Functionalized culture medium, culture medium composition and culture method for NK (Natural Killer) cell amplification

Abstract

The invention discloses a functionalized culture medium, a culture medium composition and a culture method for NK cell amplification, and belongs to the field of biological medicine. Aiming at the problem that the activity and the purity of the NK cells cannot be considered at the same time in the existing NK cell in-vitro amplification, the invention provides a functionalized culture medium for NK cell amplification, and the functionalized culture medium comprises a proliferation culture medium and an activation adaptation culture medium; the proliferation culture medium and the activated adaptive culture medium both comprise IL-10, and the concentration of the IL-10 in the proliferation culture medium is lower than that of the IL-10 in the activated adaptive culture medium. IL-10 is added into a proliferation culture medium and an activation adaptation culture medium, the concentration of IL-10 in the two culture mediums is different, and the synergistic effect of high purity (larger than 95%) and high activity (the killing rate is larger than 75%) is achieved by inhibiting T cell proliferation and enhancing the anti-tumor microenvironment capacity of NK cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and more specifically, relates to a functionalized culture medium, a culture medium combination and a culture method for NK cell expansion. Background Art

[0002] Natural Killer cells (NK cells), as key members of the innate immune system, play an irreplaceable role in the body's immune defense and immune surveillance. They are particularly valuable in tumor immunotherapy. Compared to traditional tumor treatments (such as surgery, chemotherapy, and radiotherapy), NK cell therapy offers unique advantages: it can specifically identify and kill tumor cells while causing less damage to normal tissue cells. Furthermore, in certain specific cases, NK cell therapy can overcome the drug resistance of tumor cells, bringing new hope to cancer patients who are insensitive to traditional treatments. NK cells also perform exceptionally well in the treatment of viral infections. For example, in diseases such as chronic hepatitis B, hepatitis C, and AIDS, NK cells can accurately identify and eliminate virus-infected cells, effectively inhibiting viral replication and spread, thereby playing a positive role in controlling viral infection and disease progression.

[0003] However, when applying NK cells to tumor immunotherapy, the in vitro expansion of NK cells faces problems such as low expansion multiples (<50 times with traditional methods), activity attenuation (decreased CD16 / NKG2D expression), and T cell contamination (purity <70%), resulting in unsatisfactory expansion efficiency, cell activity, and NK cell purity. Existing technologies use feeder cells (such as K562) with a tumorigenic risk, while single cytokines (such as IL-2) can easily lead to cell exhaustion, and do not effectively address the interference of immunosuppressive factors (such as TGF-β) in the tumor microenvironment.

[0004] Corresponding improvements have also been made to address the above-mentioned problems, such as the Chinese patent publication number CN119552816A, published on March 4, 2025, which discloses a serum-free culture system and culture method for NK cells. The present invention first discloses a serum-free culture system for NK cells, including a coating solution, an expansion culture medium, an activation culture medium, and autologous plasma; and further discloses a method for culturing NK cells using the above-mentioned serum-free culture system. The method for culturing NK cells using the serum-free culture system of the present invention reduces the cost of NK cell culture, and can simply, quickly, efficiently, and safely obtain a large number of NK cells with high activity, high purity, and strong killing ability in vitro, providing a better source of cells for the clinical application of NK cells and promoting the further development of NK cell therapy technology. The shortcomings of this patent are: the component concentration design is too broad, and there may be problems of over-activation and T cell contamination; and there are uncontrollable risks in autologous plasma.

[0005] Another example is Chinese patent application number CN119286781A, published on January 10, 2025. This patent discloses a large-scale culture method for NK cells and the culture medium used, which belongs to the field of NK cell culture technology. Fetal bovine serum, penicillin, streptomycin, and magnetic microspheres that stimulate amplification are added to the ACTM culture medium, and the mixture is stirred and mixed evenly to prepare a culture medium. The present invention provides a large-scale culture method for NK cells, which can efficiently activate and amplify NK cells, does not require the use of exogenous serum or autologous serum, is safe and effective, can better promote the proliferation of NK cells, prolong their vitality, and is safe and has no side effects, and has broad application prospects. The disadvantage of this patent is that there is a risk of animal-derived ingredients. Summary of the Invention

