Culture method of DC cell loaded with pancreatic cancer antigen
By adding specific cytokines and antigen loading strategies, the maturation and expansion of pancreatic cancer-associated dendritic cells (DCs) are promoted, which solves the problems of insufficient DC cell maturation rate and antigen presentation capacity in existing technologies, and achieves more efficient pancreatic cancer immunotherapy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-20
- Publication Date
- 2026-04-10
AI Technical Summary
Existing technologies for preparing dendritic cell vaccines loaded with pancreatic cancer antigens have limited efficacy, are difficult to effectively overcome the immunosuppression of the tumor microenvironment, and have insufficient maturation rate and antigen presentation capacity of DC cells.
Factors such as GM-CSF, IL-4, IFN-γ, TNF-α, IL-1β, IL-6, PGE2, Poly I:C, and R848 were used to promote the activation of monocytes into dendritic cells (DCs). These DCs were then loaded with pancreatic cancer antigens PANC-1, MIA PaCa-2, BxPC-3, KP4, and SW1990HM. High-purity monocytes were separated by multiple centrifugations and lymphocyte separation fluid. The DCs were then cultured with cationic liposomes and fetal bovine serum to improve their maturation rate and antigen presentation capacity.
It significantly improved the maturation rate and antigen presentation capacity of DC cells, increased the expression rate of CD83 and CD86 by 94.16%, and increased the secretion of IL-12p70 to 645.38 pg/mL, thereby enhancing the targeting of the immune response and the immune activation capacity of DC cells.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of cell biology. Specifically, it promotes the activation of monocytes into dendritic cells (DCs) by adding factors such as GM-CSF, IL-4, IFN-γ, TNF-α, IL-1β, IL-6, and PGE2, and loads pancreatic cancer antigens PANC-1, MIA PaCa-2, BxPC-3, KP4, and SW1990HM, thereby improving the activation and maturation rate of DCs and loading them with pancreatic cancer-related antigens. This method can be used to culture pancreatic cancer DCs. Background Technology
[0002] Pancreatic cancer is one of the most malignant tumors, with an extremely low 5-year survival rate. The main reasons include difficulty in early diagnosis, its deep anatomical location, high invasiveness, easy early metastasis, and insensitivity to conventional chemotherapy and radiotherapy. The pancreatic tumor contains a highly immunosuppressive microenvironment, filled with myeloid-derived suppressor cells and regulatory T cells, which act like "security guards," preventing the body's own immune cells (such as T cells) from entering and attacking cancer cells. In recent years, tumor immunotherapy, represented by immune checkpoint inhibitors, has achieved revolutionary success in many cancers. However, single-agent immunotherapy has shown limited efficacy in pancreatic cancer, partly due to the aforementioned immunosuppressive microenvironment.
[0003] Dendritic cells (DCs) are the most powerful professional antigen-presenting cells. When DCs capture antigens (such as tumor antigens), they mature and migrate to lymph nodes, acting as "commanders" to present processed antigen information to T cells. This activates and educates naive T cells to differentiate into cytotoxic T cells capable of specifically recognizing and killing cancer cells. The basic principle of DC vaccines is to load tumor antigens in vitro, sensitize and mature them, and then reinfuse them into the patient, thereby activating an antigen-specific T-cell immune response. By manufacturing a large number of mature DCs loaded with pancreatic cancer antigens in vitro and then reinfusing them into the patient, we are essentially sending a specially trained "elite training team" to the immune system to directly guide T cells to attack pancreatic cancer cells carrying the corresponding antigens.
[0004] The preparation process mainly includes the following steps: Mononuclear cells are isolated from the patient's peripheral blood and induced to differentiate into immature dendritic cells (DCs). Tumor antigens are loaded onto the DCs, commonly using methods such as tumor cell lysates, known tumor-associated antigen peptides (e.g., CEA, MUC1, telomerase), or tumor antigen-encoded mRNA. DCs are stimulated to mature using a specific combination of cytokines or Toll-like receptor agonists, enabling them to acquire full function. Mature DCs are then reinfused into the patient to initiate a T-cell immune response against the tumor in vivo.
[0005] Poly I:C is a synthetic double-stranded RNA molecule formed by base pairing of polyinosinic acid and polycytidylic acid. It mimics the double-stranded RNA intermediate produced by viruses during replication. In nature, double-stranded RNA is a common feature in the life cycle of many viruses and is therefore considered a "danger signal" by the immune system. When dendritic cells (DCs) recognize Poly I:C via TLR3 and / or cytoplasmic receptors, they receive a strong "viral invasion" signal. This prompts DCs to acquire migratory abilities, secrete large amounts of cytokines, and enhance antigen presentation. Ultimately, this promotes the transformation of DCs from simple antigen capturers into fully functional T-cell activators.
