[0014] S2.1.1, cell inoculation: the NK cell subpopulation obtained by the flow sorting is inoculated into a cell culture bottle at a density of 1×10 6 / mL, a special culture medium A is used, the added amount of the cytokines in the special culture medium A is 1000 U / mL of IL-2, 50 ng / mL of IL-15 and 10 ng / mL of IL-21, so as to promote the proliferation and activation of the NK cells;

[0015] S2.1.2, culture conditions: the culture bottle is continuously cultured in a constant-temperature incubator at 37℃ and 5% CO2 for 8 days, the liquid is replaced by half of the volume every other day, and the cell density is adjusted to 1×10 7 / mL in time according to the cell growth; after the cultivation is completed, all the culture liquids are combined to obtain a first-stage cell liquid for standby;

[0016] S2.2, NK cell density gradient centrifugation sorting:

[0017] S2.2.1, centrifugal preparation: transfer the first stage cell liquid to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant after combining; resuspend with PBS liquid after passing through a 400 mesh sieve to remove cell clumps, cell debris clumps, unsoluble culture medium and other large particle impurities; take a small amount of cell suspension for trypan blue staining after filtration, count the viable cell concentration, and dilute the cell suspension with PBS liquid to 1×10 6 / mL to 1×10 7 / mL cell density;

[0018] S2.2.2, density gradient centrifugation:

[0019] Add Percoll separation solution with a density of 1.085 g / ml to the centrifuge tube first, then slowly add the diluted cell suspension along the wall of the tube, the volume ratio of cell suspension to separation solution is 1:1; ensure that the interface of the two layers of liquid is clear; 400xg, room temperature, centrifuge for 20 minutes; aspirate the white membrane layer and transfer it to the special culture medium B; repeat the steps until all the first stage cell liquid is processed;

[0020] S2.3, two-stage culture;

[0021] S2.3.1, cell inoculation: inoculate the cell liquid after density gradient centrifugation in the cell culture bottle at a density of 1×10 6 / mL, use special culture medium B, the cytokine addition amount in special culture medium B is: 800 U / mL of IL-2, 80 ng / mL of IL-15 and 20 ng / mL of IL-21;

[0022] S2.3.2, culture conditions: place the culture bottle in a constant temperature incubator at 37°C, 5% CO2 for continuous culture for 10 days, replace half of the liquid every other day, and adjust the cell density to 1×10 6 / mL in time according to the cell growth; after the end of the culture period, sample each group of culture liquid, use trypan blue staining method to determine that the cell activity is ≥90% (viable cells are not stained), flow cytometry detects CD56 + CD16 + NK cell purity ≥95%, which is qualified;

[0023] Combine all qualified culture liquids to obtain two-stage cell liquid for standby.

[0024] Preferably, in the step S2.2.2, the preparation method of the Percoll solution with a density of 1.085 g / ml is as follows: accurately take 73.5 ml of Percoll stock solution into a measuring cylinder with a pipette, slowly add 26.5 ml of physiological saline diluent, and mix uniformly; transfer the mixed solution to a beaker, and stir at low speed with a magnetic stirrer for 10-15 minutes to ensure uniform dispersion of particles and avoid air bubbles; vertically immerse the densimeter into the mixed solution, and record the density value after the reading is stable;

[0025] If the measured density is >1.085 g / ml: it indicates that Percoll is excessive, and physiological saline diluent needs to be added, with an increment of 1-2 ml each time, and re-measured after stirring;

[0026] If the measured density is <1.085 g / ml: Percoll stock solution needs to be added, with an increment of 0.5-1 ml each time, and re-measured after stirring;

[0027] Until the densimeter reading is stable in the range of 1.085±0.002 g / ml.

[0028] Preferably, the special medium A is composed of X-VIVO 15 medium, 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 1000 U / mL of IL-2, 50 ng / mL of IL-15 and 10 ng / mL of IL-21.

[0029] Preferably, the special medium B is composed of X-VIVO 15 medium, 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 800 U / mL of IL-2, 80 ng / mL of IL-15 and 20 ng / mL of IL-21.

