Method for activating and expanding iNKT cells and culture medium combination thereof

Abstract

The present disclosure relates to a method for activating and expanding iNKT cells and a culture medium combination. The culture medium combination includes an APC cell culture medium and an iNKT activation culture medium. Among them, the APC cell culture medium includes the following components: GM-CSF, IL-4, α-GalCer, CoCl2, and NK basal medium; the iNKT activation culture medium includes the following components: IL-2, IL7, IL-21, α-GalCer, N-acetyl-cysteine, platelet lysate, and NK basal medium. By using the method and culture medium combination of the present disclosure, irradiation is not required, the time consumption is short, multiple additions of APC are not required, and the operation is simple. The prepared iNKT cells have a high cell purity.

Description

Technical Field

[0001] The present invention relates to the field of biology, and particularly to a method for activating and expanding iNKT cells with high efficiency and a culture medium combination. Background Art

[0002] Type I NKT cells, namely iNKT (invariant natural killer T, iNKT) cells, can specifically recognize lipid antigens presented by CD1d molecules expressed on the surface of antigen-presenting cells (APCs). Among them, α-galactosyl ceramide (α-GalCer) is a classical antigen for activating iNKT cells. iNKT cells account for only about 0.1% (0.001% - 3%) of human circulating T cells on average, and there are significant individual differences in their numbers, belonging to a rare population (Abel Trujillo-Ocampo et al. IL-7 During Antigenic Stimulation Using Allogeneic Dendritic Cells Promotes Expansion of CD45RA-CD62L+CD4+ Invariant NKT Cells With Th-2 Biased Cytokine Production Profile. Front Immunol. 2020 Nov 27;11:567406). iNKT cells can play an important immunomodulatory role in the body and participate in immune responses against anti-tumor, anti-infection, and anti-autoimmune diseases. For example, iNKT cell therapy has obvious curative effects on the treatment of various tumors such as lung cancer, head and neck squamous cell carcinoma, sarcoma carcinoma, and acute leukemia. Currently, preclinical treatment with iNKT cells is mainly used in combination with surgery, chemotherapy, and radiotherapy to solve the problem of tumor recurrence. Among them, the number and killing function of iNKT cells are directly related to the curative effect. Therefore, it is very important to obtain a large number of highly pure iNKT cells.

[0003] The current in vitro activation and expansion process of iNKT cells mainly includes the following steps:

[0004] (1) Enrich iNKT cells.

[0005] (2) Prepare autologous or allogeneic APCs:

[0006] There are usually two methods for preparing APC cells. One is to use irradiated peripheral blood mononuclear cells (PBMCs) as APCs, and the other is to use irradiated dendritic cells (DCs) as APCs. Specifically as follows:

[0007] Method 1: PBMC loaded with α-GalCer after irradiation as APC: PBMC from autologous or allogeneic sources or PBMC cells purified without iNKT are irradiated, such as with X-rays or γ-rays (40 Gy), and the treated cells are cultured in a medium containing α-GalCer at 37 °C for 1-2 h.

[0008] Method 2: Monocyte-derived dendritic cells (DC) loaded with α-GalCer after irradiation as APC: i. Enrich CD14+ cells from raw materials such as whole blood, apheresis blood, or PBMC by magnetic bead method; or ii. Collect adherent cells with differentiation ability from PBMC by adherent method, that is, fresh or cryopreserved and thawed PBMC are statically cultured in a TC-treated culture flask at 37 °C for 1 h, the supernatant and non-adherent cells are removed, and the adherent cells are collected.

[0009] The cells obtained by the above two methods are induced and cultured in a medium containing GM-CSF and IL-4 for 5-7 days to obtain immature or mature DC. After the DC cells are irradiated or treated with mitomycin (the function is to prevent DC cell proliferation), they are cultured in a medium containing α-GalCer at 37 °C for 1 h to obtain APC cells.

[0010] (3) Activate iNKT cells:

[0011] According to the ratio of APC: iNKT = 1.5-50:1, the treated APC and iNKT cells are co-cultured in a medium containing lipid antigens (such as α-GalCer, etc.) and cytokines that can activate iNKT, and APC is added once every 7-10 days.

