A CIK cell culture method and application thereof

Abstract

The present invention relates to the field of cell culture technology, and in particular to a CIK cell culture method and application thereof. The culture method comprises the following steps: (1) isolating peripheral blood mononuclear cells (PBMC); (2) preparing culture medium A; (3) preparing culture medium B; (4) resuspending and inoculating peripheral blood mononuclear cells; and (5) collecting cell culture. The culture method of the present invention can improve the yield of CIK cells, and wherein CD3 + CD56 + The proportion of double-positive cells is high, which increases cell proliferation activity and promotes the increase of CIK cell proliferation and killing activity.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a CIK cell culture method and application thereof. Background Art

[0002] CIK cells (cytokine induced killer) are immune active cells. They are autologous peripheral blood mononuclear cells that are induced and cultured by multiple cytokines to produce a type of killer cell that co-expresses two membrane protein molecules, CD3+ and CD56+. CIK cells have anti-tumor activity and non-MHC tumor killing properties. They are effector cells with the characteristics of rapid proliferation, high tumor killing activity, and a wide spectrum of tumor killing. CIK cells have the characteristics of low toxicity to normal tissues and high in vitro proliferation. They have become an important program for clinical tumor adoptive immunotherapy and play an important role in preventing tumor recurrence and inhibiting tumor metastasis. However, the content of CIK cells in normal peripheral blood is low. Therefore, in in vitro culture, increasing the absolute number of CIK cells is crucial to improving the clinical treatment effect. CIK cells can proliferate in large numbers through in vitro induction. The general CIK preparation method is to treat the isolated peripheral blood mononuclear cells (PBMC) with IFN-γ for 24 hours, then add CD3mAb, IL-1α and IL-2 factors for stimulation and induction, and finally obtain a certain number of CIK cells. However, the proliferation multiples and cytotoxic activity of the CIK cells obtained are not ideal. Summary of the invention

[0003] In view of the deficiencies in the prior art, the present invention provides a CIK cell culture method and application thereof.

[0004] The present invention is achieved through the following technical solutions:

[0005] A method for culturing CIK cells comprises the following steps:

[0006] (1) Isolation of peripheral blood mononuclear cells (PBMC):

[0007] (11) Peripheral blood was drawn and centrifuged at 1800 rpm for 15 min to obtain the upper plasma layer and the lower blood cell sediment. The upper plasma layer was placed in a 56°C water bath for 30 min, placed at 4°C for 30 min, and centrifuged at 3000 rpm for 5 min. The supernatant was obtained to obtain autologous plasma.

[0008] (12) resuspending the blood cell precipitate obtained in step (11) with an equal volume of 0.9% saline to obtain diluted blood, slowly adding it to the lymphocyte separation solution, and centrifuging at 2000 rpm for 20 min;

[0009] (13) After centrifugation in step (12), the middle buffy coat layer (PBMC layer) was aspirated, washed twice with 0.9% saline, and the supernatant was discarded to obtain peripheral blood mononuclear cells.

[0010] (2) Preparation of medium A:

[0011] (21) Dissolve trehalose and oxalic anhydride in acetonitrile, add NaOH, and stir at 70 °C and 60 rpm for 7 h. -3 Mpa to remove the solvent under reduced pressure, and recrystallize in a mixed solution of ethanol and ether to obtain carboxylated trehalose;

[0012] (22) The carboxylated trehalose obtained in step (21) was dispersed in ultrapure water, ultrasonicated at 150 W for 2-3 h, stirred at 100 rpm for 30 min, 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxysuccinimide NHS were added, reacted in an ice bath at 0°C for 1-1.5 h, fibronectin was added, reacted in an ice bath at 0°C for 1-1.5 h, removed from the ice bath, reacted at 25°C for 24 h, dialyzed in deionized water using a 3500 Da dialysis bag for 12 h, the deionized water was replaced every 4 h, and freeze-dried at -20°C for 18 h to obtain modified fibronectin;

[0013] (23) 4 μg / mL of the modified fibronectin obtained in step (22), 1000 IU / mL of IL-2, 600 IU / mL of IL-12, 900 IU / mL of IFN-γ, and 20 ng / mL of CD3 monoclonal antibody were added to RPMI-1640 medium to obtain medium A.