[0030] Preferably, the step S1 comprises:

[0031] S1.1, obtaining single nuclear cells;

[0032] Collect 50-100 mL of peripheral blood, separate the collected sample by density gradient centrifugation, centrifuge at 400 x g for 30 minutes at room temperature, and then suck the single cells located in the white membrane layer into a new sterile centrifuge tube; add 5-10 times the volume of PBS solution to the centrifuge tube containing the single nuclear cells for washing, centrifuge at 250 x g for 10 minutes, discard the supernatant, and repeat the washing for 2-3 times to remove the residual plasma and PBS solution components to obtain single nuclear cells;

[0033] S1.2, immunomagnetic bead sorting;

[0034] S1.2.1, magnetic bead labeling: resuspend the washed mononuclear cells with PBS containing 0.5% bovine serum albumin (BSA) and 2mM EDTA, and adjust the cell concentration to 1×10 7 / mL; add antibody magnetic beads at a volume ratio of 1:100, the antibody magnetic beads include CD3 antibody magnetic beads, CD14 antibody magnetic beads, and CD19 antibody magnetic beads at a mass ratio of 1:1:1, and incubate at 4°C for 30 minutes in the dark to allow the magnetic beads to fully bind to the cells;

[0035] S1.2.2, magnetic separation: slowly add the labeled cell suspension into a separation column placed on a magnetic stand, and after the liquid naturally flows out, wash the separation column with PBS containing 0.5% BSA and 2mM EDTA for 3-5 times, 3-5mL each time, to remove the cells not combined with the magnetic beads; then remove the separation column from the magnetic stand, add an appropriate amount of PBS containing 0.5% BSA, and quickly push out the cells in the column with a syringe to collect the NK cell enrichment liquid not combined with the antibody magnetic beads, and remove T cells, B cells and monocytes; centrifuge the enrichment liquid at 300xg for 10 minutes, remove the supernatant, and obtain the enriched NK cells;

[0036] S1.3, flow cytometry sorting;

[0037] S1.3.1, antibody labeling: after washing the enriched NK cells with PBS, add fluorescently labeled anti-NK cell specific antibodies CD56-PE and CD16-APC, and incubate at 4°C in the dark for 20-30 minutes; the amount of antibody added is 10μL per 1×10 6 cells;

[0038] S1.3.1, flow sorting: after labeling, wash the cells with PBS for 2-3 times to remove the unbound antibodies, then resuspend the cells in an appropriate amount of PBS, and adjust the cell concentration to 1×10 6 ; put the cell suspension into a flow cytometry for sorting, and according to the set fluorescence parameters, select the CD56 + and CD16+ NK cell subpopulation for sorting and collection, and place the sorted cells in a sterile centrifuge tube containing a special culture medium A.

[0039] Preferably, step S3, harvest the NK cells;

[0040] After the two-stage culture is completed, centrifuge the two-stage cell liquid at 250xg for 10 minutes, discard the supernatant, wash the cells with PBS for 2-3 times to remove the residual culture medium components; then resuspend the cells with an appropriate amount of PBS, and according to the experimental use requirements, store at low temperature.

[0041] The beneficial effects of the present application are as follows:

[0042] 1. Improve cell purity: adopt two-stage culture combined with density gradient centrifugation method, can remove senescent, non-functional cells and mixed cells according to cell density difference, efficiently enrich advantage NK cell subpopulation, make the final obtained NK cell purity significantly improve, provide better cell product for clinical application.

[0043] 2. Enhance cell activity: regulate culture system in stages, the first stage is rapid proliferation, the second stage is optimized function, combined with the advantage cells screened by centrifugation, effectively avoid cell overactivation or exhaustion, maintain high anti-tumor activity of NK cells, guarantee their ability to kill tumor cells.

[0044] 3. Reduce cost and operation difficulty: use specific density of density gradient centrifugation method between the first and second stage culture, simple operation, and controllable cost, more suitable for large-scale production, help to promote the clinical popularization and application of high-purity anti-tumor NK cell therapy. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 The flowchart of the present application is shown in the figure; DETAILED DESCRIPTION

[0046] As shown in the figure, Figure 1 The present application is a method for expanding high-purity anti-tumor NK cells, mainly used for rapid in vitro expansion of high-purity NK cells, while ensuring the anti-tumor activity of NK cells. Through the two-stage culture mechanism with middle-stage screening and optimization of the culture medium, the rapid in vitro expansion of NK cells is realized. This method not only has fast expansion speed, but also can effectively guarantee the killing activity of NK cells to tumor cells.

