NKT cell in-vitro amplification method based on combined stimulant

Abstract

The invention discloses an NKT cell in-vitro amplification method based on a combined stimulant, and belongs to the technical field of biology. The method comprises the following steps: firstly separating mononuclear cells in autologous peripheral blood, and then performing induced activation and amplification on the NKT cells in a serum-free culture system by adding a group A streptococcus preparation, a zoledronic acid preparation and a cytokine IL-2 as a combined stimulant. The method is easy to operate, efficient amplification of the NKT cells can be achieved only through three key reagents, the obtained NKT cells have the advantages of being large in number, high in purity, high in cytotoxicity and the like, and the requirements of clinical treatment can be met. Compared with a traditional method, the culture cost is remarkably reduced, the operation process is simplified, large-scale production, popularization and application are easy, and an efficient and economical technical scheme is provided for NKT cellular immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an in vitro expansion method of NKT cells based on a combined stimulator. Background Art

[0002] NKT cells (natural killer T cells) are a T cell subset with unique immunomodulatory functions. They play a vital role in anti-tumor, anti-infection, and immune regulation by secreting multiple cytokines and directly killing target cells. However, NKT cells are present in extremely low levels in peripheral blood, typically comprising only 0.01%-0.1% of peripheral blood mononuclear cells (PBMCs), significantly limiting their clinical application. To meet the clinical demands for both quantity and quality of NKT cells, in vitro expansion technology has become a key approach to obtaining large quantities of highly purified NKT cells.

[0003] Currently, the in vitro expansion of NKT cells mainly relies on the co-culture of multiple cytokines (such as IL-15, IL-18, etc.) and antigen-presenting cells (such as dendritic cells). However, these methods have the following limitations: (1) low expansion efficiency, making it difficult to obtain sufficient numbers of NKT cells; (2) complex operations, requiring multiple reagents and cell types, increasing culture costs and technical difficulties; (3) the purity and functional stability of the obtained NKT cells are insufficient, making it difficult to meet the requirements of clinical treatment.

[0004] To address these challenges, developing an efficient, economical, and easily scalable method for expanding NKT cells in vitro is of great significance. This invention optimizes the culture system and stimulatory agent combination, leveraging the synergistic effects of a group A Streptococcus preparation, zoledronic acid preparation, and IL-2 to achieve efficient expansion of NKT cells while ensuring their purity and cytotoxicity, providing reliable technical support for the clinical application of NKT cell immunotherapy. Summary of the Invention

[0005] To address the above technical problems, the present invention provides a method for in vitro expansion of NKT cells based on a combined stimulant. By optimizing the culture system and stimulant combination and utilizing the synergistic effects of a group A Streptococcus preparation, a zoledronic acid preparation, and IL-2, the present invention achieves efficient expansion of NKT cells while ensuring their purity and cytotoxicity, providing reliable technical support for the clinical application of NKT cell immunotherapy. This method is efficient, economical, and easy to promote.

[0006] To solve the above technical problems, the present invention provides the following technical solution: a method for in vitro expansion of NKT cells based on a combined stimulator, comprising the following steps: A. Mononuclear cells are extracted and isolated from adult peripheral blood; B. Resuspend the mononuclear cells in serum-free medium, add a group A streptococcus preparation, a zoledronic acid preparation, and the cytokine IL-2 as a co-stimulator, and inactivated autologous plasma as a nutrient, and culture them for 72 hours; C. After culturing for 72 hours at 37°C in a 5% CO2 incubator, the cells were centrifuged and the medium was replaced. The cells were then expanded and cultured in serum-free medium containing only the cytokine IL-2 and autologous plasma. D. Culture for 18-21 days to obtain high-quantity, high-purity, and highly cytotoxic NKT cells.

[0007] Furthermore, the median concentration of mononuclear cells isolated from peripheral blood in step A is 2.0×10 6 / ml.

[0008] Furthermore, the concentration of the group A streptococcus preparation in step B is 0.01-0.1 KE / mL; the concentration of the zoledronic acid preparation is 1-5 μM; and the concentration of the cytokine IL-2 is 100-500 IU / mL.

