Hybrid immune cells, culture systems and in vitro culture methods thereof

Abstract

The application discloses a kind of mixed immune cells and its culture system and in vitro culture method.The mixed immune cells include 75%~80% of γδT cell, 10%~15% of NK cell and 1~2% of NKT cell.The in vitro culture method of the application includes, when peripheral blood mononuclear cell is inoculated with activation culture medium, adding immune cell first activation component, adding immune cell reactivation component after 0~6 hours of inoculation, continuous activation culture;Then the activated culture cell is transferred into the coated culture plate or culture bottle, and the mixed immune cell is obtained by expansion culture.The mixed immune cell of the application has higher liver cancer cell HepG2 killing effect.The mixed immune cell in vitro culture method of the application has high expansion efficiency, and the expansion multiple can reach 2000-8000 times.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell culture, and particularly relates to a mixed immune cell, a culture system thereof and an in-vitro culture method. BACKGROUND

[0002] Natural killer cells, namely NK cells, are a kind of innate lymphocytes, which have the ability to quickly recognize and kill tumor cells, virus-infected cells and stressed cells without prior sensitization. T cells are the core components of the adaptive immune system, which play a key role in anti-infection, anti-tumor and tissue repair. NKT cells are a unique lymphocyte subpopulation with characteristics of both T cells and NK cells. They play an irreplaceable key role in tumor clearance, pathogen defense and immune balance regulation.

[0003] These lymphocyte subpopulations achieve effective killing of tumor cells through different recognition mechanisms and effector ways from multiple pathways. Therefore, the combined application of multiple immune cells can form functional complementation and synergy, further enhance cell proliferation activity and improve overall anti-tumor efficacy. The mixed immune cell treatment strategy not only helps to expand the coverage of immune response, but also optimizes the killing effect through the mutual regulation between cells, providing a more comprehensive and more durable mechanism for tumor immunotherapy.

[0004] Most of the existing immune cell in-vitro production technologies are to expand single immune cells, and the expansion of mixed immune cells has problems such as unstable in-vitro expansion efficiency, low desired cell purity ratio and difficulty in standardizing production process. So far, no literature or patent has proposed a mixed immune cell system that can simultaneously expand high proportion of γδT cells and certain proportion of NK cells and NKT cells in about 20 days with high efficiency. SUMMARY

[0005] The present application aims to solve the technical problem of low in-vitro culture purity of desired cells in the prior art mixed immune cells, and one of the purposes is to provide a mixed immune cell culture system.

[0006] The mixed immune cell culture system provided by the present application comprises an activation culture medium, an immune cell first activation component, an immune cell second activation component and an expansion culture medium.

[0007] Among them,

[0008] The activation culture medium comprises a cell basic culture medium, a serum substitute and a cytokine;

[0009] The immune cell first activation component is a bisphosphonate;

[0010] The immune cell second activation component is a biological reaction regulator;

[0011] The expansion medium comprises a cell basal medium, a serum substitute and a cytokine.

[0012] wherein,

[0013] The biphosphonate is added to the activation medium at a final concentration of 1-10 μmol / L when the cells are cultured in vitro.

[0014] The biological response modifier is added to the activation medium at a final concentration of no more than 5 mg / mL when the cells are cultured in vitro.

[0015] In the activation medium, the serum substitute is UltraGro; the UltraGro accounts for 5-10% of the total volume of the activation medium; the cytokine comprises human recombinant IL2, human recombinant IL15 and human recombinant IL18; the human recombinant IL2 has a final concentration of 1-100 ng / mL in the activation medium, the human recombinant IL15 has a final concentration of 1-100 ng / mL in the activation medium, and the human recombinant IL18 has a final concentration of 1-100 ng / mL in the activation medium.

[0016] In the expansion medium, the serum substitute is UltraGro; the UltraGro accounts for 5-10% of the total volume of the expansion medium; the cytokine comprises human recombinant IL2 and human recombinant IL15; the human recombinant IL2 has a final concentration of 1-100 ng / mL in the expansion medium, and the human recombinant IL15 has a final concentration of 1-100 ng / mL in the expansion medium.

