A method for efficiently inducing peripheral blood-derived monocytes into immature dendritic cells

Abstract

The present invention provides a method for inducing immature dendritic cells from monocytes. The present invention cold-treats peripheral blood mononuclear cells from different donors and then induces and cultures them in a one-step manner, thereby achieving the induction of mononuclear cells into immature dendritic cells. The present invention is simple to operate and has a wide range of applications. The present invention can achieve efficient induction of monocytes derived from peripheral blood in different populations, with good induced cell activity, a high induced cell yield, and a high proportion of the main cell population in cytometry analysis, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of cell biology, and in particular to a method for efficiently inducing peripheral blood-derived monocytes into immature dendritic cells. Background Art

[0002] Dendritic cells (DCs) play an important role in immune presentation in the body. They are mainly formed by the differentiation of monocytes under the action of cytokines such as interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). They have important applications in pharmaceutical development and clinical applications. Dendritic cells are divided into immature dendritic cells (iDCs) and mature dendritic cells (mDCs). mDCs are induced by iDCs.

[0003] Dendritic cells are the most powerful antigen presenting cells in the body and the initiators of immune responses. They have attracted much attention because of their unique ability to stimulate the activation of initial T cells. Currently, many studies on regulatory DCs are conducted in vitro, such as adding various immunosuppressive factors to the original DC culture conditions to induce hematopoietic precursor cells, mature or immature DCs into regulatory DCs with negative regulatory effects.

[0004] Mature DCs express high levels of costimulatory molecules on their surface, which can effectively stimulate T cell proliferation and induce immune response. Immature DCs express low levels of costimulatory molecules on their surface, and have a strong ability to take up and process antigens, but a weak ability to stimulate T cell proliferation. They are also called tolerant DCs or regulatory DCs. Before organ transplantation, immature dendritic cells from donors or loaded with donor antigens are introduced into the recipient's body, which significantly prolongs the survival time of the graft. That is, iDCs can induce antigen-specific transplant immune tolerance. Immature dendritic cells (iDCs) play a key role in inducing and maintaining the body's immune tolerance because of their low expression of major histocompatibility complexes and costimulatory molecules.

[0005] Since iDCs are very unstable, they are easily activated by various risk factors in the internal environment. On the one hand, activated DCs highly express surface molecules (iDC maturation), and on the other hand, they secrete pro-inflammatory cytokines such as interleukin 12. In this case, activated DCs not only fail to induce tolerance, but can promote immune response, leading to failure of tolerance induction. Therefore, obtaining stable iDCs has become a difficulty and hot spot in current research.

[0006] Currently, the industry mainly uses human peripheral blood mononuclear cells to separate monocytes, add IL-4 and GM-CSF to induce, and promote the differentiation of monocytes into iDCs. However, a large number of practices have shown that the induction of iDCs is easily affected by differences in cell sources in terms of key biomarkers and biological activity. Most previous induction schemes have huge differences in induction results on monocytes from different individual sources. Therefore, how to achieve the induction of most individual source cells to the ideal state of iDC is a technical problem to be solved by the present invention. Summary of the invention

[0007] The purpose of the present invention is to provide a method for efficiently inducing peripheral blood-derived monocytes into immature dendritic cells in order to solve the problem in the prior art that there are huge differences in the induction results on monocytes from different individuals.

[0008] The purpose of the present invention is to establish a method for efficiently inducing iDC cells, so as to achieve successful induction of iDC cells in most individual derived cells.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0010] The present invention discloses a method for inducing immature dendritic cells from monocytes, the method comprising:

[0011] 1) Isolation and culture of monocytes;

[0012] 2) subjecting the cultured monocytes to cold treatment at a temperature of 2-8° C. for a period of 30 to 90 minutes;

[0013] 3) Inducing and culturing the cold-treated monocytes to obtain immature dendritic cells, wherein the induction medium for the induction culture is a basal medium supplemented with IL-4 to a final concentration of 10-50 ng / mL, GM-CSF to a final concentration of 20-100 ng / mL, and IL-15 to a final concentration of 10-20 ng / mL, and the basal medium is: RPMI 1640+10% FBS+1% penicillin / streptomycin.