[0047] The present application will be described in more detail below with examples.

[0048] Example 1

[0049] Preparation of Percoll separation solution

[0050] Preparation of Percoll separation solution with density of 1.085±0.002 / mlg:

[0051] Accurately take 73.5ml Percoll stock solution with a pipette into a graduated cylinder, slowly add 26.5ml physiological saline diluent, mix evenly; transfer the mixed solution to a beaker, stir with a magnetic stirrer at low speed for 10-15 minutes to ensure uniform dispersion of particles and avoid air bubbles; vertically immerse the density meter into the mixed solution, and record the density value after the reading is stable.

[0052] If the measured density is >1.085g / ml: it means that Percoll is excessive, add physiological saline diluent by 1-2ml each time, stir and measure again;

[0053] If the measured density <1.085 g / ml: need to add Percoll stock solution, 0.5-1 ml each time, stir and re-measure;

[0054] Until the density meter reading is stable in the range of 1.085±0.002 g / ml.

[0055] Example 2

[0056] Preparation of special medium A:

[0057] Special medium A focuses on the rapid proliferation of NK cells, using X-VIVO 15 medium: add 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin to the medium, feeder cells and cytokines;

[0058] Feeder cells are K562, density is 1×10 5 cells / mL; through the binding of membrane surface ligands (such as MHC-I deletion, ULBP, etc.) with NK cell receptors, provide activation signals, and auxiliary cytokines promote the rapid proliferation of NK cells;

[0059] Cytokines are: 1000 U / mL of IL-2, 50 ng / mL of IL-15, and 20 ng / mL of IL-21.

[0060] Example 3

[0061] Preparation of special medium B:

[0062] Special medium B focuses on the activity and toxicity of NK cells while ensuring the proliferation efficiency; uses X-VIVO 15 medium: add 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin to the medium; at the same time, add cytokines and ROCK inhibitors;

[0063] The ROCK inhibitor Y-27632 is used at a concentration of 10 μM; by inhibiting Rho-associated kinase (ROCK), it enhances the migration ability and target cell contact efficiency of NK cells, thereby improving cytotoxicity, and has no obvious inhibition on NK cell phenotype;

[0064] The CD16 antibody concentration is 5 μg / mL, which cross-links the CD16 receptor (FcγRIII) to simulate the antibody-dependent cell-mediated cytotoxicity (ADCC) signal, directly activate NK cell degranulation, and improve its killing efficiency on target cells;

[0065] Cytokines are: 800 U / mL of IL-2, 80 ng / mL of IL-15, and 20 ng / mL of IL-21.

[0066] Example 4

[0067] Obtaining of starting NK cells:

[0068] Obtaining mononuclear cells;

[0069] 50-100 mL of peripheral blood of the patient was collected, and the collected sample was separated by ordinary density gradient centrifugation, centrifuged at 400 x g for 30 minutes at room temperature, and the mononuclear cells located in the white membrane layer were sucked into a new sterile centrifuge tube after centrifugation; 5-10 times the volume of PBS was added to the centrifuge tube containing the mononuclear cells for washing, and centrifuged at 250 x g for 10 minutes, and the supernatant was discarded, and the washing was repeated for 2-3 times to remove the residual plasma and PBS components, and the mononuclear cells were obtained;

[0070] Immunomagnetic bead sorting;

[0071] First, magnetic bead labeling: the washed mononuclear cells were resuspended with PBS containing 0.5% bovine serum albumin (BSA) and 2 mM EDTA, and the cell concentration was adjusted to 1 x 10 7 / mL; antibody magnetic beads were added at a volume ratio of 1:100, and the antibody magnetic beads included CD3 antibody magnetic beads, CD14 antibody magnetic beads, and CD19 antibody magnetic beads at a mass ratio of 1:1:1, and were incubated at 4°C for 30 minutes in the dark to allow the magnetic beads to fully bind to the cells;

