DC cell in-vitro induction amplification method and amplification culture medium

Abstract

The invention discloses a DC (dendritic cell) in-vitro induction and amplification method. The method comprises the following steps: (1) extracting CD14 < + >-CD28 < + > monocytes from PBMCs (peripheral blood mononuclear cells) by using anti-human CD14 and CD28 monoclonal antibodies coated with magnetic beads; (2) induction culture of DC cells; and (3) culturing the DC cells. The invention also discloses a culture medium for amplifying the DC cell prepared by the method. The culture medium comprises the following components: (1) a basic culture medium which is an RPMI-1640 culture medium containing 10% of fetal calf serum; and (2) adding components: 2mM of L-glutamine, 50mu M of beta-mercaptoethanol and 50ng / ml of LPS (Lipopolysaccharide). The DC cells prepared by the DC cell in-vitro induction amplification method disclosed by the invention have good performance and are suitable for large-scale preparation of the DC cells. Meanwhile, the culture medium for DC cell amplification disclosed by the invention can realize DC cell amplification on the premise of not containing cytokines, and provides a new direction for large-scale amplification of DC cells.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a DC cell in vitro induction and expansion method and an expansion culture medium. Background Art

[0002] Dendritic cells (DCs) are key cells in the immune system. They are professional antigen-presenting cells that can activate naive T cells, thus playing a vital role in initiating immune responses. DCs are widely distributed in the body, including blood, lymph, liver, spleen, and skin tissues. They can activate functional lymphocytes, induce cell-mediated cytotoxicity, and improve the body's cellular immunity. Therefore, DC cells have broad application prospects in tumor immunotherapy, vaccine development, and other fields.

[0003] However, DCs have a short lifespan in vivo and are difficult to amplify in large quantities in vitro, which limits their application and research in terms of antigen presentation function and expression ability. To address this problem, scientists have tried a variety of methods to amplify DCs, including using a specific combination of cytokines to promote the proliferation and maturation of DCs. For example, an in vitro efficient amplification system uses a specific factor combination to directly amplify DC precursor cells from peripheral blood mononuclear cells, and then uses a specific factor combination II to promote the differentiation and maturation of these precursor cells.

[0004] Although a variety of methods have been developed for the expansion of DCs, the process still faces some challenges and problems. For example, the expansion process may affect the antigen presentation ability of DCs, or the expanded DCs may be functionally different from the naturally existing DCs in the body. In addition, the expansion process may require the use of technologies such as viral or oncogene transformation, which may bring safety concerns.

[0005] In summary, there is still room for improvement in efficient methods and culture media for in vitro expansion and induction of maturation, especially in increasing the number, safety and function of DC cells. Summary of the invention

[0006] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a method for inducing and expanding DC cells in vitro and an expansion culture medium.

[0007] Therefore, the present invention discloses a method for inducing and expanding DC cells in vitro, the method comprising the following steps:

[0008] (1) Extraction of CD14 from PBMCs using magnetic beads coated with anti-human CD14 and CD28 monoclonal antibodies + -CD28 + of monocytes;

[0009] (2) DC cell induction culture;

[0010] (3) DC cell culture;

[0011] Wherein, the anti-human CD14 and CD28 monoclonal antibody in step (1) is monoclonal antibody C18, and the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody C18 are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0012] Preferably, the amino acid sequence of the monoclonal antibody C18 of the present invention that specifically binds to the CD14 protein is: KELTLEDLKIT; the amino acid sequence of the monoclonal antibody C18 that specifically binds to the CD28 protein is: DELTKNQVSLT.