[0014] Preferably, adding 1% penicillin / streptomycin reduces the contamination rate, which is a routine operation.

[0015] Preferably, the penicillin / streptomycin is a conventional dual antibody.

[0016] Preferably, the monocytes are peripheral blood mononuclear cells.

[0017] Preferably, the peripheral blood mononuclear cells are cells with a high proportion of CD14+.

[0018] Preferably, the cold treatment time in step 2) is 30 min to 60 min.

[0019] Preferably, in step 2), the peripheral blood mononuclear cells after the cold treatment are washed with DPBS solution to remove the non-adherent cells, and the adherent cells are monocytes.

[0020] Preferably, monocytes are sorted using a sorting kit.

[0021] Preferably, the monocytes are sorted using a stemcell negative selection CD14 separation kit.

[0022] Preferably, in step 3), the final concentration of IL-4 is 50 ng / mL, the final concentration of GM-CSF is 100 ng / mL, and the final concentration of IL-15 is 10 ng / mL.

[0023] Preferably, the induction culture conditions in step 3) are 37° C., 5% CO 2 .

[0024] Preferably, the culture time in step 3) is 3 days.

[0025] Preferably, the induction process is performed without changing the culture medium.

[0026] Preferably, the induced cells are further treated with trypsin digestion.

[0027] Preferably, the monocytes in step 1) are derived from different donors.

[0028] The present invention uses cold treatment on peripheral blood mononuclear cells from different donors and then induces and cultures them in a one-step manner, thereby achieving the induction of mononuclear cells into immature dendritic cells. The present invention uses the scheme to induce PBMCs from different donors into iDC cells, and the induced iDC cells have the correct phenotype, good viability, and a high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Micrograph of adherent monocytes.

[0030] Figure 2 Micrographs of induced iDC cells.

[0031] Figure 3 Induced cell clustering.

[0032] Figure 4 The percentage of CD14 positive cells.

[0033] Figure 5 The percentage of CD83 positive cells.

[0034] Figure 6Analysis of the proportion of CD209 positive cells.

[0035] Figure 7 The percentage of CD11c positive cells.

[0036] Figure 8 Flow cytometric analysis of iDCs induced by monocytes from different donors. DETAILED DESCRIPTION

[0037] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0038] Example 1 Induction of iDCs from monocytes derived from peripheral blood mononuclear cells (PBMC)

[0039] 1. Select PBMC for recovery, resuspend the recovered PBMC in DPBS, adjust the cell density to 10E6 cells / mL, and spread 10 mL of PBMC with adjusted density into T75 culture flasks. In order to optimize the cold treatment time, use a laboratory 2-8℃ refrigerator to cold treat the recovered PBMC for different time periods. The cold treatment time is set to 0 min (no cold treatment); 30 min; 60 min; 90 min; 2 h; 4 h; 8 h; 16 h; 8 time points. The other experimental conditions remain the same. Induce the recovered PBMC cells. The induction conditions are as follows;

[0040] 2. Take out the cold-treated PBMC, gently shake the T75 culture flask, pour out the non-adherent suspended cells, discard them, and use DPBS to wash the T75 culture flask to wash away the non-adherent cells. The adherent cells are monocytes;

[0041] 3. Use a microscope to observe the monocytes at the bottom of the T75 culture flask. The cells are oval and evenly attached to the bottom of the culture flask. The microscopic photo of monocytes is shown in Figure 2. Figure 1 As shown, the cells have an oval morphology;

[0042] 4. Prepare the induction medium: basal medium: RPMI 1640 + 10% FBS + 1PS. Add IL-4 to the basal medium to a final concentration of 50 ng / mL, GM-CSF to a final concentration of 100 ng / mL, and IL-15 to a final concentration of 10 ng / mL.

[0043] 5. Add the prepared culture medium to the T75 culture flask, adding 25 ml / flask, and place the culture flask at 37°C, 5% CO2 for 3 days of induction. There is no need to change the culture medium during the induction process.