[0014] (3) Preparation of medium B:

[0015] (31) Wash the Jerusalem artichoke and dry it at 80°C to constant weight, grind it through a 100-mesh sieve to obtain Jerusalem artichoke powder, mix it evenly with deionized water, add L-valine nitrate, mix well, raise the water temperature to 50°C, keep it warm for 60 min, and filter it through a 120-mesh sieve to obtain the filtrate;

[0016] (32) Wash dandelion, marigold, purslane and white peony root, dry them at 80°C, grind them through a 100-mesh sieve to obtain a Chinese medicine powder, mix them evenly with the filtrate obtained in step (31), sterilize them at 121°C for 10 min, and obtain a fermentation substrate;

[0017] (33) Activated Streptomyces coelicolor was cultured at 5 × 10 6The inoculum amount of CFU / mL was inoculated into the fermentation substrate of step (32), and cultured in a shaking platform at 28°C and 180 rpm for 96 h, and centrifuged at 10000 rpm for 30 min. The supernatant was filtered through a 0.22 μm filter membrane, and freeze-dried at -40°C for 14 h to obtain a fermentation preparation;

[0018] (34) 5 μg / mL of glutamine, 3 μg / mL of GM-CSF, 10 μg / mL of glucose, and 15 μg / mL of the fermentation preparation described in step (33) were added to RPMI-1640 medium to obtain medium B.

[0019] (4) Resuspend and inoculate peripheral blood mononuclear cells:

[0020] (41) adding the autologous plasma obtained in step (11) to medium A and filtering through a 0.22 μm filter membrane for sterilization;

[0021] (42) Resuspend the peripheral blood mononuclear cells obtained in step (13) in the medium A with autologous plasma added as described in step (41), and inoculate them into a culture bottle at a density of 1×10 6 cells / mL, the inoculation time was taken as the 0th day of culture, and the cells were cultured under 5% CO2 and 37℃;

[0022] (5) Cell culture collection:

[0023] (51) Sterilize medium B by filtering through a 0.22 μm filter membrane. After 72 h of incubation in step (42), add medium B until the cell density reaches 1×10 6 cells / mL;

[0024] (52) Continue culturing as in step (51), adding medium B every 2 days to maintain a cell density of 1×10 6 cells / mL, and the cells were collected after 13 days of culture.

[0025] Furthermore, in step (12), the volume ratio of the diluted blood to the lymphocyte separation fluid is 1:1.

[0026] Furthermore, the washing operation in step (13) is to mix by blowing with 0.9% saline, centrifuge at 1500 r / min for 10 min, and discard the supernatant.

[0027] Furthermore, in step (21), the dosage ratio of trehalose, oxalic anhydride, acetonitrile and NaOH is 0.2 mol:0.1 mol:200 mL:0.3 mol.

[0028] Furthermore, the volume ratio of the ethanol and ether mixed solution in step (21) is 1:3.

[0029] Furthermore, in step (22), the mass ratio of carboxylated trehalose, ultrapure water, EDC, NHS and fibronectin is 10:200:4:3:20.

[0030] Furthermore, the mass ratio of the Jerusalem artichoke powder to deionized water in step (31) is 1:1.

[0031] Furthermore, the mass ratio of L-valine nitrate to Jerusalem artichoke powder in step (31) is 1:50.

[0032] Furthermore, in step (32), the mass ratio of dandelion, marigold, purslane and white peony root is 1:1:0.8:0.5.

[0033] Furthermore, the dosage ratio of the Chinese medicine powder to the filtrate in step (32) is 1 g:10 mL.

[0034] Furthermore, the Streptomyces coelicolor in step (33) was purchased from China General Microbiological Culture Collection Center with a number of CGMCC 4.3587.

[0035] Furthermore, in step (41), the volume ratio of the autologous plasma to culture medium A is 1:50.