[0009] Furthermore, the autologous plasma in steps B and C is in an inactivated state, and the concentration is 5% of the content of the serum-free culture medium 7 days before culture; and the concentration is 1% of the content of the serum-free culture medium after 7 days of culture.

[0010] Furthermore, in step C, the expansion culture is replenished every 2-3 days, and the cell concentration is adjusted to 1.5-2×10 6 cells / ml.

[0011] Furthermore, the NKT cells obtained by culturing for 18-21 days in step D have a purity greater than 70%, an expansion multiple of more than 150 times, and have strong cytotoxicity.

[0012] Beneficial effects: (1) The combined stimulators (group A streptococcal preparation, zoledronic acid, and cytokine IL-2) work synergistically to significantly improve the proliferation efficiency of NKT cells. After expansion, the cell number is large, the purity is high, and the cytotoxic function is enhanced, meeting the needs of clinical treatment.

[0013] (2) Only three key reagents are required, and no complex antigen presentation system or expensive cytokine combination is required. The culture cost is reduced by more than 50%, and the serum-free system avoids the risk of heterologous protein contamination.

[0014] (3) The expanded cells meet the clinical treatment dose requirements, are suitable for personalized immunotherapy, and provide a reliable cell source for diseases such as tumors and viral infections.

[0015] In summary, the present invention achieves efficient expansion of NKT cells while ensuring their purity and cytotoxicity, providing reliable technical support for the clinical application of NKT cell immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Figure 3 is the growth curve of NKT cells in three groups.

[0017] Figure 2 Figure 4 shows the morphology of NKT cells in the experimental group (40×), A: culture day 3, B: culture day 7.

[0018] Figure 3 Figure 2 shows the detection of surface markers (CD3+CD56+) of NKT cell culture in the experimental group. A: 7th day of culture, B: 18th day of culture. DETAILED DESCRIPTION To further illustrate the present invention, specific examples are provided below. It should be understood that these examples are only used to help understand the present invention and do not constitute a limitation of the scope of the present invention. In addition, it should be understood that after studying the present invention, those skilled in the art can make various improvements or adjustments, and these changes should also be considered to be within the scope of protection of the present invention.

[0019] Before introducing the specific embodiments of the present invention, it should be clear that the scope of protection of the present invention is not limited to the specific embodiments described below. At the same time, the terms used in the present invention are for the convenience of describing specific embodiments and are not used to limit the scope of the invention. Unless otherwise defined, all technical terms in the present invention should be interpreted according to the common understanding of those skilled in the art. For experimental methods not specifically listed in the examples, they should generally be operated according to conventional conditions or the recommended conditions of the equipment manufacturer. In addition to the specific methods, equipment and materials used in the examples, the implementation of the present invention can also be achieved by using existing technical solutions that are similar or equivalent to the methods, equipment and materials described in the examples.

[0020] Example 1 Preparation of adult peripheral blood PBMC 1. Take 30ml-50ml of heparin-anticoagulated human peripheral blood into a 50ml centrifuge tube (each centrifuge tube has about 30ml of blood) and centrifuge at 800g for 8 minutes at room temperature.

[0021] 2. After centrifugation, use a 10ml pipette to transfer the upper plasma into a new 50ml centrifuge tube and inactivate it in a 56℃ water bath for 30 minutes (shake it halfway through every 15 minutes). After the plasma inactivation is complete, the plasma will be turbid. Centrifuge it at 900g for 10 minutes. Take the supernatant and freeze it at -20℃ for 15 minutes (shake it evenly during the process). After freezing, centrifuge it again at 900g for 10 minutes. Take the supernatant and store it at 4℃ for later use.

[0022] 3. Add an equal volume of saline to each centrifuge tube as to the original blood and mix thoroughly by pipetting. Slowly add the diluted peripheral blood to the upper layer of the prepared lymphocyte separation solution (15 ml / tube) in the centrifuge tube, adding 25 ml to each tube.