[0072] Then, magnetic separation: the labeled cell suspension was slowly added to the separation column placed on the magnetic stand, and after the liquid naturally flowed out, the separation column was washed with 3-5 mL of PBS containing 0.5% BSA and 2 mM EDTA for 3-4 times to remove the cells that did not bind to the magnetic beads; then the separation column was removed from the magnetic stand, and an appropriate amount of PBS containing 0.5% BSA was added, and the cells in the column were quickly pushed out with a syringe, and the NK cell enrichment liquid that did not bind to the antibody magnetic beads was collected, and T cells, B cells, and monocytes were removed; the enrichment liquid was centrifuged at 300 x g for 10 minutes, and the supernatant was removed to obtain the enriched NK cells;

[0073] Flow cytometry sorting;

[0074] First, advanced antibody labeling: after the enriched NK cells were washed with PBS, fluorescently labeled anti-NK cell-specific antibodies CD56-PE and CD16-APC were added, and incubated at 4°C for 20-30 minutes in the dark; the amount of antibody added was 10 μL per 1 x 10 6 cells;

[0075] Then, flow sorting: after labeling was completed, the cells were washed with PBS for 2-3 times to remove unbound antibodies, and then the cells were resuspended in an appropriate amount of PBS, and the cell concentration was adjusted to 1 x 10 6; the cell suspension is subjected to flow cytometry sorting, and CD56 + and CD16+ NK cell subgroups are selected and collected according to the set fluorescence parameters; and the density is adjusted to 1×10 6 / mL with standard PBS solution.

[0076] Sample library construction: 50 ml of blood sources of 10 random patients are treated according to the above method, and each blood source is divided into three groups of ABC to construct a sample library, which is sample 1A, sample 1B, sample 1C, sample 2A, sample 2B, sample 2C, and so on. 5 ml of each sample in the sample library is taken, and the collected CD56 + and CD16+ NK cells are verified again by flow cytometry, and the purity is all ≥95%; the cell activity is all ≥90% measured by trypan blue staining (viable cell staining rejection), and each sample can be used as a qualified sample.

[0077] Example 5

[0078] The following method is used to culture 5 ml of sample cell solution randomly taken from the sample library.

[0079] First-stage culture: NK cell subgroups are inoculated in a cell culture bottle at a density of 1×10 6 / mL, and the special medium A in Example 2 is used for first-stage culture; the culture bottle is placed in a constant temperature incubator at 37°C and 5% CO2 for continuous culture for 8 days, and the liquid is changed by half every other day, and the cell density is adjusted to 1×10 7 / mL in time according to the cell growth; after the culture ends, all the culture solutions are combined to obtain a first-stage cell solution for standby; after the first-stage culture ends, about 10.2 ml of cell culture solution with a NK cell density of 1×10 7 / mL is obtained, and the expansion multiple is about 20 times;

[0080] Density gradient centrifugation cell screening: first, transfer the first-stage cell solution to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and combine; resuspend with PBS solution and pass through a 400-mesh screen to remove cell clumps, cell fragment clumps, unsolubilized culture medium and other large particle impurities; dilute the cell suspension to a cell density of 1×10 6 / mL to 1×10 7 / mL with PBS solution; first add the Percoll separation solution with a density of 1.085 g / ml as described in Example 1 to the centrifuge tube, and then slowly add the diluted cell suspension along the wall of the tube, and the volume ratio of the cell suspension to the separation solution is 1:1; ensure that the interface of the two layers of liquid is clear; centrifuge at 400×g at room temperature for 20 minutes; absorb the white membrane layer and transfer it to the special medium B described in Example 3; repeat the steps until all the first-stage cell solutions are processed; adjust the NK cell density to 1×107 / mL, and the total volume of the screened cell solution was about 6.4 ml, and the screening elimination rate was 37.2%.

[0081] Two-stage culture: the NK cells after density gradient centrifugation were inoculated into cell culture bottles at a density of 1 x 10 6 / mL, and the culture period was 10 days. The two-stage culture used the special medium B in Example 3. Except for the medium, the other culture conditions were similar to those in the first stage. The cell culture bottles were continuously cultured in a constant temperature incubator at 37°C and 5% CO2for 10 days, and the medium was replaced every other day, and the cell density was adjusted to 1 x 10 6 / mL in time according to the cell growth. After the end of the culture period, samples were taken from each group of culture solution, and the cell viability was determined by trypan blue staining method, and the CD56 + CD16 + NK cell purity was ≥95%, and all were qualified. The culture solutions were combined, and a two-stage cell solution with a NK cell density of 1 x 10 6 / mL was obtained for standby, and the total volume was about 1262 ml. Compared with the NK cells after density gradient centrifugation, the expansion multiple was about 20 times, and compared with the original NK cells, the total expansion multiple was about 252 times.