[0017] In the activation medium, the UltraGro accounts for 5-10% of the total volume of the activation medium; the human recombinant IL2 has a final concentration of 50-100 ng / mL in the activation medium, the human recombinant IL15 has a final concentration of 10-50 ng / mL in the activation medium, and the human recombinant IL18 has a final concentration of 10-50 ng / mL in the activation medium. Preferably, in the activation medium, the UltraGro accounts for 10% of the total volume of the activation medium; the human recombinant IL2 has a final concentration of 50-100 ng / mL in the activation medium, the human recombinant IL15 has a final concentration of 10-50 ng / mL in the activation medium, and the human recombinant IL18 has a final concentration of 10-50 ng / mL in the activation medium.

[0018] In the expansion medium, the UltraGro accounts for 5-10% of the total volume of the expansion medium; the final concentration of the human recombinant IL2 in the expansion medium is 50-100 ng / mL, and the final concentration of the human recombinant IL15 in the expansion medium is 10-50 ng / mL. As preferred, in the expansion medium, the UltraGro accounts for 5% of the total volume of the expansion medium; the final concentration of the human recombinant IL2 in the expansion medium is 50-100 ng / mL, and the final concentration of the human recombinant IL15 in the expansion medium is 10-50 ng / mL.

[0019] In the expansion medium, the cytokine further comprises human recombinant IL18, and the final concentration of the human recombinant IL18 in the expansion medium is not more than 100 ng / mL. Preferably, the final concentration of the human recombinant IL18 in the expansion medium is not more than 20 ng / mL.

[0020] In the formula, the bisphosphonate is selected from one or more of pamidronate sodium, alendronate sodium, ibandronate sodium and zoledronate; and the biological response modifier is selected from one or more of BCG (Bacillus Calmette-Guérin), colytoxin, OK432 (lyophilized preparation of hemolytic streptococcus group A type III low-toxicity mutant, trade name Picibanil) and poly I:C.

[0021] Another object of the present application is to provide a method for culturing mixed immune cells in vitro, which comprises:

[0022] Step S1, inoculating peripheral blood mononuclear cells with the activation medium in the mixed immune cell culture system of the present application, adding the immune cell first activation component in the mixed immune cell culture system of the present application at the time of inoculation, adding the immune cell second activation component in the mixed immune cell culture system of the present application after 0-6 hours of inoculation, and continuously activating and culturing for 8 days; during the activation and culture, the activation medium is supplemented every other day;

[0023] Step S2, coating the culture plate or culture bottle with a coating solution containing soluble anti-CD3 antibody and soluble anti-CD28 antibody overnight at the day before the end of the activation and culture;

[0024] Step S3, transferring the cells obtained in step S1 into the coated culture plate or culture bottle in step S2, and using the expansion medium in the mixed immune cell culture system of the present application for expansion and culture for 18-22 days to obtain mixed immune cells.

[0025] In step S1, the final concentration of the bisphosphonate added to the activation medium is 1~10 μmol / L; the final concentration of the biological response modifier added to the activation medium does not exceed 5 mg / mL.

[0026] In step S1, the cell seeding density is (1~5)×10⁻⁶. 6 cells / mL;

[0027] In step S2, the coating solution is prepared by adding soluble anti-CD3 antibody and soluble anti-CD28 antibody to DPBS at a final concentration of 0.5~2μg / mL, and then coating culture plates or culture flasks overnight at 4°C.

[0028] In step S3, during days 11-22 of the amplification culture, the cell count is performed every other day, and the amplification medium in the mixed immune cell culture system is replenished to adjust the cell density to (0.4-1.5)×10⁻⁶. 6 Between [number] cells / mL.

[0029] Another object of the present invention is to provide mixed immune cells cultured by the in vitro culture method of the present invention. In the present invention, the mixed immune cells comprise 75%~80% γδT cells, 10%~15% NK cells and 1%~2% NKT cells.

[0030] The positive and progressive effects of this invention are as follows:

[0031] 1. The mixed immune cells of this invention comprise 75%–80% γδT cells, 10%–15% NK cells, and 1%–2% NKT cells, with other types of immune cells accounting for a lower proportion. Among these, NKT cells co-expressing CD3 and CD56 account for 1%–2% of the total cells. The mixed immune cells of this invention exhibit a high killing effect on HepG2 liver cancer cells.