[0006] 1. Problems to be solved

[0007] To address the problem that existing NK cell in vitro expansion cannot balance NK cell activity and purity, the present invention provides a functionalized culture medium, culture medium combination, and culture method for NK cell expansion. By adding IL-10 to the proliferation medium and activation adaptation medium at different concentrations, IL-10 is used to inhibit T cell proliferation and enhance the anti-tumor microenvironment ability of NK cells to achieve a synergistic effect of high purity (>95%) and high activity (killing rate >75%).

[0008] 2. Technical solution

[0009] To solve the above problems, the present invention adopts the following technical solutions.

[0010] A functionalized culture medium for NK cell expansion, comprising a proliferation culture medium and an activation adaptation culture medium; both the proliferation culture medium and the activation adaptation culture medium comprise IL-10, and the concentration of IL-10 in the proliferation culture medium is lower than that in the activation adaptation culture medium.

[0011] Furthermore, the proliferation medium components include basal medium and IL-10, IL-15, IL-12, and IL-18;

[0012] The activation adaptation medium components include basal medium and metabolic support agent, IL-10, short form IL-18 and purification reagents;

[0013] The basic culture medium includes recombinant human serum albumin and a mixed solution containing RPMI-1640, GT-T551 and X-VIVO.

[0014] Furthermore, the concentration of IL-10 in the proliferation medium is 5-8 ng / mL, the concentration of IL-15 is 8-12 ng / mL, the concentration of IL-12 is 10-15 ng / mL, and the concentration of IL-18 is 8-12 ng / mL;

[0015] The concentration of IL-10 in the activation adaptation medium is 8-12 ng / mL, and the concentration of the short form IL-18 is 5-10 ng / mL;

[0016] The volume ratio of RPMI-1640, GT-T551 and X-VIVO in the mixed solution of RPMI-1640, GT-T551 and X-VIVO in the basic culture medium is 1:1.5:2.

[0017] A culture medium combination for NK cell expansion, comprising an induction culture medium and a functionalized culture medium for NK cell expansion as described in any of the above items; the induction culture medium components include a basal culture medium and an activator, the activator being allicin and paclitaxel; the basal culture medium includes recombinant human serum albumin and a mixture containing RPMI-1640, GT-T551 and X-VIVO.

[0018] Furthermore, the induction culture medium also includes IL-10 and a natural extract, and the natural extract is Lentinan.

[0019] Furthermore, the concentration of IL-10 in the induction culture medium is 5-10 ng / mL, and the concentration of Lentinan is 60-70 μg / mL.

[0020] A method for culturing NK cells, comprising the steps of performing induction culture, proliferation culture, and activation application culture on peripheral blood mononuclear cells or umbilical cord blood mononuclear cells separated from peripheral blood or umbilical cord blood using a culture medium combination for NK cell expansion as described in any one of the above items, wherein the separated peripheral blood mononuclear cells or umbilical cord blood mononuclear cells are contacted with the induction culture medium on the first day.

[0021] Furthermore, the following steps are included:

[0022] S1: Isolation of peripheral blood mononuclear cells or umbilical cord blood mononuclear cells from peripheral blood or umbilical cord blood;

[0023] S2: placing peripheral blood mononuclear cells or umbilical cord blood mononuclear cells in an induction medium for a fixed induction culture period;

[0024] S3: After induction culture for a fixed time, proliferation medium is added and culture is continued. Proliferation medium is added at specific intervals to maintain cytokine activity and proliferation culture is continued for a fixed time.

[0025] S4: After proliferation culture for a fixed time, add activation adaptation medium and continue culture for a fixed time, then collect the cells.

[0026] Furthermore, the following steps are specifically included:

[0027] S1: Isolation of peripheral blood mononuclear cells or umbilical cord blood mononuclear cells from peripheral blood or umbilical cord blood;

[0028] S2: Place peripheral blood mononuclear cells or umbilical cord blood mononuclear cells in induction medium at a density of 4 × 10 cells / mL and incubate in a 37°C, 5% CO incubator for 2–4 days.

