Serum-free culture solution for dendritic cells and culture method thereof

Abstract

The invention discloses a serum-free culture solution for dendritic cells and a culture method thereof. The culture solution comprises a solution A and a solution B, the solution A comprises a serum-free basic culture medium, GM-CSF, IL-4, vitamin C, curcumin, huperzine A and rosmarinic acid, and the solution B comprises a serum-free basic culture medium, TGF-beta, PGE2, lipopolysaccharide and rutin. The component A of the serum-free culture solution, such as huperzine A, can enhance cell activity and promote mononuclear cells to induce differentiation of DC cells; rosmarinic acid has antioxidant ability and can enhance the immunocompetence of cells; curcumin can promote the antigen presentation capability of the dendritic cells and promote the differentiation of the dendritic cells; a serum-free culture medium is used as a basic culture medium, so that risks caused by potential pathogens and immunoreactions in serum are avoided.

Description

Technical Field

[0001] The invention relates to the preparation of cell culture fluid, in particular to a serum-free culture fluid of dendritic cells and a culture method thereof. Background Art

[0002] Dendritic cells (DCs) are important immune cells that are responsible for capturing, processing and presenting antigens, thereby activating T cells and B cells and participating in the body's immune response. Due to their key roles in immune regulation and anti-tumor immunity, DCs have received extensive attention in immunotherapy and vaccine development.

[0003] At present, the conventional in vitro DC cell preparation method is to use cytokines to induce, differentiate and expand DC cell precursor cells in peripheral blood or umbilical cord blood. The cultivation and separation of such dendritic cells mostly rely on the addition of serum, such as fetal bovine serum (FBS), which can promote cell growth and differentiation, but also brings problems such as batch differences, immunogenicity and high cost; and such conventionally prepared DC cells contain a large part of immature DC cells; such cells have strong antigen uptake and processing capabilities, but weak antigen presentation capabilities, poor ability to activate T cells, and also inhibit T cell proliferation. Summary of the invention

[0004] Based on the above problems, the problem to be solved by the present invention is to provide a serum-free culture medium that can improve the differentiation and maturity of dendritic cells, has good growth and expansion effects, and is safe and reliable to use, as well as a culture method for culturing dendritic cells using the culture medium.

[0005] The technical solution 1 of the present invention is as follows:

[0006] A dendritic cell serum-free culture medium, comprising liquid A and liquid B, wherein:

[0007] The A solution includes a serum-free basal medium, which contains GM-CSF with a final concentration of 10 to 500 ng / mL, IL-4 with a final concentration of 1 to 500 ng / mL, vitamin C with a final concentration of 50 to 1000 μg / mL, curcumin with a final concentration of 5 to 50 μM, huperzine A with a final concentration of 0.1 to 10 μg / mL, and rosmarinic acid with a final concentration of 0.1 to 5 μM;

[0008] The B solution comprises a serum-free basal culture medium, which contains TGF-β with a final concentration of 0.1-20 ng / mL, PGE2 with a final concentration of 1-200 μg / ml, lipopolysaccharide with a final concentration of 0.1-10 μg / ml, and rutin with a final concentration of 1-20 μM.

[0009] In one embodiment, the dendritic cell serum-free culture medium comprises:

[0010] The serum-free basal medium of liquid A contained GM-CSF at a final concentration of 100 ng / mL, IL-4 at a final concentration of 200 ng / mL, vitamin C at a final concentration of 500 μg / mL, curcumin at a final concentration of 10 μM, huperzine A at a final concentration of 5 μg / mL, and rosmarinic acid at a final concentration of 1 μM;

[0011] The serum-free basal medium of liquid B contains TGF-β at a final concentration of 10 ng / mL, PGE2 at a final concentration of 50 μg / ml, lipopolysaccharide at a final concentration of 5 μg / ml, and rutin at a final concentration of 8 μM.

[0012] In one embodiment, in the serum-free culture medium for dendritic cells, the serum-free basal culture medium is IMDM, RPMI-1640, 581 or DMEM.

[0013] The present invention also provides a method for culturing dendritic cells using the serum-free culture medium, comprising the following steps:

[0014] On day 0, the mononuclear cells were added to a culture flask containing the above-mentioned serum-free culture medium A for dendritic cells, and cultured in an incubator at 37° C. and 5% CO2 for 6 hours; the cell culture waste liquid and suspended cells were discarded, and an equal amount of the above-mentioned serum-free culture medium A for dendritic cells was added, and static culture was continued in an incubator at 37° C. and 5% CO2;

[0015] On the third day, according to the volume of the culture medium in the culture flask, an equal amount of the above dendritic cell serum-free culture medium A was added to the culture flask, and the culture was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0016] On the fifth day, the cells in the culture flask were transferred to a culture bag, and according to the volume of the culture medium in the culture flask, the dendritic cell serum-free culture medium B was added to the culture bag at a volume ratio of 1:2, and the culture was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0017] On the 7th day, according to the volume of the culture medium in the culture bottle, the B solution of the above dendritic cell serum-free culture medium was added to the culture bag at a volume ratio of 1:2, and the culture was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0018] On day 9, cells were collected and centrifuged at 500 g for 8 min. The supernatant was removed and the cell pellet was washed repeatedly 2 to 3 times with PBS to obtain dendritic cells.