[0012] APC is the key to the successful activation and expansion of iNKT cells. However, the above process still has the following disadvantages: (1) Cell irradiation is required when preparing APC cells, and the investment and maintenance costs of related equipment, sites, etc. are high; (2) Mitomycin is an antibiotic and belongs to an anti-tumor prescription drug, and its residue needs to be controlled during clinical application; (3) CD14+ magnetic beads and cell sorting processes are required, increasing the complexity of the process and production costs; (4) The preparation time of DC cells is long, and iNKT cells cannot be activated and expanded in time, prolonging the production cycle; (5) APC cells need to be added multiple times to stimulate the proliferation of iNKT; (6) The purity of the prepared iNKT cells is low, <60%.

[0013] Therefore, considering the needs and dosages of clinical treatment, it is necessary to provide a method for activating and expanding iNKT cells that does not require irradiation, takes a short time, does not require multiple additions of APC, and is simple to operate. Summary of the Invention

[0014] To solve the problems existing in the prior art, the object of the present disclosure is to provide a method for activating and expanding iNKT cells that does not require irradiation, has a short time consumption, does not require multiple additions of APC, and is simple to operate. The iNKT cells prepared by the method of the present disclosure have a high cell purity.

[0015] To achieve the above object, the solution of the present disclosure is as follows:

[0016] On the one hand, the present disclosure provides a medium combination for activating and expanding iNKT cells, which includes an APC cell medium and an iNKT activation medium, wherein,

[0017] The APC cell medium includes the following components: GM-CSF, IL-4, α-GalCer, CoCl2, and NK basal medium;

[0018] The iNKT activation medium includes the following components: IL-2, IL7, IL-21, α-GalCer, N-acetyl-cysteine, platelet lysate, and NK basal medium.

[0019] On the other hand, the present disclosure provides a method for activating and expanding iNKT cells, which includes the following steps:

[0020] (1) Enrich iNKT cells;

[0021] (2) Prepare APC cells:

[0022] Culture PBMC cells in the aforementioned APC medium to obtain APC cells for activating iNKT;

[0023] (3) Activate and expand iNKT:

[0024] Co-culture the iNKT cells in step (1) with the APC cells in step (2) in the aforementioned iNKT activation medium.

[0025] On the other hand, the present disclosure provides the use of the aforementioned medium combination and / or the aforementioned method to expand and activate iNKT cells.

[0026] On the other hand, the present disclosure provides the use of the aforementioned medium combination, the aforementioned method, and / or the aforementioned iNKT cells in the preparation of immunotherapy agents.

[0027] The beneficial effects obtained by the present disclosure are at least as follows:

[0028] The present disclosure optimizes the preparation method of APC cells by optimizing the culture medium components and culture conditions of APC cells. Adding cobalt chloride, GM-CSF, and IL-4 to the culture medium can improve the antigen presentation ability of APCs and efficiently activate iNKT. In addition, adjusting the culture temperature to 40-50 °C and culturing in combination with the above-mentioned culture medium can effectively inhibit the proliferation of APCs, ensure the purity of iNKT after amplification, and can also shorten the preparation time of APCs and effectively shorten the production cycle of iNKT. The APC cells prepared by the method of the present disclosure only need to be added initially once and do not need to be added repeatedly. The preparation of APC cells does not require irradiation or mitomycin treatment, has low cost, and is safer.

[0029] Meanwhile, the present invention activates and amplifies iNKT cells by using a specific culture medium. After adding IL-2, IL-7, IL-21, NAC, and hPL components to the culture medium, iNKT with the function of killing tumor cells can be efficiently activated and continuously amplified.

[0030] In summary, using the amplification method of the present invention, the purity of the cells after iNKT activation and amplification on the 8th day of culture (proportion of CD3+) > 90%, reaching 99.8%, and the purity of iNKT cells on the 16th day of culture is 99.9%. Brief Description of the Drawings

[0031] Figure 1 : Purity of cells after iNKT cell activation and amplification (proportion of CD3+).

[0032] Figure 2 : Growth curve of NKT cells.

[0033] Figure 3 : Influence of different APC treatment temperatures / time on the purity of iNKT cells.

[0034] Figure 4 : Influence of different cobalt chloride concentrations on the purity of iNKT cells.

[0035] Figure 5 : Influence of different NAC and hPL concentrations on iNKT amplification.

[0036] Figure 6 : In vitro cytotoxicity of iNKT amplified in Example 1 against CD1d+ Jurkat target cells.

