A culture medium for NK cells and a preparation method thereof

Abstract

The present invention relates to the biological field, and in particular to a culture medium and preparation method for NK cells. The culture medium comprises the following components, based on the total volume of the culture medium: 85-98% by volume of serum-free lymphocyte culture medium, 2-15% by volume of an immune cell serum replacement, and a final concentration of 100-500 IU / ml of interleukin. This culture medium formulation, combined with a transduction-promoting reagent, significantly improves the efficiency of lentiviral infection of NK cells, resulting in high amplification rates, high NK cell purity, and a significant increase in CAR-positive rates, significantly reducing costs. The method is simple, efficient, and conducive to large-scale production.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a culture medium for NK cells and a preparation method thereof. Background Art

[0002] NK cells are a type of lymphocyte that has a potent and MHC-independent cytotoxic effect on tumor cells. Their recognition of tumor cells primarily relies on the cross-regulation of activating and inhibitory receptors on their surface. After recognizing tumor cells, NK cells kill them through various pathways, including the release of the cytotoxic mediators perforin and granzyme, which induce apoptosis of target cells, the expression of membrane TNF family molecules, and antibody-dependent cytotoxicity. Modifying NK cells with CARs is expected to enhance their ability to target and kill tumor cells and develop effector cells with potent anti-tumor effects.

[0003] The most commonly used method on the market for stably integrating CAR genes into cells is the lentiviral / retroviral method. Currently, the methods for integrating CAR genes into the T cell genome in CAR T cell drugs that have been marketed both domestically and internationally all use the lentiviral / retroviral method. However, the infection efficiency of lentivirus / retrovirus on NK cells is very low. Summary of the Invention

[0004] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a culture medium and a preparation method for NK cells to solve the problems in the prior art.

[0005] To achieve the above-mentioned and other related purposes, the present invention provides a complete culture medium for culturing NK cells. The complete culture medium comprises the following components, based on the total volume of the complete culture medium: a lymphocyte serum-free culture medium with a volume fraction of 85-98%, an immune cell serum replacement (SR) with a volume fraction of 2-15%, and IL-2 with a final concentration of 180-220 IU / ml.

[0006] The present invention also provides use of the complete culture medium in culturing NK cells or preparing a lentiviral or retroviral transduction reagent.

[0007] The present invention also provides a method for culturing NK cells, wherein the preparation method comprises culturing NK cells using the complete culture medium.

[0008] The present invention also provides a method for transducing NK cells with a lentivirus or retrovirus, the method comprising the following steps: adding a lentivirus or retrovirus to the NK cells obtained by the aforementioned culture method, adding a transduction-promoting reagent, and obtaining NK cells transduced with the lentivirus or retrovirus after culture.

[0009] As described above, the culture medium and preparation method of NK cells of the present invention have the following beneficial effects: the culture medium formula combined with the transduction-promoting reagent greatly improves the efficiency of lentiviral infection of NK cells. Flow cytometer detection shows that compared with the culture medium formula with a lower CAR positivity rate, the CAR positivity rate of the highest culture medium formula is increased by 3-4 times, the amplification multiple is high, the NK cell purity is high, and the CAR positivity rate is significantly increased, which greatly reduces the cost. The method is simple, efficient, and conducive to large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Shown is a graph of the expansion fold during the NK cell culture process of the present invention.

[0011] Figure 2 Shown is a graph of the lentiviral transduction efficiency of the present invention.

[0012] Figure 3 Shown is a graph of NK cell killing efficiency of the present invention.

[0013] Figure 4 Shown are the results of perforin secretion detection after co-culture of the CAR-NK cells of the present invention with K562 cells. DETAILED DESCRIPTION

[0014] The present invention provides a complete culture medium for culturing NK cells. The complete culture medium comprises the following components, based on the total volume of the complete culture medium: a lymphocyte serum-free culture medium with a volume fraction of 85-98%, an immune cell serum replacement (SR) with a volume fraction of 2-15%, and an interleukin with a final concentration of 50-500 IU / ml.