[0019] In one embodiment, in the method for culturing dendritic cells, on day 0, the number of mononuclear cells is 1.0 to 5.0×10 6 indivual.

[0020] In one embodiment, in the dendritic cell culture method, on day 0, the volume of liquid A contained in the culture bottle is 100 mL; after 6 hours, the volume of liquid A added is 000 mL.

[0021] In one embodiment, in the dendritic cell culture method, on the third day, the volume of the A solution added is 200 mL.

[0022] In one embodiment, in the dendritic cell culture method, on the fifth day, the volume of the B solution added is 800 mL.

[0023] In one embodiment, in the dendritic cell culture method, on the 7th day, the volume of the B solution added is 1600 mL.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1. Dendritic cell serum-free culture medium A can induce mononuclear cells to rapidly differentiate into dendritic cells. For example, huperzine A can enhance cell activity and promote the differentiation of mononuclear cells into DC cells; rosmarinic acid has antioxidant capacity, which can protect cells from damage during dendritic cell differentiation and enhance the immune activity of cells; curcumin can promote the antigen presentation ability of dendritic cells, improve cell biological activity and promote the differentiation of dendritic cells;

[0026] 2. The serum-free culture medium B of dendritic cells can promote the growth of dendritic cells and transform immature dendritic cells into mature dendritic cells. For example, rutin combined with TGF-β can promote the transformation of immature dendritic cells into mature dendritic cells.

[0027] 3. In dendritic cell culture medium, serum-free culture medium is used as the basic culture medium, which avoids the risks caused by potential pathogens and immune responses in serum, and improves the safety of dendritic cells and the feasibility of clinical application;

[0028] 4. During the dendritic cell culture process, solution A is added in the first 3 days to promote the induction and differentiation of mononuclear cells into dendritic cells and to increase the activity of dendritic cells; after the 5th day, solution B is added to promote the differentiation of immature dendritic cells into mature dendritic cells and to further enhance the rapid proliferation and growth of dendritic cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a logistic diagram of the marker ratios of immature DCs and mature DCs in Example 1;

[0030] Figure 2 This is a logistic diagram of the marker ratios of immature DCs and mature DCs in Example 2;

[0031] Figure 3 This is a logistic diagram of the marker ratios of immature DCs and mature DCs in Example 3;

[0032] Figure 4 This is a logistic diagram of the marker ratios of immature DCs and mature DCs in Example 4;

[0033] Figure 5 This is a logistic diagram of the marker ratios of immature DCs and mature DCs in Example 5;

[0034] Figure 6 The bar graphs are concentrations of IL-2 and IFN-γ secreted by DC cells in Examples 1 to 5;

[0035] FIG. 7 is a microscopic picture of DC cells observed on the 9th day of culture in Examples 1 to 5. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0037] The dendritic cell culture fluid prepared by the present invention includes fluid A and fluid B; fluid A and fluid B are both based on serum-free culture medium (such as IMDM, RPMI-1640, 581 or DMEM), and corresponding cytokines or components are added to each of them, such as GM-CSF (cytokines include recombinant human granulocyte-macrophage colony stimulating factor), IL-4, vitamin C, curcumin, huperzine A and rosmarinic acid are added to fluid A; TGF-β, PGE2 (prostaglandin E2), lipopolysaccharide and rutin are added to fluid B.

[0038] IMDM, RPMI-1640, 581 or DMEM serum-free is used as the basic culture medium. This culture medium is rich in amino acids, vitamins and minerals, which can support the growth and reproduction of cells; and can avoid the safety risks caused by potential pathogens and immune responses in serum, thereby improving the safety of dendritic cells and the feasibility of clinical application.

[0039] In solution A, GM-CSF and IL-4, as cell growth factors, can promote the differentiation and maturation of monocytes into dendritic cells; Huperzine A has antioxidant and immunomodulatory capabilities, which can enhance cell activity and promote the induction of mononuclear cells into DC cells; Rosmarinic Acid has antioxidant capacity, which can protect cells from damage during dendritic cell differentiation and enhance the immune activity of cells; Curcumin can promote the antigen presentation ability of exporting cells, improve cell biological activity and promote dendritic cell differentiation; Vitamin C acts as an antioxidant to maintain cell activity and function.