[0037] Figure 7 : Cytokine secretion of iNKT cells in Example 1. Detailed Description of the Embodiments

[0038] In the following examples of the present invention, the experimental methods without specific conditions noted are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. All common chemical reagents used in the examples are commercially available products.

[0039] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0040] The terms "comprising" and "having" and any variations thereof in the present invention are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product or device that comprises a series of steps is not limited to the listed steps or modules, but optionally further includes steps not listed, or optionally further includes other steps inherent to these processes, methods, products or devices.

[0041] As used in the present invention, "a plurality of" means two or more. "And / or" describes the association relationship of associated objects and indicates that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist simultaneously, and B exists alone. The character " / " generally represents an "or" relationship between the associated objects before and after.

[0042] The term "PBMC" used in the present disclosure refers to peripheral blood mononuclear cells, which is the general term for cells with a single nucleus in peripheral blood, including but not limited to lymphocytes (T cells, B cells, NK cells, NKT cells), monocytes or dendritic cells.

[0043] There are two definitions of NKT cells, namely broad sense and narrow sense. NKT cells in the broad sense refer to a cell population that expresses both NK cell surface markers (CD161c in mice and CD56 in humans) and T cell surface markers. With the development of lipid antigen-binding CD1d tetramer technology, researchers found a cell subset in mice that can bind to CD1d tetramers loaded with lipid antigen α-GalCer. Most of this population expresses both NK cell surface markers and T cell surface markers, and secretes a large amount of cytokines to play an immunomodulatory role. According to the definition of Godfrey, this group of cells is type I NKT cells, that is, narrow-sense iNKT cells. Currently, this group of cells has been the most widely and deeply studied, so this group of cells is called classical NKT cells. The NKT cells mentioned in the existing literature mainly refer to narrow-sense NKT cells. In the present disclosure, unless otherwise specified, when referring to NKT cells, it generally refers to narrow-sense iNKT cells.

[0044] The term "TC treatment" used in the present disclosure refers to Tissue culture treated. TC treatment indicates that the vessel has been surface-modified and is suitable for culturing adherent cells. However, cell culture dishes and the like after surface modification are generally also suitable for culturing suspension cells.

[0045] The term "effector-to-target ratio" used in the present disclosure refers to the quantity ratio of effector cells to target cells.

[0046] The term "CD1d+Jurkat cells" used in the present disclosure refers to Jurkat cells expressing CD1d, where Jurkat cells are an immortalized human T lymphocyte cell line of human acute T lymphocyte leukemia, used for studying acute T cell leukemia, T cell signal transduction, and the expression of various chemokine receptors susceptible to virus entry, especially HIV.

[0047] In one aspect, the present disclosure provides a medium combination for iNKT cell activation and expansion, which includes an APC cell medium and an iNKT activation medium, wherein,

[0048] The APC cell medium includes the following components: GM-CSF, IL-4, α-GalCer, CoCl2, and NK basal medium;

[0049] The iNKT activation medium includes the following components: IL-2, IL7, IL-21, α-GalCer, N-acetyl-cysteine, platelet lysate, and NK basal medium.

[0050] In some embodiments of the present disclosure, in the APC cell medium, the concentration of GM-CSF is about 20 - 100 ng / mL, preferably about 30 - 80 ng / mL, and more preferably about 50 ng / mL.

[0051] In some embodiments of the present disclosure, in the APC cell medium, the concentration of IL-4 is about 50 - 200 ng / mL or 500 - 2000 U / mL, preferably about 80 - 120 ng / mL or about 800 - 1200 U / mL, and more preferably about 100 ng / mL or about 1000 U / mL.

[0052] In some embodiments of the present disclosure, in the APC cell medium, the concentration of α-GalCer is about 0.5 - 5 μg / mL, preferably about 0.8 - 2 μg / mL, and more preferably about 1 μg / mL.

[0053] In some embodiments of the present disclosure, in the APC cell medium, CoCl 2The concentration is about 0.5 to 4 mM, preferably about 0.8 to 2 mM, more preferably about 1 mM.

[0054] In some embodiments of the present disclosure, in the iNKT activation medium, the concentration of IL-2 is about 200 to 1000 IU / mL, preferably about 300 to 600 IU / mL, more preferably about 400 IU / mL.

[0055] In some embodiments of the present disclosure, in the iNKT activation medium, the concentration of IL-7 is about 1 to 50 ng / mL, preferably about 5 to 30 ng / mL, more preferably about 10 ng / mL.

