A high-efficiency induction culture kit for DC cells and an induction culture method thereof

Abstract

The application provides a high-efficiency induction culture kit of DC cells and an induction culture method thereof, and belongs to the technical field of cell biology. The kit comprises reagent A and reagent B; wherein the reagent A is a GM-CSF solution, an IL-4 solution, an IL-1 beta solution, an AK1 solution, an ACK1 solution, an MLK3 solution, a PLD1 solution, a type II cytoskeleton 1 solution and a type I cytoskeleton 16 solution; and the reagent B comprises a PD-L1 protein solution, a CTLA-4 protein solution, a TIM-3 protein solution, a LAG-3 protein solution, a CD44 protein solution, a CEA protein solution, a neuron-specific enolase solution and a squamous cell carcinoma-associated antigen protein solution. The kit shortens the cell culture time, improves the induction and maturation rate of DC cells and cell activity, and enhances the killing activity of T cells on lung cancer cells.

Description

Technical Field

[0001] This invention relates to the field of cell biology technology, specifically to a highly efficient induction culture kit for DC cells and its induction culture method. Background Technology

[0002] Cellular immunotherapy is a treatment method that combats diseases by modifying or enhancing the function of the body's own immune cells. It can induce long-term immune memory and reduce relapse. It mainly includes CAR-T cell therapy, TCR-T cell therapy, dendritic cell (DC) vaccines, NK cell therapy, and tumor-infiltrating lymphocyte (TIL) therapy. Among them, dendritic cell vaccines involve culturing DC cells in vitro and loading them with tumor antigens. After reinfusion, T cells are activated. DCs are currently the most potent antigen-presenting cells (APCs). Compared with other APCs (such as monocytes / macrophages, B cells, etc.), which can only stimulate activated or memory T cells, DCs are also the only APCs that can significantly stimulate the proliferation of naive T cells (Tn). Therefore, DCs play an extremely important role in tumor immunity as the initiator of the body's adaptive T cell immune response.

[0003] Currently, common methods for inducing and culturing DC cells mainly involve using cytokines such as GM-CSF and IL-4 to induce monocytes to differentiate into DC cells. Although this can yield a sufficient number of mature DC cells, it still has many limitations. For example, the induced DC cells have insufficient maturity and functional stability, and cannot effectively activate T cells, or the activated T cells do not have strong enough killing activity against tumor cells, affecting the effect of immunotherapy. Secondly, the culture cycle is long, which not only increases the culture cost but may also lead to cell contamination or other adverse changes during the culture process. Moreover, the cell yield is unstable, and the results of induction culture from different batches may vary greatly, making it difficult to guarantee that a sufficient number and quality of DC cells can be obtained each time, thus limiting the application of DC cells in clinical practice.

[0004] Therefore, there is an urgent need for a kit that can efficiently induce DC cells. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a kit for efficiently cultivating dendritic cells (DCs) and a method for inducing their culture. This kit not only has a short culture cycle, but the DCs induced and cultured using it can enhance the killing activity of T cells against lung cancer cells.

[0006] To achieve the above objectives, the present invention provides the following technical solution to address the technical problem:

[0007] In a first aspect, the present invention provides a high-efficiency induction and culture kit for DC cells, the kit comprising reagent A and reagent B;

[0008] Among them, reagent A is GM-CSF solution, IL-4 solution, IL-1β solution, AK1 solution, ACK1 solution, MLK3 solution, PLD1 solution, type II cytoskeleton 1 solution, and type I cytoskeleton 16 solution;

[0009] Reagent B includes PD-L1 protein solution, CTLA-4 protein solution, TIM-3 protein solution, LAG-3 protein solution, CD44 protein solution, CEA protein solution, neuron-specific enolase solution, and squamous cell carcinoma-associated antigen protein solution.

[0010] In some preferred embodiments, the concentrations of the GM-CSF solution are 500-600 ng / mL, the IL-4 solution is 1000-3000 U / mL, the IL-1β solution is 5000-7000 IU / mL, the AK1 solution is 50-200 ng / mL, the ACK1 solution is 50-200 ng / mL, the MLK3 solution is 30-70 ng / mL, the PLD1 solution is 30-70 ng / mL, the type II cytoskeleton 1 solution is 50-200 ng / mL, and the type I cytoskeleton 16 solution is 50-200 ng / mL.