[0015] In certain embodiments of the present invention, the volume fraction of the lymphocyte serum-free culture medium is selected from any of the following ranges: 85-87%, 87-89%, 89-91%, 91-93%, 93-95%, and 95-98%.

[0016] In certain embodiments of the present invention, the serum-free lymphocyte culture medium is HIPP-T009 serum-free lymphocyte culture medium with product number FG0103801 or RG0101302 produced by Shanghai Bio-Tech Biotechnology Co., Ltd.

[0017] In certain embodiments of the present invention, the volume fraction of the immune cell serum substitute is selected from any of the following ranges: 2-5%, 5-7%, 7-9%, 9-11%, 11-13%, and 13-15%.

[0018] In certain embodiments of the present invention, the brand and product number of the immune cell serum replacement are Gibco and A2596101, respectively.

[0019] In certain embodiments of the present invention, the interleukin is selected from any one or more combinations of IL-2, IL-15, IL-18, and IL-21.

[0020] In certain embodiments of the present invention, the interleukin is selected from IL-2 with a final concentration of 180-220 IU / ml.

[0021] In certain embodiments of the present invention, the final concentration of IL-2 is selected from any of the following ranges: 100-150 IU / ml, 150-180 IU / ml, 180-190 IU / ml, 190-200 IU / ml, 200-210 IU / ml, 210-220 IU / ml, 220-300 IU / ml, 300-400 IU / ml, 400-500 IU / ml.

[0022] In certain embodiments of the present invention, the complete culture medium is selected from any one of the following formulas:

[0023] 1) 95% serum-free lymphocyte culture medium, 5% serum replacement, and IL-2 at a final concentration of 200 IU / ml;

[0024] 2) 95% lymphocyte serum-free medium, 5% serum replacement, and 400 IU / ml final concentration of IL-2;

[0025] 3) 98% lymphocyte serum-free medium, 2% serum replacement, and 200 IU / ml final concentration of IL-2;

[0026] 4) 98% lymphocyte serum-free medium, 2% serum replacement, and 400 IU / ml final concentration of IL-2.

[0027] The present invention also provides use of the complete culture medium in culturing NK cells or preparing a lentiviral or retroviral transduction reagent.

[0028] The lentiviral or retroviral transduction reagent is a reagent used in the process of transducing lentivirus or retrovirus into NK cells to improve the transduction efficiency of the lentivirus or retrovirus.

[0029] The present invention also provides a method for culturing NK cells, wherein the preparation method comprises culturing NK cells using the complete culture medium.

[0030] The NK cells are commercial NK cells or NK cells obtained by sorting from PBMC.

[0031] In certain embodiments of the present invention, before using the complete medium to culture NK cells, the method further comprises co-culturing IL21-41BBL-K562 feeder cells with the NK cells to activate the NK cells.

[0032] The number of IL21-41BBL-K562 trophoblasts is in excess relative to the number of NK cells. In certain embodiments of the present invention, the number of IL21-41BBL-K562 trophoblasts is 1.5 times or more, preferably 2 times or more, for example 2 times, 2.5 times or 3 times or more, the number of NK cells.

[0033] In certain embodiments of the present invention, when NK cells are cultured using the complete culture medium, the initial cell concentration of NK cells is 1-3×10^6 cells / ml, preferably, 1.25-2.5×10^6 cells / ml.

[0034] In certain embodiments of the present invention, the NK cells are cultured using the complete medium for 5 to 8 days.

[0035] In certain embodiments of the present invention, during the process of culturing NK cells using the complete culture medium, complete culture medium is added every 2 to 3 days until the NK cell density is 5×10^5 cells / ml to 1×10^6 cells / ml.

[0036] In certain embodiments of the present invention, the culture conditions are 36-38° C. and 4.5-5.5% CO 2 .

[0037] The present invention also provides a method for transducing NK cells with a lentivirus or retrovirus, the method comprising the following steps: adding a lentivirus or retrovirus to the NK cells obtained by the aforementioned culture method, adding a transduction-promoting reagent, and obtaining NK cells transduced with the lentivirus or retrovirus after culture.