[0032] 2. The mixed immune cell culture system and in vitro culture method of the present invention have high amplification efficiency and can be harvested stably at 20-22 days; compared with the inoculation quantity, the amplification multiple can reach 2000-8000 times, which can meet clinical needs. The in vitro culture method of the present invention has the characteristics of simple operation, low culture technical difficulty, high cell viability, large quantity, and strong cell killing toxicity, which greatly reduces the culture cost and has broad clinical application prospects. Attached Figure Description

[0033] Figure 1 This is a proliferation curve of mixed immune cells from batch 0304;

[0034] Figure 2Flow cytometry images of mixed immune cells in 0304 batch at 16 days of expansion culture;

[0035] Figure 3 Killing efficiency of mixed immune cells in 0304, 0401, 0402, 0403, 0501 batches on hepatoma cells HepG2 at 16 days of expansion culture, at an effector-target ratio of 5:1. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present application are described in detail below. The raw materials and equipment used in the present application are known products, which are obtained by purchasing commercially available products, wherein the cell base medium is OptiVitro®NK cell serum-free base medium P01, purchased from Suzhou Yikesai Biotechnology Co., Ltd.; the serum substitute UltraGro mesenchymal stem cell nutritional additive is purchased from AventaCell BioMedical Company; the recombinant human IL2, IL15 and IL18 are purchased from Shanghai Jinan Technology Co., Ltd.; zoledronic acid is zoledronic acid for injection, and the manufacturer is Guojia Group Guorui Pharmaceutical Co., Ltd.; Anti-CD3 antibody and anti-CD28 antibody are purchased from Biaopu Saisi Biological Technology Co., Ltd.

[0037] Example 1 Mixed immune cell culture system

[0038] The preparation method of the activation medium is as follows: add serum substitute UltraGro to the cell base medium, accounting for 10% of the total volume of the activation medium; add human recombinant IL2, with a final concentration of 50 ng / mL; add human recombinant IL15, with a final concentration of 10 ng / mL; and add human recombinant IL18, with a final concentration of 10 ng / mL.

[0039] The first activation component of the immune cells is zoledronic acid;

[0040] The second activation component of the immune cells is BCG;

[0041] The preparation method of the expansion medium is as follows: add serum substitute UltraGro to the cell base medium, with an addition amount accounting for 5% of the total volume of the expansion medium; add human recombinant IL2, with a final concentration of 50 ng / mL; and add human recombinant IL15, with a final concentration of 10 ng / mL.

[0042] Example 2 In vitro expansion culture of mixed immune cells (0304 batch)

[0043] Isolation of peripheral blood mononuclear cells (PBMCs): Fresh human peripheral blood is collected, and PBMC cells are isolated using density gradient centrifugation method. The upper plasma is discarded, and the white membrane layer cells are carefully collected. After washing twice with DPBS, the PBMC cells are obtained after centrifugation.

[0044] Inoculation and activation of cells: Resuspend the PBMC cells prepared in Example 1 with the activation medium prepared in Example 1 and count, and adjust the density of the PBMC cells to 2-3 x 10 6 6 hours after inoculation, add BCG to the cell suspension to a final concentration of 2 mg / mL for stimulation, and place in a 37°C, 5% CO2 incubator for continued activation culture for 8 days. On days 2-7 of the activation culture, according to visual observation of the cell aggregation state, use the activation medium prepared in Example 1 to supplement the liquid every other day (where the cell inoculation date is designated as day 0, and supplement 1 mL of the activation medium on days 2 and 4, and 2 mL of the activation medium on day 6, and transfer the cells to a 6-well plate on day 6).

[0045] Culture bottle coating: On day 7 of inoculation, add soluble anti-CD3 antibody to a final concentration of 1 μg / mL and soluble anti-CD28 antibody to a final concentration of 1 μg / mL to DPBS to prepare a coating solution, and add to the T225 culture bottle for overnight coating of the culture bottle at 4°C.