[0006] R848 is a synthetic small-molecule imidazoquinoline compound. It can mimic the guanosine / uridine enrichment region of viral single-stranded RNA. Due to its efficient induction of IL-12, it is often used to prepare Th1-polarized dendritic cells (DCs) for cancer treatment. It induces key Th1-type cytokines and exerts a strong maturation-promoting effect on DCs.
[0007] Dendritic cell (DC) vaccines represent a promising direction for precision immunotherapy in pancreatic cancer. Current evidence supports their safety and ability to induce immune responses, but their efficacy as monotherapy is limited. Future breakthroughs depend on overcoming tumor microenvironment suppression through rational combination strategies and enhancing targeting through personalized neoantigen technology. DC vaccines hold promise for playing a specific role in future comprehensive pancreatic cancer treatment systems. Summary of the Invention
[0008] This invention aims to provide a culture method that promotes the activation of monocytes into dendritic cells (DCs) by adding factors such as GM-CSF, IL-4, IFN-γ, TNF-α, IL-1β, IL-6, PGE2, Poly I:C, and R848, and loads pancreatic cancer antigens PANC-1, MIA PaCa-2, BxPC-3, KP4, and SW1990HM onto the DCs. This method activates DCs while increasing their maturation rate, thereby promoting the maturation and expansion of DCs loaded with pancreatic cancer antigens.
[0009] To achieve the above objectives, the present invention adopts the following technical solution: A method for culturing dendritic cells loaded with pancreatic cancer antigen, characterized by the following culturing steps: Step 1: Resuscitation and culture of pancreatic cancer cell lines: The purchased pancreatic cancer cell lines PANC-1, MIAPaCa-2, BxPC-3, KP4, and SW1990HM were cultured in a separate clean laboratory. Each pancreatic cancer cell line was removed from the liquid nitrogen tank and rapidly thawed in a 37°C water bath. The cells were then centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in DMEM complete medium. After centrifugation at 1000 rpm for 10 min and discarding the supernatant, the cells were resuspended in RPMI-1640 complete medium containing 10% fetal bovine serum and seeded into cell culture flasks. The flasks were then incubated at 37°C under 5% CO2 conditions. The initial culture density for the pancreatic cancer cell lines PANC-1, MIAPaCa-2, BxPC-3, KP4, and SW1990HM was 1 × 10⁶ cells / flask.
[0010] Step 2: Preparation of pancreatic cancer cell antigens: Discard the culture medium from pancreatic cancer cells PANC-1, MIA PaCa-2, BxPC-3, KP4, and SW1990HM. Wash twice with PBS solution. Digest the pancreatic cancer cells with 0.05% trypsin for approximately 3 minutes. Discard the digestion solution. Stop digestion by adding RPMI-1640 medium containing 10% fetal bovine serum. Discard the washing medium. Wash the cells with PBS solution and resuspend them. Collect the pancreatic cancer cells into 5 mL centrifuge tubes. Under ice bath conditions, perform multiple short sonication cycles using an ultrasonic disruptor. Set the ultrasonic disruptor power to 200W, sonicate for 3 seconds, pause for 5 seconds, and repeat 10-15 times. Centrifuge the tubes at 4°C and 14000 rpm for 15-30 minutes. Collect the supernatant, which is the tumor cell lysate.
[0011] Step 3: Mononuclear cell isolation: A quantitative peripheral blood sample was placed into a 50 mL centrifuge tube and centrifuged, discarding the residue at the bottom. The obtained blood cells were diluted with physiological saline and slowly added to a 50 mL centrifuge tube containing 20%-30% of the blood sample volume of lymphocyte separation medium. After centrifugation, the supernatant was discarded. The white blood cell layer was slowly transferred to a new centrifuge tube, and 5 mL of physiological saline was added to the centrifuge tube containing the white blood cell layer. The mixture was stirred well to obtain a white blood cell diluent. The final white blood cell diluent was 70%-80% of the blood sample volume. The white blood cell diluent was centrifuged and the supernatant was discarded. The cells were resuspended in 5%-10% of the blood sample volume of physiological saline, and then centrifuged again with 70%-80% of the blood sample volume of physiological saline. The supernatant was discarded to obtain the cell pellet.
[0012] Step 4: DC cell culture: The cell pellet obtained in the previous step was resuspended in RPMI-1640 medium containing 10% fetal bovine serum and added to a 75 cm² cell culture flask. GM-CSF and IL-4 were then added to a final concentration of 500 U / mL. The flask was incubated at 37°C with 5% CO2 and saturated humidity. On day 3 of culture, half of the medium was replaced by slowly aspirating half of the supernatant. RPMI-1640 medium containing 10% fetal bovine serum was added, and GM-CSF and IL-4 were replenished to a final concentration of 500 U / mL.