[0036] Furthermore, the present invention also provides the use of the CIK cells in preparing tumor treatment preparations.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention provides a method for culturing CIK cells. The method provided by the present invention can improve the yield of CIK cells, and wherein CD3 + CD56 +The ratio of double-positive cells is high, which improves cell proliferation activity and promotes the improvement of CIK cell proliferation and killing activity. The present invention prepares modified fibronectin, adds it to the culture medium, and cooperates with IL-2, IL-12, IFN-γ and CD3 monoclonal antibodies to stimulate cell growth and proliferation, improve cell proliferation activity, and improve induction efficiency. The present invention prepares a fermentation preparation, and the fermentation preparation is added during culture to further stimulate cell growth and proliferation, increase the proliferation rate, and then increase the number of cell proliferation. The serum-free culture medium containing the fermentation preparation is continuously added during the induction culture process, so that the CIK cells obtained by amplification maintain good cell activity, have a good killing effect on tumor cells, and improve its value in clinical applications. Fibronectin (FN) has physiological activities involved in cell attachment, extension, differentiation and proliferation, and can promote the adhesion and proliferation of lymphocytes. The present invention prepares modified fibronectin by covalently linking fibronectin with trehalose. Trehalose is a non-reducing disaccharide formed by the combination of two glucose molecules, which can promote the rapid proliferation of CIK cells, enhance cell metabolism, have a non-specific protective effect on biological macromolecules such as hormones, growth factors and enzymes, and can also be used as an energy substance for cell metabolism. Trehalose has good stability, can improve the stability of fibronectin, prolong its biological activity, further enhance biocompatibility, and promote cell adhesion. The modification of trehalose can improve the structure of fibronectin, so that it can more effectively promote the adhesion of cells and matrix, thereby enhancing cell growth and function; trehalose-modified fibronectin can promote cell proliferation by enhancing cell signal transduction; trehalose itself has antioxidant properties, and the modified fibronectin can enhance the resistance of cells to oxidative stress and protect cells from damage. In addition, the present invention carries out killing activity detection and TGF-β and IL-10 content detection on CIK cells, and the results show that the killing activity and secretion of TGF-β and IL-10 content of CIK cells are increased. At the same time, modified fibronectin and other components are added to induce culture of mononuclear cells in peripheral blood to improve the induction efficiency, and then fermentation preparations, glutamine and GM-CSF and other components are added to improve the proliferation efficiency of CIK cells. The in vitro amplification efficiency of CIK cells is effectively improved by the coordination of the above-mentioned components, a large number of CIK cells can be harvested in a short time, and the cell viability is high, which can fully meet the clinical use requirements. The present invention adopts the culture medium and adds it in stages to effectively improve the proliferation effect of CIK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 The killing activity of CIK cells against A549 cells described in Examples 1-2 and Comparative Examples 1-3 of the present invention;

[0041] Figure 2 The killing activity of CIK cells against K562 cells described in Examples 1-2 and Comparative Examples 1-3 of the present invention;

[0042] Figure 3 The contents of TGF-β and IL-10 secreted by CIK cells described in Examples 1-2 and Comparative Examples 1-3 of the present invention. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments. It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention are purchased through commercial channels.

[0044] Embodiment 1: This embodiment provides a method for culturing CIK cells, comprising the following steps:

[0045] (1) Isolation of peripheral blood mononuclear cells (PBMC):

[0046] (11) Draw 50 mL of peripheral blood and centrifuge at 1800 rpm for 15 min to obtain the upper plasma layer and the lower blood cell sediment. The upper plasma layer was incubated at 56°C for 30 min, placed at 4°C for 30 min, and centrifuged at 3000 rpm for 5 min. The supernatant was obtained to obtain autologous plasma.

[0047] (12) resuspending the blood cell precipitate obtained in step (11) with 50 mL of 0.9% saline to obtain diluted blood, slowly adding it to 50 mL of Ficoll lymphocyte separation solution, and centrifuging at 2000 r / min for 20 min;

[0048] (13) After centrifugation in step (12), the middle buffy coat layer (PBMC layer) was aspirated, mixed with 0.9% saline, centrifuged at 1500 r / min for 10 min, washed twice, and the supernatant was discarded to obtain peripheral blood mononuclear cells.

[0049] (2) Preparation of medium A:

[0050] (21) Dissolve trehalose and oxalic anhydride in acetonitrile, add NaOH, the ratio of trehalose, oxalic anhydride, acetonitrile and NaOH is 0.2 mol:0.1 mol:200 mL:0.3 mol, and stir at 70 °C and 60 rpm for 7 h. -3The solvent was removed under reduced pressure at 4000 Mpa, and the mixture was recrystallized in a mixed solution of ethanol and ether, wherein the volume ratio of the mixed solution of ethanol and ether was 1:3, to obtain carboxylated trehalose;

[0051] (22) The carboxylated trehalose obtained in step (21) was dispersed in ultrapure water, ultrasonicated at 150 W for 3 h, stirred at 100 rpm for 30 min, 1-ethyl-(3-dimethylaminopropyl) carbodiimide EDC and N-hydroxysuccinimide NHS were added, and the reaction was carried out in an ice bath at 0°C for 1.5 h. Fibronectin was added, and the mass ratio of carboxylated trehalose, ultrapure water, EDC, NHS and fibronectin was 10:200:4:3:20. The reaction was carried out in an ice bath at 0°C for 1.5 h. The ice bath was removed and the reaction was carried out at 25°C for 24 h. The reaction was then performed in deionized water using a 3500 Da dialysis bag for 12 h. The deionized water was replaced every 4 h. The modified fibronectin was freeze-dried at -20°C for 18 h to obtain the modified fibronectin.

