Immunotherapy cell culture kits, NK cell culture methods and applications

Abstract

This invention provides an immune cell culture kit, a method for culturing NK cells, and its applications. The immune cell culture kit includes a basal culture medium, recombinant antibodies, and cytokines. The basal culture medium includes RPMI-1640, thioglycerol, L-ascorbic acid, dexamethasone, and β-mercaptoethanol. The recombinant antibodies include two or more of CD16, CD56, NKp30, NKp44, NKp46, NKG2D, and 2B4 antibodies. The cytokines include IL-2, IL-15, IL-18, IL-21, and FLT3L. The immune cell culture kit of this invention is low-cost, and the cultured NK cells have high purity and viability, as well as good tumor-killing activity. The NK cells cultured by this method exhibit high killing activity against both A549 and K562 cells.

Description

Technical Field

[0001] This invention relates to the field of NK cell culture technology, and in particular to an immune cell culture kit, a method for culturing NK cells, and their applications. Background Technology

[0002] Immune cells are fundamental components of the body's immune system. They recognize and eliminate pathogens that invade the body, including lymphocytes and macrophages, playing a crucial role in the immune system. NK cells (natural killer cells) are an important type of innate lymphocyte that play a vital role in immune surveillance and anti-tumor immune responses. NK cells can directly recognize abnormal molecular signals on the cell surface and release cytotoxic molecules such as perforin to kill these abnormal cells, including tumor cells and virus-infected cells, demonstrating potent anti-tumor and antiviral activity.

[0003] Although NK cell immunotherapy has shown great application potential, the current culture of NK cells suffers from high costs and low expansion rates. How to achieve large-scale in vitro production of NK cells with high expansion rates is an urgent problem to be solved. Summary of the Invention

[0004] This invention provides an immune cell culture kit, a method for culturing NK cells, and their applications. The main objective of this invention is to solve the technical problems mentioned in the background section of the prior art.

[0005] The contents of this invention are as follows:

[0006] The first aspect of this invention provides an immune cell culture kit, comprising: a basal culture medium, a recombinant antibody, and cytokines; wherein the basal culture medium comprises RPMI-1640, thioglycerol, L-ascorbic acid, dexamethasone, and β-mercaptoethanol; wherein the recombinant antibody comprises two or more of CD16 antibody, CD56 antibody, NKp30 antibody, NKp44 antibody, NKp46 antibody, NKG2D antibody, and 2B4 antibody; and wherein the cytokines comprise IL-2, IL-15, IL-18, IL-21, and FLT3L.

[0007] In this invention, the immune cell culture kit comprises a basal culture medium component A, a recombinant antibody component B, and a cytokine component C. The basal culture medium component A provides nutritional support and a suitable growth environment for cell proliferation, thereby promoting cell expansion. The recombinant antibody component B can activate NK cells and promote their proliferation, enhancing their immune response and improving cell killing efficiency. The cytokine component C promotes the expansion, activation, and maturation of NK cells; IL-2, IL-15, and FLT3L have a synergistic effect, significantly enhancing the proliferative capacity of NK cells.

[0008] In this invention, the basal culture medium component A uses RPMI-1640 medium combined with thioglycerol, L-ascorbic acid, dexamethasone, and β-mercaptoethanol. This provides the necessary nutrients for NK cell culture, reduces the growth of non-NK cells, facilitates the purification and enrichment of NK cells, and increases NK cell survival and expansion rates. Furthermore, using RPMI-1640 as the basal culture medium offers the advantage of lower cost compared to AIM-V basal medium, etc.

[0009] In an optional embodiment of the first aspect of the present invention, the concentration of thioglycerol in the basal culture medium is 1-10 μg / mL, the concentration of L-ascorbic acid is 10-100 μg / mL, the concentration of dexamethasone is 0.1-1 μM, and the concentration of β-mercaptoethanol is 0.1-0.6 μM.

[0010] In an optional embodiment of the first aspect of the present invention, among the cytokines, the concentration of IL-2 is 50,000 to 500,000 U / mL, the concentration of IL-15 is 10 to 100 μg / mL, the concentration of IL-18 is 25 to 75 μg / mL, the concentration of IL-21 is 1 to 10 μg / mL, and the concentration of FLT3L is 10 to 100 μg / mL.