[0013] Step 5: Activation and antigen-loaded culture of DC cells: Five days after culturing DC cells, pancreatic cancer cell lysate was added for co-culturing in serum-free RPMI-1640 medium, along with 20 μg / mL of cationic liposome Lipofectamine 2000. The medium was also supplemented with the following concentrations: 50 ng / mL IFN-γ, 50 ng / mL IFN-α, 10 ng / mL TNF-α, 10 ng / mL IL-1β, 10 ng / mL IL-6, 1 μg / mL PGE2, 20 μg / mL Poly I:C, and 1 μmol / L R848. The culture flasks were then placed in a 37°C, 5% CO2, saturated humidity incubator.
[0014] Step 6: Collecting Cells After 7 days of culture, the cell culture flasks were sterilized with 75% alcohol and placed in a biosafety cabinet. The culture medium was discarded, and the cells were washed twice with PBS solution. The mature DC cells were then digested with 0.05% trypsin for 2-3 minutes to detach them from the culture flasks. RPMI-1640 medium containing 10% fetal bovine serum was added, and all mature DC cells at the bottom of the culture flasks were detached using an electric pipette. The cell suspension was transferred to a 50 mL centrifuge tube and centrifuged at 2000 rpm for 10 minutes. The supernatant culture medium was discarded, and the cell pellet was resuspended with PBS solution. The pellet was centrifuged at 1500 rpm for 10 minutes, and the supernatant was discarded. The cells were washed again with PBS solution to obtain a mature DC tumor vaccine loaded with pancreatic cancer antigen.
[0015] This method promotes the activation of monocytes into dendritic cells (DCs) by adding factors such as GM-CSF, IL-4, IFN-γ, TNF-α, IL-1β, IL-6, and PGE2, and loads pancreatic cancer PANC-1, MIA PaCa-2, BxPC-3, KP4, and SW1990HM antigens, thereby enhancing the activation and maturation rate of pancreatic cancer DCs.
[0016] This invention adds different concentrations of factors such as GM-CSF, IL-4, IFN-γ, TNF-α, IL-1β, IL-6, PGE2, PolyI:C, and R848 to promote the activation of monocytes into dendritic cells (DCs). These DCs are then loaded with pancreatic cancer antigens PANC-1, MIA PaCa-2, BxPC-3, KP4, and SW1990HM. Flow cytometry is used to detect the expression rates of maturation markers CD83 and CD86 in the DCs. This patented method can increase the double-positive expression rate of CD83 and CD86 from 78.53% in conventional methods to 94.16%, and increase the secretion of IL-12p70 from 32.47 pg / mL in conventional culture methods to 645.38 pg / mL. Attached Figure Description
[0017] Figure 1 Microscopic images of DC cells cultured using conventional methods and the method of this invention.
[0018] Figure 2 The expression rates of CD83 and CD86 were detected by flow cytometry of DC cells using conventional methods and the culture method of this invention.
[0019] Figure 3 This is a comparison of the secretion levels of IL-12p70 in DC cells cultured using conventional methods and the method of this invention. Detailed Implementation
[0020] The present invention will be further described below with reference to the accompanying drawings: Example:
[0021] A method for culturing dendritic cells loaded with pancreatic cancer antigen, characterized by the following culturing steps: Step 1: Resuscitation and culture of pancreatic cancer cell lines.
[0022] The purchased pancreatic cancer cell lines PANC-1, MIAPaCa-2, BxPC-3, KP4, and SW1990HM were cultured in a separate clean laboratory. Each pancreatic cancer cell line was removed from the liquid nitrogen tank and rapidly thawed in a 37°C water bath. The cells were then centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in DMEM complete medium. After centrifugation at 1000 rpm for 10 min and discarding the supernatant, the cells were resuspended in RPMI-1640 complete medium containing 10% fetal bovine serum and seeded into cell culture flasks. The flasks were then incubated at 37°C under 5% CO2 conditions. The initial culture density for the pancreatic cancer cell lines PANC-1, MIAPaCa-2, BxPC-3, KP4, and SW1990HM was 1 × 10⁶ cells / flask.
[0023] Step 2: Preparation of pancreatic cancer cell antigens.
[0024] Discard the culture medium from pancreatic cancer cells PANC-1, MIA PaCa-2, BxPC-3, KP4, and SW1990HM. Wash twice with PBS solution. Digest the pancreatic cancer cells with 0.05% trypsin for approximately 3 minutes. Discard the digestion solution. Stop digestion by adding RPMI-1640 medium containing 10% fetal bovine serum. Discard the washing medium. Wash the cells with PBS solution and resuspend them. Collect the pancreatic cancer cells into 5 mL centrifuge tubes. Under ice bath conditions, perform multiple short sonication cycles using an ultrasonic disruptor. Set the ultrasonic disruptor power to 200W, sonicate for 3 seconds, pause for 5 seconds, and repeat 10-15 times. Centrifuge the tubes at 4°C and 14000 rpm for 15-30 minutes. Collect the supernatant, which is the tumor cell lysate.