[0056] In some embodiments of the present disclosure, in the iNKT activation medium, the concentration of IL-21 is about 1 to 50 ng / mL, preferably about 5 to 30 ng / mL, more preferably about 10 ng / mL.

[0057] In some embodiments of the present disclosure, in the iNKT activation medium, the concentration of α-GalCer is about 0.5 to 5 μg / mL, preferably about 0.8 to 2 μg / mL, more preferably about 1 μg / mL.

[0058] In some embodiments of the present disclosure, in the iNKT activation medium, the concentration of N-acetyl-cysteine is about 1 to 10 mM, preferably about 2 to 8 mM, more preferably about 5 mM.

[0059] In some embodiments of the present disclosure, in the iNKT activation medium, the concentration of platelet lysate is about 1 to 10% (v / v), preferably about 2 to 8% (v / v), more preferably about 5% (v / v).

[0060] In some embodiments of the present disclosure, the platelet lysate in the iNKT activation medium is human platelet lysate.

[0061] On the other hand, the present disclosure provides a method for activating and expanding iNKT cells, which includes the following steps:

[0062] (1) Enrich iNKT cells;

[0063] (2) Prepare APC cells:

[0064] Culture PBMC cells in the APC medium described above to obtain APC cells for activating iNKT.

[0065] (3) Activate and expand iNKT:

[0066] Co-culture the iNKT cells in step (1) with the APC cells in step (2) in the iNKT activation medium.

[0067] In some embodiments of the present disclosure, the temperature during the cultivation in step (2) of the method is about 37 - 52 °C, preferably about 40 - 50 °C, and more preferably 42 - 48 °C.

[0068] In some embodiments of the present disclosure, the cultivation time in step (2) of the method is about 1 - 8 h, preferably about 1 - 4 h, and preferably 2 h.

[0069] In some embodiments of the present disclosure, the number ratio of APC cells to the iNKT cells during the co - cultivation in step (3) of the method is about (1 - 10):1.

[0070] In some embodiments of the present disclosure, the number ratio of APC cells to the iNKT cells during the co - cultivation in step (3) of the method is about (3 - 6):1, preferably about 5:1.

[0071] In some embodiments of the present disclosure, the enrichment of iNKT cells in step (1) of the method includes: sorting and enriching iNKT cells from PBMCs using iNKT magnetic beads.

[0072] In some embodiments of the present disclosure, in step (3) of the method, the medium is changed every 2 - 3 days, preferably all of the iNKT activation medium is replaced.

[0073] On the other hand, the present disclosure provides for the amplification and activation of iNKT cells using the aforementioned medium combination and / or the aforementioned method.

[0074] On the other hand, the present disclosure provides for the use of the aforementioned medium combination, the aforementioned method, and the aforementioned iNKT cells in the preparation of immunotherapeutic agents.

[0075] The medium combination of the present disclosure can be made into a set of media, wherein the APC cell medium and the iNKT activation medium are provided in a form of separate administration. The APC cell medium can be placed in one container, and the iNKT activation medium can be placed in another container.

[0076] Examples

[0077] The technical solutions of the present disclosure will be further described below by means of specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present disclosure and should not be regarded as specific limitations to the present disclosure.

[0078] Example 1

[0079] (1) Enrichment of iNKT cells:

[0080] Obtain whole blood or apheresis blood samples, isolate PBMCs, and use Miltenyi Biotec anti-iNKT magnetic beads (Cat. 130-091-221) to sort and enrich iNKT cells from PBMCs. Perform the sorting operation according to the product manual, and collect iNKT cells and PBMC cells without iNKT for later use.

[0081] (2) Preparation of APC:

[0082] Add GM-CSF, IL-4, α-GalCer, and CoCl to the NK basal medium (manufacturer Irvine Scientific, PRIME-XV NK Cell CDM, catalog number 91215-1L). 2 , to obtain an NK basal medium containing 50 ng / mL GM-CSF, 100 ng / mL or 1000 U / mL IL-4, 1000 ng / mL α-GalCer, and 1000 μM CoCl. 2 Add PBMCs or PBMC cells without iNKT sorted by magnetic beads in step (1) to the NK basal medium containing GM-CSF, IL-4, α-GalCer, and CoCl. 2 Harvest the cells after culturing at 45 °C for 2 h to obtain APCs for activating iNKT.