[0013] Preferably, step (1) of the present invention comprises the following steps:

[0014] 1) Resuspend human PBMCs in PBS buffer containing 1% HSA, take samples and count, and take 1×10 7 Transfer cells to 1.5 mL EP tube and centrifuge at 1500 rpm for 5 min;

[0015] 2) Discard the supernatant, resuspend in 80 μL RPMI 1640 medium, add 20 μL magnetic bead-coated anti-human CD14 and CD28 monoclonal antibodies, mix well, and incubate in a 2-8°C refrigerator for 15 min;

[0016] 3) Place the LS column in the MACS separator and rinse twice with 1 mL of RPMI 1640 medium;

[0017] 4) After incubation, take the sample out of the 2-8°C refrigerator, add 1 mL of RPMI 1640 medium, centrifuge at 1500 rpm for 5 min, and discard the supernatant;

[0018] 5) Add 1 mL of RPMI 1640 medium to resuspend, add the sample to the separation column, wait for it to flow out naturally, then add RPMI 1640 medium twice, 1 mL each time, and collect the effluent in a 15 mL tube;

[0019] 6) After all the RPMI 1640 medium has flowed out, remove the separation column from the MACS separator and place it in another new 15 mL centrifuge tube. Add 3 mL of RPMI 1640 medium to the separation column and use the piston of the separation column to directly pump out the liquid.

[0020] 7) Place the 15 mL centrifuge tube containing the collected solution into a horizontal centrifuge and centrifuge at 1500 rpm for 5 min;

[0021] 8) After centrifugation, discard the supernatant and take 1 mL of RPMI1640 complete medium containing 10% fetal bovine serum, 2 mmol / L-glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin for culture, count and perform subsequent culture.

[0022] Preferably, step (2) of the present invention comprises the following steps:

[0023] 1) The monocytes screened in step (1) were divided into 1×10 6 cells / mL were inoculated into 6-well plates and cultured with 3 mL of RPMI1640 complete medium containing 10% fetal bovine serum, 2 mmol / L-glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin, and induction factors 1000 U / mL GM-CSF, 500 U / mL IL-4, 5% CO2, and 37°C;

[0024] 2) On the third day of culture, the culture plate was tilted, 50% of the supernatant was discarded, and an equal amount of RPMI 1640 complete medium containing 10% fetal bovine serum, 2 mmol / L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 1000 U / mL GM-CSF, and 500 U / mL IL-4 was added;

[0025] 3) On the 5th day of culture, add RPMI 1640 complete medium containing 100 IU / mL TNF-α, 1000 U / mL GM-CSF, and 500 U / mL IL-4;

[0026] 4) On the 8th day of culture, the culture supernatant was discarded with a pipette, the cells were digested with trypsin, and the cells were collected to obtain the prepared DC cells.

[0027] Preferably, the step (3) of the present invention is: diluting the cells in step (2) to 1×10 cells / mL using RPMI 1640 complete medium containing 10% fetal bovine serum, 2 mmol / L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 500 U / mL GM-CSF, and 100 U / mL IL-4. 6 cells / mL, inoculated in 25cm 3 The cells were cultured in a culture flask at 5% CO2 and 37°C for 2 to 3 days. When the cells grew to a confluence of about 80%, they were passaged and cultured.

[0028] In another aspect, the present invention further discloses a culture medium for expanding DC cells prepared by the method of the present invention, wherein the culture medium comprises the following components:

[0029] (1) Basic culture medium: RPMI-1640 medium containing 10% fetal bovine serum;

[0030] (2) Added ingredients: 2 mM L-glutamine, 50 μM β-mercaptoethanol, 50 ng / ml LPS.

[0031] In another aspect, the present invention also discloses the application of the method in inducing and expanding DC cells in vitro.

[0032] In another aspect, the present invention also discloses a use of the culture medium in DC cell expansion.

[0033] The DC cells prepared by the in vitro induction and expansion method of DC cells disclosed in the present invention have good performance and are suitable for large-scale preparation of DC cells. At the same time, the culture medium for DC cell expansion disclosed in the present invention can achieve DC cell expansion without containing cytokines, providing a new direction for large-scale expansion of DC cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Western blot detection results of CD14 protein and CD28 protein using monoclonal antibody C18. DETAILED DESCRIPTION

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0036] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0037] Example 1: Method for inducing and expanding DC cells in vitro

[0038] 1. Material Preparation

[0039] (1) Human peripheral blood mononuclear cells (PBMCs) were purchased from Wuhan Prosai Life Science Co., Ltd.

