Method for inducing DC precursor cells in PBMC to differentiate into pDC cells in vitro and application

Abstract

The invention provides a method for inducing DC precursor cells in PBMC (peripheral blood mononuclear cells) to differentiate into pDC cells in vitro, which comprises the following steps: the PBMC is placed in a culture medium for culture, and the culture medium comprises the following components: GM-CSF (granulocyte-macrophage colony-stimulating factor), Poly (I: C), Flt3l, TNF-alpha, ODN2006, Compound 4, autologous plasma and an AIM-V basal culture medium; replacing or supplementing the culture medium based on the culture progress until the preset culture time is reached; and collecting the cultured cells. According to the method, the PBMC serves as a culture object, the DC precursor cells in the PBMC are finally differentiated into the pDC cells through culture, the culture method is simple, a large number of pDC cells can be conveniently and efficiently obtained in vitro, and guarantee is provided for basic research and clinical application of the pDC cells. Meanwhile, the pDC cells obtained after the culture medium is used for culturing for 7 days can secrete IFN-alpha with a relatively high level under the condition of no target cell stimulation, so that the application of the culture medium in antiviral and anti-tumor treatment is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of immune cell culture, and in particular to a method for inducing DC precursor cells in PBMC to differentiate into pDC cells in vitro and an application thereof. Background Art

[0002] Dendritic cells (DCs) are the most important and powerful professional antigen-presenting cells in the body. DCs are mainly divided into two subtypes according to their sources: myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs).

[0003] pDCs are the main interferon (IFN) producing cells in the human body, and can produce more than 95% of type I IFN (IFN-α) in the body. IFN-α produced by pDCs can inhibit the replication and spread of viruses and is a powerful antiviral interferon. IFN-α can also play an immunomodulatory role by activating other immune cells.

[0004] The existing in vitro culture and induction of pDC mainly rely on sorting from peripheral blood mononuclear cells (PBMC) using flow cytometry technology. However, the number of pDC in human PBMC is extremely small, generally accounting for only 0.2% to 0.8% or even less, and there are individual differences. Simple sorting is difficult to obtain a sufficient number of cells for in vitro and in vivo and clinical application research, thus limiting the further development of pDC research and application. Summary of the invention

[0005] In order to overcome the deficiencies in the prior art, the present invention provides a method and application of in vitro inducing PBMC to differentiate into pDC cells.

[0006] The present invention provides a method for inducing DC precursor cells in PBMC to differentiate into pDC cells in vitro, comprising the steps of: placing PBMC in a culture medium for culturing, wherein the culture medium comprises the following components: 1-100 ng / mL GM-CSF, 10-1000 ng / mL Poly(I:C), 10-100 ng / mL Flt31, 10-1000 UI / mL TNF-α, 0.1-10 μM ODN2006, 0.1-10 μM Compound 4, 1-10% autologous plasma and AIM-V basic culture medium, wherein the CAS number of the Compound 4 is 2115702-83-3;

[0007] replacing or supplementing the culture medium based on the culture progress until a predetermined culture time is reached;

[0008] The cultured cells were collected.

[0009] Among them, the present invention uses PBMC as the culture object, and the DC precursor cells in the PBMC are finally differentiated into pDC cells through culture. PBMC is easy to obtain and has few restrictions, and there is no need to sort it in the early stage and then expand and culture it, and there is no need to change different culture media many times. The culture method of the present invention is simple and easy to operate, and is convenient for subsequent promotion and use.

[0010] In a preferred embodiment, the culture medium comprises the following components: 20 ng / mL GM-CSF, 100 ng / mL Poly (I: C), 50 ng / mL Flt31, 100 UI / mL TNF-α, 1 μM ODN2006, 0.5 μM Compound 4, 10% autologous plasma, and AIM-V basal medium.

[0011] In a preferred embodiment, the PBMCs are separated from human peripheral venous blood by density gradient centrifugation.

[0012] In a preferred embodiment, the predetermined culture time is 7 days.

[0013] In a preferred embodiment, the culturing of PBMC in a culture medium specifically includes the steps of resuspending PBMC cells using AIM-V basal culture medium preheated at 37°C, inoculating them in a 6-well plate, culturing them in a 37°C carbon dioxide incubator for 2 hours, washing away non-adherent cells, and replacing them with complete culture medium for culturing.

