A culture medium, a culture method and an application thereof for inducing type C1 iNKT cells

Abstract

The present invention provides a culture medium for inducing type C1 iNKT cells, a culture method thereof and applications thereof, relating to the technical field of cell culture. The culture medium comprises a DC culture medium and an iNKT culture medium. The DC culture medium comprises GM-CSF, IL-4, TNF-α, LPS, 7DW8-5 and autologous plasma. The iNKT culture medium comprises IL-2, IL-7, IL-15, IL-21, 7DW8-5, lupeol, betulinic acid, resveratrol and autologous plasma. Meanwhile, a culture method corresponding to the culture medium is also provided. The type C1 iNKT cells obtained after being cultured with the culture medium of the present invention for 14 days can secrete a relatively high level of IFN-γ without the stimulation of target cells, and the proportion of type C1 iNKT cells in CD3+ T cells increases significantly. Such a culture medium and a culture method can be used in the fields of inducing the growth and expansion of type C1 iNKT cells, exosome extraction, etc., so as to efficiently obtain target cells and target factors.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and particularly relates to a culture medium for inducing type C1 iNKT cells, a culture method thereof, and applications thereof. Background Art

[0002] iNKT cells (invariant natural killer T cells) are unconventional T cells. Besides having the common characteristics of traditional T cells and natural killer cells, the TCR of iNKT cells can recognize glycolipid antigens on CD1d molecules. Although the number of such cells is scarce, they play a quite important role in resisting cancer and pathogenic microorganism infections in organs such as the liver, lungs, intestines, and urogenital tract where they are present in relatively large amounts. Their powerful functions come from their ability to directly kill target cells, and also to rapidly secrete a large number of cytokines and inflammatory factors and activate and enhance the functions of other immune cells in the tumor microenvironment. Recently, some scholars classified iNKT into three subtypes according to the expression of CD244 and CXCR6: C0 iNKT, C1 iNKT, and C2 iNKT. Among them, C0 is the precursor cell of C1 and C2; C0 and C1 have the potential to develop into C2 in the presence of IL-15; C2 exhibits more NK cell characteristics, while C1 exhibits more T cell characteristics; both C1 and C2 type iNKT cells can secrete relatively high levels of IFN-γ, granzyme B, perforin, etc., providing a basis for the clinical application of iNKT cells.

[0003] In order to obtain a certain therapeutic quantity and therapeutically effective iNKT cells, effective methods are needed to expand and activate these cells. Currently, a variety of methods have been reported, including using α-galactosylceramide (α-GalCer) and interleukin-2 (IL-2), using CD1d molecules, using DCs (dendritic cells), and using exosomes, etc. However, the culture effects of these methods are not high, and there may be problems such as induced anergy, low amplification efficiency, low purity, and unstable functions. For example, the iNKT cells obtained by culturing in Patent CN 116004533 A secrete IFN-γ at a level <150 pg / mL under the stimulation of target cells. Also, for example, the iNKT cells obtained by culturing in Patent CN 113462646 A secrete IFN-γ at a level <30 pg / mL under the stimulation of target cells. In Patent CN 116590229 A, the purity of the iNKT cells obtained in this patent is 70.6%, and in Patent CN116716246A, the purity of the iNKT cells obtained by culturing is 53.14%. Therefore, among the currently disclosed patent methods, the content of iNKT cells with high IFN-γ secretion is extremely low, making it difficult to meet the needs of clinical applications.

[0004] Therefore, there is an urgent need for simple and effective media, methods, etc. for inducing and expanding highly IFN-γ-secreting type C1 iNKT cells to increase their quantity, purity, and function, and to provide higher-quality immune cells for immunotherapy. Summary of the Invention

[0005] The present invention provides a medium, a culture method, and their applications for inducing type C1 iNKT cells to solve the problem of insufficient efficiency of the current medium for inducing and expanding IFN-γ-secreting iNKT cells and to achieve efficient induction of type C1 iNKT cells.

[0006] The present invention provides a medium for inducing type C1 iNKT cells, and the medium comprises a DC medium and an iNKT medium;

[0007] The DC medium comprises 10 - 1000 ng / mL GM-CSF, 1 - 100 ng / mL IL-4, 10 - 1000 U / mL TNF-α, 1 - 100 ng / mL LPS, 10 - 1000 ng / mL 7DW8-5, and 1 - 10% autologous plasma;

[0008] The iNKT medium comprises 10 - 1000 U / mL IL-2, 1 - 100 ng / mL IL-7, 1 - 100 ng / mL IL-15, 1 - 100 ng / mL IL-21, 10 - 1000 ng / mL 7DW8-5, 0.1 - 10 μM lupeol, 0.1 - 10 μM betulinic acid, 0.1 - 10 μM resveratrol, and 1 - 10% autologous plasma.

