Culture method of DC-CIK cells sensitive to nasopharyngeal carcinoma cells

Abstract

The invention provides a culture method of DC-CIK cells sensitive to nasopharyngeal carcinoma cells, and belongs to the technical field of cell culture. The culture method of the DC-CIK cells sensitive to nasopharyngeal carcinoma cells comprises the following steps: (1) adding an astragalus mongholicus fermentation extract into a co-culture medium of DC cells and CIK cells to obtain a cell culture medium; and (2) mixing DC cells and CIK cells with the cell culture medium obtained in the step (1), and culturing to obtain the DC-CIK cells sensitive to nasopharyngeal carcinoma cells. The radix astragali fermentation extract is added into the co-culture medium of the DC cells and the CIK cells, so that the killing activity of the DC-CIK cells obtained through culture on nasopharynx cancer CNE2 cells is remarkably improved.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to a method for culturing DC-CIK cells which are sensitive to nasopharyngeal carcinoma cells. Background Art

[0002] Nasopharyngeal carcinoma (NPC) is an epithelial malignancy originating from the nasopharyngeal mucosa. Its occurrence is related to Epstein-Barr virus infection, environmental factors, and genetic factors. Currently, the treatment of NPC is mainly radiotherapy, but the clinical effect of chemotherapy is limited, and some patients still face the risk of recurrence or metastasis. Therefore, it is urgent to find new safe and effective methods to treat NPC.

[0003] Cytokine-induced Killer (CIK) cells are a heterogeneous cell population obtained by in vitro culture, which has the characteristics of strong in vitro proliferation ability and high tumor killing activity. Dendritic cells (DC) are special immune cells with powerful antigen presentation function, which play an important role in the immune system. Studies have shown that after co-culturing CIK cells with DC cells, the co-cultured DC-CIK cells have a significantly higher killing effect on a variety of tumor cells than CIK cells alone, but the effect of DC-CIK co-cultured cells on nasopharyngeal carcinoma cells is not very ideal. Therefore, it is necessary to provide a culture method that can improve the sensitivity of DC-CIK cells to nasopharyngeal carcinoma cells, so as to provide a theoretical basis for the use of DC-CIK cells to efficiently kill nasopharyngeal carcinoma cells in clinical treatment. Summary of the invention

[0004] The purpose of the present invention is to provide a method for culturing DC-CIK cells that are sensitive to nasopharyngeal carcinoma cells, and to provide a theoretical basis for using DC-CIK cells to efficiently kill nasopharyngeal carcinoma cells in clinical treatment.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for culturing DC-CIK cells sensitive to nasopharyngeal carcinoma cells, comprising the following steps:

[0007] (1) adding the fermented Astragalus extract to a co-culture medium of DC cells and CIK cells to obtain a cell culture medium;

[0008] (2) DC cells and CIK cells are mixed with the cell culture medium obtained in step (1) and cultured to obtain DC-CIK cells that are sensitive to nasopharyngeal carcinoma cells.

[0009] Preferably, the co-culture medium in step (1) is a serum-free medium containing 4-6% autologous plasma and 400-600 IU / mL IL-2.

[0010] Preferably, the serum-free culture medium is Lonza X-VIVO 15 serum-free culture medium.

[0011] Preferably, the fermented astragalus extract in step (1) is obtained by fermenting an astragalus extract with Bacillus subtilis and Bacillus licheniformis.

[0012] Preferably, the astragalus extract is an astragalus water extract.

[0013] Preferably, the final concentration of Bacillus subtilis in the extract during fermentation is 1×10 7 CFU / mL~1×10 9 CFU / mL, the final concentration of Bacillus licheniformis was 1×10 5 CFU / mL~1×10 7 CFU / mL; the fermentation temperature is 35-40°C and the fermentation time is 20-28h.

[0014] Preferably, the concentration of the Astragalus fermentation extract in the co-culture medium in step (1) is 0.1-1.0 mg / mL.

[0015] Preferably, in step (2), the ratio of the number of DC cells to CIK cells is 0.8-1.2:8-12, the temperature during the culture is 35-40° C., and the time is 4-10 days.

[0016] The present invention provides DC-CIK cells cultured according to the method.

[0017] The present invention also provides an application of the DC-CIK cells in preparing products against nasopharyngeal carcinoma.

