A culture medium, an in vitro amplification method and an application for enhancing the proliferation and tumor killing effect of TIL cells

Abstract

The present invention discloses a culture medium, an in vitro amplification method and an application for improving the proliferation and tumor killing effects of TIL cells. The TIL cell culture medium provided by the present invention comprises the following components: pulchinenoside B4 1.2 - 50 μg / ml, caffeine 1.2 - 2.8 μg / ml, tremella polysaccharide 0.8 - 6.4 μg / ml, and the balance of X-VIVO-15 culture medium. By combining pulchinenoside B4, caffeine and tremella polysaccharide, and integrating the functions of regulating cell metabolism, providing energy for immune cells and inducing apoptosis of tumor cells, and culturing with a fixed compatibility ratio, the present invention can significantly improve the proliferation effect and tumor killing effect of TIL cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and specifically relates to a culture medium, an in vitro amplification method and an application for improving the proliferation and tumor killing effect of TILs. Background Art

[0002] Tumor-infiltrating lymphocytes (TILs) are a group of heterogeneous lymphocytes present in the stroma of patients' own tumor tissues, mainly composed of CD4+ and CD8+ T lymphocytes. Affected by the tumor microenvironment, the immune function of TILs is usually inhibited.

[0003] In recent years, adoptive T cell therapy (ACT) using TILs has become an immunotherapy method that has attracted much attention. The basic principle of TIL-ACT is to extract TILs from patients' tumor tissues, restore the tumor killing activity of TILs after activation and amplification in vitro, and after a large number of proliferated TILs are transfused back to the patients, specific immune responses can be generated against tumor cells, thereby more effectively killing tumor cells. Since a huge number of cells are required during the treatment process, studying how to quickly and massively obtain TIL cells with good tumor killing effects plays an important role in the treatment effect.

[0004] CN 115645453A discloses a composition for improving the proliferation and tumor killing effect of TIL cells, which is a mixture of Shenqi Fuzheng Injection and NMN, and on this basis, stevioside is added for combined use; the function of improving the immune cell function of Shenqi Fuzheng Injection and the function of ATP providing energy for cell proliferation are combined, so as to directly improve the proliferation effect and tumor killing effect of TIL cells. However, the proliferation effect of this method is limited.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a culture medium and an in vitro amplification method for cells that can improve the proliferation and tumor killing effect of TIL cells, and this culture medium can significantly improve the proliferation effect and tumor killing effect of TIL cells.

[0007] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a TIL cell culture medium, which includes the following components: pulchinenoside B4 1.2 - 50 μg / ml, caffeine 1.2 - 2.8 μg / ml, tremella polysaccharide 0.8 - 6.4 μg / ml, and the balance is X-VIVO-15 culture solution.

[0009] Research shows that Pulsatilla root is the dried root of Pulsatilla chinensis (Bge.) Regel of the Ranunculaceae family, which has the effects of clearing heat and detoxifying, killing insects and stopping dysentery. Among them, pulsatilla saponin B4 has preventive and therapeutic effects on malignant tumors, and its mechanism of action is mainly to inhibit tumor cell growth, block the tumor cell cycle, inhibit signal pathways, induce tumor cell apoptosis, inhibit angiogenesis, regulate cellular energy metabolism, reverse drug resistance, induce tumor cell autophagy, and regulate immunity, etc. Caffeine is a xanthine alkaloid compound with a mild excitatory effect, which can be extracted from plants such as coffee beans and tea leaves, and has the effects of refreshing and relieving fatigue. Research shows that caffeine can accelerate the cell proliferation process by activating mitochondrial function. Tremella polysaccharide is the main functional component of Tremella, which has antioxidant, anti-tumor, intervention in diabetes, immune regulation and neuroprotective effects.

[0010] In the present invention, by combining pulsatilla saponin B4, caffeine and tremella polysaccharide, the effects of regulating cell metabolism, providing energy for immune cells, and inducing tumor cell apoptosis of the three are combined, and through cultivation with a fixed compatibility ratio, the proliferation effect and tumor killing effect of TIL cells can be significantly improved.

[0011] In a preferred embodiment of the present invention, the culture medium for improving the proliferation effect and tumor killing effect of TIL cells is composed of the following components: pulsatilla saponin B4 1.2 - 6 μg / ml, caffeine 2 - 2.8 μg / ml, tremella polysaccharide 0.8 - 1 μg / ml, and the balance of X-VIVO-15 culture medium.