[0029] S3: After 2-4 days of induction culture, add proliferation medium and continue culture at a density of 1×10 6 cells / mL, and proliferation medium was added every 2 days to maintain cytokine activity, and proliferation culture was carried out for 6 to 8 days;

[0030] S4: After 6-8 days of proliferation culture, add activation adaptation medium and continue culturing at a density of 2 × 10 cells / mL. Culture continuously for 2-4 days, and then collect the cells.

[0031] 3. Beneficial effects

[0032] (1) The present invention adds IL-10 to the proliferation medium and the activation adaptation medium. The IL-10 in the proliferation medium mainly inhibits T cell proliferation, reduces the expansion of residual T cells in the culture system, and then inhibits T cell contamination to ensure the purity of NK cells; the IL-10 in the activation adaptation medium mainly further inhibits the activity of residual T cells and further improves the purity of NK cells; at the same time, there are differences in the concentrations of IL-10 in the two culture media. Low-concentration IL-10 inhibits the proliferation of residual T cells through the STAT3 pathway and maintains the mitochondrial metabolic homeostasis of NK cells; high-concentration IL-10 resists the inhibitory effect of TGF-β in the tumor microenvironment by downregulating the expression of PD-1 on the surface of NK cells, thereby enhancing the killing activity; together, they achieve the synergistic effect of high purity (>95%) and high activity (killing rate>75%) by inhibiting T cell proliferation and enhancing the anti-tumor microenvironment ability of NK cells, and ultimately achieve both cell activity and NK cell purity during NK cell amplification;

[0033] (2) The proliferation culture medium of the present invention changes the traditional method of relying on a single cytokine such as IL-2 to achieve proliferation, so there is no problem that a single cytokine easily leads to cell exhaustion; and IL-10 and IL-15 are combined in the proliferation culture medium to achieve efficient purification by inhibiting T cell proliferation (residual <0.5%) and promoting NK cell expansion (multiplier >100 times); high concentrations of IL-10 and short forms of IL-18 are used in the activation adaptation culture medium, and the STAT1 / STAT3 dual pathway is activated to increase the secretion of IFN-γ by 2 times, thereby achieving an improvement in killing ability;

[0034] (4) The synergy of the natural extract and IL-10 in the induction culture medium of the culture method of the present invention activates the early signal of NK cells through the TLR4 / MyD88 pathway; the synergy of IL-10 and IL-15 in the proliferation culture medium achieves efficient purification by inhibiting T cell proliferation (residual <0.5%) and promoting NK cell expansion (multiples >100 times); the synergy of IL-10 and short form IL-18 in the activation adaptation culture medium activates the STAT1 / STAT3 dual pathway, thereby increasing the secretion of IFN-γ by about 2 times; the final NK cell product is free of T cell contamination (CD3+ <0.1%), avoiding the risk of graft-versus-host disease (GVHD); in the PD-L1+ tumor microenvironment, the killing activity is maintained at >70%, while the traditional method is only <40%; the viability after cryopreservation and resuscitation is >90%. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of cell killing results;

[0036] Figure 2 Schematic diagram of cell expansion results;

[0037] Figure 3 Schematic diagram of cell purity results. DETAILED DESCRIPTION

[0038] The present invention is further described below with reference to specific embodiments and accompanying drawings.

[0039] In this application, the sample source is frozen peripheral blood PBMC sample (total cell count 1×10, CD34+ ratio 1.2%); required reagents: thawing solution: PBS containing 10% human AB serum + 0.1% glucose; magnetic bead sorting reagents: CD3 MicroBeads (Miltenyi Biotec, cat. no. 130-050-101), CD19 MicroBeads (cat. no. 130-050-301); all recombinant proteins in all culture media were purchased from PeproTech; unless otherwise specified, the reagents, methods, and equipment used in this application are conventional reagents, methods, and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0040] Example 1

[0041] A method for culturing NK cells, comprising the following steps:

[0042] S1: Isolation of peripheral blood mononuclear cells from peripheral blood; specifically, including:

[0043] Thawing and pretreatment of PBMCs: Quickly place the cryovial in a 37°C water bath and gently shake until completely thawed (<2 minutes). Immediately add 10 mL of pre-chilled thawing buffer and centrifuge (300 × g, 10 minutes, 4°C). Discard the supernatant, resuspend in PBS containing 2% HSA, pass through a 70 μm cell sieve, and count to adjust the density to 5 × 10 cells / mL.