[0025] Step 3: Mononuclear cell isolation.
[0026] A quantitative peripheral blood sample was placed into a 50 mL centrifuge tube and centrifuged, discarding the residue at the bottom. The obtained blood cells were diluted with physiological saline and slowly added to a 50 mL centrifuge tube containing 20%-30% of the blood sample volume of lymphocyte separation medium. After centrifugation, the supernatant was discarded. The white blood cell layer was slowly transferred to a new centrifuge tube, and 5 mL of physiological saline was added to the centrifuge tube containing the white blood cell layer. The mixture was stirred well to obtain a white blood cell diluent. The final white blood cell diluent was 70%-80% of the blood sample volume. The white blood cell diluent was centrifuged and the supernatant was discarded. The cells were resuspended in 5%-10% of the blood sample volume of physiological saline, and then centrifuged again with 70%-80% of the blood sample volume of physiological saline. The supernatant was discarded to obtain the cell pellet.
[0027] Step 4: DC cell culture.
[0028] The cell pellet obtained in the previous step was resuspended in RPMI-1640 medium containing 10% fetal bovine serum and added to a 75 cm² cell culture flask. GM-CSF and IL-4 were then added to a final concentration of 500 U / mL. The flask was incubated at 37°C with 5% CO2 and saturated humidity. On day 3 of culture, half of the medium was replaced by slowly aspirating half of the supernatant. RPMI-1640 medium containing 10% fetal bovine serum was added, and GM-CSF and IL-4 were replenished to a final concentration of 500 U / mL.
[0029] Step 5: Activation and antigen-loaded culture of DC cells.
[0030] Five days after culturing DC cells, pancreatic cancer cell lysate was added for co-culturing in serum-free RPMI-1640 medium, along with 20 μg / mL of cationic liposome Lipofectamine 2000. The medium was also supplemented with the following concentrations: 50 ng / mL IFN-γ, 50 ng / mL IFN-α, 10 ng / mL TNF-α, 10 ng / mL IL-1β, 10 ng / mL IL-6, 1 μg / mL PGE2, 20 μg / mL Poly I:C, and 1 μmol / L R848. The culture flasks were then placed in a 37°C, 5% CO2, saturated humidity incubator.
[0031] Step 6: Collect the cells.
[0032] After 7 days of culture, the cell culture flasks were sterilized with 75% alcohol and placed in a biosafety cabinet. The culture medium was discarded, and the cells were washed twice with PBS solution. The mature DC cells were then digested with 0.05% trypsin for 2-3 minutes to detach them from the culture flasks. RPMI-1640 medium containing 10% fetal bovine serum was added, and all mature DC cells at the bottom of the culture flasks were detached using an electric pipette. The cell suspension was transferred to a 50 mL centrifuge tube and centrifuged at 2000 rpm for 10 minutes. The supernatant culture medium was discarded, and the cell pellet was resuspended with PBS solution. The pellet was centrifuged at 1500 rpm for 10 minutes, and the supernatant was discarded. The cells were washed again with PBS solution to obtain a mature DC tumor vaccine loaded with pancreatic cancer antigen.
[0033] Control group: Step 1: Resuscitation and culture of pancreatic cancer cell lines.
[0034] The purchased pancreatic cancer cell lines PANC-1, MIA PaCa-2, BxPC-3, KP4, and SW1990HM were cultured in a separate clean laboratory. Each pancreatic cancer cell line was removed from the liquid nitrogen tank and rapidly thawed in a 37°C water bath. The cells were then centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in DMEM complete medium. After centrifugation at 1000 rpm for 10 min and discarding the supernatant, the cells were resuspended in RPMI-1640 complete medium containing 10% fetal bovine serum and seeded into cell culture flasks. The flasks were then incubated at 37°C under 5% CO2 conditions.
[0035] Step 2: Preparation of pancreatic cancer cell antigens.
[0036] Discard the culture medium from pancreatic cancer cells PANC-1, MIA PaCa-2, BxPC-3, KP4, and SW1990HM. Wash twice with PBS solution. Digest the pancreatic cancer cells with 0.05% trypsin for approximately 3 minutes. Discard the digestion solution. Stop digestion by adding RPMI-1640 medium containing 10% fetal bovine serum. Discard the washing medium. Wash the cells with PBS solution and resuspend them. Collect the pancreatic cancer cells into 5 mL centrifuge tubes. Under ice bath conditions, perform multiple short sonication cycles using an ultrasonic disruptor. Set the ultrasonic disruptor power to 200W, sonicate for 3 seconds, pause for 5 seconds, and repeat 10-15 times. Centrifuge the tubes at 4°C and 14000 rpm for 15-30 minutes. Collect the supernatant, which is the tumor cell lysate.
