Culture medium for improving TIL cell proliferation and tumor killing effect, in-vitro amplification method and application

Abstract

The invention discloses a culture medium for improving TIL cell proliferation and tumor killing effects, an in-vitro amplification method and application. The TIL cell culture medium provided by the invention comprises the following components: 1.2 to 50 [mu] g / ml of pulsatilla saponin B4, 1.2 to 2.8 [mu] g / ml of caffeine, 0.8 to 6.4 [mu] g / ml of tremella polysaccharide, and the balance of an X-VIVO-15 culture solution. The anemoside B4, the caffeine and the tremella polysaccharide are combined for use, the effects of regulating cell metabolism, providing energy for immune cells and inducing tumor cell apoptosis of the anemoside B4, the caffeine and the tremella polysaccharide are combined, and the proliferation effect and the tumor killing effect of TIL cells can be remarkably improved through culture in a fixed compatibility proportion.

Description

Technical Field

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

[0002] Tumor-infiltrating lymphocytes (TIL) are a group of heterogeneous lymphocytes present in the interstitial tissue of the patient's own tumor, mainly composed of CD4+ and CD8+ T lymphocytes. Affected by the tumor microenvironment, the immune function of TIL is usually suppressed.

[0003] In recent years, adoptive T cell therapy (ACT) using TIL has become a highly-regarded immunotherapy method. The basic principle of TIL-ACT is to extract TIL from the patient's tumor tissue, restore the tumor killing activity of TIL after activation and amplification in vitro, and return a large number of proliferated TIL to the patient to produce a specific immune response to tumor cells, thereby killing tumor cells more effectively. 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 is important for the treatment effect.

[0004] CN 115645453A discloses a composition for improving TIL cell proliferation and tumor killing effect, which is a combination of Shenqi Fuzheng injection and NMN, and stevioside is added on this basis for combined use; the immune cell function improvement effect of Shenqi Fuzheng injection and the role of ATP in providing energy for cell proliferation are combined, thereby directly improving the proliferation effect of TIL cells and the tumor killing effect. However, the value-added effect of this method is limited.

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

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

[0007] In order to achieve the above object, the present invention adopts the following technical solution: In a first aspect, the present invention provides a TIL cell culture medium, comprising the following components: 1.2-50 μg / ml of pulsatilla saponin B4, 1.2-2.8 μg / ml of caffeine, 0.8-6.4 μg / ml of Tremella polysaccharide, and the remainder of X-VIVO-15 culture medium.

[0008] Studies have shown that Pulsatilla chinensis (Bge.) Regel is a dried root of the Ranunculaceae plant, which has the effects of clearing away heat and detoxifying, killing insects and stopping diarrhea. Among them, Pulsatilla saponin B4 has preventive and therapeutic effects on malignant tumors. Its mechanism of action is mainly to inhibit tumor cell growth, block tumor cell cycle, inhibit signal pathways, induce tumor cell apoptosis, inhibit angiogenesis, regulate cell energy metabolism, reverse drug resistance, induce tumor cell autophagy, and regulate immunity. Caffeine is a xanthine alkaloid compound with mild excitatory effects. It can be extracted from plants such as coffee beans and tea leaves, and has the effects of refreshing and relieving fatigue. Studies have shown that caffeine can accelerate cell proliferation by activating mitochondrial function. Tremella polysaccharide is the main functional component of Tremella, which has antioxidant, anti-tumor, diabetes intervention, immunomodulatory and neuroprotective effects.

[0009] The present invention combines pulsatilla saponin B4, caffeine and tremella polysaccharide to combine the functions of the three in regulating cell metabolism, providing energy for immune cells and inducing tumor cell apoptosis, and can significantly improve the proliferation effect and tumor killing effect of TIL cells through cultivation at a fixed compatibility ratio.