[0044] Magnetic bead sorting (double-negative T / B cell depletion): Add 10 μL of CD3 / CD19 magnetic beads per 1 × 10 cells and incubate at 4°C for 15 minutes. Add 5 mL of PBS + 2% HSA and centrifuge (300 × g, 5 minutes). Collect the negative fraction (CD3-CD19- cells). Purity is >98%.

[0045] S2: Place peripheral blood mononuclear cells in induction medium for induction culture on Days 0-3; specifically, including:

[0046] The sorted cells were seeded at a density of 4 × 10 cells / mL into a T75 culture flask containing induction medium (static culture) at 37°C, 5% CO, and gently shaken three times daily (30 rpm, 2 minutes each time). On the second day, 50% of the induction medium was replaced with fresh induction medium. The induction medium consisted of a mixture of RPMI-1640 / GT-T551 / X-VIVO at a volume ratio of 1:1.5:2; recombinant human serum albumin (rHSA): 250 μg / mL (allicin (35 μg / mL), low-dose paclitaxel (28 μg / mL), lentinan (65 μg / mL), and IL-10 (5 ng / mL).

[0047] S3: After 3 days of induction culture, proliferation medium was added and cultured continuously at an initial density of 1×10 cells / mL in a volume of 500 mL. IL-15 (5 ng / mL), IL-10 (2 ng / mL), and 5% fresh proliferation medium were added every 48 hours to maintain cell viability. The cells were cultured for fixed periods of time from Day 4 to Day 10. The proliferation medium included a mixture of RPMI-1640 / GT-T551 / X-VIVO at a volume ratio of 1:1.5:2; recombinant human serum albumin (rHSA): 250 μg / mL (instead of BSA, without animal-derived risk); IL-12 (12 ng / mL), IL-15 (10 ng / mL), IL-18 (10 ng / mL), IL-21 (10 ng / mL), and IL-10: 8 ng / mL.

[0048] S4: After 7 days of proliferation culture, activated adaptation medium was added and cultured continuously at an initial density of 2×10 cells / mL. Culture was continued for 72 hours. On the 14th day, cells were collected and washed three times with PBS + 1% HSA to a final volume of 50 mL. The activation adaptation medium includes a mixture of RPMI-1640 / GT-T551 / X-VIVO: volume ratio of 1:1.5:2; recombinant human serum albumin (rHSA): 250 μg / mL (instead of BSA, no animal source risk); insulin (3 μg / mL), transferrin (3 μg / mL), glutamine (2 mM); IL-10 (10 ng / mL), short form IL-8 (6 ng / mL); purification reagent: CD3 / CD19 magnetic bead double negative sorting: remove T / B cells, purity >98%, the short form IL-8 refers to an IL-18 variant (e.g., amino acids 1-157) with the C-terminal domain truncated by genetic engineering means, with a molecular weight of 18 kDa (full-length IL-18 is 24 kDa); in this embodiment, short form IL-8 is used for culture, which has a stronger STAT1 pathway activation ability than IL-8, and its half-life is shortened to 4-6 hours, which is suitable for short-term stimulation to avoid cell exhaustion.

