Low-serum transition multiplication culture method and culture medium for CD16 high-expression Vgamma9Vdelta2T cells

Abstract

The invention provides a low-serum transition multiplication culture method and a culture medium for CD16 high-expression V [gamma] 9V [delta] 2T cells, and belongs to the technical field of cell culture.The culture method comprises the steps that the serum concentration of a culture environment is firstly gradually reduced, induction of interleukin-15 and regulation and control of an antioxidant are combined, then the V [gamma] 9V [delta] 2T cells are massively amplified through a low-serum culture environment, and a large amount of CD16 is expressed; the low-serum culture environment is a culture environment with the serum content equal to or lower than 2%, and by combining low-serum transition, IL-15 induction of CD16 expression, antioxidant reduction of ROS and efficient amplification, high-proportion CD16 + phenotypes are maintained in the amplification process, and the ROS level in cells is reduced. The compound can be used for immune cell therapy and immune cell combined therapy related to antibody dependent cytotoxic action (ADCC), and has potential of function optimization and clinical application.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, and particularly relates to a low-serum over-expansion culture method for CD16-overexpressing Vγ9Vδ2T cells, and the culture medium used in this culture method. Background Technology

[0002] Vγ9Vδ2 T cells are the main γδT subset in peripheral blood, possessing the natural ability to kill tumor and infected cells and mediate antibody-dependent cytotoxicity (ADCC). High expression of CD16 in Vγ9Vδ2 T cells can enhance ADCC function and increase the potential for combined use of Vγ9Vδ2 T cells with antibody drugs.

[0003] However, existing γδ cell expansion methods are mostly aimed at expanding the efficiency of whole γδ T cells, and have not been optimized specifically for the function and phenotype of the Vγ9Vδ2 subset, resulting in the failure to fully realize the function of the expanded Vγ9Vδ2 T cells.

[0004] In addition, low-serum culture systems can reduce the uncertainty of serum sources, which is beneficial for clinical applications and reduces costs, but direct use may lead to insufficient amplification efficiency or phenotype retention. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention provides a low-serum transitional expansion culture method for CD16-overexpressing Vγ9Vδ2T cells. This method involves first gradually reducing the serum concentration in the culture environment, combined with interleukin-15 (IL-15) induction and antioxidant regulation, and then massively expanding Vγ9Vδ2T cells to express CD16 in a low-serum culture environment. The low-serum culture environment is defined as a culture environment with a serum content equal to or lower than 2%, and the detailed steps include: Step S1: Activate the culture, resuscitate frozen peripheral blood cells, resuspend them in initial culture medium, and adjust the cell density to 0.5-2 × 10⁻⁶ cells / year. 6 Add zoledronic acid to activate γδT cells, then add interleukin-15 (IL-15) and interleukin-2 (IL-2), and culture for 2-3 days. During the culture process, add antioxidants according to the cell status to reduce intracellular oxidative stress. Step S2 transition culture: Transfer the cells obtained in step S1 to the transition culture medium. The serum concentration of the transition culture medium is equal to or less than 5%. Then add interleukin-15 and interleukin-2 and culture for 3-4 days. During the culture process, add antioxidants according to the cell status to reduce intracellular oxidative stress. Step S3: Amplification and culture. The cells obtained in step S2 are transferred to a late-stage culture medium with a serum concentration equal to or less than 2%. Interleukin-15 and interleukin-2 are then added, and the cells are cultured for 4-7 days. During the culture process, fresh late-stage culture medium, interleukin-15, and interleukin-2 are added every 2-3 days to maintain the concentration of interleukin-15 and interleukin-2. Antioxidants are added according to the cell state to reduce intracellular oxidative stress. After the culture is completed, the cells are harvested and CD16 is obtained.

[0006] In step S1, the initial serum concentration of the culture medium is 5%-10%, the final concentration of zoledronic acid is 2-5 μM, and the antioxidant is 2-5 mM N-acetylcysteine ​​(NAC).

[0007] In steps S1-S3, the final concentration of interleukin-15 is 1-40 ng / mL, the final concentration of interleukin-2 is 100-200 IU / mL, the culture temperature is 35-45°C, and the carbon dioxide concentration is 1%-10% CO2.

[0008] In step S2 and S3, the antioxidants are 2–5 mM N-acetylcysteine ​​and 1–2 μM MitoTEMPO.