[0037] Step 3: Mononuclear cell isolation.
[0038] A quantitative peripheral blood sample was placed into a 50 mL centrifuge tube and centrifuged, discarding the residue at the bottom. The obtained blood cells were diluted with physiological saline and slowly added to a 50 mL centrifuge tube containing 20%-30% of the blood sample volume of lymphocyte separation medium. After centrifugation, the supernatant was discarded. The white blood cell layer was slowly transferred to a new centrifuge tube, and 5 mL of physiological saline was added to the centrifuge tube containing the white blood cell layer. The mixture was stirred well to obtain a white blood cell diluent. The final white blood cell diluent was 70%-80% of the blood sample volume. The white blood cell diluent was centrifuged and the supernatant was discarded. The cells were resuspended in 5%-10% of the blood sample volume of physiological saline, and then centrifuged again with 70%-80% of the blood sample volume of physiological saline. The supernatant was discarded to obtain the cell pellet.
[0039] Step 4: DC cell culture.
[0040] Resuspend the cell pellet obtained in the previous step in RPMI-1640 medium containing 10% fetal bovine serum, and add it to a 75 cm³ culture medium. 2 The cells were cultured in a flask, and GM-CSF and IL-4 were added to a final concentration of 500 U / mL. The flasks were then incubated in a 37°C, 5% CO2, saturated humidity incubator. On day 3 of incubation, half the medium was replaced by slowly aspirating half of the supernatant, adding RPMI-1640 medium containing 10% fetal bovine serum, and supplementing with GM-CSF and IL-4 to a final concentration of 500 U / mL.
[0041] Step 5: Activation and antigen-loaded culture of DC cells Five days after culturing DC cells, pancreatic cancer cell lysate was added for co-culturing. 10 ng / mL TNF-α, 10 ng / mL IL-1β, and 10 ng / mL IL-6 were added to the culture medium. The culture flasks were placed in a constant temperature incubator at 37°C and 5% CO2 with saturated humidity.
[0042] Step 6: Collect the cells.
[0043] After 7 days of culture, the cell culture flasks were sterilized with 75% alcohol and placed in a biosafety cabinet. The culture medium was discarded, and the cells were washed twice with PBS solution. The mature DC cells were then digested with 0.05% trypsin for 2-3 minutes to detach them from the culture flasks. RPMI-1640 medium containing 10% fetal bovine serum was added, and all mature DC cells at the bottom of the culture flasks were detached using an electric pipette. The cell suspension was transferred to a 50 mL centrifuge tube and centrifuged at 2000 rpm for 10 minutes. The supernatant culture medium was discarded, and the cell pellet was resuspended with PBS solution. The pellet was centrifuged at 1500 rpm for 10 minutes, and the supernatant was discarded. The cells were washed again with PBS solution to obtain a mature DC tumor vaccine loaded with pancreatic cancer antigen.
[0044] When comparing the embodiments of the present invention with the experimental control group, we can see significant differences between the two in the specific cell culture methods, the culture factors used, and the selection of other reagents. The following is a detailed analysis of the differences between the two: Compared to the embodiments of the present invention, the experimental control group used only three immune factors: TNF-α, IL-1β, and IL-6. These factors can stimulate immune responses, but they are not as comprehensive as the multiple immune factors in the embodiments of the present invention, and may not be able to fully activate the antigen presentation and immune response capabilities of dendritic cells.
[0045] In this invention, pancreatic cancer cell lysate is added to DC cells (dendritic cells) after culturing for 5 days for co-culturing. This cell lysate can provide antigenic information of cancer cells, helping DC cells to recognize and activate immune responses. By using serum-free RPMI-1640 medium for co-culturing, the interference of serum components on cells can be minimized, providing a more controllable environment.