[0014] In a preferred embodiment, the seeding density of PBMC is 8×10 6 Pieces / mL.

[0015] As a second aspect of the present invention, the present invention claims a culture medium for inducing DC precursor cells in PBMC to differentiate into pDC cells in vitro, the culture medium comprising the following components: 1-100 ng / mL GM-CSF, 10-1000 ng / mL Poly (I: C), 10-100 ng / mL Flt31, 10-1000 UI / mL TNF-α, 0.1-10 μM ODN2006, 0.1-10 μM Compound 4, 1-10% autologous plasma and AIM-V basal culture medium.

[0016] The culture medium of the present invention acts on PBMC, is easy to obtain and has few restrictions, and can induce DC precursor cells in PBMC to differentiate into pDC cells in vitro, so that the proportion of pDC cells obtained after culture in total cells is greatly increased.

[0017] Among them, the preferred embodiment of the culture medium for inducing DC precursor cells in PBMC to differentiate into pDC cells in vitro is that the culture medium comprises the following components: 20ng / mL GM-CSF, 100ng / mL Poly(I:C), 50ng / mL Flt3l, 100UI / mL TNF-α, 1μM ODN2006, 0.5μM Compound 4, and 10% autologous plasma.

[0018] Furthermore, as a third aspect of the present invention, protection is sought for a kit, which comprises any one of the above-mentioned culture media, and is used for in vitro induction of differentiation of DC precursor cells in PBMC into pDC cells for culture or / and preparation of exosomes.

[0019] The present invention also claims the use of pDC cells obtained by the above method in anti-virus and anti-tumor. Among them, the present invention differentiates DC precursor cells in PBMC into pDC cells by culturing them, produces a large amount of interferon to respond to viruses, and plays a core role in anti-virus and anti-tumor immunity.

[0020] The present invention provides a method for inducing DC precursor cells in PBMC to differentiate into pDC cells in vitro, using PBMC as the culture object, inducing DC precursor cells in PBMC to differentiate into pDC, and the pDC cells obtained after 7 days of culture based on the culture medium of the present invention account for 21.24% of the total cells, which is about 193 times higher than before culture; and the culture method of the present invention is simple, and a large number of pDC cells can be obtained in vitro conveniently and efficiently, providing a guarantee for the basic research and clinical application of pDC cells. At the same time, the pDC cells obtained after 7 days of culture with the culture medium of the present invention can secrete a higher level of IFN-α without target cell stimulation, which is beneficial to its application in antiviral and anti-tumor treatment.

[0021] The present invention also provides a culture medium for inducing DC precursor cells in PBMC to differentiate into pDC cells in vitro and its application, and the corresponding culture medium can effectively induce DC precursor cells in PBMC to differentiate into pDC cells, so that pDC cells can play a greater role in antiviral or anti-tumor treatment. The corresponding kit can be applied to kits such as DC precursor cell culture and differentiation in PBMC, pDC cell culture, and exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 This is a cell morphology diagram during the cell culture process of Example 1;

[0024] Figure 2 This is a flow cytometry chart of the percentage of pDC cells in PBMC in Comparative Example 1;

[0025] Figure 3 This is a flow cytometry image of the pDC cell ratio in Example 1;

[0026] Figure 4 This is the flow cytometry analysis of pDC cell ratio in Comparative Example 2;

[0027] Figure 5 This is the flow cytometry analysis of pDC cell ratio in Comparative Example 3;

[0028] Figure 6 This is the flow cytometry analysis of pDC cell ratio in Comparative Example 4;

[0029] Figure 7 The flow cytometry results of IFN-α content in Comparative Example 1 and Example 1 are shown in FIG. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The present invention aims to achieve in vitro acquisition of pDC cells in large quantities by inducing DC precursor cells in PBMC to differentiate into pDC cells in vitro.

[0032] The method of inducing DC precursor cells in PBMC to differentiate into pDC cells in vitro of the present invention comprises the steps of:

[0033] The PBMCs were cultured in culture medium;

[0034] replacing or supplementing the culture medium based on the culture progress until a predetermined culture time is reached;

[0035] The cultured cells were collected.