[0009] The present invention also provides a low-serum transitional expansion medium for CD16-overexpressing Vγ9Vδ2T cells, comprising a basal medium for Vγ9Vδ2T cell expansion culture, wherein interleukin-15 is also added to the basal medium.

[0010] The basal culture medium also contains interleukin-2 and antioxidants.

[0011] The serum content in the basal culture medium is less than 5%; the antioxidant is N-acetylcysteine, or the antioxidant is a combination of N-acetylcysteine ​​and a mitochondrial-targeting antioxidant in a ratio of 1:1000.

[0012] The serum content in the basal culture medium is less than 2%; the content of interleukin-15 is 1-40 ng / mL; the content of interleukin-2 is 100-200 IU / mL; the content of N-acetylcysteine ​​is 1-5 mM; and the antioxidant is added during the culture process.

[0013] The content of interleukin-15 is 10 ng / mL, and the content of interleukin-2 is 150 IU / mL. The beneficial effects of this invention are: by combining low serum transition, IL-15-induced CD16 expression, antioxidants to reduce reactive oxygen species (ROS), and efficient amplification, it can maintain a high proportion of CD16+ phenotype and reduce intracellular reactive oxygen species (ROS) levels during the amplification process. It can be used for immunocellular therapy and combination immunocellular therapy related to antibody-dependent cytotoxicity (ADCC), and has potential for functional optimization and clinical application. Attached Figure Description

[0014] Figure 1 This is a graph showing the cell expansion efficiency results donated by donor 1 in Example 5.

[0015] Figure 2 This is a graph showing the cell expansion efficiency results donated by donor 2 in Example 5.

[0016] Figure 3 This is a flow cytometry plot showing the changes in the proportion of Vγ9Vδ2 T cells in each group in Example 5.

[0017] Figure 4 This is a flow cytometry diagram of CD16 expression in Vγ9Vδ2 T cells of each group in Example 5. Detailed Implementation

[0018] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0019] Example 1: A low-serum transitional expansion culture medium for CD16-overexpressing Vγ9Vδ2T cells.

[0020] A low-serum transition expansion medium for CD16-overexpressing Vγ9Vδ2T cells includes a basal medium for Vγ9Vδ2T cell expansion culture. The basal medium can be an existing commercially available medium such as LONZA X-VIVO 15 serum-free medium, a commonly used basal cell culture medium such as RPIM-1640 medium, or other self-prepared basal cell culture media. In this embodiment, LONZA X-VIVO 15 serum-free medium is used as the basal medium. The basal medium contains interleukin-15, interleukin-2, an antioxidant, and human serum. The human serum concentration is 10%, the final concentration of interleukin-15 is 10 ng / mL, the final concentration of interleukin-2 is 150 IU / mL, and the antioxidant is N-acetylcysteine ​​at a concentration of 2 mM.

[0021] The culture medium provided in this embodiment contains 10% human serum, a concentration sufficient for normal cell culture. It can be used as the initial culture medium for the entire Vγ9Vδ2T cell low-serum transition culture system to activate cryopreserved cells. During culture, N-acetylcysteine ​​can be added according to the cell state to reduce excessively high reactive oxygen species levels.

[0022] Example 2: A low-serum transitional expansion culture medium for CD16-overexpressing Vγ9Vδ2T cells.

[0023] The difference between this embodiment and Example 1 lies in the content of human serum and the addition of antioxidants. The culture medium provided in this embodiment contains 5% human serum, and the antioxidant is a combination of N-acetylcysteine ​​and the mitochondrial-targeting antioxidant MitoTEMPO. The concentration of N-acetylcysteine ​​is 4 mM, and the concentration of MitoTEMPO is 1 μM.

[0024] The culture medium provided in this embodiment contains 5% human serum and can be used as a transition medium for the entire Vγ9Vδ2T cell low-serum transition culture system, allowing the cells to gradually adapt to the low-serum environment. During the culture process, N-acetylcysteine ​​can be added according to the cell state to reduce excessively high reactive oxygen species content.

[0025] Example 3: A low-serum transitional expansion culture medium for CD16-overexpressing Vγ9Vδ2T cells.

[0026] The difference between this embodiment and Example 2 lies in the content of human serum and the addition of antioxidants. The human serum content of the culture medium provided in this embodiment is 2%, and the antioxidant is a combination of N-acetylcysteine ​​and the mitochondrial-targeting antioxidant MitoTEMPO. The concentration of N-acetylcysteine ​​is 5 mM, and the concentration of MitoTEMPO is 2 μM.