[0046] This invention utilizes multiple immune factors, including IFN-γ, IFN-α, TNF-α, IL-1β, IL-6, PGE2, Poly I:C, and R848. The interaction of these factors can comprehensively and strongly activate dendritic cells (DCs) and enhance the immune response. IFN-γ and IFN-α are two potent immune activators that can enhance the antigen-presenting function of dendritic cells through different mechanisms and enhance the anti-tumor immune response by activating cell-mediated immune responses. They promote antigen presentation efficiency by enhancing the expression of major histocompatibility complex molecules in dendritic cells. The simultaneous addition of these two interferons can significantly improve the function of dendritic cells, increase the presentation of tumor antigens, and enhance the immune system's recognition and attack on tumors. TNF-α is a potent inflammatory factor that plays multiple roles in anti-tumor immune responses, including promoting the activation of immune cells, increasing the secretion of cytokines, and directly inducing tumor cell apoptosis. It not only enhances the immune response of dendritic cells but also directly enhances the effect of tumor immune clearance through its anti-tumor activity. IL-1β and IL-6 are important pro-inflammatory cytokines that play a regulatory role in the immune response. IL-1β can promote the activation and proliferation of immune cells, while IL-6 has a strong immunomodulatory effect, promoting T cell proliferation and antibody production, and regulating the balance of the immune response. These two cytokines enhance the immune response by regulating the function of immune cells and helping dendritic cells play a greater role in tumor immunity. PGE2 is an important lipid molecule that plays a bidirectional regulatory role in immune responses. In tumor immunity, PGE2 can promote immunosuppressive responses by regulating the immune microenvironment. However, in this technical approach, the addition of PGE2 helps to regulate the balance of the immune system, avoiding excessive immune responses, thereby enabling dendritic cells to more effectively activate the immune system. PGE2 can finely regulate the immune response, helping to improve the immune activation capacity of DC cells and prevent immunosuppression caused by excessive immune responses. Poly I:C and R848 are two potent immunostimulatory molecules that induce immune responses by mimicking viral infection and activating Toll-like receptors (such as TLR3 and TLR7 / 8). Poly I:C enhances antiviral immune responses via the TLR3 pathway, while R848 activates intracellular immune responses via the TLR7 / 8 pathway. These two immunostimulatory molecules strongly activate dendritic cells, enhancing their immune-presenting function and further activating downstream T-cell responses. The use of these molecules can significantly improve the role of dendritic cells in antitumor immune responses, especially in the immune recognition and attack of viruses or tumor cells.
[0047] These immune factors are indispensable in this invention because they work synergistically through different mechanisms to enhance the antigen presentation and immune activation capabilities of dendritic cells (DCs), thereby significantly improving the efficacy of tumor immunotherapy. The absence of any single factor may prevent the immune response from achieving optimal results, especially in the context of tumor immune escape; the lack of any single immune factor can weaken the immune response and lead to a reduction in therapeutic efficacy.
[0048] This invention utilizes pancreatic cancer cell lysate as an antigen, ensuring that dendritic cells can present antigens specific to pancreatic cancer. This targeted approach enhances the specificity of the immune response, providing a more precise target for cancer immunotherapy. Co-culturing pancreatic cancer cell lysate with dendritic cells (DCs) using cationic liposomes helps improve antigen effectiveness and the uptake capacity of DCs. The use of GM-CSF and IL-4 during DC culture, both cytokines that promote DC proliferation and maturation, effectively increases the number and function of DCs. Flow cytometry analysis of DC maturation status and antigen loading capacity allows for real-time, quantitative assessment of cell function, a capability difficult to achieve with traditional methods, thus increasing experimental reliability and reproducibility.
[0049] Furthermore, this invention effectively separates high-purity mononuclear cells through multiple centrifugations and the application of lymphocyte separation medium, improving the efficiency of subsequent DC cell culture; the use of RPMI-1640 medium with fetal bovine serum provides DC cells with abundant nutrients, effectively supporting cell growth and maturation; enhanced immune activation capacity: by activating DC cells and loading them with specific antigens, the immune activation capacity of DC cells can be significantly enhanced, thereby improving their ability to induce T cell responses and increasing their potential to combat pancreatic cancer.
[0050] The culture method described in this invention can be used not only for loading pancreatic cancer antigens, but also to adjust the antigen source as needed. It is applicable to other types of tumor immunotherapy research, has good scalability, and can adjust the culture scale according to experimental needs, making it suitable for large-scale cell culture and preclinical research.
[0051] Through experiments, the beneficial effects of this invention are as follows: (1) Increase the positive rate of CD83 and CD86 in DC cells.
[0052] The CD83 and CD86 positivity rates of DC cells cultured using conventional methods and those cultured using the DC cell activation and maturation promotion method of this invention were detected by flow cytometry. The CD83 positivity rate of DC cells cultured using conventional methods was (0.7906±0.0432), while that of DC cells cultured using the DC cell activation and maturation promotion method of this invention was (0.9532±0.0475). The CD86 positivity rate of DC cells cultured using conventional methods was (0.7557±0.0463), while that of DC cells cultured using the DC cell activation and maturation promotion method of this invention was (0.9376±0.0491). The CD83+CD86 positivity rate of DC cells cultured using conventional methods was (0.7853±0.0471), while that of DC cells cultured using the DC cell activation and maturation promotion method of this invention was (0.9416±0.0518). Figure 2 ).
[0053] (2) Increase the secretion of IL-12p70 by DC cells.