[0009] According to the medium for inducing type C1 iNKT cells provided by the present invention, the DC medium comprises 50 ng / mL GM-CSF, 10 ng / mL IL-4, 100 U / mL TNF-α, 20 ng / mL LPS, 20 ng / mL 7DW8-5, and 10% autologous plasma.

[0010] According to the medium for inducing type C1 iNKT cells provided by the present invention, the iNKT medium comprises 500 U / mL IL-2, 5 ng / mL IL-7, 5 ng / mL IL-15, 5 ng / mL IL-21, 20 ng / mL 7DW8-5, 2 μM lupeol, 2 μM betulinic acid, 2 μM resveratrol, and 3% autologous plasma.

[0011] The culture method for inducing type C1 iNKT cells provided by the present invention comprises:

[0012] When obtaining the cells to be processed, culture the cells to be processed based on the DC medium described in any one of claims 1 or 2 according to a preset first culture duration to obtain dendritic cells;

[0013] When obtaining the induced cells, resuspend the induced cells with the iNTK medium described in any one of claims 1 or 3 according to a preset second culture duration, and co-culture the resuspended induced cells with the dendritic cells.

[0014] According to a culture method for inducing C1-type iNKT cells provided by the present invention, when culturing the cells to be processed with the DC medium, the concentration of the cells to be processed is 1-10×10 6 cells / mL.

[0015] According to a culture method for inducing C1-type iNKT cells provided by the present invention, when co-culturing the dendritic cells with the induced cells, the concentration of the induced cells is 1-10×10 6 cells / mL.

[0016] According to a culture method for inducing C1-type iNKT cells provided by the present invention, when obtaining the cells to be processed, culturing the cells to be processed based on the DC medium described in any one of claims 1 or 2 according to a preset first culture duration to obtain dendritic cells includes:

[0017] Culturing the cells to be processed with a preset basal medium;

[0018] After the cells to be processed adhere to the wall, culture the cells to be processed based on the DC medium described in any one of claims 1 or 2 according to a preset first culture duration to obtain dendritic cells.

[0019] According to a culture method for inducing C1-type iNKT cells provided by the present invention, the cells to be processed are PBMCs, and the induced cells are PBMCs.

[0020] According to a culture method for inducing C1-type iNKT cells provided by the present invention, the quantity ratio of the dendritic cells to the induced cells is 1:1-1:10.

[0021] According to a culture method for inducing C1-type iNKT cells provided by the present invention, a kit provided by the present invention, the kit includes the DC medium described above and the iNKT medium described above, and the kit is used for iNKT cell culture and / or preparation of supernatant exosomes.

[0022] The present invention provides a culture medium for inducing type C1 iNKT cells, which comprises a DC culture medium and an iNKT culture medium. The DC culture medium comprises GM-CSF, IL-4, TNF-α, LPS, 7DW8-5 and autologous plasma. The iNKT culture medium comprises IL-2, IL-7, IL-15, IL-21, 7DW8-5, lupeol, betulinic acid, resveratrol and autologous plasma. Meanwhile, a culture method corresponding to this culture medium is also provided. The type C1 iNKT cells obtained after culturing with the culture medium of the present invention for 14 days can secrete a relatively high level of IFN-γ without the stimulation of target cells, and the proportion of type C1 iNKT cells in CD3+ T cells is 94.6%, showing a substantial increase compared with the traditional method. Such a culture medium and culture method can be used in the fields of inducing the growth and expansion of type C1 iNKT cells, exosome extraction, etc., so as to efficiently obtain target cells and target factors. Brief Description of the Drawings

[0023] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 It shows the effects of different concentrations of 7DW8-5 in the DC culture medium and the iNKT culture medium provided by the present invention on cell viability;

[0025] Figure 2 It shows the effects of different combinations of traditional Chinese medicine monomers in the iNKT culture medium provided by the present invention on cell viability;

[0026] Figure 3 It shows the image of culturing cells to be treated with the DC culture medium provided by the present invention;

[0027] Figure 4 It shows the image of co-culturing induced cells and DC cells with the iNKT culture medium provided by the present invention;

[0028] Figure 5 It shows the content diagram of iNKT cells in the experimental group and the control group analyzed by flow cytometry provided by the present invention;

[0029] Figure 6 It shows the content diagram of cytokines in experimental group 1 and the control group analyzed by flow cytometry provided by the present invention. Detailed Embodiments

[0030] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions in the present invention in conjunction with the accompanying drawings in the present invention. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without any creative work fall within the scope of protection of the present invention.