[0040] In fluid B, TGF-β can regulate the immune function of dendritic cells, promote the formation of their specific subtypes, and promote the differentiation of immature dendritic cells into mature dendritic cells; PGE2 can increase the production, maturity, migration ability and immune activation ability of DCs, promote the maturation of DCs, and induce the high expression of surface chemokine receptors of mature DCs; lipopolysaccharide provides nutrition for cell survival to maintain cell activity; rutin can combine with TGF-β and promote the transformation of immature dendritic cells into mature dendritic cells.

[0041] The specific implementation scheme of the present invention is as follows.

[0042] A dendritic cell serum-free culture medium, comprising liquid A and liquid B, wherein:

[0043] Solution A includes a serum-free basal medium, wherein the serum-free medium contains GM-CSF at a final concentration of 10 to 500 ng / mL, IL-4 at a final concentration of 1 to 500 ng / mL, vitamin C at a final concentration of 50 to 1000 μg / mL, curcumin at a final concentration of 5 to 50 μM, huperzine A at a final concentration of 0.1 to 10 μg / mL, and rosmarinic acid at a final concentration of 0.1 to 5 μM;

[0044] Solution B includes serum-free basal medium containing TGF-β at a final concentration of 0.1-20 ng / mL, PGE2 at a final concentration of 1-200 μg / ml, lipopolysaccharide at a final concentration of 0.1-10 μg / ml, and rutin at a final concentration of 1-20 μM.

[0045] Preferably, the dendritic cell serum-free culture medium contains:

[0046] The serum-free basal medium of liquid A contains GM-CSF at a final concentration of 50 to 400 ng / mL, IL-4 at a final concentration of 50 to 400 ng / mL, vitamin C at a final concentration of 100 to 800 μg / mL, curcumin at a final concentration of 10 to 40 μM, huperzine A at a final concentration of 1 to 8 μg / mL, and rosmarinic acid at a final concentration of 0.5 to 4 μM;

[0047] The serum-free basal medium of liquid B contains TGF-β at a final concentration of 0.5 to 15 ng / mL, PGE2 at a final concentration of 10 to 150 μg / ml, lipopolysaccharide at a final concentration of 1 to 80 μg / ml, and rutin at a final concentration of 5 to 18 μM.

[0048] Furthermore, in the serum-free culture medium of dendritic cells:

[0049] The serum-free basal medium of liquid A contains GM-CSF at a final concentration of 100-300 ng / mL, IL-4 at a final concentration of 100-300 ng / mL, vitamin C at a final concentration of 200-600 μg / mL, curcumin at a final concentration of 20-30 μM, huperzine A at a final concentration of 2-6 μg / mL, and rosmarinic acid at a final concentration of 1-3 μM;

[0050] The serum-free basal medium of liquid B contains TGF-β at a final concentration of 1 to 10 ng / mL, PGE2 at a final concentration of 50 to 120 μg / ml, lipopolysaccharide at a final concentration of 5 to 60 μg / ml, and rutin at a final concentration of 10 to 15 μM.

[0051] In one embodiment, the dendritic cell serum-free culture medium comprises:

[0052] The serum-free basal medium of liquid A contained GM-CSF at a final concentration of 200 ng / mL, IL-4 at a final concentration of 300 ng / mL, vitamin C at a final concentration of 800 μg / mL, curcumin at a final concentration of 20 μM, huperzine A at a final concentration of 1 μg / mL, and rosmarinic acid at a final concentration of 3 μM;

[0053] The serum-free basal medium of liquid B contains TGF-β at a final concentration of 12 ng / mL, PGE2 at a final concentration of 100 μg / ml, lipopolysaccharide at a final concentration of 1 μg / ml, and rutin at a final concentration of 15 μM.

[0054] In one embodiment, the dendritic cell serum-free culture medium comprises:

[0055] The serum-free basal medium of liquid A contained GM-CSF at a final concentration of 60 ng / mL, IL-4 at a final concentration of 400 ng / mL, vitamin C at a final concentration of 100 μg / mL, curcumin at a final concentration of 40 μM, huperzine A at a final concentration of 8 μg / mL, and rosmarinic acid at a final concentration of 0.5 μM;

[0056] The serum-free basal medium of liquid B contains TGF-β at a final concentration of 15 ng / mL, PGE2 at a final concentration of 150 μg / ml, lipopolysaccharide at a final concentration of 0.5 μg / ml, and rutin at a final concentration of 2 μM.

[0057] In one embodiment, the dendritic cell serum-free culture medium comprises:

[0058] The serum-free basal medium of liquid A contains GM-CSF at a final concentration of 300 ng / mL, IL-4 at a final concentration of 250 ng / mL, vitamin C at a final concentration of 600 μg / mL, curcumin at a final concentration of 30 μM, huperzine A at a final concentration of 5 μg / mL, and rosmarinic acid at a final concentration of 2 μM.