[0052] (23) 4 μg / mL of the modified fibronectin obtained in step (22), 1000 IU / mL of IL-2, 600 IU / mL of IL-12, 900 IU / mL of IFN-γ, and 20 ng / mL of CD3 monoclonal antibody were added to RPMI-1640 medium to obtain medium A.

[0053] (3) Preparation of medium B:

[0054] (31) Wash the Jerusalem artichoke and dry it at 80°C to constant weight, grind it through a 100-mesh sieve to obtain Jerusalem artichoke powder, mix it evenly with deionized water, the mass ratio of Jerusalem artichoke powder to deionized water is 1:1, add L-valine nitrate, the mass ratio of L-valine nitrate to Jerusalem artichoke powder is 1:50, mix evenly, increase the water temperature to 50°C, keep it warm for 60 min, and filter it through a 120-mesh sieve to obtain the filtrate;

[0055] (32) Wash dandelion, marigold, purslane and white peony root, dry them at 80°C, grind them through a 100-mesh sieve to obtain Chinese medicine powder, the mass ratio of dandelion, marigold, purslane and white peony root is 1:1:0.8:0.5, mix them evenly with the filtrate obtained in step (31), the dosage ratio of Chinese medicine powder to filtrate is 1 g:10 mL, sterilize at 121°C for 10 min, and obtain a fermentation substrate;

[0056] (33) Activated Streptomyces coelicolor was cultured at 5 × 10 6CFU / mL inoculated into the fermentation substrate of step (32), wherein the Streptomyces coelicolor was purchased from China General Microbiological Culture Collection Center and is numbered CGMCC 4.3587, cultured on a shaking table at 28°C and 180 rpm for 96 h, centrifuged at 10,000 rpm for 30 min, the supernatant was filtered through a 0.22 μm filter membrane, and freeze-dried at -40°C for 14 h to obtain a fermentation preparation;

[0057] (34) 5 μg / mL of glutamine, 3 μg / mL of GM-CSF, 10 μg / mL of glucose, and 15 μg / mL of the fermentation preparation described in step (33) were added to RPMI-1640 medium to obtain medium B.

[0058] (4) Resuspend and inoculate peripheral blood mononuclear cells:

[0059] (41) adding the autologous plasma obtained in step (11) to culture medium A, the volume ratio of autologous plasma to culture medium A being 1:50, and filtering through a 0.22 μm filter membrane for sterilization;

[0060] (42) Resuspend the peripheral blood mononuclear cells obtained in step (13) in the medium A with autologous plasma added as described in step (41), and inoculate them into a T25 culture flask at a seeding density of 1×10 6 cells / mL, the culture volume was 15 mL, the inoculation time was taken as the 0th day of culture, and the culture was placed in 5% CO2 and 37℃;

[0061] (5) Cell culture collection:

[0062] (51) Sterilize medium B by filtering through a 0.22 μm filter membrane. After 72 h of incubation in step (42), add medium B until the cell density reaches 1×10 6 cells / mL;

[0063] (52) Continue culturing as in step (51), adding medium B every 2 days to maintain a cell density of 1×10 6 cells / mL. If the culture volume exceeds 30 mL, expand it to a T75 flask. If the volume exceeds 100 mL, expand it to a T175 flask. If the volume exceeds 300 mL, divide the cells into different flasks. After 11 days of cell culture, transfer the cells to a 2 L cell culture bag for culture. Collect the cells after 13 days of cell culture.

[0064] Embodiment 2: This embodiment provides a method for culturing CIK cells, comprising the following steps:

[0065] (1) Isolation of peripheral blood mononuclear cells (PBMC):

[0066] (11) Draw 50 mL of peripheral blood and centrifuge at 1800 rpm for 15 min to obtain the upper plasma layer and the lower blood cell sediment. The upper plasma layer was incubated at 56°C for 30 min, placed at 4°C for 30 min, and centrifuged at 3000 rpm for 5 min. The supernatant was obtained to obtain autologous plasma.