[0012] In a preferred embodiment of the present invention, the culture medium for improving the proliferation effect and tumor killing effect of TIL cells is composed of the following components: pulsatilla saponin B4 45 - 50 μg / ml, caffeine 1.2 - 1.6 μg / ml, tremella polysaccharide 6 - 6.4 μg / ml, and the balance of X-VIVO-15 culture medium.

[0013] In a second aspect, the present invention provides an in vitro amplification method for TIL cells, which includes the following steps: taking TIL cells in the logarithmic growth phase, adjusting the cell density, and adding them to the above-mentioned culture medium for proliferation culture.

[0014] The TIL cells are autologous cells.

[0015] The proliferation culture is carried out according to the following operation: taking TIL cells in the logarithmic growth phase, adjusting the cell density to 0.5×10 6 -1×10 6 cells / ml, culturing in a 24-well plate, and respectively adding IL-2, feeder cells and the above-mentioned culture medium, and placing them in a CO 2 incubator for culture.

[0016] Among them, the addition amount range of the IL-2 is 2900 - 3100 iu / ml, specifically such as 3000 iu / ml; the addition amount ratio of the trophoblast cells is: TIL cells:trophoblast cells = 1:150 - 1:300; the addition amount range of the culture medium is 1 - 2 ml; the CO 2 The culture time in the incubator is 13 days.

[0017] Thirdly, the present invention also provides the application of the above TIL cell culture medium in the preparation of an immune cell preparation for adoptive immunotherapy.

[0018] Fourthly, the present invention also provides an immune cell preparation for adoptive immunotherapy, which contains TIL cells amplified by the above method.

[0019] In the immune cell preparation, the number of the TIL cells is 0.5×10 6 -1×10 6 cells / ml.

[0020] Compared with the prior art, the beneficial effects obtained by the present invention are:

[0021] By combining pulchinenoside B4, caffeine and tremella polysaccharide, the present invention combines the functions of regulating cell metabolism, providing energy for immune cells and inducing apoptosis of tumor cells of the three, and through cultivation with a fixed compatibility ratio, can significantly improve the proliferation effect and tumor killing effect of TIL cells. Description of the Drawings

[0022] Figure 1 Shows the influence of the combined use of the culture medium provided by the present invention and TIL cells on the LDH release amount of the organoid model; among them, ABC are co-cultured for 24 h, 48 h and 72 h respectively; *P < 0.05, **P < 0.01, **P < 0.001, ****P < 0.0001 represent compared with the TIL cell group.

[0023] Figure 2 Shows the influence of the combined use of the culture medium provided by the present invention and TIL cells on the cytokine secretion amount in the organoid culture system; among them, ABC are co-cultured for 24 h, 48 h and 72 h respectively; *P < 0.05, **P < 0.01, **P < 0.001, ****P < 0.0001 represent compared with the TIL cell group. Detailed Embodiments

[0024] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0025] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0026] Unless otherwise specified, the reagents, materials, instruments, etc. used in the following examples are all commercially available.

[0027] The pulchinenoside B4 (product number B20060), caffeine (product number B81798), and tremella polysaccharide (product number T25935) used in the following examples were purchased from Shanghai Yuanye Bio-Technology Co., Ltd., and their purities were ≥95%, ≥95%, and ≥90% respectively.

[0028] The tissues of non-small cell lung cancer patients used in the following examples were obtained from Peking University People's Hospital.

[0029] The X-VIVO-15 medium (serum-free medium for immune cells) used in the following examples was purchased from LONZA, Switzerland.

[0030] Example 1 (Formulation 4)

[0031] This example provides a medium for improving the proliferation effect and tumor killing effect of TIL cells, which is composed of the following components: pulchinenoside B4 1.2 μg / ml, caffeine 2 μg / ml, tremella polysaccharide 0.8 μg / ml, and the balance is X-VIVO-15 culture solution.

[0032] Example 2 (Formulation 5)

[0033] This example provides a medium for improving the proliferation effect and tumor killing effect of TIL cells, which is composed of the following components: pulchinenoside B4 6 μg / ml, caffeine 2.8 μg / ml, tremella polysaccharide 0.8 μg / ml, and the balance is X-VIVO-15 culture solution.