[0059] The serum-free basal medium of liquid B contains TGF-β at a final concentration of 10 ng / mL, PGE2 at a final concentration of 80 μg / ml, lipopolysaccharide at a final concentration of 2 μg / ml, and rutin at a final concentration of 5 μM.

[0060] In one embodiment, in the serum-free culture of dendritic cells, the serum-free basal medium is IMDM, RPMI-1640, 581 or DMEM; preferably IMDM serum-free basal medium.

[0061] The present invention also provides a method for culturing dendritic cells using the serum-free culture medium, comprising the following steps:

[0062] On day 0, first, mononuclear cells were added to a culture flask containing the above-mentioned serum-free dendritic cell culture medium A, and cultured in an incubator at 37°C and 5% CO2 for 6 hours; secondly, the cell culture waste liquid and suspended cells were aspirated and an equal amount of 100 mL of the above-mentioned serum-free dendritic cell culture medium A was added, and static culture was continued in an incubator at 37°C and 5% CO2.

[0063] On the third day, according to the volume of the culture medium in the culture flask, an equal amount of the above dendritic cell serum-free culture medium A was added to the culture flask, and the culture was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0064] On the fifth day, the cells in the culture flask were transferred to a culture bag, and according to the volume of the culture medium in the culture flask, the dendritic cell serum-free culture medium B was added to the culture bag at a volume ratio of 1:2, and the culture was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0065] On the 7th day, according to the volume of the culture medium in the culture bottle, the B solution of the above dendritic cell serum-free culture medium was added to the culture bag at a volume ratio of 1:2, and the culture was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0066] On day 9, cells were collected and centrifuged at 500 g for 8 min. The supernatant was removed and the cell pellet was washed repeatedly 2 to 3 times with PBS to obtain dendritic cells.

[0067] In one embodiment, in the method for culturing dendritic cells, on day 0, the number of mononuclear cells is 1.0 to 5.0×10 6 The number of mononuclear cells is 1.5-3.0×10 6 or 1.0×10 6 pcs, 1.5×10 6 pcs, 2.0×10 6 2.3×10 6 pcs, 3.0×10 6 pcs, 3.5×106 pcs, 4.0×10 6 4.5×10 6 pcs, 5.0×10 6 The preferred number of mononuclear cells is 2.0×10 6 indivual.

[0068] In one embodiment, in the culture method of dendritic cells, preferably, on day 0, the volume of liquid A contained in the culture bottle is 100 mL; after 6 hours, 100 mL of the liquid A is added; on day 3, 200 mL of the liquid A is added; on day 5, 800 mL of the liquid B is added; on day 7, 1600 mL of the liquid B is added.

[0069] The mononuclear cells used in the present invention are obtained by the following separation method:

[0070] Fresh peripheral blood drawn from a healthy person is slowly transferred to a 50 ml centrifuge tube containing Ficoll solution. The ratio of peripheral blood to Ficoll solution is 1:2. Try not to destroy the boundary layer between the two liquids during the operation.

[0071] Transfer the centrifuge tube to the centrifuge, set the setting to 5°C and 0°C, and centrifuge at 800g for 20 minutes at room temperature after the centrifuge is balanced;

[0072] After centrifugation, the mononuclear cells of PBMC in the middle buffy coat layer were transferred to another 50 ml centrifuge tube, and DPBS solution was added for washing. The washed cells were centrifuged at 500 g for 10 min at room temperature. The washing and centrifugation were repeated 2 to 3 times to collect the mononuclear cells (PBMC) for use in the following examples.

[0073] The technical solution of the present invention is described in detail below through some specific examples, and the initial number of mononuclear cells is 2.0×10 6 indivual.

[0074] 1. Dendritic Cell Culture

[0075] Example 1

[0076] 1.1. Preparation of serum-free culture medium

[0077] Prepare 500 mL of serum-free culture medium A: use IMDM serum-free medium as the basal medium, add GM-CSF at a final concentration of 100 ng / mL, IL-4 at a final concentration of 200 ng / mL, vitamin C at a final concentration of 500 μg / mL, curcumin at a final concentration of 10 μM, huperzine A at a final concentration of 5 μg / mL, and rosmarinic acid at a final concentration of 1 μM;

[0078] Prepare 2500 mL of serum-free culture medium B: use IMDM serum-free medium as the basal medium, add TGF-β at a final concentration of 10 ng / mL, PGE2 at a final concentration of 50 μg / ml, lipopolysaccharide at a final concentration of 5 μg / ml, and rutin at a final concentration of 8 μM.