[0046] Expansion culture: On day 8 of inoculation, discard the coating solution in the culture bottle, transfer the cell suspension to the coated culture bottle, and use the expansion medium prepared in Example 1 to supplement the liquid and adjust the cell density to 1 x 10 6 On days 9 and 10 after inoculation, supplement with an equal volume of the expansion medium every day. On day 11 after inoculation, transfer the cells to a cell culture bag for expansion culture, take a sample for counting, supplement with the expansion medium and adjust the cell density to (0.8-1.0) x 10 6 On days 12-20, take a sample of the cells for counting every other day, supplement with the expansion medium, and adjust the cell density to (0.8-1.0) x 10 6 On days 12-20, take a sample of the cells for counting every other day, supplement with the expansion medium, and adjust the cell density to (0.8-1.0) x 10

[0047] 0304The proliferation curve of the mixed immune cells of batch is shown in Figure 1 From Figure 1 it can be concluded that the expansion can reach 2000-8000 times, and the multiples vary from 2000 times to 8000 times according to the slight difference in the harvest time point (20-22 days).

[0048] Example 3 In vitro expansion culture of mixed immune cells (0401 batch)

[0049] The same culture method as in Example 2 was adopted, except that BCG was added to the cell suspension to a final concentration of 2 mg / mL for stimulation at 0 hour after PBMC cell inoculation.

[0050] Example 4 Mixed immune cell in vitro expansion culture (0402 batch)

[0051] The same culture method as in Example 2 was adopted, except that BCG was added to the cell suspension to a final concentration of 2 mg / mL for stimulation at 2 hours after PBMC cell inoculation.

[0052] Example 5 Mixed immune cell in vitro expansion culture (0403 batch)

[0053] The same culture method as in Example 2 was adopted, except that BCG was added to the cell suspension to a final concentration of 2 mg / mL for stimulation at 4 hours after PBMC cell inoculation.

[0054] Example 6 Mixed immune cell in vitro expansion culture (0201 batch)

[0055] The same culture method as in Example 2 was adopted, except that the first activation component of the immune cells was ibandronate sodium, and ibandronate sodium was added to a final concentration of 10 μmol / L for stimulation after PBMC inoculation.

[0056] Example 7 Mixed immune cell in vitro expansion culture (0202 batch)

[0057] The same culture method as in Example 2 was adopted, except that the second activation component of the immune cells was OK432, and OK432 was added to the cell suspension to a final concentration of 1 μg / mL for stimulation at 6 hours after PBMC cell inoculation.

[0058] Comparative Example 1 Mixed immune cell in vitro expansion culture (0501 batch)

[0059] The same culture method as in Example 2 was adopted, except that BCG was added to the cell suspension to a final concentration of 2 mg / mL for stimulation at 24 hours after PBMC cell inoculation.

[0060] Comparative Example 2 Mixed immune cell in vitro expansion culture (0502 batch)

[0061] The same culture method as in Example 2 was adopted, except that no second activation component was added for stimulation after PBMC cell inoculation.

[0062] Example 8 Flow cytometry detection of cell phenotype

[0063] The cells were collected at the 11th, 14th and 16th day of the mixed immune cell expansion culture in Examples 2-7 (counted from the inoculation as the 0th day), and the CD3+TCRgama+, CD3-CD56+and CD3+TCRgama-CD56+phenotype indicators of the cells were detected using a BD FACSLyric flow cytometer.

[0064] In the 0304 batch of mixed immune cells, the proportion of CD3+TCRgama+cells was about 77.37% at the 16th day of the culture time, i.e., the proportion of γδT cells was about 77.37%; the proportion of CD3-CD56+cells was about 13.49%, i.e., the proportion of NK cells was about 13.49%, and the proportion of CD3+TCRgama-CD56+cells was about 1.43%, i.e., the proportion of NKT cells was about 1.43%; as shown in Table 1, Figure 2

[0065] In the 0401 batch of mixed immune cells, the proportion of γδT cells was about 77.11% at the 16th day of the culture time; the proportion of NK cells was about 10.78%, and the proportion of NKT cells was about 1.75%; as shown in Table 1.

[0066] In the 0402 batch of mixed immune cells, the proportion of γδT cells was about 76.25% at the 16th day of the culture time; the proportion of NK cells was about 12.02%, and the proportion of NKT cells was about 1.47%; as shown in Table 1.

[0067] In the 0403 batch of mixed immune cells, the proportion of γδT cells was about 76.73% at the 16th day of the culture time; the proportion of NK cells was about 13.10%, and the proportion of NKT cells was about 1.37%; as shown in Table 1.