[0018] The present invention provides a method for culturing DC-CIK cells that are sensitive to nasopharyngeal carcinoma cells. By adding an astragalus fermentation extract to the co-culture medium of DC cells and CIK cells, the killing activity of the cultured DC-CIK cells on nasopharyngeal carcinoma CNE2 cells is significantly improved. The method of the present invention provides a new idea for culturing DC-CIK cells that are highly sensitive and have killing activity to nasopharyngeal carcinoma cells. DETAILED DESCRIPTION

[0019] The present invention provides a method for culturing DC-CIK cells sensitive to nasopharyngeal carcinoma cells, comprising the following steps:

[0020] (1) adding the fermented Astragalus extract to a co-culture medium of DC cells and CIK cells to obtain a cell culture medium;

[0021] (2) DC cells and CIK cells are mixed with the cell culture medium obtained in step (1) and cultured to obtain DC-CIK cells that are sensitive to nasopharyngeal carcinoma cells.

[0022] In the present invention, the co-culture medium in step (1) is a serum-free medium containing 4-6% autologous plasma and 400-600 IU / mL IL-2, preferably a serum-free medium containing 4.5-5.5% autologous plasma and 450-550 IU / mL IL-2, and further preferably a serum-free medium containing 5% autologous plasma and 500 IU / mL IL-2.

[0023] In the present invention, the serum-free culture medium is Lonza X-VIVO 15 serum-free culture medium.

[0024] In the present invention, the fermented astragalus extract in step (1) is obtained by fermenting the astragalus extract with Bacillus subtilis and Bacillus licheniformis.

[0025] In the present invention, the astragalus extract is an astragalus water extract.

[0026] In the present invention, the final concentration of Bacillus subtilis in the extract during fermentation is 1×10 7 CFU / mL~1×10 9 CFU / mL, preferably 5×10 7 CFU / mL~5×10 8 CFU / mL, more preferably 1×10 8 CFU / mL, the final concentration of Bacillus licheniformis was 1×10 5 CFU / mL~1×10 7 CFU / mL, preferably 5×10 5 CFU / mL~5×10 6 CFU / mL, more preferably 1×10 6 CFU / mL; the fermentation temperature is 35-40°C, preferably 36-38°C, more preferably 37°C, and the fermentation time is 20-28h, preferably 22-26h, more preferably 24h.

[0027] In the present invention, the concentration of the Astragalus fermentation extract in step (1) in the co-culture medium is 0.1-1.0 mg / mL, preferably 0.2-0.8 mg / mL, more preferably 0.4-0.6 mg / mL, and more preferably 0.5 mg / mL.

[0028] In the present invention, the ratio of the number of DC cells to CIK cells in step (2) is 0.8-1.2:8-12, preferably 0.9-1.1:9-11, and more preferably 1:10. The temperature during the culture is 35-40°C, preferably 36-38°C, and more preferably 37°C. The time is 4-10 days, preferably 5-9 days, more preferably 6-8 days, and more preferably 7 days.

[0029] The present invention provides DC-CIK cells cultured according to the method.

[0030] The present invention also provides an application of the DC-CIK cells in preparing products against nasopharyngeal carcinoma.

[0031] The technical solutions provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention. The Bacillus subtilis and Bacillus licheniformis used in the following examples were purchased from the China Industrial Microbiological Culture Collection Administration Center.

[0032] Example 1 Acquisition of DC cells and CIK cells

[0033] 1. Preparation of peripheral blood mononuclear cells

[0034] Mix the peripheral blood with an equal volume of PBS and slowly add it to a centrifuge tube containing cell separation solution (Ficoll), centrifuge at 2000rpm for 20min, use a pipette to draw the peripheral blood mononuclear cells and add them to a centrifuge tube containing PBS, centrifuge at 8500rpm for 10min, remove the supernatant, resuspend the precipitate with PBS and centrifuge at 2000rpm for 5min, the resulting precipitate is the peripheral blood mononuclear cell (PBMC). Resuspend the obtained peripheral blood mononuclear cells in LonzaX-VIVO 15 serum-free medium and adjust the cell concentration to 1×10 6 Pieces / mL.

[0035] 2. Culture of DC cells

[0036] The above-mentioned resuspended PBMC cells were inoculated into a T75 culture flask and statically cultured for 3 hours to obtain adherent cells (non-adherent cells were collected for later use); LonzaX-VIVO 15 serum-free medium containing 50 ng / mL IL-4 and 100 ng / mL GM-CSF was added to the culture flask of adherent cells, and cultured in a 37°C, 5% CO2 incubator. After 5 days of culture, GM-CSF was added to a final concentration of 50 ng / mL. After continuing to culture for 1 day, IFN-γ was added to a final concentration of 1000 IU / mL and LPS was added to a final concentration of 100 ng / mL. The culture was continued for 7 days to obtain DC cells.