[0079] 1.2. Cultivation of dendritic cells

[0080] On day 0, first, take 2.0×10 6 The mononuclear cells are added into a T75C culture flask, the culture flask contains 100 mL of serum-free culture medium A for dendritic cells prepared in step 1.1 of this embodiment, and cultured in an incubator at 37° C. and 5% CO2 for 6 h; the culture waste liquid and suspended cells are discarded, 100 mL of solution A is added, and static culture is continued in an incubator at 37° C. and 5% CO2 to induce the mononuclear cells to differentiate into dendritic cells;

[0081] On the third day, 200 mL of serum-free dendritic cell culture medium A prepared in step 1.1 of this example was added to the culture flask, and the culture was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0082] On the fifth day, the cells in the culture flask were transferred to a culture bag, 800 mL of the serum-free dendritic cell culture medium B prepared in step 1.1 of this example was added to the culture bag, and the bag was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0083] On the 7th day, 1600 mL of serum-free dendritic cell culture medium B prepared in step 1.1 of this example was added to the culture bag, and the culture was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0084] On day 9, cells were collected and centrifuged at 500 g for 8 min. The supernatant was removed and the cell pellet was washed repeatedly 2 to 3 times with PBS to obtain dendritic cells.

[0085] Example 2

[0086] 2.1. Preparation of serum-free culture medium

[0087] Prepare 350 mL of serum-free culture medium A: use DMEM serum-free medium as the basal medium, add GM-CSF at a final concentration of 10 ng / mL, IL-4 at a final concentration of 500 ng / mL, vitamin C at a final concentration of 50 μg / mL, curcumin at a final concentration of 50 μM, huperzine A at a final concentration of 10 μg / mL, and rosmarinic acid at a final concentration of 0.1 μM;

[0088] Prepare 2000 mL of serum-free culture medium B: use DMEM serum-free medium as the basal medium, add TGF-β at a final concentration of 20 ng / mL, PGE2 at a final concentration of 1 μg / ml, lipopolysaccharide at a final concentration of 10 μg / ml, and rutin at a final concentration of 1 μM.

[0089] 2.2. Cultivation of dendritic cells

[0090] On day 0, first, take 2.0×10 6 The mononuclear cells were added into a T75C culture flask, which contained 80 mL of serum-free culture medium A for dendritic cells prepared in step 2.1 of this embodiment, and cultured in an incubator at 37° C. and 5% CO2 for 6 h; the culture waste liquid and suspended cells were discarded, and 80 mL of solution A was added, and the culture was continued in an incubator at 37° C. and 5% CO2 to induce the mononuclear cells to differentiate into dendritic cells;

[0091] On the third day, 160 mL of serum-free dendritic cell culture medium A prepared in step 2.1 of this example was added to the culture flask, and the flask was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0092] On the fifth day, the cells in the culture flask were transferred to a culture bag, 640 mL of the dendritic cell serum-free culture medium B prepared in step 2.1 of this example was added to the culture bag, and the bag was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0093] On the 7th day, 1280 mL of serum-free culture medium B for dendritic cells prepared in step 2.1 of this example was added to the culture bag, and the bag was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0094] On day 9, cells were collected and centrifuged at 500 g for 8 min. The supernatant was removed and the cell pellet was washed repeatedly 2 to 3 times with PBS to obtain dendritic cells.

[0095] Example 3

[0096] 3.1. Preparation of serum-free culture medium

[0097] Prepare 350 mL of serum-free culture medium A: use RPMI-1640 serum-free culture medium as the basal medium, add GM-CSF at a final concentration of 500 ng / mL, IL-4 at a final concentration of 1 ng / mL, vitamin C at a final concentration of 1000 μg / mL, curcumin at a final concentration of 5 μM, huperzine A at a final concentration of 0.1 μg / mL, and rosmarinic acid at a final concentration of 5 μM;

[0098] Prepare 2500 mL of serum-free culture medium B: use RPMI-1640 serum-free culture medium as the basal culture medium, add TGF-β at a final concentration of 0.1 ng / mL, PGE2 at a final concentration of 200 μg / ml, lipopolysaccharide at a final concentration of 0.1 μg / ml, and rutin at a final concentration of 20 μM.

[0099] 3.2. Cultivation of dendritic cells

[0100] On day 0, first, take 2.0×10 6 The mononuclear cells were added into a T75C culture flask, which contained 80 mL of serum-free dendritic cell culture medium A prepared in step 3.1 of this embodiment, and cultured in an incubator at 37° C. and 5% CO2 for 6 h; the culture waste liquid and suspended cells were discarded, and 80 mL of solution A was added, and the culture was continued in an incubator at 37° C. and 5% CO2 to induce the mononuclear cells to differentiate into dendritic cells;

[0101] On the third day, 160 mL of serum-free dendritic cell culture medium A prepared in step 3.1 of this example was added to the culture flask, and the flask was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0102] On the fifth day, the cells in the culture flask were transferred to a culture bag, 720 mL of the serum-free dendritic cell culture medium B prepared in step 3.1 of this example was added to the culture bag, and the bag was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0103] On the 7th day, 1440 mL of serum-free culture medium B for dendritic cells prepared in step 3.1 of this example was added to the culture bag, and the bag was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0104] On day 9, cells were collected and centrifuged at 500 g for 8 min. The supernatant was removed and the cell pellet was washed repeatedly 2 to 3 times with PBS to obtain dendritic cells.