[0049] In this example, IL-10 is added to the proliferation medium and the activation adaptation medium. The IL-10 in the proliferation medium mainly inhibits T cell proliferation, reduces the expansion of residual T cells in the culture system, and then inhibits T cell contamination to ensure the purity of NK cells; the IL-10 in the activation adaptation medium mainly further inhibits the activity of residual T cells and further improves the purity of NK cells; at the same time, there are differences in the concentrations of IL-10 in the two culture media. Low-concentration IL-10 inhibits the proliferation of residual T cells through the STAT3 pathway, while maintaining the mitochondrial metabolic homeostasis of NK cells; high-concentration IL-10 resists the inhibitory effect of TGF-β in the tumor microenvironment by downregulating PD-1 expression on the NK cell surface, thereby enhancing the killing activity; together, they achieve the synergistic effect of achieving high purity (>95%) and high activity (killing rate>75%) by inhibiting T cell proliferation and enhancing the anti-tumor microenvironment ability of NK cells, ultimately achieving both cell activity and NK cell purity during NK cell amplification.

[0050] In order to illustrate the effect more intuitively, NK cells cultured with the culture medium combination described in this application, such as Figures 1 to 3 As shown, Figure 1 Schematic diagram of cell killing results; Figure 2 Schematic diagram of cell expansion results; Figure 3 Schematic diagram of cell purity structure; Donor1, Donor2 and Donor3 in the figure are all PBMCs derived from frozen peripheral blood;

[0051] Killing result test plan: collect the expanded NK cells and co-incubate them with K562-luc cells at the ratio of 10:1 and 5:1. After 4 hours, collect the cell pellet and use the firefly luciferase reporter gene detection kit to calculate the killing effect. Figure 1 shown.

[0052] Amplification result test: count every 2-3 days during the amplification process and calculate the amplification multiple, such as Figure 2 shown.

[0053] Purity test: During the expansion process, CD3 / CD56 antibody staining was performed on DAY7, DAY10, and DAY14, and the ratio of CD3-CD56+ cells was detected by flow cytometry to determine the purity of NK cells. Figure 3 shown.

[0054] pass Figures 1 to 3 It can be clearly seen that the use of the culture medium combination of the present application for in vitro culture of NK cells has strong killing ability, fast expansion speed and high cell purity.

[0055] Comparative Example 1

[0056] The other steps were the same as in Example 1, except that the induction medium in step S2 did not contain lentinan. The test results were as follows: T cell contamination increased significantly: the proportion of residual CD3+ cells was >5% (compared to <0.5% in the group containing lentinan); and the expansion factor was significantly reduced: the expansion factor was only 100-fold (compared to 1000-fold in the group containing lentinan).

[0057] Comparative Example 2

[0058] The other steps were the same as in Example 1, except that IL-10 was not included in the proliferation medium in step S3. The test results were as follows: T cell contamination was significantly increased: the proportion of residual CD3+ cells was >10% (compared to <0.5% in the IL-10 group); functional inhibition sensitivity was increased: in a tumor microenvironment containing TGF-β (10 ng / mL), NK cell killing activity decreased to 40% (compared to 75% in the IL-10 group); metabolic homeostasis was unbalanced: the cell apoptosis rate increased to 15% (compared to <5% in the IL-10 group).

[0059] Comparative Example 3

[0060] The other steps were the same as in Example 1, except that the IL-10 concentration in the proliferation medium in step S3 was kept consistent with the IL-10 concentration in the activation and adaptation medium in step S4. The test results were as follows: CIK cell contamination increased significantly: the proportion of residual CD3+CD56+ cells was >20% (compared to <5% in groups with different concentrations), and cell viability decreased to 70% (compared to >90% in groups with different concentrations).

[0061] Comparative Example 4

[0062] The remaining steps were the same as in Example 1, except that the IL-10 concentration in the proliferation medium in step S3 was higher than the IL-10 concentration in the activation adaptation medium in step S4. The test results were as follows: T cell contamination increased significantly: the proportion of residual CD3+ cells was >15% (compared to <0.5% in the groups with different concentrations), and NK cell cytotoxicity decreased to 40% (compared to 75% maintained in the group with 2 ng / mL IL10 in S3).

[0063] The examples described in the present invention are merely descriptions of the preferred embodiments of the present invention and are not intended to limit the concept and scope of the present invention. Without departing from the design concept of the present invention, various modifications and improvements made to the technical solutions of the present invention by engineers and technicians in this field should fall within the scope of protection of the present invention.