[0037] 3. CIK cell culture

[0038] Take the cells that did not adhere to the wall during the DC cell culture process, transfer them to a new T75 culture flask, and adjust the cell concentration to 1×10 6 / mL, add IFN-γ to 100 ng / mL and OKT-3 monoclonal antibody to 50 ng / mL in Lonza X-VIVO 15 serum-free medium, and culture in a 37°C, 5% CO2 incubator; after 24 hours of culture, add IL-2 to 500 IU / mL, and continue to culture for 3 days and then add Lonza X-VIVO 15 serum-free medium to maintain the cell concentration at 1×10 6 / mL, and IL-2 was added to 500IU / mL, and then cultured for 3 days to obtain CIK cells.

[0039] Example 2

[0040] A method for preparing an astragalus fermented extract comprises the following steps:

[0041] (1) Grind the root of Astragalus membranaceus and pass it through a 20-mesh sieve. Take the sieved material and add 10 times the weight of water to boil for 1 hour. Filter the residue and add 5 times the weight of water to boil for another 1 hour. Combine the filtrates after the two decoctions to obtain an Astragalus membranaceus water extract.

[0042] (2) Inoculate Bacillus subtilis and Bacillus licheniformis into the Astragalus membranaceus water extract obtained in step (1) to adjust the final concentration of Bacillus subtilis to 1×10 8 CFU / mL, the final concentration of Bacillus licheniformis was 1×10 6 CFU / mL, fermented at 37℃ for 24h to obtain the Astragalus fermentation extract.

[0043] (3) The astragalus fermentation extract obtained in step (2) was centrifuged at 3000 rpm for 10 min, and the liquid portion was concentrated under reduced pressure at a temperature of 45° C. and a vacuum degree of 0.1 MPa to a solid content of about 30%, and then vacuum dried at a temperature of 120° C. and a pressure of 20 KPa for 10 min to obtain an astragalus fermentation extract.

[0044] Example 3

[0045] A method for culturing DC-CIK cells sensitive to nasopharyngeal carcinoma cells, comprising the following steps:

[0046] (1) The Astragalus fermentation extract obtained in Example 2 was added to Lonza X-VIVO 15 serum-free culture medium containing 5% autologous plasma and 500 IU / mL IL-2 to adjust the concentration of the Astragalus fermentation extract to 0.5 mg / mL to obtain a cell culture medium.

[0047] (2) The DC cells and CIK cells cultured in Example 1 were counted and added to the cell culture medium prepared in step (1) at a ratio of 1:10 to adjust the cell concentration to 1×10 6 / mL, and cultured in a 37°C, 5% CO2 incubator for 7 days to obtain DC-CIK cells sensitive to nasopharyngeal carcinoma cells.

[0048] Comparative Example 1

[0049] A method for culturing DC-CIK cells, comprising the following steps:

[0050] The DC cells and CIK cells cultured in Example 1 were counted and added to Lonza X-VIVO 15 serum-free medium containing 5% autologous plasma and 500 IU / mL IL-2 at a ratio of 1:10, and cultured in a 37° C., 5% CO 2 incubator for 7 days to obtain DC-CIK cells.

[0051] Comparative Example 2

[0052] A method for culturing DC-CIK cells, comprising the following steps:

[0053] (1) Grind the root of Astragalus membranaceus and pass it through a 20-mesh sieve. Take the sieved material and add 10 times the weight of water to boil for 1 hour. Filter the residue and add 5 times the weight of water to boil for another 1 hour. Combine the filtrates after the two decoctions to obtain an Astragalus membranaceus water extract.

[0054] (2) Inoculate Bacillus licheniformis into the Astragalus membranaceus water extract obtained in step (1) to adjust the final concentration of Bacillus licheniformis to 1×10 8 CFU / mL, fermented at 37℃ for 24h to obtain the Astragalus fermentation extract.

[0055] (3) The astragalus fermentation extract obtained in step (2) was centrifuged at 3000 rpm for 10 min, and the liquid portion was concentrated under reduced pressure at a temperature of 45° C. and a vacuum degree of 0.1 MPa to a solid content of about 30%, and then vacuum dried at a temperature of 120° C. and a pressure of 20 KPa for 10 min to obtain an astragalus fermentation extract.

[0056] (4) Add the Astragalus fermentation extract obtained in step (3) to Lonza X-VIVO 15 serum-free culture medium containing 5% autologous plasma and 500 IU / mL IL-2, and adjust the concentration of the Astragalus fermentation extract to 0.5 mg / mL to obtain a cell culture medium.

[0057] (5) The DC cells and CIK cells cultured in Example 1 were counted and added to the cell culture medium prepared in step (4) at a ratio of 1:10 to adjust the cell concentration to 1×10 6 / mL, and cultured in a 37°C, 5% CO2 incubator for 7 days to obtain DC-CIK cells sensitive to nasopharyngeal carcinoma cells.