[0082] The cell solution after two-stage culture of the sample was centrifuged at 250 x g for 10 minutes, and the supernatant was discarded. The cells were washed with PBS solution for 2-3 times to remove residual medium components. Then the cells were resuspended with an appropriate amount of PBS, and were aliquoted and stored at low temperature.

[0083] Example 6

[0084] The method described in Example 5 was used to take 5 ml of samples randomly selected from the sample bank for two-stage culture. The only difference was that when density gradient centrifugation was performed between the first-stage culture and the second-stage culture, the Percoll separation solution with a density of 1.075 g / ml was used.

[0085] After the end of the first-stage culture, about 9.8 ml of cell culture solution with a NK cell density of 1 x 10 7 / mL was obtained, and the expansion multiple was about 19.6 times.

[0086] After density gradient centrifugation, about 7.8 ml of screened cell solution was obtained, and the screening elimination rate was 20.4%.

[0087] After the end of the two-stage culture, about 1248 ml of cell culture solution with a NK cell density of 1 x 10 6 / mL was obtained, and the expansion multiple was about 16 times compared with the NK cells after density gradient centrifugation, and the total expansion multiple was about 249.6 times compared with the original NK cells.

[0088] Example 7

[0089] The method described in Example 5 was used to take 5 ml samples from the sample bank for two-stage culture; the only difference was that Percoll separation solution with a density of 1.090 g / ml was used for density gradient centrifugation between the first-stage culture and the second-stage culture.

[0090] After the first-stage culture, about 10.8 ml of cell culture solution with a NK cell density of 1 x 10 7 / mL was obtained, with an expansion ratio of about 21.6 times;

[0091] After density gradient centrifugation, about 4.3 ml of screened cell solution was obtained, with a screening rejection rate of 60.1%.

[0092] After the second-stage culture, about 975 ml of cell culture solution with a NK cell density of 1 x 10 6 / mL was obtained, with an expansion ratio of about 22.6 times compared with the NK cells after density gradient centrifugation, and a total expansion ratio of about 195 times compared with the original NK cells.

[0093] Comparative Example 1

[0094] The method described in Example 5 was used to take 5 ml of sample 2 for two-stage culture; the only difference was that no density gradient centrifugation was performed between the first-stage culture and the second-stage culture.

[0095] After the first-stage culture, about 10.5 ml of cell culture solution with a NK cell density of 1 x 10 7 / mL was obtained, with an expansion ratio of about 21 times;

[0096] After the second-stage culture, about 1470 ml of cell culture solution with a NK cell density of 1 x 10 6 / mL was obtained, with an expansion ratio of 14 times compared with the first-stage culture, and a total expansion ratio of about 294 times compared with the original NK cells.

[0097] Comparative Example 2

[0098] The method described in Example 5 was used to take 5 ml samples from the sample bank for two-stage culture; the only difference was that special culture medium B was used for the first-stage culture and special culture medium A was used for the second-stage culture.

[0099] After the first-stage culture, about 6.8 ml of cell culture solution with a NK cell density of 1 x 10 7 / mL was obtained, with an expansion ratio of about 13.6 times;

[0100] After density gradient centrifugation, about 3.7 ml of screened cell solution was obtained, with a screening rejection rate of 45.6%.

[0101] After the end of the two-stage culture, about 802 ml of cell culture solution with NK cell density of 1 x 10 6 / mL was obtained, which was about 21.6 times of the NK cell expansion ratio after density gradient centrifugation, and about 160.4 times of the total expansion ratio compared with the original NK cells.