[0068] In the 0201 batch of mixed immune cells, the proportion of γδT cells was about 75.36% at the 16th day of the culture time; the proportion of NK cells was about 14.26%, and the proportion of NKT cells was about 1.63%; as shown in Table 1.

[0069] In the 0202 batch of mixed immune cells, the proportion of γδT cells was about 77.53% at the 16th day of the culture time; the proportion of NK cells was about 10.08%, and the proportion of NKT cells was about 1.05%; as shown in Table 1.

[0070] Table 1: Phenotype flow cytometry proportion of each batch of mixed immune cells at the 16th day of expansion culture

[0071]

[0072] In addition, the 0501 and 0502 batches of mixed immune cells of Comparative Examples 1 and 2 were also detected.

[0073] ​The proportion of CD3+TCRgama+ cells in the mixed immune cells of the 0501 batch was about 50.56% at the 16th day of the culture time, that is, the proportion of γδT cells was about 50.56%; the proportion of CD3-CD56+ cells was about 3.03%, that is, the proportion of NK cells was about 3.03%, and the proportion of CD3+TCRgama-CD56+ cells was about 17.59%, that is, the proportion of NKT cells was about 17.59%; as shown in Table 2.

[0074] The proportion of γδT cells in the mixed immune cells of the 0502 batch was about 35.54% at the 16th day of the culture time; the proportion of NK cells was about 23.44%, and the proportion of NKT cells was about 13.18%; as shown in Table 2.

[0075] Table 2: Proportions of mixed immune cells of 0501 and 0502 batches at the 16th day of the expansion culture

[0076]

[0077] Conclusion: According to the comparison of the data of the 0304, 0401, 0402, 0403, 0201, and 0202 batches of cells with the 0501 and 0502 batches of cells, it can be concluded that the mixed immune cells with the following cell proportion ranges can be considered as the desired mixed immune cell type of the present application, wherein the proportion of γδT cells is about 75% to 80%, the proportion of NK cells is about 10% to 15%, and the proportion of NKT cells is about 1% to 2%.

[0078] Example 9: Killing experiment of mixed immune cells

[0079] The mixed immune cells of the 0304, 0401, 0402, 0403, and 0501 batches were collected at the 16th day of the expansion culture, transferred into a 12-well culture plate, and co-cultured with liver cancer cells HepG2 (HepG2 was labeled with CFSE staining before co-culture) for 4 h. After 4 h, all cells were collected, centrifuged, the supernatant was discarded, resuspended with DPBS, stained with 7-AAD to distinguish between dead and live cells, and the killing efficiency of the mixed immune cells of each batch on the liver cancer cells HepG2 was detected using a BD FACSLyric flow cytometer, and calculated and analyzed, as shown in Table 3. Figure 3

[0080] ​Conclusion: From the above examples, it can be concluded that the proportion of gammadelta T cells and NK cells of the mixed immune cells (0304, 0401, 0402, 0403 batches) obtained by the present application is significantly higher than that of the comparative example (0501 batch). There is a difference in cell killing toxicity, and the killing efficiency of the 0304, 0401, 0402, 0403 batch cells is obviously better than that of the 0501 batch. That is, the mixed immune cells cultured according to the culture system and culture method provided by the present application have high proportion of target cell types, high expansion fold, and strong killing toxicity.

[0081] The above embodiments of the present application are described in detail in combination with the drawings, and those of ordinary skill in the art can make various changes to the present application according to the above description. Therefore, certain details in the embodiments should not constitute a limitation on the present application, and the scope of protection of the present application will be defined by the appended claims.