[0058] Test example

[0059] The purpose of this test example is to determine the killing activity of cultured DC-CIK cells against nasopharyngeal carcinoma CNE2 cells using the MTT method.

[0060] CNE2 cells in the logarithmic growth phase were inoculated in a 96-well plate and cultured in a 37°C, 5% CO2 incubator for 24 hours. The DC-CIK cells and CNE2 cells obtained by culture in Example 3 and Comparative Examples 1 to 2 were mixed in a 96-well plate at an effector-target ratio of 1:10, 1:20 and 1:30, respectively, and 3 replicates were set for each treatment. After 24 hours of culture, 30 μL of 5 mg / mL MTT was added to each well and incubated for 4 hours. After the incubation, the supernatant was removed, 100 μL DMSO was added to each well and shaken for 30 minutes, and the absorbance (A) was measured at 590 nm using an enzyme marker, and the killing rate of each DC-CIK cell on nasopharyngeal carcinoma CNE2 cells was calculated. The results are shown in Table 1.

[0061] Killing rate (%) = 1 - {(experimental well A590 - effector well A590) / target cell well A590} × 100%

[0062] Table 1 Killing rate of DC-CIK cells on nasopharyngeal carcinoma CNE2 cells (%)

[0063] Treatment Group 1：10 1：20 1：30 Example 3 56.69±3.23 77.25±6.28 92.24±4.57 Comparative Example 1 41.67±4.36 58.41±9.10 73.62±2.94 Comparative Example 2 49.53±5.14 70.68±4.97 85.02±6.25

[0064] As can be seen from Table 1, within the effect-target ratio range of 1:10 to 30, the killing activity of DC-CIK cells cultured by the methods of Example 3 and Comparative Example 2 on nasopharyngeal carcinoma CNE2 cells is significantly higher than that of DC-CIK cells cultured by the method of Comparative Example 1 at the same effect-target ratio, indicating that the addition of Astragalus fermentation extract when DC cells and CIK cells are co-cultured in the present invention is beneficial to improving the sensitivity of DC-CIK cells to nasopharyngeal carcinoma cells; at the same time, the Astragalus fermentation broth extract prepared by inoculating Astragalus water extract with Bacillus subtilis and Bacillus licheniformis for mixed fermentation in Example 3 also has the effect of significantly improving the killing activity of cultured DC-CIK cells on nasopharyngeal carcinoma CNE2 cells compared to the Astragalus fermentation broth extract prepared by inoculating Bacillus licheniformis alone for fermentation in Comparative Example 2. The method of the present invention provides a new idea for culturing DC-CIK cells that efficiently kill nasopharyngeal carcinoma cells.

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for culturing DC-CIK cells sensitive to nasopharyngeal carcinoma cells, characterized in that: The steps include: (1) adding the fermented Astragalus extract to a co-culture medium of DC cells and CIK cells to obtain a cell culture medium; (2) DC cells and CIK cells are mixed with the cell culture medium obtained in step (1) and cultured to obtain DC-CIK cells that are sensitive to nasopharyngeal carcinoma cells.

2. The method according to claim 1, characterized in that The co-culture medium in step (1) is a serum-free medium containing 4-6% autologous plasma and 400-600 IU / mL IL-2.

3. The method according to claim 2, characterized in that The serum-free culture medium is Lonza X-VIVO 15 serum-free culture medium.

4. The method according to claim 3, characterized in that The astragalus fermented extract in step (1) is obtained by fermenting the astragalus extract with Bacillus subtilis and Bacillus licheniformis.

5. The method according to claim 4, characterized in that The astragalus extract is an astragalus water extract.

6. The method according to claim 5, characterized in that The final concentration of Bacillus subtilis in the extract during fermentation was 1×10 7 CFU / mL~1×10 9 CFU / mL, the final concentration of Bacillus licheniformis was 1×10 5 CFU / mL~1×10 7 CFU / mL; the fermentation temperature is 35-40°C and the fermentation time is 20-28h.

7. The method according to claim 6, characterized in that The concentration of the Astragalus fermentation extract in the co-culture medium in step (1) is 0.1-1.0 mg / mL.

8. The method according to any one of claims 1 to 7, characterized in that: In step (2), the ratio of the number of DC cells to CIK cells is 0.8-1.2:8-12, the temperature during the culture is 35-40° C., and the time is 4-10 days.

9. DC-CIK cells cultured according to the method of any one of claims 1 to 8.

10. Use of the DC-CIK cells according to claim 9 in preparing a product against nasopharyngeal carcinoma.