[0102] As can be seen from Examples 5-7, the present application can effectively ensure the quality of the original cells in the two-stage culture by carrying out two-stage culture on the NK cells and gradient centrifugation on the cell population at a specific density of the separation liquid after the end of the first-stage culture to screen the NK cells with full cytoplasm. This method can effectively ensure the quality of the original cells in the two-stage culture, and the screening is extremely sensitive to the density of the separation liquid. Too high density of the separation liquid will greatly increase the rejection rate, although the quality of the original cells in the two-stage culture can be fully ensured, but due to the too high rejection rate, it is not conducive to the rapid expansion of the two-stage culture. Too low density of the separation liquid also makes it difficult to effectively screen the high-quality original cells from the NK cell population in the first-stage culture, which is also not conducive to the two-stage culture. The Percoll separation liquid with a density of 1.085 g / ml is the best, which can screen the high-quality NK cells and the rejection rate in two directions, and provides a solid foundation for the expansion of the two-stage culture.

[0103] As can be seen by comparing Comparative Example 1 with Example 5, after the density gradient separation is cancelled between the first-stage and the two-stage culture, although the number of NK cells after the first-stage culture is not subjected to the rejection treatment in theory, the cell source is larger, but due to the large number of inferior cell population (cytoplasm is not full, density is small), the expansion speed is limited, and it will compete with the dominant cell population, which will adversely affect the overall expansion.

[0104] As can be seen by comparing Comparative Example 2 with Example 5, since the special culture medium A and B have obvious differences in the effect of cell expansion in each stage, when the culture media are cross-used, the overall culture system will be unbalanced, which will greatly reduce the expansion number. The inventor believes that the possible reason is that in the first stage, the excessive toxicity of the special culture medium B to the NK cells will greatly slow down the division and proliferation speed of the dominant cells, so that a large number of inferior cells exist after the end of the first-stage culture. Such a situation will cause the rejection rate to be too high during the cell screening between the two stages, thereby causing adverse effects on the overall expansion system.

[0105] In order to further verify the above conclusion, the present application also takes another sample, which is subjected to 2 times of culture by the methods described in Comparative Examples 1, 2 and Examples 5-7, respectively. The conclusion obtained is also consistent with the above conclusion, which will not be described in detail in the present application.

[0106] Anti-tumor activity test

[0107] 1. Experimental group setting:

[0108] Group A (amplified by the method described in Example 5)

[0109] Group B (amplified by the method described in Example 6)

[0110] Group C (amplified by the method described in Example 7)

[0111] Group E (amplified by the method described in Comparative Example 1)

[0112] Group F (amplified by the method described in Comparative Example 2)

[0113] Blank control group 1 (randomly selected non-amplified primary NK cells)

[0114] 2. Target cell selection:

[0115] Human hepatocarcinoma cells (HepG2), lung cancer cells (A549), breast cancer cells (MCF-7);

[0116] 3. Killing activity detection method:

[0117] The experimental group and the target cells were co-incubated at an effector-to-target ratio of 10:1 at 37°C and a carbon dioxide concentration of 5% for 24 hours. The apoptosis rate of the target cells was detected by flow cytometry to evaluate the killing activity.

[0118] 4. The results are shown in Tables 1-3.

[0119] Table 1. Human hepatocarcinoma cell (HepG2) apoptosis rate statistics

[0120] grouping HepG2 cell apoptosis rate A 57.4％ B 48.2％ C 58.4％ D 44.5％ E 37.4％ blank 27.8％

[0121] Table 2. Lung cancer cell (A549) apoptosis rate statistics

[0122] grouping A549 cell apoptosis rate A 45.6％ B 38.4％ C 46.7％ D 33.2％ E 28.5％ blank 17.5％

[0123] Table 3. Breast cancer cell (MCF-7) apoptosis rate statistics

[0124]

[0125]

[0126] As can be seen from Tables 1-3, the NK cells separated by the two-stage culture and specific density screening of the present application have good broad-spectrum antitumor activity. Although Group C has similar (or even better) antitumor activity compared to Group A, there is a large difference in the overall amplification factor between the two groups, which to some extent limits the potential for industrialization and promotion, and is expected to be addressed in future research.