[0083] (3) Activation and expansion of iNKT:

[0084] According to the ratio of APC: positive cells = 5:1, culture the APC obtained in step (2) and the iNKT cells obtained in step (1) in a serum-free medium containing 400 IU / mL IL-2, 10 ng / mL IL-7, 10 ng / mL IL-21, 1000 ng / mL α-GalCer, 5 mM N-acetyl-cysteine (NAC), and 5% (v / v) human platelet lysate (hPL). Perform medium replacement or supplementation culture every 2 - 3 days, and the culture medium is the same as the previous one. During the culture period, detect the cell purity (i.e., the proportion of CD3+) of iNKT cells after activation and expansion by flow cytometry every two days, and record the growth of NKT cells.

[0085] The experimental results are as Figure 1 and Figure 2 shown. It can be seen from Figure 1 that the cell purity of iNKT cells after activation and expansion is 99.8% on the 8th day of culture. The cell growth curve of days 1 - 20 of culture is as Figure 2 shown. After activation, iNKT expands rapidly, and the purity can be maintained. The cell purity of iNKT cells after activation and expansion reaches 99.9% on the 16th day. The amplification multiple of iNKT reaches 20788 times after 20 days of culture.

[0086] Example 2

[0087] Referring to the activation and amplification method of Example 1, the difference between this example and Example 1 is that the culture temperature or time of APC in step (2) of Example 1 is changed. Five different experimental groups are set up respectively, controlling a single variable to verify the influence of different APC treatment temperatures or times on the purity of iNKT cells. The specific details of the experiments in different experimental groups are different from those in Example 1 as follows:

[0088] Group 1: Change the culture temperature in step (2) of Example 1 from 45 °C to 37 °C;

[0089] Group 2: Change the culture temperature in step (2) of Example 1 from 45 °C to 42 °C;

[0090] Group 3: Change the culture temperature in step (2) of Example 1 from 45 °C to 50 °C;

[0091] Group 4: Change the culture time in step (2) of Example 1 from 2 h to 4 h;

[0092] Group 5: Change the culture time in step (2) of Example 1 from 2 h to 1 h.

[0093] After co-culturing the APCs treated in Groups 1-5 with iNKT cells for 8 days respectively, detect the cell purity after iNKT activation and amplification.

[0094] The experimental results are shown in Figure 3 , and it can be seen from Figure 3 that except for the iNKT purity in Groups 1 and 5 being lower than 90%, the iNKT purity in the remaining groups is > 90%.

[0095] Example 3

[0096] Referring to the activation and amplification method of Example 1, the difference between this example and Example 1 is that the concentration of cobalt chloride in the culture medium in step (2) of Example 1 is changed. Five different experimental groups are set up respectively, controlling a single variable to verify the influence of different cobalt chloride concentrations on the purity of iNKT cells. The specific details of the experiments in different experimental groups are different from those in Example 1 as follows:

[0097] Group 1: Change the concentration of CoCl 2 in the culture medium in step (2) of Example 1 from 1000 μM to 500 μM;

[0098] Group 2: Change the concentration of CoCl 2 in the culture medium in step (2) of Example 1 from 1000 μM to 2000 μM;

[0099] Group 3: Change the concentration of CoCl 2The concentration was changed from 1000 μM to 4000 μM;

[0100] Group 4: In step (2) of Example 1, CoCl was not added to the culture medium 2 .

[0101] After culturing the APCs treated in Groups 1-4 with iNKT cells for 8 days respectively, the cell purity after iNKT activation and amplification was detected.

[0102] The experimental results are shown in Figure 4 , and it can be seen from Figure 4 that compared with Group 4 of Example 3 without adding cobalt chloride, adding cobalt chloride can effectively improve the purity of iNKT, and the purity of iNKT in Group 2 of Example 1 and Example 3 > 90%.