Claims

1. A mixed immune cell culture system, characterized by, The culture system consists of an activation medium, a first immune cell activation component, a second immune cell activation component, and an expansion medium; wherein, The activation medium consists of a cell basal medium, a serum substitute, and a cytokine; The first immune cell activation component is a bisphosphonate; The second immune cell activation component is a biological response modifier selected from one or more of BCG, Coley's toxin, OK432, and poly I:C; The expansion medium consists of a cell basal medium, a serum substitute, and a cytokine; In the activation medium, the serum substitute accounts for 5-10% of the total volume of the activation medium; the cytokine includes human recombinant IL2, human recombinant IL15, and human recombinant IL18; the final concentration of human recombinant IL2 in the activation medium is 1-100 ng / mL, the final concentration of human recombinant IL15 in the activation medium is 1-100 ng / mL, and the final concentration of human recombinant IL18 in the activation medium is 1-100 ng / mL; In the expansion medium, the serum substitute accounts for 5-10% of the total volume of the expansion medium; the cytokine includes human recombinant IL2 and human recombinant IL15; the final concentration of human recombinant IL2 in the expansion medium is 1-100 ng / mL, and the final concentration of human recombinant IL15 in the expansion medium is 1-100 ng / mL; The final concentration of the biological response modifier added in the activation medium is not more than 5 mg / mL.

2. The mixed immune cell culture system of claim 1, wherein, In the activation medium, the serum substitute is UltraGro; In the expansion medium, the serum substitute is UltraGro.

3. The mixed immune cell culture system of claim 2, wherein, In the activation medium, the UltraGro accounts for 5-10% of the total volume of the activation medium; the final concentration of human recombinant IL2 in the activation medium is 50-100 ng / mL, the final concentration of human recombinant IL15 in the activation medium is 10-50 ng / mL, and the final concentration of human recombinant IL18 in the activation medium is 10-50 ng / mL; In the expansion medium, the UltraGro accounts for 5-10% of the total volume of the expansion medium; the final concentration of human recombinant IL2 in the expansion medium is 50-100 ng / mL, and the final concentration of human recombinant IL15 in the expansion medium is 10-50 ng / mL.

4. The mixed immune cell culture system of claim 3, wherein the immune cell is a T cell. In the expansion medium, the cytokine further includes human recombinant IL18, and the final concentration of human recombinant IL18 in the expansion medium is not more than 100 ng / mL.

5. The mixed immune cell culture system of claim 4, wherein the immune cell is a T cell. The final concentration of human recombinant IL18 in the expansion medium is not more than 20 ng / mL.

6. The mixed immune cell culture system of claim 1, wherein, The bisphosphonate is selected from one or more of pamidronate, alendronate, ibandronate, and zoledronate.

7. A method for mixed lymphocyte culture in vitro, characterized by, The method comprises: Step S1, peripheral blood mononuclear cells are inoculated with the activation medium in the mixed immune cell culture system according to any one of claims 1-6, the first activation component of immune cells in the mixed immune cell culture system according to any one of claims 1-6 is added at the time of inoculation, the second activation component of immune cells in the mixed immune cell culture system according to any one of claims 1-6 is added after 0-6 hours of inoculation, and continuous activation culture is performed for 8 days; during the activation culture, the activation medium is supplemented every other day; Step S2, at the last day before the end of the activation culture, the culture plate or culture bottle is coated overnight with a coating solution containing soluble anti-CD3 antibody and soluble anti-CD28 antibody; Step S3, the cells obtained in step S1 are transferred into the coated culture plate or culture bottle in step S2, and the expansion medium in the mixed immune cell culture system according to any one of claims 1-6 is used for expansion culture for 18-22 days to obtain mixed immune cells.

8. The mixed immune cell in vitro culture method according to claim 7, wherein, In step S1, the final concentration of the bisphosphonate added in the activation medium is 1-10 μmol / L; and the final concentration of the biological reaction regulator added in the activation medium is not more than 5 mg / mL.

9. The mixed immune cell in vitro culture method according to claim 7, wherein, In step S1, the cell seeding density is (1-5) x 10 6 cells / mL; In step S2, the coating solution is prepared by adding soluble anti-CD3 antibody at a final concentration of 0.5-2 μg / mL and soluble anti-CD28 antibody at a final concentration of 0.5-2 μg / mL into DPBS, and coating the culture plate or culture bottle overnight at 4°C; In step S3, the expansion culture medium in the mixed immune cell culture system according to any one of claims 1 to 6 is counted and supplemented every other day during the period of 11th to 22nd day of the expansion culture, and the cell density is adjusted to be between (0.4 to 1.5) x 10 6 cells / mL.

10. The mixed immune cells obtained by the method of claim 7, wherein the mixed immune cells are obtained by the method of claim 7. The mixed immune cells comprise 75%-80% γδ T cells, 10%-15% NK cells, and 1-2% NKT cells.

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