Claims

1. A method for amplifying high-purity anti-tumor NK cells, characterized in that: The following steps are involved: S1. Acquisition of initial NK cells; Density gradient centrifugation is used to separate mononuclear cells from peripheral blood obtained from humans. Immunomagnetic bead sorting is used to remove T cells, B cells, and monocytes from the mononuclear cells to enrich and separate NK cells. NK cell-specific antibodies were used to purify the enriched NK cells by flow cytometry; S2, dual-stage culture; A two-stage culture method was used. The first stage culture cycle was 8 days, and the cytokine addition amounts in the culture medium used were: 1000U / mL IL-2, 50ng / mL IL-15, and 10ng / mL IL-21; the second stage culture cycle was 10 days, and the cytokine addition amounts in the culture medium used were: 800U / mL IL-2, 80ng / mL IL-15, and 20ng / mL IL-21. After the completion of the first stage culture, NK cells with full cytoplasm were separated by density gradient centrifugation for the second stage culture. S3. Harvest NK cells.

2. The method for amplifying high-purity anti-tumor NK cells according to claim 1, characterized in that: The step S2 double-stage culture comprises the following steps: S2.1, one-stage cultivation; S2.1.

1. Cell inoculation: NK cell subsets obtained by flow cytometry were seeded at a rate of 1×10 6 / mL density was seeded in a cell culture flask, and a special medium A was used. The cytokine addition amount in the special medium A was: 1000U / mL IL-2, 50ng / mL IL-15 and 10ng / mL IL-21 to promote the proliferation and activation of NK cells; S2.1.2 Culture conditions: Place the culture flask in a constant temperature incubator at 37°C and 5% CO2 for 8 consecutive days, change the medium by half every other day, and adjust the cell density to 1×10 7 / mL; after the incubation, all the culture medium was combined to obtain the first-stage cell solution for standby use; S2.

2. NK cell density gradient centrifugation separation: Percoll solution with a density adjusted to 1.085 g / ml was used as the separation solution to separate the first-stage cell fluid; S2.3, second-stage cultivation; S2.3.

1. Cell inoculation: Cells were centrifuged at a density gradient of 1 × 10 6 / mL density was inoculated into cell culture flasks, and a special medium B was used. The cytokine addition amounts in the special medium B were: 800U / mL of IL-2, 80ng / mL of IL-15, and 20ng / mL of IL-21; S2.3.2 Culture conditions: Place the culture flask in a constant temperature incubator at 37°C and 5% CO2 for 10 consecutive days, replace half of the medium every other day, and adjust the cell density to 1×10 6 / mL; After the incubation period, samples of culture medium were taken from each group, and the cell activity was determined to be ≥90% by trypan blue staining, and CD56 was detected by flow cytometry. + CD16 + NK cell purity ≥95% is qualified; Combine all qualified culture fluids to obtain the second-stage cell fluid for use.

3. The method for amplifying high-purity anti-tumor NK cells according to claim 2, characterized in that: The step S2.2 includes the following steps: S2.2.

1. Centrifugation Preparation: Transfer the first-stage cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and combine the supernatants. Resuspend in PBS and pass through a 400-mesh sieve to remove cell clumps, cell debris, undissolved culture medium, and other large particles. Dilute the cell suspension with PBS to 1×106 / mL to 1×10 7 / mL cell density; S2.2.2 Density Gradient Centrifugation: First, add Percoll separation solution adjusted to a density of 1.085g / ml to the centrifuge tube, then slowly add the diluted cell suspension along the tube wall, with a volume ratio of cell suspension to separation solution of 1:1; ensure a clear interface between the two liquid layers; centrifuge at 400×g, room temperature, for 20 minutes; aspirate the buffy coat layer and transfer it to special culture medium B; repeat the steps until all the first-stage cell fluids are processed.

4. The method for amplifying high-purity anti-tumor NK cells according to claim 3, characterized in that: In step S2.2.2, the preparation method of the Percoll separation solution with a density of 1.085 g / ml is as follows: 73.5 ml of the Percoll stock solution is accurately measured into a graduated cylinder using a pipette, 26.5 ml of the physiological saline diluent is slowly added, and mixed evenly; the mixture is transferred to a beaker and stirred at low speed with a magnetic stirrer for 10 to 15 minutes to ensure that the particles are evenly dispersed and to avoid the generation of bubbles; a hydrometer is vertically immersed in the mixture, allowed to stand until the reading stabilizes, and the density value is recorded; If the measured density is greater than 1.085 g / ml, it means that there is an excess of Percoll. You need to add more saline diluent in increments of 1 to 2 ml each time, stir, and then measure again. If the measured density is less than 1.085 g / ml: add 0.5-1 ml of Percoll stock solution each time, stir and re-measure; Until the density meter reading stabilizes within the range of 1.085±0.002g / ml.