[0011] In some preferred embodiments, the volume ratio of the GM-CSF solution, IL-4 solution, IL-1β solution, AK1 solution, ACK1 solution, MLK3 solution, PLD1 solution, type II cytoskeleton 1, and type I cytoskeleton 16 solution is (3-7):(1-3):(0.5-1.5):(1-3):(0.5-1.5):(0.5-1.5):(1-3):(2-4):(1-3).

[0012] In some preferred embodiments, the concentrations of the PD-L1 protein solution are 200-400 ng / mL, the CTLA-4 protein solution is 50-200 ng / mL, the TIM-3 protein solution is 50-200 ng / mL, the LAG-3 protein solution is 50-200 ng / mL, the CD44 protein solution is 300-500 ng / mL, the CEA protein solution is 300-700 ng / mL, the neuron-specific enolase solution is 100-200 ng / mL, and the squamous cell carcinoma-associated antigen protein solution is 100-200 ng / mL.

[0013] In some preferred embodiments, the volume ratio of the PD-L1 protein solution, CTLA-4 protein solution, TIM-3 protein solution, LAG-3 protein solution, CD44 protein solution, CEA protein solution, neuron-specific enolase solution, and squamous cell carcinoma-associated antigen protein solution is (1-3):(0.5-1.5):(0.5-1.5):(0.5-1.5):(2-4):(1-3):(0.5-1.5):1.

[0014] Secondly, the present invention provides a method for inducing and culturing DC cells using the above-mentioned high-efficiency DC cell induction and culture kit, comprising the following steps:

[0015] S1: PBMCs were seeded in 1640 medium and cultured. The suspension cells were then discarded, and adherent cells were obtained, which are DC cells.

[0016] S2: Add serum-free RPMI 1640 medium and reagent A to DC cells and continue culturing;

[0017] S3: Change the medium once on the second day, add reagent A and continue culturing;

[0018] S4: On the third day, add reagent B and continue culturing;

[0019] S4: Day 4, collect mature DC cells.

[0020] In some preferred embodiments, the inoculation density of the PBMCs is 1×10⁻⁶. 7 per mL.

[0021] In some preferred embodiments, the culture conditions are 5% CO2 and 37°C.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention provides a highly efficient DC cell induction and culture kit, which not only effectively improves DC cell-specific expression and enhances the expression of related cytokines in DC cell culture supernatant, but also shortens cell culture time, increases DC cell induction maturation rate and cell activity, and enhances the killing activity of T cells against lung cancer cells, providing a new approach for DC cell induction and culture. Attached Figure Description

[0024] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a microscope image taken at 100x magnification of DC cells induced and cultured by the high-efficiency induction culture kit in Example 1 of the present invention.

[0026] Figure 2 This is a microscope image taken at 100x magnification of DC cells induced and cultured by the high-efficiency induction culture kit in Comparative Example 1 of this invention.

[0027] Figure 3 This is a microscope image taken at 100x magnification of the DC cells induced and cultured by the high-efficiency induction culture kit in Comparative Example 2 of this invention.

[0028] Figure 4 This is a graph showing the detection results of IL-12 content secreted by DC cells in this invention.

[0029] Figure 5 This is a graph showing the detection results of the killing activity of T cells presented by mature DC cells against lung cancer cells in this invention. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0032] Unless otherwise specified, the experimental methods used in this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0033] In this invention, the cell culture conditions are 5% CO2 and 37°C.

[0034] In this invention, the preparation of PBMCs (peripheral blood mononuclear cells) specifically includes the following steps:

[0035] 1. Collect 50 mL of peripheral venous blood from a person as a blood sample. After centrifugation at 3000 rpm for 30 min, separate the upper plasma layer and the lower blood cell layer. First, inactivate the upper plasma layer in a 56℃ water bath for 40 min. Then, centrifuge at 3000 rpm for 20 min at 4℃. Remove the lower precipitate to obtain inactivated plasma. Store it in a 4℃ refrigerator for later use.

[0036] 2. Dilute the lower layer of blood cells with physiological saline to 50 mL, mix gently, and then prepare two lymphocyte separation tubes, each containing 5 mL of Ficoll lymphocyte separation solution. Add the blood cell and physiological saline mixture evenly to the two separation tubes. After centrifugation at 3000 rpm for 20 min, the liquid will separate into layers. Take the white, cloudy liquid layer and collect it into a new 50 mL centrifuge tube to obtain the PBMC suspension.

[0037] 3. Centrifuge the PBMC suspension at 2000 rpm for 10 min and discard the supernatant; add 30 ml of physiological saline to the cell pellet and wash, centrifuge at 2000 rpm for 10 min and discard the supernatant. Repeat the washing process 3 times to obtain PBMC.