[0103] Example 4

[0104] Referring to the activation and amplification method of Example 1, the difference between this example and Example 1 is that the concentration of N-acetyl-cysteine (NAC) or human platelet lysate (hPL concentration) in the culture medium for activating and amplifying iNKT in step (3) of Example 1 was changed. Seven different experimental groups were set up respectively, controlling a single variable to verify the influence of different NAC concentrations or hPL concentrations on the amplification fold of iNKT cells. The specific details of the experiments in different experimental groups are different from those of Example 1 as follows:

[0105] Group 1: The concentration of NAC in the culture medium of step (3) of Example 1 was changed from 5 mM to 2 mM;

[0106] Group 2: The concentration of NAC in the culture medium of step (3) of Example 1 was changed from 5 mM to 10 mM;

[0107] Group 3: The concentration of NAC in the culture medium of step (3) of Example 1 was changed from 5 mM to 0 mM;

[0108] Group 4: The hPL concentration in the culture medium of step (3) of Example 1 was changed from 5% (v / v) to 2%;

[0109] Group 5: The hPL concentration in the culture medium of step (3) of Example 1 was changed from 5% (v / v) to 7%;

[0110] Group 6: The hPL concentration in the culture medium of step (3) of Example 1 was changed from 5% (v / v) to 10%;

[0111] Group 7: The hPL concentration in the culture medium of step (3) of Example 1 was changed from 5% (v / v) to 0%.

[0112] The amplification fold of iNKT after culturing in the culture media of Groups 1-7 was detected.

[0113] The experimental results are shown in Figure 5 , and it can be seen from Figure 5 that taking the amplification multiple in Example 1 as 100%, compared with Group 3 and Group 7 without adding NAC or hPL in this example, adding NAC and hPL to the culture medium can effectively increase the amplification multiple of iNKT.

[0114] Example 5

[0115] The cytotoxicity of the amplified iNKT cells in Example 1 against CD1d+ target cells was detected by the LDH method. CD1d+ Jurkat cells (a kind of human acute T lymphocyte leukemia cells, purchased from ATCC, catalog number TIB-152) in the logarithmic growth phase were collected, centrifuged, counted, and adjusted to a density of 1×10 6 cells / mL. In the test wells, 100 μL of effector cells (iNKT cells) and target cells (Jurkat cells) were added in a one-to-one correspondence according to the ratios of 0.5:1, 1:1, and 3:1, and co-cultured in an incubator at 37°C and 5% CO 2 for 24 h. After centrifugation at 250 g for 4 min, 50 μL of the supernatant was aspirated into a new 96-well plate, and then 50 μL of CytoTox Reagent (purchased from Promega, catalog number G1780) was added, and incubated in the dark for 30 min. Then 50 μL of the reaction termination solution (Stop Solution) was added, and the absorbance was detected at 490 nm using an enzyme-linked immunosorbent assay detector.

[0116] The formula for calculating cytotoxicity is: Cytotoxicity (%) = (Absorbance of the experimental group - Spontaneous absorbance of effector cells - Spontaneous absorbance of target cells × 100) / (Maximum absorbance of target cells - Spontaneous absorbance of target cells)

[0117] In addition, the ELISA method was used to detect the levels of cytokines secreted in the cell supernatant after iNKT was stimulated by target cells for 24 h and without stimulation.

[0118] The experimental results showed that the iNKT amplified by the method of Example 1 had obvious cytotoxicity against CD1d+ target cells, and the cytotoxicity gradually increased with the increase of the effector-to-target ratio (see Figure 6 ), and the cytokine secretion of iNKT cells is shown in Figure 7 . After being stimulated by target cells, iNKT could significantly secrete cytokines related to cytotoxicity, indicating that the iNKT amplified by the method of the present disclosure had biological activity.

[0119] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0120] The above-described embodiments merely represent several implementation manners of the present disclosure. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present disclosure, several variations and improvements can still be made, and these all fall within the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure patent shall be subject to the appended claims.

Claims

1. A medium combination for iNKT cell activation and expansion, which is composed of an APC cell medium and an iNKT activation medium, wherein, The APC cell culture medium consists of the following components: GM-CSF, IL-4, α-GalCer, CoCl 2 and NK basal medium; the iNKT activation medium is composed of the following components: IL-2, IL7, IL-21, α-GalCer, N-acetyl-cysteine, platelet lysate and NK basal medium; In the described APC cell culture medium, the concentration of GM-CSF is 20-100 ng / mL, the concentration of IL-4 is 50-200 ng / mL or 500-2000 U / mL, the concentration of α-GalCer is 0.5-5 μg / mL, and the concentration of CoCl 2 is 0.5-4 mM; in the iNKT activation medium, the concentration of IL-2 is 200-1000 IU / mL, the concentration of IL-7 is 1-50 ng / mL, the concentration of IL-21 is 1-50 ng / mL, the concentration of α-GalCer is 0.5-5 μg / mL, the concentration of N-acetyl-cysteine is 1-10 mM, and the concentration of human platelet lysate is 1-10% (v / v).