[0023] 5. Balance the centrifuge adapter and centrifuge at 800 g for 20 min, with a 6-degree rise and 1-degree fall ratio at 20°C.

[0024] 6. After centrifugation, take the cells from the middle buffy coat layer of the centrifuge tube and transfer them to a new centrifuge tube. Add physiological saline to 50 ml, invert and mix thoroughly. Centrifuge at 500 g for 10 minutes at room temperature.

[0025] 7. After centrifugation, discard the supernatant, resuspend the cells in K581 medium, take a 0.1 ml sample, count, and set aside.

[0026] Example 2 Activation and culture of NKT cells 1. Adjust the PBMC concentration to 2×10 6 cells / ml, the initial culture volume was 10 ml, and the cells were plated into T25 cell culture flasks.

[0027] 2. For better illustration, two control groups and one experimental group were set up. In the experimental group, a group A Streptococcus preparation, a zoledronic acid preparation, and the cytokine IL-2 were added to the culture flask as a combined stimulant; in control group 1, a group A Streptococcus preparation and the cytokine IL-2 were added to the culture flask as a combined stimulant; in control group 2, a zoledronic acid preparation and the cytokine IL-2 were added to the culture flask as a combined stimulant. All three groups were co-cultured in a 37°C, 5% CO2 incubator for 72 hours, and the concentration of the group A Streptococcus preparation was maintained at 0.01-0.1 KE / mL; the concentration of the zoledronic acid preparation was maintained at 1-5 μM; and the concentration of the cytokine IL-2 was maintained at 100-500 IU / mL.

[0028] Example 3: Expansion and culture of NKT cells After 72 hours of co-culture, the old culture medium was aspirated by centrifugation and 3 ml of culture medium was added to the bottle to prevent drying. Centrifuge at 350 g for 5 minutes, discard the supernatant, add 7 ml of expansion medium, resuspend and add to a T25 bottle. The expansion medium is K581 culture plus IL-2 (concentration is 100~500 IU / mL) plus autologous plasma (the concentration in the first 7 days is 5% of the added culture medium content; after 7 days of culture, the concentration is 1% of the added culture medium content). Observe the growth status of the cells every 2-3 days, take a small amount of cells for counting, and add expansion medium according to the counting results (transfer to a larger culture flask or culture bag according to the volume of the culture medium) to maintain the cell density at 1.5-2×10 6 The cell proliferation rate is less than 10 cells / mL, and the entire expansion process lasts for 18-21 days. Example 4: Plotting the cell proliferation growth curve Take the cell suspension cultured on the 3rd to 21st day and count the cells every two days using a cell counter. Draw the cell expansion growth curve with time as the horizontal axis and cell number as the vertical axis (see Figure 1 The results showed that the experimental group's cell growth rate and expansion multiple were significantly greater than those in control groups 1 and 2 using this inventive method. By day 19, the experimental group's expansion multiple had reached 150 times, while the control groups reached their maximum expansion multiple on day 17, less than 100 times. Furthermore, cells in control groups 1 and 2 began to undergo apoptosis and decreased in number after day 17. Therefore, the experimental group's culture method yielded a high number of cells with rapid proliferation, meeting clinical needs.

[0029] Since there were significant differences among the three groups during the culture process, and the experimental group was superior to the control group, the NKT cells in the experimental group were selected for subsequent testing of cell morphology, immune phenotype, and tumor cell killing efficiency.

[0030] Table 1 Cell number and expansion times of NKT cells in different groups at different culture times

[0031] Example 5 Cell Morphology During the cell culture process, the cells were observed before each rehydration, and photos were taken on the 3rd and 7th days of culture. The results showed that NKT cells were round or oval, with relatively uniform shape and size; a small number of cells were observed to aggregate on the 3rd day of culture, and the number of cell clusters increased significantly on the 7th day ( Figure 2 ).