[0010] 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 ingredients: pulsatilla saponin B4 1.2-6 μg / ml, caffeine 2-2.8 μg / ml, tremella polysaccharide 0.8-1 μg / ml, and the remainder X-VIVO-15 culture medium.

[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 ingredients: 45-50 μg / ml of pulsatilla saponin B4, 1.2-1.6 μg / ml of caffeine, 6-6.4 μg / ml of Tremella polysaccharide, and the remainder of X-VIVO-15 culture medium.

[0012] In a second aspect, the present invention provides 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 the above-mentioned culture medium for proliferation culture.

[0013] The TIL cells are autologous cells.

[0014] 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.

[0015] Among them, the added amount of IL-2 ranges from 2900-3100 iu / ml, specifically 3000 iu / ml; the added amount ratio of the trophoblast cells is: TIL cells: trophoblast cells = 1:150-1:300; the added amount of the culture medium ranges from 1-2 ml; and the culture time in the CO2 incubator is 13 days.

[0016] In a third aspect, the present invention also provides the use of the above-mentioned TIL cell culture medium in the preparation of an immune cell preparation for adoptive immune therapy.

[0017] In a fourth aspect, the present invention also provides an immune cell preparation for adoptive immune therapy, comprising TIL cells expanded by the above method.

[0018] In the immune cell preparation, the number of TIL cells is 0.5×10 6 -1×10 6 Pieces / ml.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention combines pulsatilla saponin B4, caffeine and tremella polysaccharide to combine the functions of the three in regulating cell metabolism, providing energy for immune cells and inducing tumor cell apoptosis, and can significantly improve the proliferation effect and tumor killing effect of TIL cells through cultivation at a fixed compatibility ratio. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of the culture medium provided by the present invention in combination with TIL cells on the amount of LDH released by the organoid model; wherein, ABC represents co-culture for 24 h, 48 h and 72 h, respectively; *P<0.05, **P<0.01, **P<0.001, ****P<0.0001 represent comparisons with the TIL cell group.

[0021] Figure 2 The effect of the culture medium provided by the present invention in combination with TIL cells on the secretion of cytokines in the organoid culture system; wherein, ABC represents co-culture for 24 h, 48 h and 72 h, respectively; *P<0.05, **P<0.01, **P<0.001, ****P<0.0001 represent comparisons with the TIL cell group. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0024] Unless otherwise specified, the reagents, materials, instruments, etc. used in the following examples can be obtained from commercial sources.

[0025] Pulsatilla saponin B4 (Cat. No. B20060), caffeine (Cat. No. B81798) and Tremella polysaccharide (Cat. No. T25935) used in the following examples were purchased from Shanghai Yuanye Company, with purities of ≥95%, ≥95% and ≥90%, respectively.

[0026] The non-small cell lung cancer patient tissues used in the following examples were obtained from Peking University People's Hospital.

[0027] The X-VIVO-15 medium (immune cell serum-free medium) used in the following examples was purchased from LONZA, Switzerland.

[0028] Example 1 (Formula 4) The present embodiment provides a culture medium for improving the proliferation effect and tumor killing effect of TIL cells, which is composed of the following ingredients: 1.2 μg / ml of pulsatilla saponin B4, 2 μg / ml of caffeine, 0.8 μg / ml of Tremella polysaccharide, and the remainder is X-VIVO-15 culture medium.

[0029] Example 2 (Formula 5) The present embodiment provides a culture medium for improving the proliferation effect and tumor killing effect of TIL cells, which is composed of the following components: 6 μg / ml of pulsatilla saponin B4, 2.8 μg / ml of caffeine, 0.8 μg / ml of Tremella polysaccharide, and the remainder is X-VIVO-15 culture medium.

[0030] Example 3 (Formula 7) This embodiment provides a culture medium for improving the proliferation effect and tumor killing effect of TIL cells, which is composed of the following ingredients: 45 μg / ml of pulsatilla saponin B4, 1.6 μg / ml of caffeine, 6.4 μg / ml of Tremella polysaccharide, and the remainder is X-VIVO-15 culture medium.