[0067] (12) resuspending the blood cell precipitate obtained in step (11) with 50 mL of 0.9% saline to obtain diluted blood, slowly adding it to 50 mL of Ficoll lymphocyte separation solution, and centrifuging at 2000 r / min for 20 min;

[0068] (13) After centrifugation in step (12), the middle buffy coat layer (PBMC layer) was aspirated, mixed with 0.9% saline, centrifuged at 1500 r / min for 10 min, washed twice, and the supernatant was discarded to obtain peripheral blood mononuclear cells.

[0069] (2) Preparation of medium A:

[0070] (21) Dissolve trehalose and oxalic anhydride in acetonitrile, add NaOH, the ratio of trehalose, oxalic anhydride, acetonitrile and NaOH is 0.2 mol:0.1 mol:200 mL:0.3 mol, and stir at 70 °C and 60 rpm for 7 h. -3 The solvent was removed under reduced pressure at 4000 Mpa, and the mixture was recrystallized in a mixed solution of ethanol and ether, wherein the volume ratio of the mixed solution of ethanol and ether was 1:3, to obtain carboxylated trehalose;

[0071] (22) The carboxylated trehalose obtained in step (21) was dispersed in ultrapure water, ultrasonicated at 150 W for 2 h, stirred at 100 rpm for 30 min, 1-ethyl-(3-dimethylaminopropyl) carbodiimide EDC and N-hydroxysuccinimide NHS were added, reacted in an ice bath at 0°C for 1 h, fibronectin was added, and the mass ratio of carboxylated trehalose, ultrapure water, EDC, NHS and fibronectin was 10:200:4:3:20. The reaction was carried out in an ice bath at 0°C for 1 h. The ice bath was removed and the reaction was carried out at 25°C for 24 h. The reaction was then dialyzed in deionized water using a 3500 Da dialysis bag for 12 h. The deionized water was replaced every 4 h. The modified fibronectin was freeze-dried at -20°C for 18 h to obtain the modified fibronectin.

[0072] (23) 4 μg / mL of the modified fibronectin obtained in step (22), 1000 IU / mL of IL-2, 600 IU / mL of IL-12, 900 IU / mL of IFN-γ, and 20 ng / mL of CD3 monoclonal antibody were added to RPMI-1640 medium to obtain medium A.

[0073] (3) Preparation of medium B:

[0074] (31) Wash the Jerusalem artichoke and dry it at 80°C to constant weight, grind it through a 100-mesh sieve to obtain Jerusalem artichoke powder, mix it evenly with deionized water, the mass ratio of Jerusalem artichoke powder to deionized water is 1:1, add L-valine nitrate, the mass ratio of L-valine nitrate to Jerusalem artichoke powder is 1:50, mix evenly, increase the water temperature to 50°C, keep it warm for 60 min, and filter it through a 120-mesh sieve to obtain the filtrate;

[0075] (32) Wash dandelion, marigold, purslane and white peony root, dry them at 80°C, grind them through a 100-mesh sieve to obtain Chinese medicine powder, the mass ratio of dandelion, marigold, purslane and white peony root is 1:1:0.8:0.5, mix them evenly with the filtrate obtained in step (31), the dosage ratio of Chinese medicine powder to filtrate is 1 g:10 mL, sterilize at 121°C for 10 min, and obtain a fermentation substrate;

[0076] (33) Activated Streptomyces coelicolor was cultured at 5 × 10 6 CFU / mL inoculated into the fermentation substrate of step (32), wherein the Streptomyces coelicolor was purchased from China General Microbiological Culture Collection Center and is numbered CGMCC 4.3587, cultured on a shaking table at 28°C and 180 rpm for 96 h, centrifuged at 10,000 rpm for 30 min, the supernatant was filtered through a 0.22 μm filter membrane, and freeze-dried at -40°C for 14 h to obtain a fermentation preparation;

[0077] (34) 5 μg / mL of glutamine, 3 μg / mL of GM-CSF, 10 μg / mL of glucose, and 15 μg / mL of the fermentation preparation described in step (33) were added to RPMI-1640 medium to obtain medium B.