Claims

1. A functionalized culture medium for NK cell expansion, comprising a proliferation culture medium and an activation adaptation culture medium; characterized in that: Both the proliferation medium and the activation adaptation medium include IL-10, and the concentration of IL-10 in the proliferation medium is lower than the concentration of IL-10 in the activation adaptation medium.

2. A functionalized culture medium for NK cell expansion according to claim 1, characterized in that: The proliferation culture medium components include basal culture medium and IL-10, IL-15, IL-12, and IL-18; The activation adaptation medium components include basal medium and metabolic support agent, IL-10, and short form IL-18; The basic culture medium includes recombinant human serum albumin and a mixed solution containing RPMI-1640, GT-T551 and X-VIVO.

3. A functionalized culture medium for NK cell expansion according to claim 2, characterized in that: The concentration of IL-10 in the proliferation medium is 5-8 ng / mL, the concentration of IL-15 is 8-12 ng / mL, the concentration of IL-12 is 10-15 ng / mL, and the concentration of IL-18 is 8-12 ng / mL; The concentration of IL-10 in the activation adaptation medium is 8-12 ng / mL, and the concentration of the short form IL-18 is 5-10 ng / mL; The volume ratio of RPMI-1640, GT-T551 and X-VIVO in the mixture of RPMI-1640, GT-T551 and X-VIVO in the basic culture medium is 1:1.5:

2.

4. A culture medium combination for NK cell expansion, characterized in that: It comprises an induction medium and a functional medium for NK cell expansion according to any one of claims 1 to 3; the induction medium components include a basal medium and an activator, and the activator is allicin and paclitaxel; the basal medium includes recombinant human serum albumin, and a mixture containing RPMI-1640, GT-T551 and X-VIVO.

5. A culture medium combination for NK cell expansion according to claim 4, characterized in that: The induction culture medium also includes IL-10 and natural extracts, and the natural extracts are lentinan.

6. A culture medium combination for NK cell expansion according to claim 5, characterized in that: The concentration of IL-10 in the induction culture medium is 5-10 ng / mL, and the concentration of lentinan is 60-70 μg / mL.

7. A method for culturing NK cells, characterized in that: The method comprises the steps of performing induction culture, proliferation culture and activation application culture on peripheral blood mononuclear cells or umbilical cord blood mononuclear cells separated from peripheral blood or umbilical cord blood using the culture medium combination for NK cell expansion according to any one of claims 4 to 6, wherein the separated peripheral blood mononuclear cells or umbilical cord blood mononuclear cells are contacted with the induction culture medium on the first day.

8. The method for culturing NK cells according to claim 7, wherein: The following steps are involved: S1: Isolation of peripheral blood mononuclear cells or umbilical cord blood mononuclear cells from peripheral blood or umbilical cord blood; S2: placing peripheral blood mononuclear cells or umbilical cord blood mononuclear cells in an induction medium for a fixed induction culture period; S3: After induction culture for a fixed time, proliferation medium is added and culture is continued. Proliferation medium is added at specific intervals to maintain cytokine activity and proliferation culture is continued for a fixed time. S4: After proliferation culture for a fixed time, add activation adaptation medium and continue culture for a fixed time, then collect the cells.

9. The method for culturing NK cells according to claim 7 or 8, wherein: The specific steps include: S1: Isolation of peripheral blood mononuclear cells or umbilical cord blood mononuclear cells from peripheral blood or umbilical cord blood; S2: Place peripheral blood mononuclear cells or umbilical cord blood mononuclear cells in induction medium at a density of 4 × 10 cells / mL and incubate in a 37°C, 5% CO incubator for 2–4 days. S3: After 2-4 days of induction culture, add proliferation medium and continue culture at a density of 1×10 6 cells / mL, and proliferation medium was added every 2 days to maintain cytokine activity, and proliferation culture was carried out for 6 to 8 days; S4: After 6-8 days of proliferation culture, add activation adaptation medium and continue culturing at a density of 2 × 10 cells / mL. Culture continuously for 2-4 days, and then collect the cells.

Images