[0036] Among them, placing PBMC in culture medium for culturing specifically includes the steps of: resuspending PBMC cells using AIM-V basal culture medium preheated at 37°C, inoculating them in a 6-well plate, placing them in a 37°C carbon dioxide incubator for adherent culture for 2 hours, washing away non-adherent cells, and replacing with complete culture medium for culturing.

[0037] In the present invention, the culture medium for inducing DC precursor cells in PBMC to differentiate into pDC cells in vitro comprises the following components: 1-100 ng / mL GM-CSF, 10-1000 ng / mL Poly(I:C), 10-100 ng / mL Flt31, 10-1000 UI / mL TNF-α, 0.1-10 μM ODN2006, 0.1-10 μM Compound 4, 1-10% autologous plasma, and AIM-V basal culture medium.

[0038] Among the components of the culture medium, GM-CSF, Poly (I: C), and Flt31 can induce pDC cell differentiation and proliferation; TNF-α can induce pDC cell maturation; ODN2006 and Compound 4 can induce pDC to secrete IFN-α. Among them, the Compound 4 specifically refers to TLR7 / 8agonist 6, which is an effective agonist of TLR7 / 8 and is an imidazoquinoline derivative compound, CAS No.: 2115702-83-3. ODN 2006 is a TLR9 agonist, CAS No.: 525625-52-9.

[0039] Specifically, in the present invention, PBMCs are used as culture cells, which are widely distributed in the body and easy to obtain. Compared with other special cells, the method is more convenient and has a wider range of applicable scenarios and fewer restrictions. The DC precursor cells in PBMCs are differentiated into pDCs through the culture medium of the present invention, and the proportion of pDC cells obtained after culture is greatly increased.

[0040] Among them, PBMCs were isolated from human peripheral venous blood using density gradient centrifugation.

[0041] Replacing or supplementing the culture medium based on the culture progress specifically refers to performing a fluid replenishment / replacement operation every 2 to 3 days according to the cell proliferation situation. The entire culture time is 7 days. After the culture is completed, all cells are collected to detect cell viability, pDC cell purity, and the content of cytokine IFN-α in the cell supernatant.

[0042] In order to better illustrate the technical effect of the technical solution of the present invention, the present invention constructed the following examples and comparative examples for testing, wherein the culture methods are the same, and the variable is that the culture medium used is different.

[0043] Example 1: The culture medium of the pDC cells of Example 1 includes the following components: 20 ng / mL GM-CSF, 100 ng / mL Poly (I: C), 50 ng / mL Flt31, 100 UI / mL TNF-α, 1 μM ODN2006, 0.5 μM Compound 4, 10% autologous plasma, and AIM-V basal medium.

[0044] The specific cultivation steps are:

[0045] PBMCs were isolated from human peripheral venous blood using density gradient centrifugation;

[0046] PBMC cells were resuspended in AIM-V basal medium preheated at 37°C and inoculated into 6-well plates at a cell density of 8 × 10 6 / mL, 2mL / well. Then place in a 37℃ carbon dioxide incubator for 2 hours, wash away the non-adherent cells, replace with 3mL of complete medium of Example 1 and continue culturing. Perform fluid replenishment / replacement every 2-3 days according to the cell proliferation, and culture for 7 days in total;

[0047] After the culture was completed, all cells were collected.

[0048] At the same time, the specific implementation of the present invention also provides the following comparative examples for verification and comparison.

[0049] Comparative Example 1: Comparative Example 1 is peripheral blood mononuclear cells (PBMC), which are not cultured.

[0050] Comparative Example 2: Compared with Example 1, the culture medium of Comparative Example 2 does not contain ODN2006. The culture medium composition of Comparative Example 2 is: 20 ng / mL GM-CSF, 100 ng / mL Poly(I:C), 50 ng / mL Flt31, 100 UI / mL TNF-α, 0.5 μM Compound 4, 10% autologous plasma, and AIM-V basal medium.

[0051] Comparative Example 3: Compared with Example 1, the culture medium of Comparative Example 3 does not contain Compound 4, and the composition of the culture medium of Comparative Example 3 is: 20 ng / mL GM-CSF, 100 ng / mL Poly(I:C), 50 ng / mL Flt31, 100 UI / mL TNF-α, 1 μM ODN2006, 10% autologous plasma, and AIM-V basal medium.