[0038] Example 1

[0039] A highly efficient method for inducing and culturing DC cells includes the following steps:

[0040] 1. Set PBMCs to 1×10 7 The cells were seeded at 20 mL of serum-free RPMI 1640 medium and cultured in a cell culture incubator for 2 h. The resulting adherent cells were dendritic cells (DCs), while the suspension cells were mainly T lymphocytes.

[0041] 2. DC cell differentiation induction:

[0042] (1) Remove non-adherent cells, add 50 mL of serum-free RPMI 1640 medium and 3 mL of high-efficiency inducer to DC cells, and culture in a cell culture incubator;

[0043] The highly effective inducers include 550 ng / mL GM-CSF solution, 2000 U / mL IL-4 solution, 6000 IU / mL IL-1β solution, 100 ng / mL AK1 solution, 100 ng / mL ACK1 solution, 50 ng / mL MLK3 solution, 50 ng / mL PLD1 solution, 100 ng / mL type II cytoskeleton 1 solution, and 100 ng / mL type I cytoskeleton 16 solution.

[0044] The volume ratio of the GM-CSF solution, IL-4 solution, IL-1β solution, AK1 solution, ACK1 solution, MLK3 solution, PLD1 solution, type II cytoskeleton 1, and type I cytoskeleton 16 solution is 5:2:1:2:1:1:2:3:2.

[0045] (2) Change half the amount of solution on the second day and replenish the high-efficiency inducer;

[0046] (3) On the third day, 3 mL of maturation activator was added and the cells were cultured in the cell culture incubator. The maturation activator included 300 ng / mL of PD-L1 protein solution, 100 ng / mL of CTLA-4 protein solution, 100 ng / mL of TIM-3 protein solution, 100 ng / mL of LAG-3 protein solution, 400 ng / mL of CD44 protein solution, 500 ng / mL of CEA protein solution, 150 ng / mL of neuron-specific enolase solution (AmyJet Scientific, 1 mg / mL solution in 20 mM Tris-HCl (pH 7.5) containing 100 mM KCl and 5 mM MgSO4) and 150 ng / mL of squamous cell carcinoma-associated antigen protein (Fischer Medical, 0.1 mg) solution, with a volume ratio of 2:1:1:1:3:2:1:1.

[0047] (4) On day 4, collect mature DC cells.

[0048] like Figure 1 As shown, the suspended cells are mature DC cells obtained through culture, indicating that the kit and induction method of this embodiment can obtain a large number of mature DC cells, which can significantly improve the proliferation rate of DC cells.

[0049] Example 2

[0050] A highly efficient method for inducing and culturing DC cells includes the following steps:

[0051] 1. Set PBMCs to 1×10 7 The cells were seeded at 20 mL of serum-free RPMI 1640 medium and cultured in a cell culture incubator for 2 h. The resulting adherent cells were dendritic cells (DCs), while the suspension cells were mainly T lymphocytes.

[0052] 2. DC cell differentiation induction:

[0053] (1) Remove non-adherent cells, add 50 mL of serum-free RPMI 1640 medium and 3 mL of high-efficiency inducer to DC cells, and culture in a cell culture incubator;

[0054] The highly effective inducers include 500 ng / mL GM-CSF solution, 3000 U / mL IL-4 solution, 7000 IU / mL IL-1β solution, 50 ng / mL AK1 solution, 50 ng / mL ACK1 solution, 70 ng / mL MLK3 solution, 30 ng / mL PLD1 solution, 50 ng / mL type II cytoskeleton 1 solution, and 200 ng / mL type I cytoskeleton 16 solution.

[0055] The volume ratio of the GM-CSF solution, IL-4 solution, IL-1β solution, AK1 solution, ACK1 solution, MLK3 solution, PLD1 solution, type II cytoskeleton 1, and type I cytoskeleton 16 solution is 5:2:1:2:1:1:2:3:2.

[0056] (2) Change half the amount of solution on the second day and replenish the high-efficiency inducer;

[0057] (3) On the third day, 3 mL of maturation activator was added and the cells were cultured in the cell culture incubator. The maturation activator included 200 ng / mL PD-L1 protein solution, 50 ng / mL CTLA-4 protein solution, 200 ng / mL TIM-3 protein solution, 200 ng / mL LAG-3 protein solution, 300 ng / mL CD44 protein solution, 700 ng / mL CEA protein solution, 100 ng / mL neuron-specific enolase solution and 200 ng / mL squamous cell carcinoma-associated antigen protein solution, with a volume ratio of 2:1:1:1:3:2:1:1.