2. The medium combination according to claim 1, wherein in the APC cell medium, the concentration of GM-CSF is 30-80 ng / mL.

3. The medium combination according to claim 2, wherein in the APC cell medium, the concentration of GM-CSF is 50 ng / mL.

4. The medium combination according to claim 1, wherein in the APC cell medium, the concentration of IL-4 is 80-120 ng / mL or 800-1200 U / mL.

5. The medium combination according to claim 4, wherein in the APC cell medium, the concentration of IL-4 is 100 ng / mL or 1000 U / mL.

6. The medium combination according to claim 1, wherein in the APC cell medium, the concentration of α-GalCer is 0.8-2 μg / mL.

7. The medium combination according to claim 6, wherein in the APC cell medium, the concentration of α-GalCer is 1 μg / mL.

8. The culture medium combination according to claim 1, wherein in the APC cell culture medium, the concentration of CoCl 2 is 0.8 to 2 mM.

9. The culture medium combination according to claim 8, wherein in the APC cell culture medium, the concentration of CoCl 2 is 1 mM.

10. The medium combination according to claim 1, wherein in the iNKT activation medium, the concentration of IL-2 is 300-600 IU / mL.

11. The medium combination according to claim 10, wherein in the iNKT activation medium, the concentration of IL-2 is 400 IU / mL.

12. The medium combination according to claim 1, wherein in the iNKT activation medium, the concentration of IL-7 is 5-30 ng / mL.

13. The medium combination according to claim 12, wherein in the iNKT activation medium, the concentration of IL-7 is 10 ng / mL.

14. The medium combination according to claim 1, wherein in the iNKT activation medium, the concentration of IL-21 is 5-30 ng / mL.

15. The medium combination according to claim 14, wherein in the iNKT activation medium, the concentration of IL-21 is 10 ng / mL.

16. The medium combination according to claim 1, wherein in the iNKT activation medium, the concentration of α-GalCer is 0.8-2 μg / mL.

17. The medium combination according to claim 16, wherein in the iNKT activation medium, the concentration of α-GalCer is 1 μg / mL.

18. The culture medium combination according to claim 1, wherein in the iNKT activation culture medium, the concentration of N-acetyl-cysteine is 2-8 mM.

19. The culture medium combination according to claim 18, wherein in the iNKT activation culture medium, the concentration of N-acetyl-cysteine is 5 mM.

20. The culture medium combination according to claim 1, wherein in the iNKT activation culture medium, the concentration of human platelet lysate is 2-8% (v / v).

21. The culture medium combination according to claim 20, wherein in the iNKT activation culture medium, the concentration of human platelet lysate is 5% (v / v).

22. A method for activating and expanding iNKT cells, which comprises the following steps: (1) Enriching iNKT cells; (2) Preparing APC cells: Culturing PBMC cells in the APC cell culture medium according to any one of claims 1-21 to obtain APC cells for activating iNKT; (3) Activating and expanding iNKT: Co-culturing the iNKT cells in step (1) with the APC cells in step (2) in the iNKT activation culture medium according to any one of claims 1-21; wherein, the temperature of the culture in step (2) is 37-52 °C, and the culture time is 1-8 h; The number ratio of the APC cells to the iNKT cells during co-culture in step (3) is (1-10):

1.

23. The method according to claim 22, wherein, the temperature of the culture in step (2) is 40-50 °C.

24. The method according to claim 23, wherein, the temperature of the culture in step (2) is 42-48 °C.

25. The method according to claim 22, wherein, the culture time in step (2) is 1-4 h.

26. The method according to claim 25, wherein, the culture time in step (2) is 2 h.

27. The method according to claim 22, wherein, The number ratio of the APC cells to the iNKT cells during co-culture in step (3) is (3-6):

1.

28. The method according to claim 27, wherein, The number ratio of the APC cells to the iNKT cells during co-culture in step (3) is 5:

1.

29. The method according to claim 22, wherein, The enrichment of iNKT cells in step (1) is: sorting and enriching iNKT cells from PBMCs using iNKT magnetic beads.

30. The method according to claim 22, wherein, In step (3), the medium is changed every 2-3 days, and all of it is replaced with the iNKT activation culture medium.

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