[0027] The culture medium provided in this embodiment contains 2% human serum and can be used as the late-stage culture medium for the entire Vγ9Vδ2T cell low-serum transition culture system to massively expand Vγ9Vδ2T cells and achieve high expression of CD16. During the culture process, basal medium, human serum, interleukin-15 and interleukin-2, N-acetylcysteine, and MitoTEMPO can be added appropriately according to the cell state to ensure that the cell culture environment is suitable for massive cell expansion.

[0028] Example 4: A method for low-serum transitional expansion culture of CD16-overexpressing Vγ9Vδ2T cells.

[0029] A low-serum transitional expansion culture method for CD16-overexpressing Vγ9Vδ2T cells involves first gradually reducing the serum concentration in the culture environment, combined with interleukin-15 induction and antioxidant regulation, and then massively expanding Vγ9Vδ2T cells to express CD16 in a low-serum culture environment. The low-serum culture environment is a culture environment with a serum content equal to or lower than 2%. In this embodiment, peripheral blood cells (PBMCs) are isolated from peripheral blood of healthy donors, frozen for later use, and the basal culture medium used in this embodiment is LONZA X-VIVO 15 serum-free medium.

[0030] The low serum transition amplification culture method specifically includes the following detailed steps: Step S1: Activate the culture. Add human serum to the basal culture medium until the serum concentration reaches 10%. Resuscitate frozen peripheral blood cells, resuspend them in culture medium, and adjust the cell density to 1×10⁻⁶. 6 Cells were cultured at a concentration of 4 μM / mL, with zoledronic acid added to activate γδT cells. Interleukin-15 and interleukin-2 were then added to support initial expansion, with a final concentration of 10 ng / mL for interleukin-15 and 150 IU / mL for interleukin-2. Cells were cultured at 37°C and 5% CO2 for 3 days (days 1-3 of the entire culture process). During culture, antioxidants were added according to cell status to reduce intracellular oxidative stress. The antioxidant was N-acetylcysteine ​​at a concentration of 2-5 mM. The culture system can be implemented using shake flasks, bag filters, or bioreactor systems. Step S2: Transition culture. Human serum was added to the basal culture medium until the serum concentration reached 5%. The cells obtained in Step S1 were transferred to the freshly prepared culture medium, and interleukin-15 and interleukin-2 were added. The final concentration of interleukin-15 was 10 ng / mL, and the final concentration of interleukin-2 was 150 IU / mL. The cells were cultured at 37°C and 5% CO2 for 4 days (days 4-7 of the entire culture process). Cell growth and activity were continuously monitored during the culture process. When the cell density reached 1–2 × 10⁻⁶ cells / mL... 6 When the cell count is 1 / mL, fresh culture medium should be added as needed. In addition, antioxidants should be added according to the cell status to reduce intracellular oxidative stress. The antioxidants are N-acetylcysteine ​​and the mitochondrial-targeting antioxidant MitoTEMPO used in combination to further inhibit mitochondrial ROS. The concentration of N-acetylcysteine ​​is 2-5 mM and the concentration of MitoTEMPO is 1-2 μM. Step S3: Amplification and Culture. Human serum was added to the basal culture medium until the serum concentration reached 2%. Cells obtained in Step S2 were transferred to freshly prepared culture medium, and interleukin-15 and interleukin-2 were added. The final concentration of interleukin-15 was 10 ng / mL, and the final concentration of interleukin-2 was 150 IU / mL. Cells were cultured at 37°C and 5% CO2 for 5 days (days 8-12 of the entire culture process). During the culture process, fresh culture medium, interleukin-15, and interleukin-2 were added every 2-3 days to maintain the concentrations of interleukin-15 and interleukin-2 and to maintain cell expansion efficiency. Antioxidants were added according to the cell state to reduce intracellular oxidative stress. The antioxidants used were N-acetylcysteine ​​and the mitochondrial-targeting antioxidant MitoTEMPO, which were used in combination to further inhibit mitochondrial ROS. The concentration of N-acetylcysteine ​​was 2-5 mM, and the concentration of MitoTEMPO was 1-2 mM. μM; after culture, cells were harvested and CD16 was obtained.

[0031] The culture method in this embodiment adopts a strategy of transitioning from commonly used serum concentrations to low serum concentrations, which can reduce the uncertainty of serum source and improve the controllability of amplification and cell quality.