[0058] (4) On day 4, collect mature DC cells.

[0059] Example 3

[0060] A highly efficient method for inducing and culturing DC cells includes the following steps:

[0061] 1. Set PBMCs to 1×10 7 The cells were seeded at 20 mL of serum-free RPMI 1640 medium and cultured in a cell culture incubator for 2 h. The resulting adherent cells were dendritic cells (DCs), while the suspension cells were mainly T lymphocytes.

[0062] 2. DC cell differentiation induction:

[0063] (1) Remove non-adherent cells, add 50 mL of serum-free RPMI 1640 medium and 3 mL of high-efficiency inducer to DC cells, and culture in a cell culture incubator;

[0064] The highly effective inducers include 600 ng / mL GM-CSF solution, 1000 U / mL IL-4 solution, 5000 IU / mL IL-1β solution, 200 ng / mL AK1 solution, 200 ng / mL ACK1 solution, 30 ng / mL MLK3 solution, 70 ng / mL PLD1 solution, 200 ng / mL type II cytoskeleton 1 solution, and 50 ng / mL type I cytoskeleton 16 solution.

[0065] The volume ratio of the GM-CSF solution, IL-4 solution, IL-1β solution, AK1 solution, ACK1 solution, MLK3 solution, PLD1 solution, type II cytoskeleton 1, and type I cytoskeleton 16 solution is 5:2:1:2:1:1:2:3:2.

[0066] (2) Change half the amount of solution on the second day and replenish the high-efficiency inducer;

[0067] (3) On the third day, 3 mL of maturation activator was added and the cells were cultured in a cell culture incubator. The maturation activator included 400 ng / mL of PD-L1 protein solution, 200 ng / mL of CTLA-4 protein solution, 200 ng / mL of TIM-3 protein solution, 200 ng / mL of LAG-3 protein solution, 500 ng / mL of CD44 protein solution, 300 ng / mL of CEA protein solution, 200 ng / mL of neuron-specific enolase solution and 100 ng / mL of squamous cell carcinoma-associated antigen protein solution, with a volume ratio of 2:1:1:1:3:2:1:1.

[0068] (4) On day 4, collect mature DC cells.

[0069] Comparative Example 1

[0070] The difference between Comparative Example 1 and Example 1 is that the high-efficiency inducer is different, but everything else is the same.

[0071] The highly efficient inducer comprises a GM-CSF solution of 550 ng / mL, an IL-4 solution of 2000 U / mL, and an IL-1β solution of 6000 IU / mL, in a volume ratio of 5:2:1.

[0072] In Comparative Example 1, the cultured DC cells were as follows: Figure 2 As shown, the number of suspended cells is very small, indicating that the DC cells in Comparative Example 1 kit proliferate at a slower rate.

[0073] Comparative Example 2

[0074] The difference between Comparative Example 1 and Example 1 is that the maturation activator is different, but everything else is the same.

[0075] The maturation activator is a 25 μg / mL tumor polypeptide antigen solution (human non-small cell lung cancer A549 cell lysate).

[0076] In Comparative Example 2, the cultured DC cells were as follows: Figure 3 As shown, the number of suspended cells is very small, indicating that the DC cells in Comparative Example 2 kit proliferate at a slower rate.

[0077] Test Example 1

[0078] Mature dendritic cells (DCs) can secrete high levels of IL-12. The state of DCs can be analyzed by detecting the IL-12 secreted by DCs.

[0079] Culture supernatants from DC cells induced and cultured in Example 1 and Comparative Examples 1-2 were collected on day 4. The IL-12 secreted by mature DC cells in the culture supernatants was detected using an interleukin-12 (IL-12) ELISA kit. Specific procedures were performed according to the ELISA instructions. Results are shown below. Figure 4 .

[0080] Depend on Figure 4 It can be seen that, compared with the kits of Comparative Examples 1-2, the IL-12 content secreted by the mature DC cells induced and cultured in Example 1 was significantly higher than that secreted by the mature DC cells induced and cultured in the comparative examples, indicating that the kit of the present invention can effectively promote the maturation of DC cells and the expression of the related cytokine IL-12.

