Efficient in-vitro culture method of clinical-grade umbilical cord blood NK (Natural Killer) cells

Abstract

The invention discloses an efficient in-vitro culture method of clinical-grade umbilical cord blood NK cells. The method comprises the following steps: carrying out in-vitro separation on umbilical cord blood mononuclear cells; nK cell activation culture: adding an NK cell activation culture medium to enable the density of the NK cells to be 2.5 * 10 < 6 > cells / mL, and transferring the NK cells into an incubator with the temperature of 37 DEG C and the CO2 content of 5% to be activated and cultured for 72 hours; and NK cell multiplication culture: adding an amplification culture medium to resuspend the cells, enabling the cell density to be 1-2 * 10 < 6 > cells / mL, and culturing in a 5% CO2 incubator at 37 DEG C. The NK cells cultured and amplified by the method have higher proliferation speed and higher purity, have stronger killing activity on various tumor cells, do not need trophoblast cell stimulation and antibody coating processes, are free of animal-derived and human-derived serum, and are simple in operation process.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro cell culture, and in particular to a high-efficiency in vitro culture method of clinical-grade umbilical cord blood NK cells. Background Art

[0002] Natural killer cells (NK cells) are one of the important components of the human immune system. They are effector cells of the innate immune system and are derived from bone marrow lymphoid stem cells. They are mainly distributed in the bone marrow, peripheral blood, liver, spleen, lungs and lymph nodes. NK cells are different from T and B cells. They do not require antigen sensitization and have no MHC restriction. They can non-specifically recognize and kill tumor cells, virus-infected cells, aging and damaged cells, and play an important role in the early anti-infection and anti-tumor immune processes.

[0003] NK cells come from a variety of sources, including NK92 cells, induced pluripotent stem cells, peripheral blood (PB), and umbilical cord blood (UCB). Among them, NK92 cells are tumor cells and need to be irradiated before clinical use, which affects their persistence and has the risk of tumorigenesis; NK cells derived from induced pluripotent stem cells have a complex preparation process, are expensive, and have the risk of tumorigenesis. NK cells have high requirements for quantity and purity in clinical applications and cannot contain too many lymphocytes such as T cells and B cells. Due to the large individual differences in the proportion of lymphocytes in peripheral blood, the purity of NK cells cultured from peripheral blood is uneven, or cell sorting processes and trophoblast cells are required to stimulate proliferation, which increases process costs and has the risk of tumorigenesis. However, the proportion of lymphocytes in umbilical cord blood is relatively stable, and the quality level of NK cells cultured by the same culture method is stable. Therefore, NK cells derived from umbilical cord blood are regarded as a potential "universal" NK cell product with multiple advantages.

[0004] However, the existing in vitro proliferation methods of umbilical cord blood NK cells are relatively complicated, and there are problems such as low proportion of effector cells (low purity), poor killing effect, and low amplification multiple. Chinese Patent 202110711376.3 cultivates umbilical cord blood NK cells based on the expansion and activation process of trophoblast cells, but its cell purity is low (85%), the amplification multiple is low, and the presence of feeder cells increases the risk of clinical application, and more product testing steps are required. Chinese Patent 202311107870.4 uses a pure cytokine stimulation method to cultivate umbilical cord blood NK cells, which has the advantages of faster amplification speed and higher purity, but it is necessary to sort out NK cells from the starting CBMC cells and then culture them, which is complicated to operate and costly. Chinese patents 202110429068.1, 202110061750.X and other feeder cell-free expansion systems based on cytokines and antibodies have high purity of umbilical cord blood NK cells after culture, but there are problems such as weak killing function, low amplification multiple, high cost, and large individual differences.

[0005] Therefore, exploring the production process of clinical-grade umbilical cord blood NK cells with high purity, high quantity, high activity, strong functionality, stability, safety, and strong operability remains a challenge currently faced. Summary of the invention

[0006] The purpose of the present invention is to provide an efficient in vitro culture method for clinical-grade umbilical cord blood NK cells, in which the amplified NK cells have a faster proliferation rate and higher purity, have stronger killing activity against a variety of tumor cells, do not require trophoblast cell stimulation and antibody coating process, do not contain animal and human serum, and have a simple operation process.

[0007] To achieve the above object, the present invention provides the following technical solution: an in vitro expansion and culture method of NK cells, comprising the following steps:

[0008] Step 1: Isolation of cord blood mononuclear cells in vitro;

[0009] Step 2: NK cell activation culture: Add NK cell activation medium to make the NK cell density 2.5×10 6 Transfer to 37°C, 5% CO 2 Activate and culture in an incubator for 72 h;

[0010] Step 3: NK cell expansion culture: Add expansion medium to resuspend the cells to a cell density of 1-2×10 6 cells / mL, 37°C, 5% CO 2 Culture in an incubator.