[0040] (2) RPMI-1640 culture medium and fetal bovine serum (FBS) were purchased from GIBCO.

[0041] (3) Human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), human recombinant interleukin-4 (IL-4), and tumor necrosis factor-α (TNF-α) were purchased from Shanghai Puxin Biotechnology Co., Ltd.

[0042] 2. Step (1) Extract CD14 from PBMCs using magnetic beads coated with anti-human CD14 and CD28 monoclonal antibodies + -CD28 + Monocytes

[0043] 1.1 Resuspend human PBMCs in PBS buffer containing 1% HSA, take samples and count, and take 1×10 7 Transfer cells to a 1.5 mL EP tube and centrifuge at 1500 rpm for 5 min.

[0044] 1.2 Discard the supernatant, take 80 μL RPMI 1640 medium to resuspend, add 20 μL magnetic bead-coated anti-human CD14 and CD28 monoclonal antibodies (prepared in Example 2), mix well and place in a 2-8°C refrigerator for incubation for 15 min.

[0045] 1.3 Take the LS separation column (Beijing Novi Biotechnology, 130-042-401) and place it in the MACS sorter (Beijing Novi Biotechnology, 130-042-303), and rinse it twice with 1 mL RPMI 1640 culture medium.

[0046] 1.4 After incubation, take the sample out of the 2-8°C refrigerator, add 1 mL of RPMI 1640 medium, centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0047] 1.5 Add 1 mL of RPMI 1640 medium to resuspend, add the sample to the separation column (Beijing Novi Biotechnology, 130-042-401), wait for it to flow out naturally, then add RPMI 1640 medium twice, 1 mL each time, and collect the effluent in a 15 mL tube.

[0048] 1.6 After all the RPMI 1640 medium has flowed out, remove the separation column from the MACS separator and place it in another new 15 mL centrifuge tube. Add 3 mL of RPMI 1640 medium to the separation column and use the piston that comes with the separation column to directly pump out the liquid.

[0049] 1.7 Place the 15 mL centrifuge tube containing the collected solution into a horizontal centrifuge and centrifuge at 1500 rpm for 5 minutes.

[0050] 1.8 After centrifugation, discard the supernatant and take 1 mL of RPMI1640 complete medium containing 10% fetal bovine serum, 2 mmol / L-glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin for culture, count and perform subsequent culture.

[0051] (2) DC cell induction culture

[0052] 1.1 The monocytes screened in step (1) were divided into 1×10 6 cells / mL were inoculated into 6-well plates and cultured with 3 mL of RPMI1640 complete medium containing 10% fetal bovine serum, 2 mmol / L-glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin, and induction factors 1000 U / mL GM-CSF, 500 U / mL IL-4 were added, and cultured at 37°C with 5% CO2.

[0053] 1.2 On the third day of culture, tilt the culture plate, discard 50% of the supernatant, and add an equal amount of RPMI 1640 complete medium containing 10% fetal bovine serum, 2 mmol / L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 1000 U / mL GM-CSF, and 500 U / mL IL-4.

[0054] 1.3 On the 5th day of culture, add RPMI 1640 complete medium containing 100 IU / mL TNF-α, 1000 U / mL GM-CSF, and 500 U / mL IL-4.

[0055] 1.4 On the 8th day of culture, discard the culture supernatant with a pipette, digest the cells with trypsin, and collect the cells to prepare dendritic cells (DCs). The cells are frozen and stored in liquid nitrogen (the freezing medium is 50% RPMI1640 culture medium + 40% fetal bovine serum + 10% DMSO) or expanded for subsequent experiments.