[0054] The secretion of IL-12p70 in the culture supernatant of DC cells in two groups on day 7 was detected by ELISA. The secretion of IL-12p70 in the supernatant of DC cells cultured using conventional methods was (32.47±5.38) pg / mL, while the secretion of IL-12p70 in the supernatant of DC cells cultured using the method of activating DC cells and promoting DC maturation in this invention was (645.38±36.49) pg / mL. Figure 3 ).
[0055] In summary, this invention compared the effects of conventional culture methods with the culture method of activating and promoting dendritic cell (DC) maturation of this invention on DC cells. The results showed that the CD83 positivity rate of DC cells cultured using this method was 0.9532±0.0475, significantly higher than the 0.7906±0.0432 of the conventional method; the CD86 positivity rate also increased to 0.9376±0.0491, a significant improvement compared to the 0.7557±0.0463 of the conventional method. Furthermore, the amount of IL-12p70 secreted by DC cells significantly increased from 32.47±5.38 pg / mL in the conventional method to 645.38±36.49 pg / mL. These results indicate that the method of this invention can effectively improve the maturation and function of DC cells.
[0056] In summary, this invention utilizes pancreatic cancer cell lysate as an antigen to ensure that dendritic cells (DCs) can specifically present pancreatic cancer antigens, enhancing the targeting of the immune response and providing a more precise approach for cancer immunotherapy. Co-culturing pancreatic cancer cell lysate with DCs, combined with the use of cationic liposomes, helps improve antigen effectiveness and the uptake capacity of DCs. GM-CSF and IL-4 are used during DC culture to promote DC proliferation and maturation, thereby increasing cell number and function. Flow cytometry is used to detect DC maturity and antigen loading capacity, allowing for quantitative assessment of cell functional status and increasing experimental reliability and reproducibility. Furthermore, this invention effectively separates high-purity monocytes through multiple centrifugations and lymphocyte separation medium, enhancing DC culture efficiency; the use of RPMI-1640 medium with fetal bovine serum provides nutritional support for cells, promoting cell growth and maturation. This culture method not only enhances the immune activation capacity of DCs but also strengthens their potential to induce T-cell responses, thereby combating pancreatic cancer. This method has good scalability, allowing the antigen source to be adjusted according to experimental needs. It is applicable to other tumor immunotherapy research and provides an ideal technical platform for large-scale cell culture and preclinical research.
Claims
1. A method for culturing dendritic cells (DCs) loaded with pancreatic cancer antigen, comprising the following steps in sequence: resuscitation and culture of pancreatic cancer cell lines, preparation of pancreatic cancer cell antigen, isolation of monocytes, DC cell culture, activation and antigen-loading culture of DCs, and collection of DCs, characterized in that, In the activation and antigen-loaded culture of DC cells, after culturing DC cells for 90-110 hours, pancreatic cancer cell lysate was added for co-culture. Serum-free RPMI-1640 medium and cationic liposomes were used, and IFN-γ, IFN-α, TNF-α, IL-1β, IL-6, PGE2, Poly I:C, and R848 were added to the medium. After co-culturing for 20-50 hours under standard cell culture conditions, the cells were further cultured.
2. The method for culturing DC cells loaded with pancreatic cancer antigen according to claim 1, characterized in that... The cationic liposomes are Lipofectamine 2000 at a concentration of 20 μg / mL. The culture medium also contains IFN-γ at a concentration of 50 ng / mL, IFN-α at a concentration of 50 ng / mL, TNF-α at a concentration of 10 ng / mL, IL-1β at a concentration of 10 ng / mL, IL-6 at a concentration of 10 ng / mL, PGE2 at a concentration of 1 μg / mL, Poly I:C at a concentration of 20 μg / mL, and R848 at a concentration of 1 μmol / L.
3. A method for culturing DC cells loaded with pancreatic cancer antigen according to claim 1 or 2, characterized in that, The pancreatic cancer cell line resuscitation and culture refers to the process of taking the pancreatic cancer cell line out of the liquid nitrogen tank in an independent clean laboratory, rapidly shaking and melting it at room temperature and centrifuging it, resuspending the cells in RPMI-1640 complete medium containing fetal bovine serum and then seeding them into cell culture flasks and culturing them under standard cell culture conditions. The preparation of the pancreatic cancer cell antigen requires discarding the culture medium in the pancreatic cancer cell culture flask, washing the cells with PBS solution, adding trypsin for digestion and terminating the digestion, collecting the cells and sonicating them, and finally centrifuging them at high speed to obtain tumor cell lysates. The mononuclear cell isolation refers to centrifuging a quantitative peripheral blood sample, diluting it, adding lymphocyte separation solution, centrifuging it again, collecting white membrane layer cells, and centrifuging it multiple times to obtain cell precipitate. The DC cell culture refers to resuspending the cell pellet in RPMI-1640 medium containing fetal bovine serum, adding GM-CSF and 5IL-4 to a final concentration, and culturing under standard cell culture conditions, followed by a half-volume medium replacement after 40-80 hours. The activation and antigen-loading culture of the DC cells were carried out after culturing the DC cells for 90-110 hours, followed by co-culturing with pancreatic cancer cell lysates using serum-free RPMI-1640 medium supplemented with cationic liposomes and tumor cell lysates. The collection and detection of DC cells refer to the following steps: after co-culture, DC cells are collected and washed with PBS solution, then digested with enzyme digestion solution and collected for centrifugation, and finally the maturation status and antigen loading capacity of DC cells are detected by flow cytometry.