[0011] The present invention also provides a culture system for in vitro expansion of umbilical cord blood NK cells, the NK cell culture system comprises an activation medium and an expansion medium, the medium comprises a basal medium and a supplement, the basal medium is selected from one or more of NK MACS medium (Miltenyi Biotec), GT-T551 H3 medium (Takara), X-VIV015 medium (Lonza), and NK cell expansion serum-free medium P01 (Suzhou Yikesai Biological), the supplement consists of 500-1000 IU / mL of IL-2 (National Medicine Standard S10970058), 0.1-50 ng / mL of IL-15 (Beijing Tongli Haiyuan), 0.1-100 ng / mL of IL-27 (Peprotech), 0.1-50 ng / mL of IL-18 (Peprotech), 0.1-50 ng / mL of anti-human CD16 monoclonal antibody (Peprotech), and 1-5% of serum replacement (EliteCell).

[0012] Preferably, the activation medium includes a basal medium and a supplement, the basal medium is a mixture of GT-T551H3 medium and NK cell expansion serum-free medium P01; the supplement consists of 1000 IU / mL IL-2, 10 ng / mL IL-15, 100 ng / mL IL-27, 5 ng / mL IL-18, 50 ng / mL anti-human CD16 monoclonal antibody, and 5% serum replacement.

[0013] Preferably, the expansion medium comprises a basal medium and a supplement, wherein the basal medium is a mixture of GT-T551H3 medium and NK cell expansion serum-free medium P01; and the supplement consists of 1000 IU / mL IL-2 and 5% serum replacement.

[0014] Preferably, during the NK in vitro proliferation culture, fresh expansion medium is added every 2 to 3 days, and the cell density is adjusted to 1 to 2×10 6 cells / mL, 37°C, 5% CO 2 Culture in an incubator for 20 to 21 days.

[0015] Preferably, the in vitro separation method of umbilical cord blood mononuclear cells described in step 1 comprises the following steps:

[0016] Step 1.1: Blood collection: Umbilical cord blood is collected from the umbilical cord of a full-term healthy newborn, anticoagulated with sodium citrate, and separated within 24 hours after collection;

[0017] Step 1.2: After the blood bag containing the umbilical cord blood is disinfected, the mononuclear cells are separated: the umbilical cord blood is diluted in a volume ratio of 1:1 with normal saline, and then slowly added to an equal volume of human lymphocyte separation solution, and centrifuged at 800×g for 20 min;

[0018] Step 1.3: Take the buffy coat layer: Collect the mononuclear cell buffy coat layer into a new centrifuge tube, add physiological saline to wash twice, centrifuge at 600×g for 5 minutes, discard the supernatant, and obtain the umbilical cord blood mononuclear cell pellet.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The in vitro expansion method of NK cells of the present invention does not require trophoblast cells and antibody coating, and NK cells can be expanded in vitro only through cytokine stimulation.

[0021] 2. The NK cells amplified by the present invention can proliferate more than 10,000 times in 21 days.

[0022] 3. The purity of the expanded NK cells of the present invention can be kept stable at more than 95%, without T cell contamination, and highly expresses activating receptors such as NKG2D and NKp46.

[0023] 4. The NK cells amplified by the present invention have stronger killing activity against various tumor cells.

[0024] 5. The NK cell expansion process of the present invention does not require animal and human serum, and does not require NK cell sorting process. The operation process is simple, meets clinical application standards, and is highly safe. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the result of the expansion of NK cells in umbilical cord blood;

[0026] Figure 2 Flow cytometry was used to detect the purity of NK cells before and after 21 days of culture;

[0027] Figure 3 The expression of CD16, NKG2D, NKp46 and CD69 activating receptors on NK cells;

[0028] Figure 4 is the killing activity of NK cells against K562 cells;

[0029] Figure 5 is the killing activity of NK cells against HepG2 cells;

[0030] Figure 6 It is the killing activity of NK cells against 5637 cells. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] See also Figure 1-6 The present invention provides a technical solution: a highly efficient in vitro culture method for clinical-grade umbilical cord blood NK cells, comprising the following steps:

[0033] Step 1: In vitro isolation of umbilical cord blood mononuclear cells.

[0034] Step 2: NK cell activation culture: Add NK cell activation medium to make the NK cell density 2.5×10 6 Transfer to 37°C, 5% CO 2 The cells were activated and cultured in an incubator for 72 h.

[0035] Step 3: NK cell expansion culture: Add expansion medium to resuspend the cells to a cell density of 1-2×10 6 cells / mL, 37°C, 5% CO 2 Culture in an incubator.