[0056] (3) DC cell culture

[0057] The cells in step (2) were diluted to 1×10 cells / mL using RPMI 1640 complete medium containing 10% fetal bovine serum, 2 mmol / L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 500 U / mL GM-CSF, and 100 U / mL IL-4. 6 cells / mL, inoculated in 25cm 3 The cells were cultured in a culture flask at 5% CO2 and 37°C for 2 to 3 days. When the cells grew to a confluence of about 80%, they were passaged and cultured for subsequent research.

[0058] Example 2: Preparation of magnetic bead-coated anti-human CD14 and CD28 monoclonal antibodies

[0059] 1. Recombinant human CD14 protein (abcam, ab290112, the amino acid sequence is shown in SEQ ID NO.1) and recombinant human CD28 protein (abcam, ab245796, the amino acid sequence is shown in SEQ ID NO.2) were mixed in equal mass ratios to prepare a vaccine, and the content of each protein in the vaccine was 0.1 mg / mL.

[0060] 2. Immunize 6-8 week old female BALB / c mice with the prepared vaccine for a total of four immunizations, all of which were subcutaneously injected at multiple points, 0.2 mL / time. The first immunization was emulsified with an equal amount of Freund's complete adjuvant, the second and third immunizations were emulsified with an equal amount of Freund's incomplete adjuvant, and the fourth immunization was mixed with an equal amount of saline and injected intraperitoneally. Blood was collected 7 days after the fourth immunization, and the titers of CD14 protein and CD28 protein antibodies were detected by indirect methods to determine whether antibodies against CD14 protein and CD28 protein were generated at the same time. The titers of different mice were compared, and mice with higher titers of CD14 protein and CD28 protein antibodies were selected for cell fusion.

[0061] 3. Through testing, two mice with a titer of 1:10000 were screened. The spleen of the screened mice was taken to extract splenocytes, and then the splenocytes were fused with myeloma cells to screen hybridoma cells. After screening and testing, a good hybridoma cell was screened. This hybridoma cell was used to prepare mouse ascites and monoclonal antibodies.

[0062] 4. Take healthy female mice aged 6 to 7 weeks and inject 0.5 mL of liquid paraffin intraperitoneally. After 7 to 10 days, inject 1×10 6 The hybridoma cells selected above were observed for 7 to 10 days, and the ascites was collected after the abdominal cavity of the mouse was obviously swollen. The ascites was centrifuged at room temperature for 15 minutes at 4000 rpm, and the supernatant was taken and purified using a Protein G column. The concentration of the purified monoclonal antibody (labeled as monoclonal antibody C18) was measured using the BCA method, and the purified monoclonal antibody was stored at -80°C after aliquoting.

[0063] 5. The prepared monoclonal antibody C18 was used to perform western blot detection on CD14 recombinant protein and CD28 recombinant protein. The monoclonal antibody C18 had good specificity and sensitivity to both CD14 protein and CD28 protein. Figure 1As shown. At the same time, the sequence of the monoclonal antibody C18 was detected and analyzed, and the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody C18 are shown in SEQ ID NO.3 and SEQ ID NO.4. At the same time, the positions of the monoclonal antibody C18 specifically binding to the CD14 protein and the CD28 protein were analyzed, wherein the amino acid sequence of the monoclonal antibody C18 specifically binding to the CD14 protein is: KELTLEDLKIT (the position in SEQ ID NO.1 is aa100 to aa110); the amino acid sequence of the monoclonal antibody C18 specifically binding to the CD28 protein is: DELTKNQVSLT (the position in SEQ ID NO.2 is aa226 to aa236).