[0038] In certain embodiments of the present invention, the lentivirus or retrovirus and its transduction concentration are not specifically limited in the present invention, and those skilled in the art can determine it based on actual needs or experience.

[0039] The transduction-promoting agent may be a conventional viral infection enhancer in the prior art, such as the agent with the miltenyi product number 130-111-163.

[0040] The final concentration of the transduction-promoting reagent is 5-10 μg / ml.

[0041] In one embodiment, the cells are cultured for 7 to 15 days after transduction.

[0042] In one embodiment, complete medium can be added during transduction, and replenished every 2-3 days.

[0043] The present invention also provides cells prepared by the method, wherein the polynucleotide of the lentivirus or retrovirus is integrated into the genome of the cells.

[0044] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0045] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0046] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention. Example 1

[0047] NK cell expansion and lentiviral transduction are performed using cryopreserved PBMCs (peripheral blood mononuclear cells) from healthy donors. The steps involved are PBMC and IL21-41BBL-K562 trophoblast recovery, NK cell sorting, trophoblast addition, lentiviral transduction, and expansion culture. The details are as follows:

[0048] 1) Thawing of PBMC and IL21-41BBL-K562 Tropoblast Cells: Cryopreserved PBMC and IL21-41BBL-K562 trophoblast cells (Jiangsu Puxin Biopharmaceutical Co., Ltd., HG-FEC001-RG) from healthy donors were thawed in a 37°C water bath. PBMCs were then washed with 5-10 volumes of preheated sorting buffer (Miltenyi Biotec BV&Co. KG: 200-070-025) and centrifuged at 400 g for 10 min. The supernatant was discarded and the cells were counted.

[0049] 2) Cell sorting: After cell counting, add 20-30 ml of sorting buffer and mix thoroughly. Centrifuge at 400 g for 10 min. Add 80 μl of sorting buffer per 1E7 total cells to the cell pellet based on the PBMC cell number. Add CD3 sorting magnetic beads at 80 μl per 1E7 total cells and incubate at 4°C for 15-20 min. After incubation, add 20-30 ml of sorting buffer and centrifuge at 300 g for 15 min. Resuspend the pellet in 500 μl of sorting buffer and sort the cells using a sorting column. The liquid retained from the column is the CD3- cells.

[0050] 3) Addition of IL21-41BBL-K562 feeder blasts: Add IL21-41BBL-K562 feeder blasts at a ratio of 1:2 CD3+ cells:IL21-41BBL-K562 feeder blasts. Supplement medium until the CD3+ cell density reaches 1.25-2.5E6 cells / ml. Feeder blasts do not need to be isolated later. At the time of transduction, their viability is already very low or their proportion is minimal, so they will not affect subsequent experimental procedures.

[0051] 4) Cell Expansion: Cells were cultured for 10–14 days in complete medium containing the following different medium formulations. SR is a serum replacement (Gibco, CTS, Immune cell SR: A2596101). Cytokines include one or more combinations of IL-2 (100–500 IU / ml) and IL-15 (50–150 IU / ml):

[0052] Formulation 1: HIPP-T009 serum-free medium (Bei Anji, FG0103801) + SR (final concentration 5%) + IL-2 (final concentration 200 IU / ml);

[0053] Formulation 2: HIPP-T009 serum-free medium + SR (final concentration 5%) + IL-2 (final concentration 400 IU / ml);

[0054] Formulation 3: HIPP-T009 serum-free medium + SR (final concentration 2%) + IL-2 (final concentration 200 IU / ml);

[0055] Formulation 4: HIPP-T009 serum-free medium + SR (final concentration 2%) + IL-2 (final concentration 400 IU / ml);

[0056] Formulation 5: CTS NK-Xpander Medium (Gibco, A5019001) + SR (final concentration 5%) + IL-2 (final concentration 200 IU / ml);