[0078] (4) Resuspend and inoculate peripheral blood mononuclear cells:

[0079] (41) adding the autologous plasma obtained in step (11) to culture medium A, the volume ratio of autologous plasma to culture medium A being 1:50, and filtering through a 0.22 μm filter membrane for sterilization;

[0080] (42) Resuspend the peripheral blood mononuclear cells obtained in step (13) in the medium A with autologous plasma added as described in step (41), and inoculate them into a T25 culture flask at a seeding density of 1×10 6 cells / mL, the culture volume was 15 mL, the inoculation time was taken as the 0th day of culture, and the culture was placed in 5% CO2 and 37℃;

[0081] (5) Cell culture collection:

[0082] (51) Sterilize medium B by filtering through a 0.22 μm filter membrane. After 72 h of incubation in step (42), add medium B until the cell density reaches 1×10 6 cells / mL;

[0083] (52) Continue culturing as in step (51), adding medium B every 2 days to maintain a cell density of 1×10 6 cells / mL. If the culture volume exceeds 30 mL, expand it to a T75 flask. If the volume exceeds 100 mL, expand it to a T175 flask. If the volume exceeds 300 mL, divide the cells into different flasks. After 11 days of cell culture, transfer the cells to a 2 L cell culture bag for culture. Collect the cells after 13 days of cell culture.

[0084] The difference between Comparative Example 1 and Example 1 is that fibronectin is used instead of modified fibronectin.

[0085] The only difference between Comparative Example 2 and Example 1 is that the modified fibronectin in medium A is replaced by the fermentation preparation, and the fermentation preparation in medium B is replaced by the modified fibronectin.

[0086] The only difference between Comparative Example 3 and Example 1 is that no fermentation preparation is added.

[0087] Experimental Example 1: The phenotype of CIK cells collected from Examples 1-2 and Comparative Examples 1-3 was detected by flow cytometry, and the expression of CD3 + CD56 + The results are shown in Table 1.

[0088] Table 1:

[0089]

[0090] The results in Table 1 show that the proportion of cells expressing CD3+CD56+ simultaneously in Examples 1-2 is higher than that in Comparative Examples 1-3, indicating that the culture method of the present invention can effectively increase the number of cells expressing CD3+CD56+ simultaneously in the cultured cells. + CD56 + cells, enhancing the anti-tumor activity.

[0091] Experimental Example 2: The trypan blue staining method was used to count the initial inoculation amount of cells in Examples 1-2 and Comparative Examples 1-3 and the total number of cells collected after culture, the proliferation times were calculated, and the viability of the collected cells was counted. The results are shown in Table 2.

[0092] Table 2:

[0093]

[0094] The results in Table 2 show that the cell proliferation activity in Examples 1-2 is good, the proliferation multiple is high, and the CIK viability in the obtained cells is high, while the cell proliferation activity in Comparative Examples 1-3 decreases to varying degrees, and the cell viability is also lower than that in Examples 1-2. This indicates that the culture method of the present invention can effectively improve the in vitro proliferation efficiency of CIK cells and promote cell survival.

[0095] Experimental Example 3: Target cells (A549 or K562) and effector cells (CIK cells) were plated at effector-target ratios of 20:1 and 10:1, with the concentration of CIK cells being 2×10 6 / mL (efficacy-target ratio 20:1), 1×10 6 / mL (efficacy-target ratio 10:1), 100 μL per well, K562 or A549 concentration 1×10 5 / mL, 100 μL per well; LDH (lactate dehydrogenase) was determined according to the method of CN201510896888.6, and the killing activity (%) was calculated. Figure 1-Figure 2 shown.

[0096] Figure 1-Figure 2 The results showed that the killing activity of Example 1-2 was higher than that of Comparative Example 1-3, indicating that the culturing method of the present invention can effectively improve the killing effect of CIK cells.

[0097] Experimental Example 4: The culture medium after cell culture in Examples 1-2 and Comparative Examples 1-3 was collected as experimental samples, and the contents of TGF-β and IL-10 were quantitatively detected by ELASA method, and the contents of cytokines secreted by CIK cells were detected. Figure 3 shown.

[0098] Figure 3 The results showed that the contents of TGF-β and IL-10 in Example 1-2 were higher than those in Comparative Example 1-3, indicating that the CIK cells harvested by the culture method of the present invention had stronger abilities to secrete IFN-γ and IL-2.