[0034] Example 3 (Formulation 7)

[0035] This example provides a medium for improving the proliferation effect and tumor killing effect of TIL cells, which is composed of the following components: pulchinenoside B4 45 μg / ml, caffeine 1.6 μg / ml, tremella polysaccharide 6.4 μg / ml, and the balance is X-VIVO-15 culture solution.

[0036] Experimental Example 1. Investigation of cell proliferation and cell killing effects

[0037] 1. Preparation of Formulations 1-10, which was carried out according to the following operating steps:

[0038] (1) Weigh appropriate amounts of pulchinenoside B4, caffeine, and tremella polysaccharide respectively, and add them to the X-VIVO-15 medium to prepare stock solutions with concentrations of 10 mM, 5 mM, and 10 mM respectively;

[0039] (2) Appropriately take the mother liquor of pulchinenoside B4 and prepare solutions with concentrations of 1, 6, 30, 150, and 250 μg / ml respectively; appropriately take the mother liquor of caffeine and prepare solutions with concentrations of 4, 5, 6, 7, and 8 mg / ml respectively; appropriately take the tremella polysaccharide solution and prepare solutions with concentrations of 1, 8, 16, 32, and 64 μg / ml respectively.

[0040] (3) According to the following table, respectively take the corresponding volumes of pulchinenoside B4, caffeine, and tremella polysaccharide solutions, add them to a 10 ml volumetric flask, and make up the volume with X-VIVO-15 culture medium solution.

[0041]

[0042] 2. Isolation of TIL cells and tumor cells and preparation of cell suspensions

[0043] Tissues from non-small cell lung cancer patients were obtained from Peking University People's Hospital. After the tissues were retrieved, they were placed in X-VIVO-15 culture medium containing penicillin, gentamicin, and amphotericin B solution and soaked for 0.5 h, and the necrotic tissues were cut off. They were rinsed clean with Hanks solution and cut into small pieces of 0.1 - 0.2 mm 3 and added to the tissue digestive juice, and digested at 37 °C for 1 - 2 h. The cell suspension was collected and separated with 100% and 75% discontinuous density gradient lymphocyte separation liquid to obtain TIL cells and tumor cells;

[0044] TIL cells were prepared into a cell suspension of 0.5×10 6 - 1×10 6 cells / ml with the culture medium containing 3000 IU / ml IL-2 (the base of the culture medium is X-VIVO-15 culture medium).

[0045] Tumor cells were cultured and prepared into a cell suspension of 0.2×10 4 - 1×10 4 cells / ml with 1640 solution containing 10% calf serum and continued to be cultured.

[0046] 3. Results of cell proliferation and tumor cell killing

[0047] Take TIL cells in the logarithmic growth phase, adjust the cell density to 0.5×10 6 - 1×10 6 cells / ml, culture them in a 24-well plate, and respectively add IL-2 (addition ratio 3000 iu / ml), feeder cells (addition ratio TIL cells:feeder cells = 1:150 - 1:300), and the culture media of the above-mentioned formula 1 - formula 10 (add 1 - 2 ml), and place them in a CO 2 incubator for 13 d, and use the cell counting method to measure the number of TIL cells.

[0048] Take a certain amount of TIL cells and non-small cell lung cancer, mix them at an effector-to-target ratio of 50:1, and place them in a CO 2 incubator for 48 h, and measure the tumor cell killing rate by the CCK method. Repeat in parallel 4 times.

[0049] Use the comprehensive score obtained by normalizing the corresponding cell number and tumor cell killing rate after treating Formulation 1 - Formulation 10 as the evaluation index.

[0050]

[0051] It can be seen from the above experimental results that when the concentrations of pulchinenoside B4, caffeine, and tremella polysaccharide are 30 μg / ml, 7 mg / ml, and 8 μg / ml respectively, and the volume ratio is 2:4:1 (Formulation 5); the concentrations are 150 μg / ml, 8 mg / ml, and 16 μg / ml respectively, and the volume ratio is 3:2:4 (Formulation 7); the concentrations are 6 μg / ml, 5 mg / ml, and 8 μg / ml respectively, and the volume ratio is 2:4:1 (Formulation 4); the concentrations are 150 μg / ml, 4 mg / ml, and 32 μg / ml respectively, and the volume ratio is 4:3:2 (Formulation 8), the comprehensive levels of the corresponding TIL cell number and tumor cell killing rate are relatively high; however, 2 out of 4 samples of Formulation 8 were contaminated during the culture process, presumably due to the too high concentration of the culture medium. Therefore, Formulation 8 will not be verified in subsequent experiments.