[0105] Example 4 (Comparative Example 1)

[0106] 4.1. Preparation of serum-free culture medium

[0107] Prepare 500 mL of serum-free culture medium A: use 581 serum-free medium as the basal medium, add GM-CSF at a final concentration of 100 ng / mL, IL-4 at a final concentration of 200 ng / mL, vitamin C at a final concentration of 500 μg / mL, and curcumin at a final concentration of 10 μM;

[0108] Prepare 2500 mL of serum-free culture medium B: use 581 serum-free medium as the basal medium, add TGF-β at a final concentration of 10 ng / mL, PGE2 at a final concentration of 50 μg / ml, and LPS at a final concentration of 5 μg / ml.

[0109] 4.2. Cultivation of Dendritic Cells

[0110] On day 0, first, take 2.0×10 6 The mononuclear cells are added into a T75C culture flask, the culture flask contains 100 mL of the serum-free culture medium A for dendritic cells prepared in step 4.1 of this embodiment, and cultured in an incubator at 37° C. and 5% CO2 for 6 h; the culture waste liquid and suspended cells are discarded, 100 mL of solution A is added, and the culture is continued in an incubator at 37° C. and 5% CO2 to induce the mononuclear cells to differentiate into dendritic cells;

[0111] On the third day, 200 mL of serum-free dendritic cell culture medium A prepared in step 4.1 of this example was added to the culture flask, and the flask was cultured in an incubator at 37° C. and 5% CO 2 ;

[0112] On the fifth day, the cells in the culture flask were transferred to a culture bag, 800 mL of the serum-free dendritic cell culture medium B prepared in step 4.1 of this example was added to the culture bag, and the bag was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0113] On the 7th day, 1600 mL of serum-free dendritic cell culture medium B prepared in step 1.1 of this example was added to the culture bag, and the culture was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0114] On day 9, cells were collected and centrifuged at 500 g for 8 min. The supernatant was removed and the cell pellet was washed repeatedly 2 to 3 times with PBS to obtain dendritic cells.

[0115] Example 5 (Comparative Example 2)

[0116] 5.1. Preparation of serum-free culture medium

[0117] Prepare 200 mL of serum-free culture medium A: use IMDM containing 10% fetal bovine serum (FBS) as the basal medium, add GM-CSF at a final concentration of 100 ng / mL, IL-4 at a final concentration of 200 ng / mL, vitamin C at a final concentration of 500 μg / mL, curcumin at a final concentration of 10 μM, huperzine A at a final concentration of 5 μg / mL, and rosmarinic acid at a final concentration of 1 μM;

[0118] Prepare 2500 mL of serum-free culture medium B: use IMDM containing 10% fetal bovine serum (FBS) as the basic culture medium, add TGF-β at a final concentration of 10 ng / mL, PGE2 at a final concentration of 50 μg / ml, lipopolysaccharide at a final concentration of 5 μg / ml and rutin at a final concentration of 8 μM.

[0119] 5.2. Cultivation of Dendritic Cells

[0120] On day 0, first, take 2.0×10 6 The mononuclear cells are added into a T75C culture flask, the culture flask contains 100 mL of the serum-free culture medium A for dendritic cells prepared in step 5.1 of this embodiment, and cultured in an incubator at 37° C. and 5% CO2 for 6 h; the culture waste liquid and suspended cells are discarded, 100 mL of solution A is added, and the culture is continued in an incubator at 37° C. and 5% CO2 to induce the mononuclear cells to differentiate into dendritic cells;

[0121] On the third day, 200 mL of serum-free culture medium A for dendritic cells prepared in step 5.1 of this example was added to the culture flask, and the culture was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0122] On the fifth day, the cells in the culture flask were transferred to a culture bag, 800 mL of the dendritic cell serum-free culture medium B prepared in step 5.1 of this example was added to the culture bag, and the bag was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0123] On the 7th day, 1600 mL of serum-free dendritic cell culture medium B prepared in step 5.1 of this example was added to the culture bag, and the culture was statically cultured in an incubator at 37° C. and 5% CO 2 ;

[0124] On day 9, cells were collected and centrifuged at 500 g for 8 min. The supernatant was removed and the cell pellet was washed repeatedly 2 to 3 times with PBS to obtain dendritic cells.