[0057] Formulation 6: CTS NK-Xpander Medium (Gibco, A5019001) + SR (final concentration 5%) + IL-2 (final concentration 400 IU / ml);

[0058] Formulation 7: CTS NK-Xpander Medium (Gibco, A5019001) + SR (final concentration 2%) + IL-2 (final concentration 200 IU / ml);

[0059] Formulation 8: CTS NK-Xpander Medium (Gibco, A5019001) + SR (final concentration 2%) + IL-2 (final concentration 400 IU / ml);

[0060] Formulation 9: NK MACS CD Medium (Miltenyi Biotec, 130-114-429) + SR (final concentration 5%) + IL-2 (200 IU / ml);

[0061] Formulation 10: NK MACS CD Medium (Miltenyi Biotec, 130-114-429) + SR (final concentration 5%) + IL-2 (400 IU / ml);

[0062] Formulation 11: NK MACS CD Medium (Miltenyi Biotec, 130-114-429) + SR (final concentration 2%) + IL-2 (200 IU / ml);

[0063] Formulation 12: NK MACS CD Medium (Miltenyi Biotec, 130-114-429) + SR (final concentration 2%) + IL-2 (400 IU / ml);

[0064] Formulation 13: NK MACS CD Medium (Miltenyi Biotec, 130-114-429) + SR (final concentration 2%) + IL-2 (400 IU / ml) + IL-15 (5 ng / ml);

[0065] During this period, complete culture medium was added every 2-3 days to 5E5 cells / ml-1E6 cells / ml according to the cell density, and lentiviral transduction was performed on the 5th-8th day of culture.

[0066] 5) Lentiviral Transduction: NK cells cultured for 5-8 days were transduced with lentiviral vectors containing an anti-CD19 CAR. The sequence and preparation method of the lentiviral vector are identical to those in the domestic patent application CN201910625785.4. The appropriate amount of virus was added, along with Vectofusin-1 (5-10 μg / ml, manufacturer: Miltenyi, catalog number 130-111-163).

[0067] 6) Cell harvesting: NK cells were harvested after 14 days of culture. The expansion times were monitored during the culture process. The NK cell expansion data of each group of CAR-NK cells after 14 days of culture are as follows: Figure 1 As shown, the results show that the NK cell expansion fold using the medium of formula 2 according to the above operation is close to 10,000 times (9,622 times). The cells harvested after 14 days of culture were sent for flow cytometry to detect the NK cell transduction efficiency.

[0068] 7) Streaming results: Figure 2 As shown, the NK cell transduction efficiency was the highest when the medium of formula 2 was used according to the above operation, which could reach more than 40%. Compared with the medium formula with the lowest transduction efficiency (formulation 7), the transduction efficiency was increased by 3 to 4 times.

[0069] Example 2 Cell Killing Experiment

[0070] 1) Take 100 μl of target cell K562 cell suspension and group them into "target cell spontaneous release group" (i.e., a system in which target cells are cultured alone and only spontaneously released LDH is detected without lysis), "maximum release group" (i.e., a group in which all target cells cultured alone are lysed and their LDH release is detected), and "killing effector group" (i.e., a group in which target cells are co-incubated with effector cells). Add the cells to a 96-well round-bottom plate, with three parallel wells set up for each group.

[0071] 2) Take 100 μl of effector CAR-NK cells and mock NK cells respectively and add them to the corresponding wells of the "killing effector group" in a 96-well round-bottom plate. Set up 3 parallel wells for each group and mix them evenly. The effector-target ratio is 5:1. At the same time, set up an "effector cell spontaneous release group", that is, only effector cells are added, and no lysis is performed during the test. Only the spontaneous release of LDH is detected.

[0072] 3) Add 100 μl of culture medium to the "spontaneous release group", "maximum release group" and "killing effect group" respectively to make the final volume 200 μl.

[0073] 4) Incubate the culture plate in a 37 ± 1°C, 5 ± 0.5% CO2 incubator. After approximately 16 hours, perform a lysis assay on the "maximum release group" and measure the OD at a wavelength of 492 nm. Terminate the reaction and complete the assay within 10 minutes.