5. The method for amplifying high-purity anti-tumor NK cells according to claim 4, characterized in that: The dedicated culture medium A consists of X-VIVO 15 culture medium, 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 1000 U / mL IL-2, 50 ng / mL IL-15 and 20 ng / mL IL-21.

6. The method for amplifying high-purity anti-tumor NK cells according to claim 5, characterized in that: The special culture medium A is also added with a density of 1×10 5 K562 cells at a concentration of 1:1 / mL were used as feeder cells.

7. The method for amplifying high-purity anti-tumor NK cells according to claim 4, characterized in that: The dedicated culture medium B consists of X-VIVO 15 culture medium, 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 800 U / mL IL-2, 80 ng / mL IL-15 and 20 ng / mL IL-21.

8. The method for amplifying high-purity anti-tumor NK cells according to claim 7, characterized in that: The special culture medium B is also added with 5 μg / mL CD16 antibody and 10 μM ROCK inhibitor.

9. The method for amplifying high-purity anti-tumor NK cells according to claim 1, wherein: The step S1 comprises: S1.

1. Obtain mononuclear cells; Collect 50-100 mL of peripheral blood and separate the collected sample by density gradient centrifugation at 400×g for 30 minutes at room temperature. After centrifugation, aspirate the single cells located in the buffy coat layer into a new sterile centrifuge tube. Add 5-10 volumes of PBS solution to the centrifuge tube containing the mononuclear cells for washing. Centrifuge at 250×g for 10 minutes, discard the supernatant, and repeat the washing process 2-3 times to remove residual plasma and PBS components to obtain mononuclear cells. S1.2, immunomagnetic bead sorting; S1.2.

1. Magnetic bead labeling: The washed mononuclear cells were resuspended in PBS containing 0.5% bovine serum albumin (BSA) and 2 mM EDTA, and the cell concentration was adjusted to 1 × 10 7 / mL; add antibody magnetic beads at a volume ratio of 1:100, including CD3 antibody magnetic beads, CD14 antibody magnetic beads, and CD19 antibody magnetic beads at a mass ratio of 1:1:1, and incubate in the dark at 4°C for 30 minutes to allow the magnetic beads to fully bind to the cells; S1.2.2, Magnetic separation: Slowly add the labeled cell suspension to a separation column placed on a magnetic stand. After the liquid has naturally drained away, wash the separation column 3-4 times with PBS containing 0.5% BSA and 2mM EDTA, 3-5 mL each time, to remove cells not bound to the magnetic beads. Then, remove the separation column from the magnetic stand, add an appropriate amount of PBS containing 0.5% BSA, and quickly push out the cells in the column with a syringe to collect the NK cell enrichment fluid that is not bound to the antibody magnetic beads, and remove T cells, B cells, and monocytes. Centrifuge the enrichment fluid at 300×g for 10 minutes, remove the supernatant, and obtain the enriched NK cells. S1.3, flow cytometry sorting; S1.3.1 Antibody labeling: After the enriched NK cells were washed with PBS, fluorescently labeled anti-NK cell specific antibodies CD56-PE and CD16-APC were added and incubated at 4°C in the dark for 20-30 minutes. The amount of antibody added was 1×10 6 Add 10 μL per cell; S1.3.1 Flow cytometry: After labeling, wash the cells 2-3 times with PBS to remove unbound antibodies, then resuspend the cells in an appropriate amount of PBS and adjust the cell concentration to 1×10 6 The cell suspension was sorted by flow cytometry, and CD56 was selected according to the set fluorescence parameters. + The NK cell subsets of CD16+ and CD16+ were sorted and collected, and the sorted cells were placed in a sterile centrifuge tube containing a special culture medium A.

10. The method for amplifying high-purity anti-tumor NK cells according to claim 1, characterized in that: Step S3, harvesting NK cells; After the second stage of culture, centrifuge the second stage cell solution at 250×g for 10 minutes, discard the supernatant, and wash the cells 2-3 times with PBS to remove residual culture medium components; then resuspend the cells with an appropriate amount of PBS and store them in aliquots at low temperature according to experimental needs.

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