[0036] Example 1: In vitro expansion and culture of NK cells derived from human umbilical cord blood:

[0037] 1. Isolation of mononuclear cells from umbilical cord blood:

[0038] (1) Umbilical cord blood is obtained from the umbilical cord of a full-term healthy newborn, anticoagulated with sodium citrate, and separated within 24 hours after collection;

[0039] (2) After the blood bag containing the umbilical cord blood is disinfected, the umbilical cord blood is aspirated and added to a 50 mL centrifuge tube, and then diluted with an equal amount of physiological saline solution;

[0040] (3) Take half the volume of lymphocyte separation solution of the resuspended umbilical cord blood and add it to a new 50 mL centrifuge tube. Slowly add the diluted umbilical cord blood to the surface of the lymphocyte separation solution to keep the contact surface stable;

[0041] (4) Centrifuge the tube at 800 × g for 20 min;

[0042] (5) After centrifugation, the white membrane between the lymphocyte separation fluid layer and the plasma layer can be clearly seen, which is the mononuclear cells. Use a pipette to suck out all the white membrane in the middle layer and place it in a new centrifuge tube. Dilute the cells to a volume of 50 mL with physiological saline solution;

[0043] (6) Centrifugation, 600 × g, 5 min;

[0044] (7) Discard the supernatant, wash again with physiological saline, centrifuge at 600 × g for 5 min, and discard the supernatant to obtain the umbilical cord blood mononuclear cell pellet.

[0045] 2. NK cell activation culture: GT-T551 H3 medium and NK cell expansion serum-free medium P01 were mixed at a volume ratio of 9:1; the NK cell density was adjusted to 2.5×10 6 / mL, and add 1000IU / mL IL-2, 10ng / mL IL-15, 100ng / mL IL-27, 5ng / mL IL-18, 50ng / mL anti-human CD16 monoclonal antibody and 5% serum replacement. The cells were transferred to 37℃, 5% CO 2 The cells were activated and cultured in an incubator for 72 h.

[0046] 3. NK cell expansion culture: GT-T551 H3 medium and NK cell expansion serum-free medium P01 were mixed at a volume ratio of 9:1, and 1000 IU / mL of IL-2 and 5% serum replacement were added. The expansion medium was added to the activated NK cells to make the cell density 1-2×10 6 / mL, at 37°C, 5% CO 2 During the NK cell proliferation culture in vitro, fresh expansion medium was added every 2 to 3 days, and the cell density was adjusted to 1 to 2 × 10 6 / mL and cultured for 20 to 21 days.

[0047] Example 2: Detection of NK cells in umbilical cord blood:

[0048] 1. The method of Example 1 was used to expand and culture the umbilical cord blood mononuclear cells in vitro, and the cells were counted using a Countstar automatic cell counter (the cell suspension was mixed with the AO / PI dye solution in a volume ratio of 1:1). The results are shown in Table 1, which shows the number and expansion multiple of umbilical cord blood NK cells (30 mL of umbilical cord blood). The umbilical cord blood NK cells expanded an average of 13122 times within 21 days.

[0049]

[0050] 2. Detection of NK cell purity and activated receptors by flow cytometry: Take the NK cells amplified according to the method of Example 1, count them, and adjust the cell density to 1×10 7 / mL, dispensed into 100μL / tube, set up control tubes and test tubes, and added anti-CD56 antibody, anti-CD3 antibody, anti-CD16 antibody, anti-NKG2D, anti-CD69 antibody and anti-NKp46 antibody (antibodies were purchased from Biolegend) respectively. After incubation in a dark environment at 4℃ for 20min, 2mL of normal saline was added to wash the cells and wash away the excess antibodies. Finally, the cells were resuspended with 200μL of normal saline and detected by flow cytometer.

[0051] NK cell phenotype is CD3 - CD56 + After activation and expansion of mononuclear cells, CD3 - CD56 + The cells increased from an average of 13.9% before culture to 98.96% after culture. Figure 2 Almost all NK cells express CD16 and activating receptors NKG2D, CD69, and NKp46 ( Figure 2 ).

[0052] 3. Flow cytometry to measure the killing activity of NK cells in umbilical cord blood:

[0053] S1: Prepare 5 μM CFSE (Biolegend) working solution;

[0054] S2: Prepare target cell suspensions, centrifuge K562, HepG2, and 5637 cells at 300×g for 10 min, discard the supernatant, resuspend in D-PBS, and count;

[0055] S3: Resuspend the cells with CFSE working solution to control the cell concentration to 1×107 / mL;

[0056] S4: Incubate the cells at 37°C in the dark for 20 min.