[0125] 2. Detection of dendritic cells (DCs)

[0126] 1) Flow cytometry phenotyping of dendritic cells

[0127] In Examples 1 to 5, DC cells were sampled on the 9th day of culture, and PBS solution was added to adjust the cell concentration to 1×10 6 / ml. Using flow cytometry, the DC precursor cell (i.e., immature DC cell) marker CD14 and mature DC cell markers CD80 and CD86 were detected by phenotypic flow cytometry. The flow cytometry diagram is shown in the figure. Figures 1 to 5 As shown in Table 1; Data and Figures 1 to 5 The two are consistent.

[0128] Figure 1 In the figure, the left picture is the flow cytometric graph of the CD14 phenotype of DC precursor cells in Example 1, and the right picture is the flow cytometric graph of the CD80 and CD86 phenotypes of DC cells in Example 1;

[0129] Figure 2 In the figure, the left picture is the flow cytometric graph of the CD14 phenotype of DC precursor cells in Example 2, and the right picture is the flow cytometric graph of the CD80 and CD86 phenotype of DC cells in Example 1;

[0130] Figure 3 In the figure, the left picture is the flow cytometric graph of the CD14 phenotype of DC precursor cells in Example 3, and the right picture is the flow cytometric graph of the CD80 and CD86 phenotype of DC cells in Example 1;

[0131] Figure 4 In the figure, the left picture is the flow cytometric graph of the CD14 phenotype of DC precursor cells in Example 4, and the right picture is the flow cytometric graph of the CD80 and CD86 phenotype of DC cells in Example 1;

[0132] Figure 5 In the figure, the left picture is the flow cytometric graph of the CD14 phenotype of DC precursor cells in Example 5, and the right picture is the flow cytometric graph of the CD80 and CD86 phenotype of DC cells in Example 1.

[0133] Table 1 Statistical table of DC cell marker ratios

[0134]

[0135] From Table 1, Figures 1 to 5 It can be seen that the markers CD80 and CD86 represent the expression levels of mature DC cells, respectively. In Examples 1 to 3 and 5, the proportions of DC precursor cell marker CD14 are 45.57%, 42.87%, 40.59% and 39.47%, respectively; the proportions of mature DC cell markers CD80 and CD86 are 91.07%, 89.94%, 86.52% and 86.38%, respectively; this shows that in Examples 1 to 3 and 5, the proportions of immature DC cells are basically the same, but the proportions of induced mature DC cells in Examples 1 to 3 and 5 (greater than 86%) are significantly higher than the proportion of mature DC cells in Example 4 (50.59%). This is because: in Examples 1 to 3 and 5, the combination of the A liquid components of the DC culture medium, huperzine A, rosmarinic acid and curcumin, can enhance cell activity, promote the induction and differentiation of mononuclear cells into DC cells, and make DC cells have better antioxidant capacity, which can protect cells from damage and enhance the immune activity and antigen presentation ability of cells.

[0136] 2) Detection of factor secretion by dendritic cells

[0137] In Examples 1 to 5, DC cells were cultured for 9 days and samples were taken. The concentrations of IL-2 and IFN-γ in the supernatant were detected by ELISA. The test results are shown in Tables 2 and Figure 6 As shown; among them, Figure 6 The data is drawn based on Table 2.

[0138] Table 2 Concentrations of IL-2 and IFN-γ secreted by DC cells

[0139]

[0140] From Table 2 and Figure 6 It can be seen that: 1) the concentrations of IL-2 and IFN-γ in Examples 1 to 3 and 5 are higher than those in Example 4; this shows that the combination of huperzine A, rosmarinic acid and curcumin can enhance cell activity, promote the induction and differentiation of mononuclear cells into DC cells and make DC cells have better antioxidant capacity, protect cells from damage, and enhance the immune activity and antigen presentation ability of cells; 2) the concentrations of IL-2 and IFN-γ in Examples 1 to 3 are higher than those in Example 5; this is because the dendritic cell culture fluid components in Examples 1 to 3 use serum-free culture medium as the basal culture medium, while Example 5 uses a serum-free culture medium containing FBS, so the DC culture fluid of the present invention ensures cell safety and non-toxicity and improves cell activity.

[0141] 3. DCs cell microscopic observation test

[0142] The DCs cells in Examples 1 to 5 were cultured, and samples were taken on the 9th day. The DCs cells were observed using an optical microscope at a magnification of 40 times. The microscope photographs are shown in Figure 7; wherein Picture a is a photograph of the DCs cells in Example 1, Picture b is a photograph of the DCs cells in Example 2, Picture c is a photograph of the DCs cells in Example 3, Picture d is a photograph of the DCs cells in Example 4, and Picture e is a photograph of the DCs cells in Example 5.