[0032] IL-15 can significantly induce high expression of CD16 in Vγ9Vδ2 T cells, thereby enhancing ADCC function and increasing its potential for combination with antibody drugs. Enhancing CD16 expression in Vγ9Vδ2 T cells is an important optimization measure of this method.

[0033] During in vitro expansion, activated T cells may produce excessive ROS, affecting cell function and metabolic stability. This method reduces ROS and protects the function of expanded cells by using NAC and mitochondrial-targeted antioxidants.

[0034] This method can achieve large-scale expansion of Vγ9Vδ2 T cells, obtaining a sufficient number of CD16+ cell populations with stable function, meeting the needs of in vitro research and potential clinical applications.

[0035] Example 5: Amplification efficiency and cell phenotype detection.

[0036] Peripheral blood cells from two healthy donors were used as experimental subjects, and the cell detection experiments were grouped as follows: Negative control group (No-Zol group (no zoledronate control)): Without zoledronic acid, but with the addition of interleukin-15 and interleukin-2, the culture system used was LONZA X-VIVO 15 serum-free medium + 10% human serum + antioxidant.

[0037] Control group for conventional amplification method (Conv group, conventional γδT amplification method): Zoledronic acid and interleukin-2 were added, and the culture system was RPMI-1640 + 10% human serum.

[0038] Normal serum control group (NS group): Zoledronic acid, interleukin-15, and interleukin-2 were added, and the culture system consisted of LONZA X-VIVO 15 serum-free medium, 10% human serum, and antioxidants.

[0039] The optimized mix system of this invention (Opt-Mix group, cultivation method of Example 4): Zoledronic acid, interleukin-15, and interleukin-2 were added, and the culture system was LONZA X-VIVO 15 serum-free medium + 10%-2% low concentration human serum transition + antioxidant.

[0040] Each group was cultured for 12 days. After the culture was completed, the cells were harvested, the cell count was calculated, and the cell phenotype was detected.

[0041] Amplification efficiency (large-scale amplification capability): The detection indicators included the total number of Vγ9Vδ2 T cells and the purity of Vγ9Vδ2 T cells. The results are shown in Tables 1-2 and 1-2. Figure 1-2 As shown: Table 1. Statistical results of amplification fold and proportion in each group of donor 1: Table 2. Statistical results of amplification fold and proportion in each group of donor 2: Table 1-2 shows that the total number of Vγ9Vδ2 T cells in the Opt-Mix group can reach more than 500 times that of the initial PBMCs, and the proportion of Vγ9Vδ2 T cells is about 90%. Figure 1-2 The curves showing the change in the expansion fold of Vγ9Vδ2 T cells over time under different culture systems are shown. The curves include four groups: negative control group (No-Zol), traditional expansion method group (Conv), normal serum group (NS), and the optimized system group of this invention (Opt-Mix). The expansion fold of the Opt-Mix group is no less than that of the NS group within 9–12 days, and the expansion efficiency is no less than that of the other groups.

[0042] Cell phenotype: The detection indicator was the CD16 expression ratio. Flow cytometry was used to detect the CD16 expression ratio (using an anti-human CD16 antibody marker), and the analysis showed whether the high expression ratio remained stable at 30–50%. The results are as follows: Figure 3 and 4 As shown, the proportion of CD16⁺ cells in the Opt-Mix group increased from the initial 0% to approximately 50%, which was significantly higher than that in the Conv group.

[0043] Figure 3 The study used flow cytometry to analyze the proportion of Vγ9Vδ2 T cells in the total cell population under different culture systems. The four groups of samples were labeled with Vγ9 and CD3 surface antigens, respectively, to show the dynamic changes in cell proportion. The Opt-Mix group can significantly increase the proportion of Vγ9Vδ2 T cells, and the final purity can reach more than 92.7%.

[0044] Figure 4 The proportion of CD16 expression in Vγ9Vδ2 T cells under the Opt-Mix system was detected by flow cytometry. The Opt-Mix group maintained a high CD16 expression level (about 50%-60%), suggesting that this method can effectively enhance ADCC function-related phenotypes.

[0045] ROS level: Intracellular ROS levels were detected using the DCFH-DA probe. Compared with the group without added antioxidants, the DCFH-DA fluorescence signal in the Opt-Mix group decreased by 40–60% compared with the control group, indicating a significant decrease in intracellular ROS levels.