[0081] Test Example 2

[0082] The analysis of the cytotoxic activity of T cells presented by mature dendritic cells against lung cancer cells includes the following steps:

[0083] 1. T lymphocytes and mature DC cells from Example 1 and Comparative Examples 1-2 were co-cultured at a ratio of 20:1 to enhance the differentiation of T lymphocytes into cytotoxic T lymphocytes (CTL cells). IL-2, anti-CD3 monoclonal antibody, and anti-CD28 monoclonal antibody were added to a final concentration of 100 ng / mL, respectively. After culturing for 4 days, serum-free RPMI 1640 medium containing the above-mentioned additives was added, and the cells were cultured for another 4 days to obtain highly active CTL cells with targeted tumor killing.

[0084] 2. Lung cancer cells A549 were seeded in Transwell 6-well plates (1×10⁻⁶). 5 / well), after normal incubation for 24 hours, at the same density (1×10 6CTL cells obtained in Example 1 and Comparative Examples 1-2 were respectively placed into Transwell 6-well plates. The A549 lung cancer cells cultured alone served as the negative control group (Group A), the CTL cells obtained in Comparative Example 2 were placed into Transwell 6-well plates as the positive control group (Group B), the CTL cells obtained in Example 1 were placed into Transwell 6-well plates as the experimental group (Group C), and the CTL cells obtained in Comparative Example 1 were placed into Transwell 6-well plates as the positive control group (Group D). After co-culturing for 24h, 48h, and 72h, the 6-well plates were removed, digested with trypsin, and the cells were placed in 96-well plates. CCK-8 solution (10μL / well) was added, and the cells were cultured for another 1h. The absorbance at 450nm was measured using a microplate reader. The results are shown in [Figure 1]. Figure 5 .

[0085] Depend on Figure 5 It is evident that the DC cell culture kit of the present invention, when used to induce the culture of activated T cells from DC cells, inhibited the growth rate of lung cancer cells A549 after co-culturing for 24h, 48h, and 72h in a time-dependent manner. The activity of lung cancer cells A549 was significantly lower than that of the negative control group A and the positive control groups B and D. This indicates that the mature DC cells induced by the kit of the present invention can significantly enhance the tumor-killing activity of CTLs. The kits for Examples 2-3 are the same as those for Example 1 and will not be described again here.

[0086] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A high-efficiency induction culture kit for DC cells, characterized by, The kit comprises reagent A and reagent B; The reagent A is a mixed solution of GM-CSF solution with a concentration of 500-600 ng / mL, IL-4 solution with a concentration of 1000-3000 U / mL, IL-1β solution with a concentration of 5000-7000 IU / mL, AK1 solution with a concentration of 50-200 ng / mL, ACK1 solution with a concentration of 50-200 ng / mL, MLK3 solution with a concentration of 30-70 ng / mL, PLD1 solution with a concentration of 30-70 ng / mL, type II cytoskeleton 1 solution with a concentration of 50-200 ng / mL, and type I cytoskeleton 16 solution with a concentration of 50-200 ng / mL, with a volume ratio of (3-7) :(1-3) :(0.5-1.5) :(1-3) :(0.5-1.5) :(0.5-1.5) :(1-3) :(2-4) :(1-3); The reagent B is a mixed solution of PD-L1 protein solution with a concentration of 200-400 ng / mL, CTLA-4 protein solution with a concentration of 50-200 ng / mL, TIM-3 protein solution with a concentration of 50-200 ng / mL, LAG-3 protein solution with a concentration of 50-200 ng / mL, CD44 protein solution with a concentration of 300-500 ng / mL, CEA protein solution with a concentration of 300-700 ng / mL, neuron-specific enolase solution with a concentration of 100-200 ng / mL, and squamous cell carcinoma-associated antigen protein solution with a concentration of 100-200 ng / mL, with a volume ratio of (1-3) :(0.5-1.5) :(0.5-1.5) :(0.5-1.5) :(2-4) :(1-3) :(0.5-1.5) :

1.

2. A method for inducing culture of DC cells using the high-efficiency induction culture kit for DC cells according to claim 1, characterized by, The method comprises the following steps: S1: inoculate PBMC in 1640 medium for culture, then discard the suspended cells to obtain adherent cells, i.e., DC cells; S2: add serum-free RPMI 1640 medium and reagent A to the DC cells for continuous culture; S3: replace the liquid once the next day, supplement reagent A for continuous culture; S4: on the third day, add reagent B for continuous culture; S4: on the fourth day, collect the mature DC cells.

3. The method of claim 2, wherein, The seeding density of the PBMCs is 1 x 10 7 cells / mL.

4. The method of claim 2, wherein, The culture condition is 5% CO2, 37℃.

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