[0064] 6. Take 1 mg of carboxyl magnetic particles (purchased from thermofisher with a diameter of 1 μm), add 0.5 mL of 0.1 M MES (pH 6.0) buffer, shake at room temperature for 1 min, centrifuge to remove the supernatant, and wash three times in succession. Resuspend with 0.5 mL of 0.1 M MES (pH 6.0) buffer, add 10 μg of monoclonal antibody C18, mix well, and incubate at room temperature for 20 min. Add 10 μL of LEDC (10 mg / mL), mix well, and incubate at room temperature for 2 h. After centrifugation, discard the supernatant, add 500 μL of TBST buffer, and repeat washing three times. Block with TBST buffer containing 1% BSA, and incubate at room temperature for 2 h. Add 500 μL of TBST buffer, wash three times, resuspend with 0.5 mL of TBST buffer containing 1% BSA, and store at 4°C for later use.

[0065] Example 3: Comparison of different induced cultures of DC cells

[0066] CD14 was captured using a commercial CD14 MicroBeads human monocyte kit (Miltenyibiotec, Germany). + The mononuclear cells were induced according to the method of Example 1 to prepare DC cells. The DC cells and the DC cells prepared in Example 1 were compared with the DC cell phenotype (surface co-stimulatory molecules CD40, CD80, CD83 and CD86 are surface markers of DC functional maturity, and HLA-DR is an antigen presenting molecule).

[0067] The cells prepared in Example 1 and the CD14 + DC cells prepared from monocytes (referred to as cells prepared in Example 3) were digested, washed with PBS, and resuspended into 1×10 6cells / mL, 0.5 mL of the suspension was taken, and PE-labeled mouse anti-human CD40 antibody, PE-labeled mouse anti-human CD80 antibody, PE-labeled mouse anti-human CD83 antibody, PE-labeled mouse anti-human CD86 antibody, and FITC-labeled mouse anti-human HLA-DR antibody (all purchased from Shanghai Biotech) were added, mixed, incubated at 4°C in the dark for 40 minutes, washed twice with PBS, and detected by flow cytometry. The results are shown in Table 1.

[0068] The results showed that the cells prepared in Example 1 expressed better surface markers than the cells prepared in Example 3, indicating that the cells prepared in Example 1 were better than the cells prepared in Example 3.

[0069] Table 1 Detection results of different induced DC cell surface markers

[0070]

[0071] Example 4: DC cell expansion culture and its application

[0072] 1. A DC cell expansion medium comprising the following components:

[0073] (1) Basic culture medium: RPMI-1640 culture medium containing 10% fetal bovine serum.

[0074] (2) Added ingredients: 2 mM L-glutamine, 50 μM β-mercaptoethanol, 50 ng / ml LPS.

[0075] 2. Comparison of different culture media

[0076] The DC cells prepared in the example were cultured using the culture medium of Example 1 (RPMI 1640 complete culture medium containing 10% fetal bovine serum, 2 mmol / L-glutamine, 500 U / mL GM-CSF, and 100 U / mL IL-4) and the culture medium of Example 4. The cells were first diluted to 0.5×10 6 cells / mL, and then inoculated into 6-well plates, 2 mL / well, 5% CO2, 37°C for 3 days, digested with trypsin, counted, washed with PBS, and resuspended into 1×10 6 Cells / mL, and each cell surface marker was detected according to the method of Example 3. The specific data are shown in Table 2.

[0077] The results showed that there was no significant difference between the two culture media in culturing DC cells, indicating that the culture medium of Example 4 still had a good culture effect on DC cells without GM-CSF and IL-4.

[0078] Table 2 Detection results of DC cell surface markers induced at different generations

[0079]

[0080] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for inducing and expanding DC cells in vitro, characterized in that: The method comprises the following steps: (1) Extraction of CD14 from PBMCs using magnetic beads coated with anti-human CD14 and CD28 monoclonal antibodies + -CD28 + of monocytes; (2) DC cell induction culture; (3) DC cell culture; Wherein, the anti-human CD14 and CD28 monoclonal antibody in step (1) is monoclonal antibody C18, and the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody C18 are shown in SEQ ID NO.3 and SEQ ID NO.

4.