[0046] This invention successfully achieved efficient expansion of Vγ9Vδ2 T cells with high CD16 expression, low ROS levels, and enhanced function by combining low serum transition, IL-2 and IL-15 induction with antioxidant regulation, providing a feasible cell preparation protocol for subsequent combined immunotherapy and ADCC applications.

[0047] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for low-serum transitional expansion culture of CD16-overexpressing Vγ9Vδ2T cells, characterized in that, First, the serum concentration in the culture environment is gradually reduced, combined with interleukin-15 induction and antioxidant regulation, and then Vγ9Vδ2T cells are expanded in large quantities and CD16 is expressed in large quantities in a low serum culture environment; the low serum culture environment is a culture environment with a serum content equal to or lower than 2%.

2. The method for low-serum transitional expansion culture of CD16-overexpressing Vγ9Vδ2T cells according to claim 1, characterized in that, It includes the following steps: Step S1: Activate the culture, resuscitate frozen peripheral blood cells, resuspend them in initial culture medium, and adjust the cell density to 0.5-2 × 10⁻⁶ cells / year. 6 Add zoledronic acid to activate γδT cells, then add interleukin-15 and interleukin-2, and culture for 2-3 days. During the culture process, add antioxidants according to the cell status to reduce intracellular oxidative stress. Step S2 transition culture: Transfer the cells obtained in step S1 to the transition culture medium. The serum concentration of the transition culture medium is equal to or less than 5%. Then add interleukin-15 and interleukin-2 and culture for 3-4 days. During the culture process, add antioxidants according to the cell status to reduce intracellular oxidative stress. Step S3: Amplification and culture. The cells obtained in step S2 are transferred to a late-stage culture medium with a serum concentration equal to or less than 2%. Interleukin-15 and interleukin-2 are then added, and the cells are cultured for 4-7 days. During the culture process, fresh late-stage culture medium, interleukin-15, and interleukin-2 are added every 2-3 days to maintain the concentration of interleukin-15 and interleukin-2. Antioxidants are added according to the cell state to reduce intracellular oxidative stress. After the culture is completed, the cells are harvested and CD16 is obtained.

3. The method for low-serum transitional expansion culture of CD16-overexpressing Vγ9Vδ2T cells according to claim 2, characterized in that, The initial serum concentration of the culture medium in step S1 is 5%-10%, the final concentration of zoledronic acid is 2-5 μM, and the antioxidant is 2-5 mM N-acetylcysteine.

4. The method for low-serum transitional expansion culture of CD16-overexpressing Vγ9Vδ2T cells according to claim 2, characterized in that, In steps S1-S3, the final concentration of interleukin-15 is 1-40 ng / mL, the final concentration of interleukin-2 is 100-200 IU / mL, the culture temperature is 35-45°C, and the carbon dioxide concentration is 1%-10% CO2.

5. The method for low-serum transitional expansion culture of CD16-overexpressing Vγ9Vδ2T cells according to claim 2, characterized in that, The antioxidants in steps S2 and S3 are 2–5 mM N-acetylcysteine ​​and 1–2 μM MitoTEMPO.

6. A low-serum transitional expansion medium for CD16-overexpressing Vγ9Vδ2T cells, comprising a basal medium for Vγ9Vδ2T cell expansion culture, characterized in that, Interleukin-15 was also added to the basal culture medium.

7. The low-serum transitional expansion culture medium for CD16-overexpressing Vγ9Vδ2T cells according to claim 6, characterized in that, The basal culture medium also contains interleukin-2 and antioxidants.

8. The low-serum transitional expansion culture medium for CD16-overexpressing Vγ9Vδ2T cells according to claim 7, characterized in that, The serum content of the basal culture medium is less than 5%; the antioxidant is N-acetylcysteine, or the antioxidant is a combination of N-acetylcysteine ​​and a mitochondrial-targeting antioxidant in a ratio of 1:1000.

9. A low-serum transitional expansion culture medium for CD16-overexpressing Vγ9Vδ2T cells according to claim 7 or 8, characterized in that, The serum content in the basal culture medium is less than 2%; the content of interleukin-15 is 1-40 ng / mL, the content of interleukin-2 is 100-200 IU / mL, the content of N-acetylcysteine ​​is 1-5 mM, and antioxidants are added during the culture process.

10. The low-serum transitional expansion culture medium for CD16-overexpressing Vγ9Vδ2T cells according to claim 9, characterized in that, The content of interleukin-15 is 10 ng / mL, and the content of interleukin-2 is 150 IU / mL.

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