[0052] Experimental Example 2. Analysis of the in vitro killing effect of TIL cells

[0053] Take the cancer cells of the patient to construct an organoid model (PDO). Using PDO as the standard control, Jurkat cells as the negative control, and TIL cells as the positive control, co-culture for 24, 48, and 72 h respectively, detect the LDH level in the co-culture system when TIL cells are co-cultured with Formulation 4, Formulation 5, and Formulation 7, and analyze the in vitro killing effect of TIL cells.

[0054] Take the supernatant of the culture medium to detect the secretion amounts of INFγ, Granzyme A, Granzyme B, Perforin, and Granulysin cytokines, and use them to evaluate the activation degree of TIL cells.

[0055] The results show that, as Figure 1 shown, at each time point, the killing effects of the Formulation 4 group, the Formulation 5 group, and the Formulation 7 group are all significantly higher than those of the Jurkat control group and the TIL control group; and the killing effect of Formulation 7 is the strongest. As the time prolongs, the LDH secretion amounts of the Formulation 4 group, the Formulation 5 group, and the Formulation 7 group gradually increase, and reach the peak when co-cultured for 72 h.

[0056] As Figure 2 shown, the activation degree of TIL cells shows that, compared with the culture of TIL cells alone, the secretion levels of INFγ, Granzyme A, and Granzyme B are the highest after adding Formulation 7, followed by adding Formulation 5 and Formulation 4, with significant differences from the negative control. With the prolongation of time, the killing of organoids by TIL cells gradually increases, and when TIL cells are combined with the formulations for drug use, the killing of organoids always remains at a relatively high level. The detection results of cytokines in the supernatant of each group are proportional to the killing power of organoids. Thus, co-culturing Formulation 7 with TIL cells can most effectively promote the proliferation of TIL cells and enhance the tumor killing effect of TIL cells.

[0057] At the same time, compared with CN115645453A, it can be seen that Formulation 7 of the present invention is superior to the composition with the best comprehensive effect (Example 1) in CN115645453A in terms of both TIL cell proliferation and tumor cell killing.

[0058] Although the present invention has been described in detail above with general descriptions and specific implementation examples, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. An in vitro expansion method of TIL cells, comprising the following steps: taking TIL cells in the logarithmic growth phase, adjusting the cell density, and adding them to a culture medium for proliferation culture; The culture medium comprises the following components: 6 μg / ml of Pulsatilla saponin B4, 2.8 mg / ml of caffeine, 0.8 μg / ml of Tremella polysaccharide, and the remainder of X-VIVO-15 culture solution; The proliferation culture was performed as follows: TIL cells in the logarithmic growth phase were taken and the cell density was adjusted to 0.5×10 6 -1×10 6 / ml, cultured in a 24-well plate, added with IL-2, trophoblast cells and the culture medium, respectively, and cultured in a CO2 incubator.

2. An in vitro expansion method of TIL cells, comprising the following steps: taking TIL cells in the logarithmic growth phase, adjusting the cell density, and adding them to a culture medium for proliferation culture; The culture medium comprises the following components: 45 μg / ml of pulsatilla saponin B4, 1.6 mg / ml of caffeine, 6.4 μg / ml of Tremella polysaccharide, and the remainder of X-VIVO-15 culture solution; The proliferation culture was performed as follows: TIL cells in the logarithmic growth phase were taken and the cell density was adjusted to 0.5×10 6 -1×10 6 / ml, cultured in a 24-well plate, added with IL-2, trophoblast cells and the culture medium, respectively, and cultured in a CO2 incubator.

3. The in vitro amplification method according to claim 1 or 2, characterized in that: The amount of IL-2 added ranges from 2900 to 3100 iu / ml; The ratio of the added trophoblast cells is: TIL cells: trophoblast cells = 1:150-1:300; The amount of the culture medium added ranges from 1 to 2 ml.

4. The in vitro amplification method according to claim 3, characterized in that: The culture time in the CO2 incubator is 13 days.

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