2. The method according to claim 1, characterized in that The amino acid sequence of the monoclonal antibody C18 specifically binding to the CD14 protein is: KELTLEDLKIT; the amino acid sequence of the monoclonal antibody C18 specifically binding to the CD28 protein is: DELTKNQVSLT.

3. The method according to claim 1, characterized in that The step (1) comprises the following steps: 1) Resuspend human PBMCs in PBS buffer containing 1% HSA, take samples and count, and take 1×10 7 Transfer cells to 1.5 mL EP tube and centrifuge at 1500 rpm for 5 min; 2) Discard the supernatant, resuspend in 80 μL RPMI 1640 medium, add 20 μL magnetic bead-coated anti-human CD14 and CD28 monoclonal antibodies, mix well, and incubate in a 2-8°C refrigerator for 15 min; 3) Place the LS column in the MACS separator and rinse twice with 1 mL of RPMI 1640 medium; 4) After incubation, take the sample out of the 2-8°C refrigerator, add 1 mL of RPMI 1640 medium, centrifuge at 1500 rpm for 5 min, and discard the supernatant; 5) Add 1 mL of RPMI 1640 medium to resuspend, add the sample to the separation column, wait for it to flow out naturally, then add RPMI 1640 medium twice, 1 mL each time, and collect the effluent in a 15 mL tube; 6) After all the RPMI 1640 medium has flowed out, remove the separation column from the MACS separator and place it in another new 15 mL centrifuge tube. Add 3 mL of RPMI 1640 medium to the separation column and use the piston of the separation column to directly pump out the liquid. 7) Place the 15 mL centrifuge tube containing the collected solution into a horizontal centrifuge and centrifuge at 1500 rpm for 5 min; 8) After centrifugation, discard the supernatant and take 1 mL of RPMI1640 complete medium containing 10% fetal bovine serum, 2 mmol / L-glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin for culture, count and perform subsequent culture.

4. The method according to claim 1, characterized in that The step (2) comprises the following steps: 1) The monocytes screened in step (1) were divided into 1×10 6 cells / mL were inoculated into 6-well plates and cultured with 3 mL of RPMI1640 complete medium containing 10% fetal bovine serum, 2 mmol / L-glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin, and induction factors 1000 U / mL GM-CSF, 500 U / mL IL-4, 5% CO2, and 37°C; 2) On the third day of culture, the culture plate was tilted, 50% of the supernatant was discarded, and an equal amount of RPMI1640 complete medium containing 10% fetal bovine serum, 2 mmol / L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 1000 U / mL GM-CSF, and 500 U / mL IL-4 was added; 3) On the 5th day of culture, add RPMI1640 complete medium containing 100 IU / mL TNF-α, 1000 U / mL GM-CSF, and 500 U / mL IL-4; 4) On the 8th day of culture, the culture supernatant was discarded with a pipette, the cells were digested with trypsin, and the cells were collected to obtain the prepared DC cells.

5. The method according to claim 1, characterized in that The step (3) is as follows: dilute the cells in step (2) to 1×10 cells / mL using RPMI 1640 complete medium containing 10% fetal bovine serum, 2 mmol / L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 500 U / mL GM-CSF, and 100 U / mL IL-4. 6 cells / mL, inoculated in 25cm 3 The cells were cultured in a culture flask at 5% CO2 and 37°C for 2 to 3 days. When the cells grew to a confluence of about 80%, they were passaged and cultured.

6. A culture medium for expanding DC cells prepared by the method of claim 1, characterized in that: The culture medium comprises the following components: (1) Basic culture medium: RPMI-1640 medium containing 10% fetal bovine serum; (2) Added ingredients: 2 mM L-glutamine, 50 μM β-mercaptoethanol, 50 ng / ml LPS.

7. Use of the method according to claim 1 in inducing and expanding DC cells in vitro.

8. Use of the culture medium as claimed in claim 5 in DC cell expansion.

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