Method for in vitro differentiation and expansion of nk cells and uses thereof

Abstract

The application belongs to the field of cell biology, and provides a method for in-vitro differentiation and expansion of NK cells and application thereof, which comprises the following steps: (1) isolating umbilical cord hematopoietic stem cells; (2) culturing in a stem cell growth medium added with FLT3L, SCF, IL-7, IL-15 and IL-21 for 14 days; and (3) culturing in a stem cell growth medium added with FLT3L, SCF, IL-7, IL-15, IL-21 and BIX-01294 for 14 days. The method has good differentiation effect, and the obtained NK cells have good tumor killing effect.

Description

Technical Field

[0001] This application belongs to the field of cell biology, specifically, this application provides a method for in vitro differentiation and expansion of NK cells. Background Technology

[0002] Natural killer cells (NK cells), as innate immune cells, are lymphocytes that can non-specifically kill tumor cells or infected cells without prior sensitization. NK cells play an important role in anti-tumor and anti-infective immunity, and in vitro differentiation and expansion is a key step in their research and application. Currently, various methods for in vitro differentiation and expansion exist, but previous studies have shown low efficiency and instability in NK cell differentiation and expansion. Therefore, there is an urgent need to explore a simple and efficient method for in vitro differentiation and expansion of NK cells. BIX-01294 is a small molecule inhibitor that targets and inhibits G9a enzyme activity, playing a crucial role in suppressing tumor development and progression, but its application in NK cell differentiation and expansion has not been studied. Summary of the Invention

[0003] On the one hand, this application provides a method for in vitro differentiation and expansion of NK cells, the method comprising: (1) Isolation of umbilical cord hematopoietic stem cells; (2) The stem cells were cultured for 14 days in a culture medium containing FLT3L, SCF, IL-7, IL-15 and IL-21; (3) The stem cells were cultured for 14 days in a stem cell growth medium containing FLT3L, SCF, IL-7, IL-15, IL-21 and BIX-01294.

[0004] Furthermore, the concentrations of FLT3L, SCF, IL-7, IL-15 and IL-21 added to the stem cell growth medium in steps (2) and (3) are 10-30 ng / ml; the concentration of BIX-01294 added to the stem cell growth medium in step (3) is 0.5-1.5 μM.

[0005] Furthermore, the concentrations of FLT3L, SCF, IL-7, IL-15 and IL-21 added to the stem cell growth medium in steps (2) and (3) are 20 ng / ml; the concentration of BIX-01294 added to the stem cell growth medium in step (3) is 1 μM.

[0006] Furthermore, the stem cell growth medium described in steps (2) and (3) also contains fetal bovine serum, antibiotics, and gentamicin.

[0007] Furthermore, the stem cell growth medium described in steps (2) and (3) also contains 10 v / v% fetal bovine serum, 1 v / v% penicillin and 25 μg / mL gentamicin.

[0008] Furthermore, in steps (2) and (3), the culture medium is partially replaced every 3 days.

[0009] Furthermore, the stem cell growth medium is CellGenix® GMP SCGM medium.

[0010] Further, step (1) includes: (1-1) Transfer the umbilical cord blood to a culture bottle and let it stand; (1-2) Add HES to the umbilical cord blood, mix well, and let stand to allow red blood cells to settle; (1-3) Transfer the supernatant after the red blood cells have settled in step (1-2) to a centrifuge tube pre-filled with lymphocyte separation medium; (1-4) Centrifugation; (1-5) Absorb the mononuclear cell layer and wash; (1-6) Sorting CD34 + cell.

[0011] Furthermore, in steps (1-3), the volume ratio of umbilical cord blood to HES is 5:1.

[0012] Furthermore, in steps (1-3), the volume ratio of the supernatant to the lymphocyte separation solution is 2:1.

[0013] On the other hand, this application provides NK cells prepared using the above method.

[0014] On the other hand, this application provides the use of the above-mentioned NK cells in the preparation of drugs for treating cancer.

[0015] Furthermore, the cancer is leukemia, preferably chronic myeloid leukemia.

[0016] The reagents used in this application are not limited to the specific types used in the examples; for example, other commercially available and homemade stem cell growth media besides CellGenix® GMPSCGM medium can also be used. (Except for Human CD34 from Medtronic) + Other kits and sorting methods besides magnetic bead sorting kits can also be used for steps (1-6). Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the differentiation and expansion of umbilical cord blood hematopoietic stem cells into NK cells.

[0018] Figure 2 Expression of NK cell markers NKp46 and NKG2D after BIX administration.