4. The method for culturing DC cells loaded with pancreatic cancer antigen according to claim 3, characterized in that... During the resuscitation and culture of the pancreatic cancer cell lines, the pancreatic cancer cell lines included PANC-1, MIA PaCa-2, BxPC-3, KP4, and SW1990HM. The pancreatic cancer cell lines were cultured in an independent clean laboratory. After being removed from the liquid nitrogen tank, each pancreatic cancer cell line was rapidly thawed by shaking in a 37°C constant temperature water bath. The thawed cells were then centrifuged at 1000 rpm for 10 minutes, the supernatant was discarded, and the cells were resuspended in DMEM complete medium. After another 10 minutes of centrifugation at 1000 rpm, the supernatant was discarded, and the cells were resuspended in RPMI-1640 complete medium containing 10% fetal bovine serum. The cells were then seeded into cell culture flasks with an initial culture quantity of 1×10^6 cells / flask and cultured at 37°C with 5% CO2.
5. The method for culturing DC cells loaded with pancreatic cancer antigen according to claim 4, characterized in that, In the preparation of the pancreatic cancer cell antigen, the culture medium in the pancreatic cancer cell culture flask is discarded, and the cells are washed twice with PBS solution. 0.05% trypsin is added to digest the pancreatic cancer cells for approximately 3 minutes. The digestion solution is discarded, and RPMI-1640 medium containing 10% fetal bovine serum is added to terminate the digestion. The washing medium is then discarded. The cells are washed with PBS solution and resuspended. The pancreatic cancer cells are collected into 5 mL centrifuge tubes. Under ice bath conditions, multiple short bursts of sonication are performed using an ultrasonic disruptor, with a power setting of 200W, a sonication time of 3 seconds, a 5-second interval, and repeated 10-15 times. The centrifuge tubes are then placed in a centrifuge and centrifuged at 4°C and 14000 rpm for 15-30 minutes. The supernatant is collected to obtain the tumor cell lysate.
6. The method for culturing DC cells loaded with pancreatic cancer antigen according to claim 5, characterized in that, For the monocyte isolation process, a quantitative peripheral blood sample is placed into a 50 mL centrifuge tube and centrifuged, discarding the residue at the bottom. The obtained blood cells are diluted with physiological saline and slowly added to a 50 mL centrifuge tube containing 20%-30% of the blood sample volume of lymphocyte separation solution. The sample is then centrifuged again, and the supernatant is discarded. White blood cell membrane cells are slowly transferred to a new centrifuge tube, and 5 mL of physiological saline is added. The mixture is thoroughly mixed to obtain a leukocyte diluent, the final volume of which is 70%-80% of the blood sample volume. The leukocyte diluent is centrifuged, the supernatant is discarded, and the cells are resuspended in 5%-10% of the blood sample volume of physiological saline. Then, 70%-80% of the blood sample volume of physiological saline is added, and the sample is centrifuged again, discarding the supernatant to obtain the cell pellet.
7. The method for culturing DC cells loaded with pancreatic cancer antigen according to claim 5, characterized in that, During DC cell culture, the cell pellet obtained in the previous step was resuspended in RPMI-1640 medium containing 10% fetal bovine serum and added to a 75 cm² cell culture flask. GM-CSF and IL-4 were added to the flask to a final concentration of 500 U / mL. The cell culture flask was then placed in a constant temperature incubator at 37°C and 5% CO2 saturated humidity. On the third day of culture, half of the medium was replaced. Half of the supernatant was slowly aspirated, and RPMI-1640 medium containing 10% fetal bovine serum was added, along with GM-CSF and IL-4 to a final concentration of 500 U / mL.
8. The method for culturing DC cells loaded with pancreatic cancer antigen according to claim 5, characterized in that, The collection of DC cells involves gently collecting the DC cells after co-culture, washing them with PBS solution to remove unbound antigens and culture medium components, digesting the cells with an enzyme digestion solution (such as trypsin) for 2-3 minutes, and then adding culture medium containing serum to stop the digestion. The digested cells are then collected into centrifuge tubes, centrifuged at 1000 rpm for 10 minutes, and the supernatant is discarded to obtain the DC cell pellet.