[0031] Experimental Example 1: Investigation of Cell Proliferation and Cell Killing Effects 1. Preparation of formula 1-10, follow the following steps: (1) Weigh appropriate amounts of pulsatilla saponin B4, caffeine and Tremella polysaccharide, and add them to X-VIVO-15 medium to prepare mother solutions with concentrations of 10 mM, 5 mM and 10 mM, respectively; (2) Take an appropriate amount of Pulsatilla saponin B4 stock solution and prepare solutions with concentrations of 1, 6, 30, 150, and 250 μg / ml, respectively; take an appropriate amount of caffeine stock solution and prepare solutions with concentrations of 4, 5, 6, 7, and 8 mg / ml, respectively; take an appropriate amount of Tremella polysaccharide solution and prepare solutions with concentrations of 1, 8, 16, 32, and 64 μg / ml, respectively; (3) Take the corresponding volumes of Pulsatilla saponin B4, caffeine and Tremella polysaccharide solution according to the table below, add them to a 10 ml volumetric flask, and make up to volume with X-VIVO-15 culture medium solution.

[0032]

[0033] 2. Isolation of TIL cells and tumor cells and preparation of cell suspension Tissues from patients with non-small cell lung cancer 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 for 0.5 h, and necrotic tissues were cut off. They were rinsed with Hanks solution and cut into pieces of 0.1-0.2 mm. 3 Add tissue digestion solution to small pieces and digest at 37°C for 1-2h. Collect the cell suspension and separate it with 100% and 75% discontinuous density gradient lymphocyte separation solution to obtain TIL cells and tumor cells; TIL cells were prepared with culture medium containing 3000 IU / ml IL-2 (the culture medium was based on X-VIVO-15 culture medium) at a concentration of 0.5×10 6 -1×10 6 / ml cell suspension.

[0034] Tumor cells were cultured in 1640 medium containing 10% calf serum at a concentration of 0.2×10 4 -1×10 4 / ml cell suspension and continue culturing.

[0035] 3. Cell proliferation and tumor cell killing results Take TIL cells in the logarithmic growth phase and adjust the cell density to 0.5×10 6 -1×10 6 The cells were cultured in 24-well plates at a ratio of 3000 iu / ml, IL-2 (added at a ratio of 3000 iu / ml), trophoblasts (added at a ratio of TIL cells: trophoblasts = 1:150-1:300) and the culture medium of the above-mentioned Formulation 1 to Formulation 10 (added 1-2 ml) were added respectively, and the cells were cultured in a CO2 incubator for 13 days. The number of TIL cells was determined by cell counting.

[0036] A certain amount of TIL cells and non-small cell lung cancer were mixed at an effector-target ratio of 50:1, placed in a CO2 incubator for 48 hours, and the tumor cell killing rate was determined by the CCK method. The process was repeated 4 times in parallel.

[0037] The comprehensive score obtained by normalizing the corresponding cell numbers and tumor cell killing rates after treatment with Formulations 1 to 10 was used as the evaluation index.

[0038]

[0039] From the above test results, it can be seen that when the concentrations of scutellaria saponin B4, caffeine and tremella polysaccharide are 30μg / ml, 7mg / ml and 8μg / ml respectively, and the volume ratio is 2:4:1 (Formula 5); the concentrations are 150μg / ml, 8mg / ml and 16μg / ml respectively, and the volume ratio is 3:2:4 (Formula 7); the concentrations are 6μg / ml, 5mg / ml and 8μg / ml respectively, and the volume ratio is 2:4:1 (Formula 4); the concentrations are 150μg / ml, 4mg / ml and 32μg / ml respectively, and the volume ratio is 4:3:2 (Formula 8), the corresponding comprehensive levels of TIL cell number and tumor cell killing rate are relatively high; however, 2 of the 4 samples of Formulation 8 were contaminated with bacteria during the culture process, which is presumably related to the excessive concentration of the culture medium. Therefore, Formulation 8 will no longer be verified in subsequent experiments.