[0052] Comparative Example 4: Compared with Example 1, the culture medium of Comparative Example 4 does not contain ODN2006 and Compound 4. The composition of the culture medium of Comparative Example 4 is: 20 ng / mL GM-CSF, 100 ng / mL Poly(I:C), 50 ng / mL Flt31, 100 UI / mL TNF-α, 10% autologous plasma, and AIM-V basal culture medium.

[0053] Among them, the culture methods and environments of comparative examples 2-4 are the same as those of embodiment 1, and the following experiments are carried out to detect the embodiments and comparative examples.

[0054] Experiment 1: Different ODN2006 concentrations were set, a single variable was controlled, and an IFN-α ELISA kit was used to detect the effects of different concentrations of ODN2006 on the total cell IFN-α secretion level obtained after 7 days of culture. The culture steps and other conditions of the experiment were the same as those in Example 1. The results are shown in Table 1.

[0055] Table 1. IFN-α secretion levels of cells with different concentrations of ODN2006

[0056] ODN2006 concentration (μM) IFN-α concentration (pg / mL) 0 15.26±0.99 0.1 26.73±1.21 0.5 59.27±1.01 1 80.32±1.74 5 81.34±1.25 10 82.17±1.12

[0057] The results in Table 1 show that the addition of ODN2006 can promote the secretion of IFN-α. When the concentration of ODN2006 is greater than 1 μM, the increase in concentration has a smaller effect on the secretion level of IFN-α. Therefore, 1 μM is selected as the optimal culture concentration of ODN2006.

[0058] Experiment 2: Different concentrations of Compound 4 were set, a single variable was controlled, and an IFN-α ELISA kit was used to detect the effects of different concentrations of Compound 4 on the total cell IFN-α secretion level obtained after 7 days of culture. The culture steps and other conditions of the experiment were the same as those in Example 1. The results are shown in Table 2.

[0059] Table 2. IFN-α secretion levels of cells with different concentrations of Compound 4

[0060]

[0061]

[0062] The results in Table 2 above show that the addition of Compound 4 can promote the secretion of IFN-α. When the concentration of Compound 4 is greater than 0.5 μM, the increase in concentration has little effect on the secretion level of IFN-α. Therefore, 0.5 μM is selected as the optimal culture concentration of Compound 4.

[0063] Experiment 3: The cell morphology during the culture process of Example 1 was recorded, and the total cells were collected for detection after 7 days of culture.

[0064] like Figure 1 As shown, Figure 1 The cell morphology diagram of the pDC cell culture process of PBMC cell culture in the embodiment of the present invention shows that from the third day, more obvious dendrites appeared in the cells, and the pDC cells with dendrites matured and increased significantly with time. That is, using the culture medium and the corresponding culture method of the present invention, a large number of pDC cells can be obtained conveniently and efficiently.

[0065] The total cells were collected after 7 days of culture, and the method for detecting the percentage of pDC cells was as follows:

[0066] After 7 days of PBMC culture, the total cells were collected to detect the percentage of pDC cells. The percentage of pDC cells in the collected cells was detected by flow cytometry. The anti-human antibodies used were: APC-Lineage (CD3 / 14 / 19 / 20 / 56) antibody, Alexa 700-HLA-DR antibody, FITC-CD303 antibody, PE-CD123 antibody, pDC cells are Lin-HLA-DR+CD123+CD303+. The test results are as follows Figure 2-6 As shown in Table 3 below.

[0067] Table 3. The proportion of pDC cells in total cells in different experimental groups

[0068]

[0069]

[0070] Combined with Table 3 and Figure 2-6 , Figure 2 This is the flow cytometry chart of the proportion of pDC cells in PBMC in Comparative Example 1. Figure 3 The pDC cell ratio flow cytometry analysis in Example 1 shows that Figure 3 It can be seen that the pDC cells obtained after culturing for 7 days using the culture medium and culture method of Example 1 of the present invention account for 21.24% of the total cells, compared with only 0.11% before culture in Comparative Example 1, and the pDC cells have expanded by about 193 times; Figure 4-6 In the absence of Compound 4 or ODN2006 or both, the culture medium cannot achieve the corresponding differentiation induction effect. Figure 3-6By comparison, Compound 4 and ODN 2006 in this solution can synergistically promote the induction and differentiation of pDC cells. Through the culture medium and culture method of Example 1 of this solution, a large number of pDC cells can be obtained in vitro conveniently and efficiently, providing a guarantee for the basic research and clinical application of pDC cells.