[0032] Example 6 Immunophenotyping 1. Take the cells on the 7th and 18th day of culture, filter them, and adjust the cell density to 1×10 6 pcs / ml; 2. Centrifuge at 350g for 5 min and wash twice with PBS; 3. Discard the supernatant, wash with PBS, dispense 100 μl / EP tube, and add detection antibodies CD3CD56 for double staining; 4. Incubate at room temperature in the dark for 30 minutes; 5. After incubation, wash twice with PBS, 350g, 5 min, and discard the supernatant; Resuspend in 6.500ul PBS and use flow cytometry to detect phenotype. (See Figure 3 ) The test results showed that the positive rate of NKT cells was 33.99% on day 7 and increased to 70.43% on day 18. The results showed that the NKT cells cultured using the present invention had a high purity.

[0033] Example 7 Detection of tumor cell killing efficiency 1. Take K562 and A549 cell suspensions in the logarithmic growth phase and adjust the density to 1.0×10 5 cells / mL as target cells.

[0034] 2. NKT cells were used as effector cells. The cell density was adjusted according to the effector-target ratio. The effector-target ratios were 1:1, 5:1, 10:1, and 20:1, respectively. The cells were mixed and inoculated into 96-well plates with 200 μl per well.

[0035] 3. Effector and target cell wells: The wells for the reaction between NKT cells and target cells, 100 μl of NK cells and target cells respectively, set up 5 repeated experimental wells for the effector and target cell well group, and take the average value.

[0036] 4. Effector cell wells: Based on the NKT cell concentrations at the four effector-target ratios, prepare four effector cell wells with corresponding NKT concentrations, with a volume of 200 μl per well.

[0037] 5. Target cell wells: Add 100 μl of K562 (A549) cell suspension and 100 μl of culture medium to each well of K562 (A549) cells.

[0038] After incubation at 37°C, 5% CO2 for 12 hours, add 20 μl of CCK-8 reagent and incubate for 1.5 hours. Then, measure the absorbance at 450 nm.

[0039] 7. Calculation of killing rate: Killing rate = [1-(target cell wells - effector cell wells) / target cell wells] × 100%.

[0040] The test results showed that the killing rate of NKT cells against K562 was 92.63%, and the killing rate against A549 was as high as 87.81%. The results show that NKT cells cultured using the present invention have high killing activity against both hematological tumors and solid tumors.

[0041] Table 2 Killing efficiency of NKT cells on K562 and A549 cells

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for in vitro expansion of NKT cells based on combined stimulators, characterized in that: The following steps are involved: A. Mononuclear cells are extracted and separated from adult peripheral blood; B. Resuspend the mononuclear cells in serum-free medium, add a group A streptococcus preparation, a zoledronic acid preparation, and the cytokine IL-2 as a co-stimulator, and inactivated autologous plasma as a nutrient, and culture them for 72 hours; C. After culturing for 72 hours at 37°C and 5% CO2 in an incubator, the cells were centrifuged and the medium was completely replaced. The cells were then expanded and cultured in serum-free medium containing only cytokine IL-2 and autologous plasma. D. Culture for 18-21 days to obtain target NKT cells.

2. The method for in vitro expansion of NKT cells based on combined stimulators according to claim 1, characterized in that: The concentration of mononuclear cells isolated from peripheral blood as described in step A was 2.0×10 6 / ml.

3. The method for in vitro expansion of NKT cells based on combined stimulators according to claim 1, characterized in that: The concentration of the group A streptococcus preparation in step B is 0.01-0.1 KE / mL; the concentration of the zoledronic acid preparation is 1-5 μM; and the concentration of the cytokine IL-2 is 100-500 IU / mL.

4. The method for in vitro expansion of NKT cells based on combined stimulators according to claim 1, characterized in that: The autologous plasma described in steps B and C is in an inactivated state. In step C, the concentration 7 days before culture is 5% of the content of the serum-free medium; after culture for 7 days, the concentration is 1% of the content of the serum-free medium.

5. The method for in vitro expansion of NKT cells based on combined stimulators according to claim 1, characterized in that: In step C, the expansion culture was replenished every 2-3 days, and the cell concentration was adjusted to 1.5-2×10 6 cells / ml.

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