[0040] Experimental Example 2: Analysis of the killing effect of TIL cells in vitro The patient's cancer cells were used to construct an organoid model (PDO). PDO was used as the standard control, Jurkat cells as the negative control, and TIL cells as the positive control. The cells were co-cultured for 24, 48, and 72 hours, respectively. The LDH level in the culture system when TIL cells were co-cultured with Formulation 4, Formulation 5, and Formulation 7 was detected to analyze the in vitro killing effect of TIL cells.

[0041] The culture medium supernatant was taken to detect the secretion of INFγ, Granzyme A, Granzyme B, Perforin and Granulysin cytokines to evaluate the degree of activation of TIL cells.

[0042] The results show that if Figure 1 As shown in the figure, at each time point, the killing effects of group 4, group 5, and group 7 were significantly higher than those of the Jurkat control and TIL control groups, and the killing effect of group 7 was the strongest. As time went on, the LDH secretion of group 4, group 5, and group 7 gradually increased, and reached a peak when co-cultured for 72 hours.

[0043] like Figure 2As shown, the activation degree of TIL cells shows that compared with TIL cells cultured alone, the secretion of INFγ, Granzyme A, and Granzyme B is the highest after adding formula 7, followed by formula 5 and formula 4, which are significantly different from the negative control. As time goes on, the killing of organoids by TIL cells gradually increases, and the killing of organoids by TIL cells combined with the formula is always at a high level. The detection results of cytokines in the supernatant of each group are proportional to the killing of organoids. It can be seen that co-culture of formula 7 and TIL cells can most effectively promote the proliferation of TIL cells and enhance the tumor killing effect of TIL cells.

[0044] At the same time, compared with CN115645453A, it can be seen that the effects of formulation 7 of the present invention in terms of TIL cell proliferation and tumor cell killing are better than the composition with the best comprehensive effect in CN115645453A (Example 1).

[0045] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A TIL cell culture medium, comprising the following components: 1.2-50 μg / ml of pulsatilla saponin B4, 1.2-2.8 μg / ml of caffeine, 0.8-6.4 μg / ml of Tremella polysaccharide, and the remainder of X-VIVO-15 culture medium.

2. The TIL cell culture medium according to claim 1, characterized in that The culture medium for improving the proliferation effect and tumor killing effect of TIL cells is composed of the following components Composition: Pulsatilla saponin B4 1.2-6μg / ml, caffeine 2-2.8μg / ml, Tremella polysaccharide 0.8-1μg / ml, and the remainder of X-VIVO-15 culture medium.

3. The TIL cell culture medium according to claim 1, characterized in that The culture medium for improving the proliferation effect and tumor killing effect of TIL cells is composed of the following components Composition: Pulsatilla saponin B4 45-50μg / ml, caffeine 1.2-1.6μg / ml, Tremella polysaccharide 6-6.4μg / ml, and the remainder of X-VIVO-15 culture medium.

4. A method for in vitro expansion of TIL cells, comprising the following steps: taking TIL cells in the logarithmic growth phase, adjusting the cell density, and adding the culture medium described in any one of claims 1 to 3 for proliferation culture.

5. The in vitro amplification method according to claim 4, characterized in that: 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.

6. The in vitro amplification method according to claim 5, 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.

7. The in vitro amplification method according to claim 5, characterized in that: The culture time in the CO2 incubator is 13 days.

8. An immune cell preparation for adoptive immune therapy, comprising TIL cells amplified by the method described in any one of claims 4 to 7.

9. The immune cell preparation according to claim 8, characterized in that The number of TIL cells in the immune cell preparation is 0.5×10 6 -1×10 6 Pieces / ml.

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