[0031] Through research, the present invention provides a culture medium for inducing type C1 iNKT cells, and this culture medium includes a DC culture medium and an iNKT culture medium. Among them, the DC culture medium is used to induce and culture the cells to be processed into DCs. When DC cells induce type C1 iNKT cells, they will produce IL-12, which will stimulate the activation of iNKT cells, thus forming a positive feedback loop. In addition, iNKT cells will also interact with dendritic cells through the CD40L-CD40 interaction. In response, DCs will produce IL-12, and then IL-12 further stimulates the activation of iNKT cells, forming a positive feedback loop.

[0032] The DC culture medium includes 10 - 1000 ng / mL GM-CSF, 1 - 100 ng / mL IL-4, 10 - 1000 U / mL TNF-α, 1 - 100 ng / mL LPS, 10 - 1000 ng / mL 7DW8-5, and 1 - 10% autologous plasma. GM-CSF and IL-4 in the DC culture medium can induce the proliferation of DC cells, TNF-α and LPS can induce the maturation of DC cells, 7DW8-5 (a derivative of α-GalCe) is a lipid antigen that can bind to the CD1d molecule expressed on the surface of DC cells. 7DW8-5 can activate iNKT cells. 7DW8-5 is loaded onto the CD1d molecule of DC cells and then presented by DC cells to iNKT cells to induce the proliferation and activation of iNKT cells. Autologous plasma can provide nutritional components.

[0033] When the iNKT medium is used for co - culturing DC cells and induced cells, it guides the differentiation of the induced cells and promotes the proliferation and activation of iNKT cells. The iNKT medium includes 10 - 1000 U / mL IL - 2, 1 - 100 ng / mL IL - 7, 1 - 100 ng / mL IL - 15, 1 - 100 ng / mL IL - 21, 10 - 1000 ng / mL 7DW8 - 5, 0.1 - 10 μM lupeol, 0.1 - 10 μM betulinic acid, 0.1 - 10 μM resveratrol and 1 - 10% autologous plasma. IL - 2, IL - 7 and IL - 15 in the iNKT medium can promote the proliferation and activation of iNKT cells, and IL - 21 can increase the cytotoxicity of iNKT cells. 7DW8 - 5 can activate iNKT cells, and it is found that lupeol, betulinic acid and resveratrol can promote the proliferation of iNKT cells and the secretion of IFN - γ. The autologous plasma in the iNKT medium also plays a role in providing nutritional components.

[0034] In addition to the above factors, for the DC medium and the iNKT medium, the basal medium can select a serum - free medium with defined components for immune cell proliferation and / or control, such as AIM - V medium. Add the above factors to the basal medium to prepare the DC medium and the iNKT medium.

[0035] As Figure 1 shown, on the premise of ensuring that other components remain unchanged, different concentrations of 7DW8 - 5 have different effects on the survival rate of total cells (including iNKT cells), and 7DW8 - 5 at 10 - 1000 ng / mL can significantly improve the cell survival rate.

[0036] For another example Figure 2 shown, on the basis of other components remaining unchanged, different concentrations of the combination of traditional Chinese medicine monomers (lupeol, betulinic acid, resveratrol) have different effects on the survival rate of total cells (including iNKT cells). In the range of 0.1 - 10 μM of the combination of traditional Chinese medicine monomers, the cell survival rate is significantly improved.

[0037] Except for the above 7DW8 - 5 and the combination of traditional Chinese medicine monomers, all other components and their concentrations have been tested and verified, and will not be described one by one here.

[0038] Furthermore, to improve the efficiency of inducing iNKT cells, in the DC medium used in this example, there are 50 ng / mL GM-CSF, 10 ng / mL IL-4, 100 U / mL TNF-α, 20 ng / mL LPS, 20 ng / mL 7DW8-5, and 10% autologous plasma. The iNKT medium includes 500 U / mL IL-2, 5 ng / mL IL-7, 5 ng / mL IL-15, 5 ng / mL IL-21, 20 ng / mL 7DW8-5, 2 μM lupeol, 2 μM betulinic acid, 2 μM resveratrol, and 3% autologous plasma.