[0019] Figure 3 Results of NK cells killing target cells K562 after BIX treatment. Detailed Implementation

[0020] The mechanism by which umbilical cord blood hematopoietic stem cells differentiate into NK cells: Umbilical cord blood is rich in CD34 + Hematopoietic stem cells, after being supplemented in vitro with appropriate human cytokines (SCF, Flt3L, IL7, IL-15, and IL-21), can induce CD34. + HSCs were directed to differentiate into NK cells. Because cells are relatively easy to reprogram at the stem cell stage, this embodiment used the small molecule inhibitor BIX-01294 to inhibit the methyltransferase activity of G9a when hematopoietic stem cells differentiated into immature NK cells (day 14) to observe the development and function of NK cells derived from umbilical cord blood, as well as the isolation of umbilical cord blood and the development and function of the cells. The isolation and enrichment of hematopoietic stem cells from umbilical cord blood, as well as subsequent culture, were all performed under aseptic conditions.

[0021] The reagents and experimental methods used in the examples are as follows: Molecular biochemical reagents: Recombinant human SCF, Flt3L, IL7, IL-15, and IL-21 were purchased from Peprotech. Flow cytometry antibodies: anti-human CD56-BV605 (Clone: ​​NCAM16.2; BD Biosciences); anti-human NKp46-APC (Clone: ​​9E2; BioLegend); anti-human NKG2D-PE (Clone: ​​1D11; BioLegend). Ghost Dye Violet 510 (#13-0870-T100) cell viability dye was purchased from Tonbo Biosciences.

[0022] Example 1: Sorting and Culture of Umbilical Cord Blood Hematopoietic Stem Cells (1) Transfer fresh umbilical cord blood to a T75 culture flask and let it stand for 5 min.

[0023] (2) Add HES to the cord blood and mix thoroughly at a ratio of cord blood to HES (erythrocyte sedimentation agent) of 5:1. Let stand at room temperature for 40 minutes to settle the red blood cells.

[0024] (3) Transfer the supernatant to a 50 mL centrifuge tube pre-filled with 15 mL of Ficoll lymphocyte separation medium, and slowly add 2 times the volume of supernatant (supernatant: Ficoll = 2:1).

[0025] (4) Centrifuge at 20℃, 500 g for 20 min (5 up, 3 down).

[0026] (5) Carefully aspirate the mononuclear cell layer (white membrane layer) and wash twice with 1×PBS containing 2 mM EDTA on ice.

[0027] (6) Cell counting, according to the Human CD34 of Meteni. + Cell sorting was performed using a magnetic bead sorting kit.

[0028] (7) Separate the CD34 + HSCS were inoculated into 24-well plates of SCGM medium (approximately 4 × 10⁻⁶). 5 (1 cell / well) and added FLT3L, SCF, IL-7, IL-15 and IL-21 cytokines to the culture medium containing cells at a final concentration of 20 ng / mL, along with 10% fetal bovine serum, 1% penicillin and antibiotics, and 25 μg / mL gentamicin. The culture was then placed in a constant temperature incubator for directed in vitro differentiation of NK cells.

[0029] (8) Change the medium halfway every 3 days. After 7 days, inoculate into a 6-well plate and change the medium every 3 days.

[0030] (9) On day 14 of the differentiation process, 1 μM of BIX-01294 was added. On day 28 (with half of the medium changed every 3 days), the surface markers (NKG2D and NKp46) of NK cells and their cytotoxic phenotypes were detected.

[0031] Example 2: Flow Cytometry Detection of NK Cell Surface Markers (1) Collect the cell suspension from each well into a centrifuge tube, centrifuge at 300g / min for 7min, and discard the supernatant; (2) Wash the cells twice with 1×PBS, and centrifuge under the same conditions as above; (3) Add 100 μL of BV510 live / dead dye to each tube (add 0.1 μL of antibody to 100 μL system), mix well and incubate at room temperature in the dark for 5 min; (4) Add 200 μL of 1×PBS to stop staining, centrifuge at 200 g / min for 7 min, and discard the supernatant; (5) Add 100 μL of fluorescently labeled CD56 NKG2D and NKp46 surface antibodies to each tube (1 μL of antibody is added to 100 μL of the system) and incubate at room temperature in the dark for 30 min; (6) Add 200 μL of 1×PBS to stop staining, centrifuge at 300 g / min for 7 min, and discard the supernatant; (7) Add 300 μL of 1×PBS to each tube to resuspend the cells, and filter into a flow cytometry tube; (8) Flow cytometry LSRFortessa™ test, FlowJo software analysis of NKG2D and NKp46 ratio.

[0032] To further investigate whether G9a plays a role in the development and maturation of human NK cells, this embodiment isolated hematopoietic stem cells from umbilical cord blood and established an in vitro NK cell induction differentiation system. Figure 1 During the first 14 days of induction, cytokines SCF, Flt3L, IL-15, IL-21, and IL-7 were added to induce hematopoietic stem cells to differentiate into NK precursor cells. After 14 days, BIX and DMSO were added (control group, BIX was lysed with DMSO) to induce the development of mature NK cells. Flow cytometry analysis revealed that, compared with the control group with added DMSO, the addition of the inhibitor BIX-01294 resulted in increased CD56 levels. + NKp46 + and CD56 + NKG2D + The number of subgroups has increased significantly. Figure 2 This indicates that G9a inactivation promotes the development and maturation of NK cells.