[0099] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0100] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for culturing CIK cells, characterized in that: The following steps are involved: (1) Isolation of peripheral blood mononuclear cells: (11) Draw peripheral blood, centrifuge to obtain the upper plasma layer and the lower blood cell sediment, place the upper plasma layer in a 56°C water bath, centrifuge at 4°C, and take the supernatant to obtain autologous plasma; (12) resuspending the blood cell precipitate obtained in step (11) with physiological saline to obtain diluted blood, slowly adding it to the lymphocyte separation solution, and centrifuging; (13) After the centrifugation in step (12), the middle buffy coat layer is aspirated, washed with physiological saline, and the supernatant is discarded to obtain peripheral blood mononuclear cells; (2) Preparation of Medium A: (21) Dissolve trehalose and oxalic anhydride in acetonitrile, add NaOH, stir, reduce pressure, and recrystallize in a mixed solution of ethanol and ether to obtain carboxylated trehalose; (22) dispersing the carboxylated trehalose obtained in step (21) in ultrapure water, ultrasonicating, stirring, adding EDC and NHS, reacting in an ice bath, adding fibronectin, reacting in an ice bath, removing from the ice bath, reacting at 25° C., dialyzing, and freeze-drying to obtain modified fibronectin; (23) adding 4 μg / mL of the modified fibronectin obtained in step (22), 1000 IU / mL of IL-2, 600 IU / mL of IL-12, 900 IU / mL of IFN-γ, and 20 ng / mL of CD3 monoclonal antibody to RPMI-1640 medium to obtain medium A; (3) Preparation of medium B: (31) Wash and dry the Jerusalem artichoke, crush and sieve to obtain Jerusalem artichoke powder, mix it evenly with deionized water, add L-valine nitrate, mix evenly, raise the water temperature to 50°C, keep warm, and filter to obtain a filtrate; (32) washing and drying dandelion, marigold, purslane and white peony root, crushing and sieving to obtain Chinese medicine powder, mixing evenly with the filtrate obtained in step (31), sterilizing and obtaining a fermentation substrate; (33) inoculating the activated Streptomyces coelicolor into the fermentation substrate of step (32), culturing on a shaking platform, centrifuging, filtering the supernatant, and freeze-drying to obtain a fermentation preparation; (34) adding 5 μg / mL of glutamine, 3 μg / mL of GM-CSF, 10 μg / mL of glucose, and 15 μg / mL of the fermentation preparation described in step (33) to RPMI-1640 medium to obtain medium B; (4) Resuspend and inoculate peripheral blood mononuclear cells: (41) adding the autologous plasma obtained in step (11) to culture medium A and filtering and sterilizing; (42) Resuspend the peripheral blood mononuclear cells obtained in step (13) in medium A with autologous plasma added in step (41), and inoculate them into a culture flask at a density of 1×10 6 cells / mL, the inoculation time was taken as the 0th day of culture, and the cells were cultured under 5% CO2 and 37℃; (5) Cell culture collection: (51) Sterilize medium B by filtering through 0.22 μm. After 72 h of incubation in step (42), add medium B until the cell density reaches 1×10 6 cells / mL; (52) Continue culturing as in step (51), adding medium B every 2 days to maintain a cell density of 1×10 6 cells / mL, cells were collected after 13 days of culture; In step (22), the mass ratio of carboxylated trehalose, ultrapure water, EDC, NHS and fibronectin is 10:200:4:3:20; In step (41), the volume ratio of the autologous plasma to culture medium A is 1:

50.

2. The method for culturing CIK cells according to claim 1, characterized in that: In step (12), the volume ratio of the diluted blood to the lymphocyte separation fluid is 1:1; in step (13), the washing operation is to mix by blowing with physiological saline, centrifuge, and discard the supernatant.

3. The method for culturing CIK cells according to claim 2, characterized in that: The dosage ratio of trehalose, oxalic anhydride, acetonitrile and NaOH in step (21) is 0.2 mol:0.1 mol:200 mL:0.3 mol; the volume ratio of the mixed solution of ethanol and ether in step (21) is 1:

3.

4. The method for culturing CIK cells according to claim 3, characterized in that: In step (31), the mass ratio of the Jerusalem artichoke powder to deionized water is 1:1; and the mass ratio of the L-valine nitrate to the Jerusalem artichoke powder in step (31) is 1:

50.

5. The method for culturing CIK cells according to claim 4, characterized in that: In step (32), the mass ratio of dandelion, marigold, purslane and white peony root is 1:1:0.8:0.5; and the dosage ratio of the traditional Chinese medicine powder to the filtrate is 1 g:10 mL.

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