[0039] To improve the efficiency of the above medium and maximize the induction and proliferation effect of the medium on iNKT cells, the present invention also provides a culture method for inducing type C1 iNKT cells with high IFN-γ secretion. The method includes:

[0040] S10. When obtaining the cells to be processed, according to the preset first culture duration, based on the DC medium described above, culture the cells to be processed to obtain dendritic cells.

[0041] First, obtain the cells to be processed. The cells to be processed refer to cells or cell populations with the ability to differentiate into DC cells, such as PBMC (peripheral blood mononuclear cell). Taking PBMC as an example, the Ficoll method (density gradient centrifugation method) can be used to extract PBMC from peripheral blood.

[0042] Use the DC medium to culture the cells to be processed, so that a large number of DC cells proliferate in the cells to be processed for subsequent co-culture. The time for culturing the cells to be processed with DC cells, that is, the first culture duration, can be determined according to factors such as the source and form of the cells to be processed. Taking PBMC from blood as an example, the culture time used in this example is 7 days. For different cells to be processed, the consumption rates of each component in the medium are different. Still taking PBMC as an example, the fresh DC medium can be replaced every 2 - 3 days. As Figure 3 shown, during the culture process with the DC medium, the DC cells adhere and grow well.

[0043] Furthermore, there are a large number of non-adherent cells in PBMC. To reduce the interference of these cells, first resuspend the PBMC cells with a basal medium and inoculate them on a well plate. After culturing with the basal medium for a period of time, the cells adhere. Then discard the non-adherent cells and replace them with the DC medium, and then culture the cells to be processed according to the preset first culture duration to obtain dendritic cells.

[0044] S20. When obtaining induced cells, resuspend the induced cells with iNTK medium according to a preset second culture duration, and co-culture the resuspended induced cells with the dendritic cells.

[0045] Induced cells are cells that differentiate and proliferate into iNTK after co-culture with DC cells, such as PBMC cells. When obtaining induced cells, first resuspend the induced cells with iNTK medium, and then add the resuspended induced cells to the culture dish for culturing DC cells in the previous step, and co-culture the induced cells with dendritic cells.

[0046] The second culture duration is the duration of co-culture of induced cells and dendritic cells. Similar to the first culture duration, it can be adjusted according to the type, source, status, etc. of the induced cells. The second culture duration adopted in this embodiment is 7 days. As Figure 4 shown, during the co-culture of induced cells and DC cells, the number of cells increases significantly.

[0047] To verify the induction and proliferation effect of the medium and culture method provided in this embodiment on iNKT, a control group and three experimental groups are set up to conduct experiments and verification on this scheme.

[0048] The control group uses uncultured PBMC cells. The DC medium used in the experimental groups includes 50 ng / mL GM-CSF, 10 ng / mL IL-4, 100 U / mL TNF-α, 20 ng / mL LPS, 20 ng / mL 7DW8-5, and 10% autologous plasma. The iNKT medium includes 500 U / mL IL-2, 5 ng / mL IL-7, 5 ng / mL IL-15, 5 ng / mL IL-21, 20 ng / mL 7DW8-5, 2 μM lupeol, 2 μM betulinic acid, 2 μM resveratrol, and 3% autologous plasma.

[0049] In experimental group 1, the seeding density of the cells to be treated is 3×106 cells / mL, and the seeding density of the induced cells is 3×10 6 cells / mL, that is, the cell number ratio of the cells to be treated to the induced cells is 1:1; in experimental group 2, the seeding density of the cells to be treated is 3×10 6 cells / mL, and the seeding density of the induced cells is 6×10 6 cells / mL, that is, the cell number ratio of the cells to be treated to the induced cells is 1:2; in experimental group 3, the seeding density of the cells to be treated is 1×10 6 cells / mL, and the seeding density of the induced cells is 10×10 6 cells / mL, that is, the cell number ratio of the cells to be treated to the induced cells is 1:10.

[0050] After the culture was completed, all the cells were collected for detection of cell viability and purity of C1-type iNKT cells, and for detection of the contents of cytokines IFN-γ, TNF-α, IL-8, IL-4, IL-12p70 and IL-17 in the cell supernatant.

[0051] Experiment 1 was the detection of cell viability. PBMCs and total cells in the control group and the experimental group were stained with AO / PI, and cell counting was performed using a cell counter to calculate the cell viability. The results are shown in Table 1. As can be seen from Table 1, after 14 days of co-culture, there were still a relatively high number of viable cells in Experimental Groups 1-3.