[0143] It can be observed from the photos shown in FIG7 that after the 9th day of the induction culture of DC cells by mononuclear cells, mature DC cells, i.e., dendritic cells, with stellate polymorphism or dendrites on the outer wall of the cells can be seen in Examples 1 to 5. It can be seen that in Examples 1 to 5, after the mononuclear cells were induced to differentiate and cultured using conventional DC culture medium or the DC culture medium provided by the present invention, mature DC cells can be obtained; however, compared with Example 4, the number of mature DC cells in Examples 1 to 3 and 5 is more; this is because in Examples 1 to 3 and 5, the combination of the A liquid components of the DC culture medium, huperzine A, rosmarinic acid, and curcumin, can enhance cell activity and promote the induction and differentiation of mononuclear cells into DC cells.

[0144] It should be understood that the above description of the preferred embodiments of the present invention is relatively detailed and cannot be regarded as limiting the scope of patent protection of the present invention. The scope of patent protection of the present invention shall be based on the attached claims.

Claims

1. A serum-free culture medium for dendritic cells, characterized in that: Including liquid A and liquid B: The A solution includes a serum-free basal medium, which contains GM-CSF with a final concentration of 10 to 500 ng / mL, IL-4 with a final concentration of 1 to 500 ng / mL, vitamin C with a final concentration of 50 to 1000 μg / mL, curcumin with a final concentration of 5 to 50 μM, huperzine A with a final concentration of 0.1 to 10 μg / mL, and rosmarinic acid with a final concentration of 0.1 to 5 μM; The B solution comprises a serum-free basal culture medium, which contains TGF-β with a final concentration of 0.1-20 ng / mL, PGE2 with a final concentration of 1-200 μg / ml, lipopolysaccharide with a final concentration of 0.1-10 μg / ml, and rutin with a final concentration of 1-20 μM.

2. The serum-free culture medium for dendritic cells according to claim 1, characterized in that The serum-free basal culture medium of the A solution contains GM-CSF with a final concentration of 100 ng / mL, IL-4 with a final concentration of 200 ng / mL, vitamin C with a final concentration of 500 μg / mL, curcumin with a final concentration of 10 μM, huperzine A with a final concentration of 5 μg / mL, and rosmarinic acid with a final concentration of 1 μM; the serum-free basal culture medium of the B solution contains TGF-β with a final concentration of 10 ng / mL, PGE2 with a final concentration of 50 μg / ml, lipopolysaccharide with a final concentration of 5 μg / ml, and rutin with a final concentration of 8 μM.

3. The serum-free culture medium for dendritic cells according to claim 1, characterized in that: The serum-free basal culture medium is IMDM, RPMI-1640, 581 or DMEM.

4. A method for culturing dendritic cells, characterized in that: The steps include: On day 0, the mononuclear cells are added to a culture flask containing the serum-free culture medium A of dendritic cells as claimed in claim 1, and cultured in an incubator at 37° C. and 5% CO2 for 6 hours; the cell culture waste liquid and suspended cells are discarded, and an equal amount of the serum-free culture medium A of dendritic cells as claimed in claim 1 is added, and the culture is continued in an incubator at 37° C. and 5% CO2; On the third day, according to the volume of the culture medium in the culture flask, an equal amount of the serum-free culture medium A of dendritic cells according to claim 1 is added to the culture flask, and the culture is statically cultured in an incubator at 37° C. and 5% CO 2 ; On the fifth day, the cells in the culture flask were transferred to a culture bag, and according to the volume of the culture medium in the culture flask, the serum-free culture medium B of dendritic cells according to claim 1 was added to the culture bag at a volume ratio of 1:2, and the culture was statically cultured in an incubator at 37° C. and 5% CO 2 ; On the 7th day, according to the volume of the culture medium in the culture bag, the serum-free culture medium B of dendritic cells according to claim 1 is added to the culture bag at a volume ratio of 1:2, and the culture is statically cultured in an incubator at 37° C. and 5% CO 2 ; On day 9, cells were collected and centrifuged at 500 g for 8 min. The supernatant was removed and the cell pellet was washed repeatedly 2 to 3 times with PBS to obtain dendritic cells.

5. The method for culturing dendritic cells according to claim 4, characterized in that: On day 0, the number of mononuclear cells was 1.0-5.0×10 6 indivual.

6. The method for culturing dendritic cells according to claim 4, characterized in that: On day 0, the volume of liquid A in the culture bottle was 100 mL; 6 hours later, 100 mL of liquid A was added.

7. The method for culturing dendritic cells according to claim 4, characterized in that: On the 3rd day, the volume of liquid A was added to 200 mL.

8. The method for culturing dendritic cells according to claim 4, characterized in that: On the 5th day, the volume of the B solution added was 800 mL.

9. The method for culturing dendritic cells according to claim 4, characterized in that: On the 7th day, the volume of the B solution added was 1600 mL.

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