[0074] 5) Calculation formula:

[0075] Killing rate %=(killing effector group-target cell spontaneous release group-effector cell spontaneous release group) / (maximum release group-target cell spontaneous release group).

[0076] 6) Results

[0077] The killing efficiency of NK cells cultured with different culture formulas on K562 is as follows Figure 3 As shown in the results, the cell killing effects of formula 2, formula 3 and formula 4 were higher than those of the other groups. The final killing efficiencies measured by the LDH release method were 78.8%, 76.5% and 77.7%, respectively. It can be concluded that the CAR-NK cells cultured with formula 2, formula 3 and formula 4 have a strong killing effect on target cells.

[0078] Example 3 Detection of perforin secretion after co-culture of CAR-NK cells and K562 cells

[0079] 1) Take 100 μl of target K562 cell suspension and add it to a 96-well round-bottom plate, setting up three parallel wells.

[0080] 2) Add 100 μl of effector CAR-NK cells to the target cells in a 96-well round-bottom plate and mix thoroughly. The effector-target ratio is 5:1. This serves as the co-incubation group. Also, set up an "effector cell autoincubation group" where only effector cells are added. Set up three parallel wells for each group.

[0081] 3) Add 100 μl of culture medium to each group to make the final volume 200 μl.

[0082] 4) After incubation in a 37±1°C, 5±0.5% CO2 incubator for approximately 20 hours, the supernatant from the co-incubation group and the effector cell self-incubation group was collected for perforin factor secretion detection using a perforin detection kit.

[0083] The secretion amount of perforin in each group is obtained by subtracting the value of the self-incubation group from the value of the co-incubation group. The specific results are as follows Figure 4 As shown, it can be concluded that the CAR-NK cells cultured with Formula 2, Formula 3 and Formula 4 have a stronger ability to promote the release of perforin protein levels when co-incubated with target cells.

[0084] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods in the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications apparent to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. Use of the complete medium described below in culturing NK cells or in preparing NK cells transduced by lentivirus or retrovirus: based on the total volume of the complete medium, the complete medium comprises: 95% by volume of a serum-free lymphocyte medium, 5% by volume of an immune cell serum replacement, and IL-2 at a final concentration of 400 IU / ml; the serum-free lymphocyte medium is HIPP-T009 serum-free lymphocyte medium with a product number of FG0103801 produced by Shanghai Bio-Tech Biotechnology Co., Ltd.; the NK cells are CD3+ cells obtained by sorting PBMCs. - cell.

2. A method for culturing NK cells, characterized in that: The culture method comprises co-culturing IL21-41BBL-K562 trophoblast cells with NK cells to activate the NK cells, and then culturing the NK cells using the following complete medium, based on the total volume of the complete medium: a lymphocyte serum-free medium with a volume fraction of 95%, an immune cell serum replacement with a volume fraction of 5%, and IL-2 with a final concentration of 400 IU / ml. The lymphocyte serum-free medium is HIPP-T009 lymphocyte serum-free medium with a product number of FG0103801 produced by Shanghai Beianji Biotechnology Co., Ltd. The NK cells are CD3 - cell.

3. The culture method according to claim 2, wherein The cell number of the IL21-41BBL-K562 trophoblast cells is in excess relative to the cell number of NK cells.

4. The culture method according to claim 3, wherein Also includes any one or more of the following features: 1) The number of IL21-41BBL-K562 trophoblast cells is 1.5 to 2.5 times the number of NK cells; 2) When culturing NK cells using the complete medium, the initial cell concentration of NK cells is 1-3×10^6 cells / ml; 3) culturing NK cells using the complete medium for 5 to 8 days; 4) During the process of culturing NK cells using the complete medium, the complete medium was supplemented every 2 to 3 days until the NK cell density reached 5×10^5 cells / ml to 1×10^6 cells / ml; 5) Culture conditions are 36-38°C and 4.5-5.5% CO2.

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