[0052] Table 1. Total cell viability of PBMCs and after 14 days of culture (%)

[0053]

[0054] Experiment 2 was the detection of the purity of C1-type iNKT cells. The purity detection method used in this example was flow cytometry analysis. The tag proteins were CD3 and CD56, and the tag proteins of NK cells included CD3+, CXCR6+ and CD244-. Therefore, the proportion of NK cells could be analyzed through the tag proteins. The antibodies used in this example were PerCP-Cy5.5-CD3 antibody, FITC-CD244 antibody, and APC-CXCR6 antibody. The detection results are as Figure 5 shown.

[0055] According to Figure 5 , it was found that the content of iNKT cells in the control group was only 0.42%, while using the above-mentioned culture medium and culture method, the content of iNTK cells was at least 93.1% and at most 94.6%, far higher than the result of the control group, approximately amplified by 225 times.

[0056] Experimental Group 1 with the highest content of iNTK cells was selected for subsequent experiments. Experiment 3 was the detection of the contents of cytokines IFN-γ, TNF-α, IL-8, IL-4, IL-12p70 and IL-17 in the cell supernatant. In this example, flow cytometry was used for detection, and the cytokines in the collected supernatant were detected through two fluorescence of PE and APC. The detection results are as Figure 6 shown.

[0057] Figure 6 In, the contents of TNF-α, IL-8, IL-4, IL-12p70 and IL-17 in the control group and Experimental Group 1 were not very different, but the IFN-γ level in Experimental Group 1 was as high as 5297.83 pg / mL, far exceeding that of the control group.

[0058] The present invention also provides a kit, which comprises the DC medium and the iNTK medium as described above. The kit can be applied to aspects such as iNKT cell culture and preparation of exosomes from supernatant. Through the DC medium and the iNKT medium, the number of type C iNKT cells can be efficiently amplified. Based on this, the exosomes are rich in IFN-γ factor, thereby increasing the production of IFN-γ.

[0059] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A group of culture media for inducing type C1 iNKT cells, characterized in that, The culture medium includes a DC culture medium and an iNKT culture medium; The DC culture medium includes 50 ng / mL GM-CSF, 10 ng / mL IL-4, 100 U / mL TNF-α, 20 ng / mL LPS, 20 ng / mL 7DW8-5, and 10% autologous plasma; The iNKT culture medium includes 500 U / mL IL-2, 5 ng / mL IL-7, 5 ng / mL IL-15, 5 ng / mL IL-21, 20 ng / mL 7DW8-5, 2 μM lupeol, 2 μM betulinic acid, 2 μM resveratrol, and 3% autologous plasma.

2. A culture method for inducing type C1 iNKT cells, characterized in that, The method includes: When obtaining the cells to be processed, culturing the cells to be processed based on the DC culture medium described in Claim 1 according to a preset first culture duration to obtain dendritic cells; When obtaining the induced cells, resuspending the induced cells with the iNTK culture medium described in Claim 1 according to a preset second culture duration, and co-culturing the resuspended induced cells with the dendritic cells, The cells to be processed are PBMCs, and the induced cells are PBMCs.

3. The culture method for inducing C1-type iNKT cells according to claim 2, characterized in that, When culturing the cells to be treated with the DC medium, the concentration of the cells to be treated is 1 to 10×10 6 cells / mL.

4. The culturing method for inducing type C1 iNKT cells according to claim 2, wherein, When the dendritic cells are co-cultured with the inducing cells, the concentration of the inducing cells is 1 to 10×10 6 cells / mL.

5. The culturing method for inducing type C1 iNKT cells according to claim 2, wherein When obtaining the cells to be processed, culturing the cells to be processed based on the DC culture medium described in Claim 1 according to a preset first culture duration to obtain dendritic cells includes: Culturing the cells to be processed with a preset basal culture medium; When the cells to be processed adhere to the wall, culturing the cells to be processed based on the DC culture medium described in Claim 1 according to a preset first culture duration to obtain dendritic cells.

6. The culture method for inducing type C1 iNKT cells according to any one of claims 2-5, characterized in that, The quantity ratio of the dendritic cells to the induced cells is 1:1 to 1:

10.

7. A kit, characterized in that, The kit includes a group of culture media for inducing type C1 iNKT cells described in Claim 1, and the kit is used for iNKT cell culture and / or preparation of exosomes from the supernatant.

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