[0033] Example 3: Detection of Cell Killing Activity (1) Calculate the required number of cells. Each well should contain at least 2 × 10⁶ target cells (NK cells). 4 Each group contains 7 replicates of 10 cells; each well contains at least 2 × 10⁶ effector cells (K562). 4 10 cells, with a total of 40 replicates, and the effector-to-target ratio was set at 5:1 and 10:1; (2) Transfer the cells used for the killing experiment to a new 15 mL centrifuge tube; (3) Centrifuge at 600g / min for 6min, discard the supernatant, and resuspend the cells in 1mL of culture medium; (4) Dilute and count the cells in a new 1.5 mL centrifuge tube according to the number of cells in each sample; (5) Calculate the required amount of cell suspension for each sample and transfer it to a new centrifuge tube; (6) Add complete culture medium to a total volume of “replica number × 100 μL”; (7) Take a 96-well U-shaped plate and add samples in sequence according to the system of 200 μL per well: experimental well (100 μL of target cells of each concentration + 100 μL of effector cells), effector cell maximum release well (100 μL of effector cells + 82 μL of culture medium), effector cell control well (100 μL of effector cells + 100 μL of culture medium). (8) After the samples are added, the 96-well plate is placed in an incubator at 37°C for 4 hours; (9) Remove the cells after 3 hours of incubation, add 18 μL of LDH release reagent to the maximum release well of the effector cells, and continue incubation at 37°C for 1 hour. (10) After the process is completed, centrifuge at 400 g / min for 5 min and transfer 120 μL of supernatant to a new 96-well plate; (11) Prepare the test reagent according to the number of sample wells. The single-well formula is 2μL INT 1× + 18μL INT diluent + 20μL enzyme solution + 20μL lactic acid solution. The preparation process must be kept away from light. (12) Add 60 μL of detection reagent to each well in the dark and incubate for 30 min in the dark; (13) After incubation, the absorbance was measured at a wavelength of 490 nm using an ELISA reader, and dual-wavelength detection was performed with 600 nm as the reference wavelength. (14) Organize the experimental data and calculate the killing rate according to the killing rate formula "[(absorbance of treated sample - absorbance of sample control well) / (absorbance of maximum cell enzyme activity - absorbance of sample control well)] × 100%".

[0034] This embodiment examined whether G9a affects the killing function of NK cells, and the results are as follows: Figure 3 As shown, the in vitro killing ability of BIX-treated NK cells was significantly enhanced when co-incubated with tumor cells, and the results were statistically significant.

Claims

1. A method for in vitro differentiation and expansion of NK cells, characterized in that, The method includes the following steps: (1) Isolation of umbilical cord hematopoietic stem cells; (2) The stem cells were cultured for 14 days in a culture medium containing FLT3L, SCF, IL-7, IL-15 and IL-21; (3) The stem cells were cultured for 14 days in a stem cell growth medium containing FLT3L, SCF, IL-7, IL-15, IL-21 and BIX-01294.

2. The method according to claim 1, characterized in that, The concentrations of FLT3L, SCF, IL-7, IL-15 and IL-21 added to the stem cell growth medium in steps (2) and (3) are 10-30 ng / ml; the concentration of BIX-01294 added to the stem cell growth medium in step (3) is 0.5-1.5 μM.

3. The method according to claim 1, characterized in that, In steps (2) and (3), the concentrations of FLT3L, SCF, IL-7, IL-15 and IL-21 added to the stem cell growth medium were 20 ng / ml; in step (3), the concentration of BIX-01294 added to the stem cell growth medium was 1 μM.

4. The method according to claim 1, characterized in that, The stem cell growth medium described in steps (2) and (3) also contains fetal bovine serum, antibiotics and gentamicin; preferably, the stem cell growth medium described in steps (2) and (3) also contains 10 v / v% fetal bovine serum, 1% v / v antibiotics and 25 μg / mL gentamicin.

5. The method according to any one of claims 1-4, characterized in that, The stem cell growth medium is CellGenix® GMP SCGM medium.

6. The method according to claim 1, characterized in that, Step (1) includes the following steps: (1-1) Transfer the umbilical cord blood to a culture bottle and let it stand; (1-2) Add HES to the umbilical cord blood, mix well, and let stand to allow red blood cells to settle; (1-3) Transfer the supernatant after the red blood cells have settled in step (1-2) to a centrifuge tube pre-filled with lymphocyte separation medium; (1-4) Centrifugation; (1-5) Absorb the mononuclear cell layer and wash; (1-6) Sorting CD34 + cell.

7. The method according to claim 6, characterized in that, In steps (1-3), the volume ratio of umbilical cord blood to HES is 5:1; in steps (1-3), the volume ratio of supernatant to lymphocyte separation fluid is 2:

1.

8. NK cells prepared using the method according to any one of claims 1-7.

9. The use of NK cells according to claim 8 in the preparation of drugs for treating cancer.

10. The application according to claim 9, wherein the cancer is leukemia, preferably chronic myeloid leukemia.

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