[0071] The samples of Example 1 and Comparative Example 1 were collected and the IFN-α content in the cell supernatant was detected by flow cytometry. The test results are as follows: Figure 7 shown.

[0072] Combined with Figure 7 ,from Figure 7 It can be seen that the pDC cells obtained after culturing with the culture medium of Example 1 of the present invention for 7 days can secrete 81.59 pg / mL of IFN-α without target cell stimulation, while the PBMC of Comparative Example 1 secretes only 0.92 pg / mL of IFN-α. The IFN-α of Example 1 is much higher than that of Comparative Example 1, that is, the pDC cells obtained after culturing with the culture medium of the present invention for 7 days can secrete a higher level of IFN-α without target cell stimulation.

[0073] The present invention also provides a kit, which includes the kit for inducing PBMC to differentiate into pDC cells in vitro as described above, and the kit can be applied to culturing PBMC to differentiate into pDC cells, preparing supernatant exosomes, and other aspects.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for inducing DC precursor cells in PBMC to differentiate into pDC cells in vitro, characterized in that: Includes steps: The PBMCs are placed in a culture medium for culture, wherein the culture medium comprises the following components: 1-100 ng / mL GM-CSF, 10-1000 ng / mL Poly(I:C), 10-100 ng / mL Flt31, 10-1000 UI / mL TNF-α, 0.1-10 μM ODN2006, 0.1-10 μM Compound 4, 1-10% autologous plasma and AIM-V basic culture medium, wherein the CAS number of Compound 4 is 2115702-83-3; replacing or supplementing the culture medium based on the culture progress until a predetermined culture time is reached; The cultured cells were collected.

2. The method for inducing DC precursor cells in PBMC to differentiate into pDC cells in vitro according to claim 1, characterized in that: The culture medium includes the following components: 20 ng / mL GM-CSF, 100 ng / mL Poly(I:C), 50 ng / mL Flt31, 100 UI / mL TNF-α, 1 μM ODN2006, 0.5 μM Compound 4, and 10% autologous plasma.

3. The method for inducing DC precursor cells in PBMC to differentiate into pDC cells in vitro according to claim 1, characterized in that: The PBMCs are separated from human peripheral venous blood by density gradient centrifugation.

4. The method for inducing DC precursor cells in PBMC to differentiate into pDC cells in vitro according to claim 1, characterized in that: The predetermined culture time is 7 days.

5. The method for inducing DC precursor cells in PBMC to differentiate into pDC cells in vitro according to claim 1, characterized in that: The step of placing PBMC in a culture medium for culturing specifically includes the following steps: resuspending PBMC cells using AIM-V basal culture medium preheated at 37°C, inoculating them in a 6-well plate, culturing them in a 37°C carbon dioxide incubator for 2 hours, washing away cells that are not attached to the wall, and replacing them with complete culture medium for culturing.

6. The method for inducing DC precursor cells in PBMC to differentiate into pDC cells in vitro according to claim 5, characterized in that: The seeding density of the PBMC was 8×10 6 Pieces / mL.

7. A culture medium for inducing DC precursor cells in PBMC to differentiate into pDC cells in vitro, characterized in that: The culture medium comprises the following components: 1-100 ng / mL GM-CSF, 10-1000 ng / mL Poly(I:C), 10-100 ng / mL Flt31, 10-1000 UI / mL TNF-α, 0.1-10 μM ODN2006, 0.1-10 μM Compound 4, 1-10% autologous plasma and AIM-V basic culture medium.

8. The culture medium for inducing DC precursor cells in PBMC to differentiate into pDC cells in vitro according to claim 7, characterized in that: The culture medium includes the following components: 20 ng / mL GM-CSF, 100 ng / mL Poly(I:C), 50 ng / mL Flt31, 100 UI / mL TNF-α, 1 μM ODN2006, 0.5 μM Compound 4, and 10% autologous plasma.

9. A kit, characterized in that: The kit comprises a culture medium as claimed in any one of claims 7 to 8, and is used for in vitro induction of differentiation of DC precursor cells in PBMC into pDC cells for culture or / and preparation of exosomes.

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