[0057] S5: Add 5 mL of culture medium and quench the staining for 10 min;

[0058] S6: Centrifuge at 300 × g for 10 min, discard the supernatant, and wash once with D-PBS;

[0059] S7: Adjust the cell density to 2×105 / mL with culture medium and dispense into culture tubes, 50 μL / tube;

[0060] S8: Take NK cells, centrifuge at 300×g for 10 min, discard the supernatant, and adjust the NK cell density to 1×106 / mL with culture medium;

[0061] S9: Dilute NK cells according to different effector-target ratios (5:1, 2.5:1, 1.25:1, 0.625:1), mix with target cell suspension, 50 μL / tube, and mix gently;

[0062] S10: 37°C, 5% CO 2 4h in culture environment;

[0063] S11: Add 7-AAD dye (Biolegend), 5 μL / tube, mix well for 10 min, and perform flow cytometry detection;

[0064] S12: Principles of gate setting: First, gate the target cell cluster 1 based on the forward scattered light signal (FSC) and the side scattered light signal (SSC). Then, based on cluster 1, target cell cluster 2 can be defined according to CFSE+. Then, the proportion of 7-AAD-positive cells in CFSE+ target cells is analyzed.

[0065] S13: Killing efficiency = (ratio of dead cells in CFSE+ cells in co-culture group - ratio of dead cells in CFSE+ cells in target cell group) / (1 - ratio of dead cells in CFSE+ cells in target cell group).

[0066] The killing effects of NK cells cultured according to the present invention targeting K562, HepG2, and 5637 cells are shown in FIG. Figure 4 , Figure 5 , Figure 6 .

[0067] 4. Endotoxin, mycoplasma and bacterial culture detection:

[0068] Bacterial endotoxin test method: Take this product and test it according to the fourth part of the 2020 edition of the Pharmacopoeia of the People's Republic of China <1143 Bacterial endotoxin test method>, which should be <0.5EU / mL. Bacterial detection uses the BacTALERT3D microbial detection system. The sample is added to the BACT / ALERT culture bottle and cultured for 5 days to detect anaerobic and aerobic bacteria. The sample test results should be negative. Mycoplasma detection uses fluorescent staining method, using Sigma's hochest 33258 staining solution for staining. Under a fluorescent microscope, only the cell nucleus shows blue fluorescence, which is negative.

[0069] After testing, the NK cell preparation cultured according to the present invention was negative for endotoxin, mycoplasma and bacteria.

[0070] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A highly efficient in vitro culture method for clinical-grade umbilical cord blood NK cells, characterized in that: Includes steps: Step 1: Isolation of cord blood mononuclear cells in vitro; Step 2: NK cell activation culture: Add NK cell activation medium to make the NK cell density 2.5×10 6 / mL, transferred to a 37°C, 5% CO2 incubator for activation and culture for 72h; Step 3: NK cell expansion culture: Add expansion medium to resuspend the cells to a cell density of 1-2×10 6 / mL, cultured in a 37°C, 5% CO2 incubator.

2. The method for efficiently culturing clinical-grade umbilical cord blood NK cells in vitro according to claim 1, characterized in that: The method for isolating umbilical cord blood mononuclear cells in vitro described in step 1 comprises the following steps: Step 1.1: Blood collection: Umbilical cord blood is collected from the umbilical cord of a full-term healthy newborn, anticoagulated with sodium citrate, and separated within 24 hours after collection; Step 1.2: After the blood bag containing the umbilical cord blood is disinfected, the mononuclear cells are separated: the umbilical cord blood is diluted in a volume ratio of 1:1 with normal saline, and then slowly added to an equal volume of human lymphocyte separation solution, and centrifuged at 800×g for 20 min; Step 1.3: Take the buffy coat layer: Collect the mononuclear cell buffy coat layer into a new centrifuge tube, add physiological saline to wash twice, centrifuge at 600×g for 5 minutes, discard the supernatant, and obtain the umbilical cord blood mononuclear cell pellet.

3. The method for efficiently culturing clinical-grade umbilical cord blood NK cells in vitro according to claim 1, characterized in that: The activation medium in step 1 includes a basal medium and a supplement, wherein the basal medium of the activation medium is a mixture of GT-T551 H3 medium and NK cell expansion serum-free medium P01; the supplement of the activation medium consists of 1000 IU / mL IL-2, 10 ng / mL IL-15, 100 ng / mL IL-27, 5 ng / mL IL-18, 50 ng / mL anti-human CD16 monoclonal antibody, and 5% serum replacement.

4. The method for efficiently culturing clinical-grade umbilical cord blood NK cells in vitro according to claim 1, characterized in that: The expansion medium in step 1 includes a basal medium and a supplement, wherein the basal medium of the expansion medium is a mixture of GT-T551H3 medium and NK cell expansion serum-free medium P01; the supplement of the expansion medium consists of 1000 IU / mL IL-2 and 5% serum replacement.

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