[0044] 6. Cell collection: Pour the supernatant of the induced cell culture medium into a 50 mL centrifuge tube, and use trypsin to digest the cells at the bottom of the culture flask and collect the cells; the micrograph of the induced iDC is shown in Figure 2. Figure 2 As shown, the cells have irregular morphology and dendrites;

[0045] 7. Induced iDC detection: First, wash the cells with FACS Buffer once, then adjust the cell density to 5E5 to 1E6 / mL, transfer them to a 96-well U-shaped plate, 100 µL / well, add 2 µL / test of RcR block, mix thoroughly and react at 4°C in the dark for 30 min; then add Isotype, CD14, CD11c, CD209 and CD83 fluorescent direct-labeled antibodies, mix thoroughly and react at 4°C in the dark for 30 min; after the reaction, wash twice with FACS Buffer, 100 µL / well, resuspend and detect;

[0046] 8. Signs of successful iDC induction: high cell viability, low expression of CD14, high expression of CD11c, high expression of CD209, and low expression of CD83 in cell marker detection;

[0047] The phenotype of each group of cells was detected by flow cytometry, and the cells of each group were counted using a cell technology instrument. The experimental results are shown in the following table:

[0048] The cell phenotypes at different cold treatment times are shown in Table 1 below.

[0049] Table 1: Cell phenotype detection results at different cold treatment times

[0050] Cold treatment time / cell marker 0min 30min 60min 90min 2h 4h 8h 16h CD14 3.49% 7.47% 0.98% 35.13% 68.25% 86.07% 85.78% 94.00% CD209 28.60% 88.61% 99.93% 77.40% 87.47% 53.78% 63.89% 35.97% CD11c 89.12% 99.72% 90.83% 91.97% 98.77% 99.79% 99.94% 99.63% CD83 0.06% 0.08% 7.29% 4.16% 2.15% 7.49% 20.50% 54.00%

[0051] The cell counts at different cold treatment times are shown in Table 2.

[0052] Table 2: Cell counting statistics at different cold treatment times

[0053] Cold treatment time Viable cell density Cell viability Collected cell volume Total number of iDCs (millions) Induced iDC yield (induced iDC / initial PBMC) 0min 1.37E5 / mL 55.60% 50mL 9.85 11.40% 30min 3.91E5 / mL 94.16% 50mL 19.5 19.50% 60min 4.82E5 / mL 90.42% 50mL 21 21.00% 90min 3.76E5 / mL 89.24% 50ml 18.8 18.80% 2h 3.12E5 / mL 85.84% 50ml 15.6 15.60% 4h 3.42E5 / mL 83.38% 50mL 17.1 17.10% 8h 2.92E5 / ml 70.52% 50ml 14.6 14.60% 16h 2.02E5 / mL 63.95% 50ml 10.1 10.10%

[0054] The experimental results showed that the iDC cells induced by cold treatment for 30min and 60min had a better phenotype and cell viability. As the cold treatment time increased, the phenotype of the induced cells did not conform to the iDC cell phenotype, and the viability of the induced cells was also poor.

[0055] Test results such as Figures 3 to 7 Among them, CD14 and CD83 of the target cells were not expressed, and the positive rate was extremely low (less than 5%), while CD11c and CD209, two key DC markers, were highly expressed, accounting for a very high proportion in the cells (more than 90%), which was consistent with the biomarker characteristics of iDC.

[0056] Cell freezing: Use a centrifuge to centrifuge the collected cells at 1500rpm for 5 minutes. After the centrifugation, completely remove the supernatant, add a certain amount of cell freezing solution to the centrifuge tube, resuspend the induced iDC cells, divide the resuspended iDC cells into cryopreservation tubes, use a cryopreservation box, and freeze the cells in a -80℃ refrigerator for >4h; preferably, use CS10 freezing solution to freeze iDC cells, and 10% autologous plasma can be added to the freezing solution; preferably, for long-term storage of iDC cells, the cells should be stored in liquid nitrogen.

[0057] Example 2 Induction of iDC cells from monocytes from different donors

[0058] In order to verify the stability of this method among different donors, PBMCs from three donors were collected for iDC induction. The specific experimental method was as follows:

[0059] ① Collect blood from three healthy people clinically, 20 mL from each donor, the three donor numbers are: XW0102161; XW0102027; XW0801240. Use gradient density centrifugation to separate and obtain PBMC cells, and use a programmed cooling device to freeze the separated PBMC cells;

[0060] ② After one week of cryopreservation, PBMCs were revived in a water bath and resuspended in DPBS to adjust the cell density to 10E6 cells / mL. 10 mL of PBMCs with adjusted density were plated into a T75 culture flask, and the T75 culture flask was placed in a 2-8°C refrigerator for 60 minutes for cold treatment.

[0061] ③Take out the cold-treated PBMC, gently shake the T75 culture flask, pour out the non-adherent suspended cells, discard them, and use DPBS to wash the T75 culture flask to wash away the non-adherent cells. The adherent cells are monocytes;

[0062] ④Use a microscope to observe the mononuclear cells at the bottom of the T75 culture flask. The cells are oval and evenly attached to the bottom of the culture flask;

[0063] ⑤ Prepare induction medium: Basic medium: RPMI 1640+10% FBS+1PS. Add IL-4 to the basic medium to a final concentration of 50 ng / mL, GM-CSF to a final concentration of 100 ng / mL, and IL-15 to a final concentration of 10 ng / mL;

[0064] ⑥ Add the prepared culture medium to the T75 culture bottle, adding 25 ml / bottle, and place the culture bottle at 37°C, 5% CO2, and induce culture for 3 days. There is no need to change the culture medium during the induction process;

[0065] ⑦Cell collection: Pour the supernatant of the induced cell culture medium into a 50 mL centrifuge tube, use trypsin to digest the cells at the bottom of the culture flask, collect the cells, and count them using a cell counter;

[0066] ⑧ Induced iDC detection: First, wash the cells with FACS Buffer once, then adjust the cell density to 5E5 to 1E6 / mL, transfer them to a 96-well U-shaped plate, 100 µL / well, add 2 µL / test of RcR block, mix thoroughly and react at 4°C in the dark for 30 minutes; then add Isotype, CD14, CD11c, CD209 and CD83 fluorescent direct-labeled antibodies, mix thoroughly and react at 4°C in the dark for 30 minutes; after the reaction, wash twice with FACS Buffer, 100 µL / well, resuspend and detect; for flow cytometry results, see Figure 8 The statistical data of iDC cells induced by different donors are shown in Table 3 below.

[0067] Table 3: Statistical results of iDC cells induced by different donors

[0068]

[0069] The present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for inducing immature dendritic cells from monocytes, characterized in that: The method comprises: 1) Isolation and culture of monocytes; 2) subjecting the cultured monocytes to cold treatment at a temperature of 2-8° C. for a period of 30 to 60 minutes; 3) Inducing and culturing the cold-treated monocytes to obtain immature dendritic cells, wherein the induction culture medium is a basal medium supplemented with IL-4 to a final concentration of 10-50 ng / mL, GM-CSF to a final concentration of 20-100 ng / mL, and IL-15 to a final concentration of 10-20 ng / mL, and the basal medium is: RPMI 1640+10% FBS+1% penicillin / streptomycin.

2. The method for inducing immature dendritic cells from monocytes according to claim 1, characterized in that: The monocytes are peripheral blood mononuclear cells.

3. The method for inducing immature dendritic cells from monocytes according to claim 2, characterized in that: In step 2), the peripheral blood mononuclear cells that have been cold treated are washed with DPBS solution to remove the non-adherent cells. The adherent cells are monocytes.

4. The method for inducing immature dendritic cells from monocytes according to claim 1, characterized in that: In step 3), the final concentration of IL-4 is 50 ng / mL, the final concentration of GM-CSF is 100 ng / mL, and the final concentration of IL-15 is 10 ng / mL.

5. The method for inducing immature dendritic cells from monocytes according to claim 1, characterized in that: In step 3), the induction culture conditions are 37°C and 5% CO2.

6. The method for inducing immature dendritic cells from monocytes according to claim 1, characterized in that: The culture time in step 3) is 3 days.

7. The method for inducing immature dendritic cells from monocytes according to any one of claims 5-6, characterized in that: The culture medium was not changed during the induction process.

8. The method for inducing immature dendritic cells from monocytes according to claim 1, characterized in that: The induced cells were further treated with trypsin.

9. The method for inducing immature dendritic cells from monocytes according to claim 1, characterized in that: The monocytes in step 1) are derived from different donors.

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