[0011] In this invention, the recombinant antigen used may include two or more of the following: 10-100 μg / mL anti-human CD16 antibody, 10-100 μg / mL anti-human CD56 antibody, 10-100 μg / mL anti-human NKp30 antibody, 10-100 μg / mL anti-human NKp44 antibody, 10-100 μg / mL anti-human NKp46 antibody, 10-100 μg / mL anti-human NKG2D antibody, and 10-100 μg / mL anti-human 2B4 antibody.

[0012] In an optional embodiment of the first aspect of the present invention, the recombinant antibody is a combination of CD56 antibody and NKG2D antibody, wherein the concentration of CD56 antibody is 10-100 μg / mL and the concentration of NKG2D antibody is 10-100 μg / mL.

[0013] As a second aspect of the present invention, the present invention provides a method for culturing NK cells, comprising:

[0014] Provide an immune cell culture kit as described in the first aspect of the present invention;

[0015] The culture vessel was coated with a mixture of recombinant antibody and buffer; the basal culture medium and cytokines were mixed to obtain the first mixed culture medium;

[0016] PBMCs were isolated from human peripheral blood.

[0017] PBMCs were seeded into coated culture containers, and cultured in the first mixed medium for a predetermined time. During the culture process, the first mixed medium was supplemented to maintain a cell concentration of 2.0–3.5 × 10⁻⁶ cells / year. 6 cells / mL;

[0018] After the first predetermined time of incubation, a second mixed culture medium is added to the culture container, wherein the second mixed culture medium consists of the basal culture medium and 500 U / mL IL-2, and the culture is carried out for a second predetermined time.

[0019] After the second predetermined incubation period, the cultured NK cells are collected from the culture container.

[0020] The culture containers referred to in this invention include culture flasks or culture bags, such as T25 or T75 culture flasks, T175 culture flasks, T225 culture flasks, and 1L culture bags, etc. They can be transferred or replaced according to the actual volume of the culture medium to ensure sufficient space for culture.

[0021] In this invention, D-PBS is used as a buffer, and the recombinant antibody is mixed with D-PBS at a ratio of 1:9 to coat the culture container. Specifically, the culture container can be a T25 or T75 culture flask.

[0022] The inoculation density of the PBMCs is 3–8 × 10⁻⁶. 6 per mL.

[0023] In an optional embodiment of the second aspect of the present invention, the first predetermined time is 12 days and the second predetermined time is 2 days.

[0024] In this invention, the object of culture is NK cells, and the total culture time of the NK cells is 14 days, with the first predetermined time being the first 12 days. A first mixed culture medium is used to ensure that PBMCs differentiate into NK cells and proliferate, while improving the anti-tumor activity of the target NK cells.

[0025] In an optional embodiment of the second aspect of the present invention, during the first predetermined time period of cultivation, autologous plasma is added to the culture container at the initial stage of cultivation, wherein the concentration of the autologous plasma is 1-10%, and the initial stage of cultivation is 0-9 days of the first predetermined time period.

[0026] In an optional embodiment of the second aspect of the present invention, the first mixed culture medium is obtained by mixing the basal culture medium with cytokines at a ratio of 1000:1.

[0027] In the initial stage of cell culture, it is preferable to add autologous plasma at a concentration of 1%-10%, preferably 10%. The addition of autologous plasma can meet the nutrient requirements of cell culture and promote cell growth and proliferation in the culture environment. Specifically, the addition of autologous plasma is only required for the first 9 days of culture; after 9 days, no further addition of autologous plasma is necessary.

[0028] During the culture process, specifically during the first predetermined time period (0-12 days), a first mixed culture medium is added. Autologous plasma can also be added to promote NK cell differentiation and proliferation. The first mixed culture medium is replenished as needed based on cell density and the color of the culture medium to maintain a cell concentration of 2.0–3.5 × 10⁻⁶ cells / day. 6 Cells were transferred sequentially to T175 culture flasks, T225 culture flasks, and 1L culture bags according to the culture medium volume to ensure sufficient space.

[0029] At the second predetermined time of culture, i.e., 13-14 days of culture, a second mixed culture medium is added to ensure the growth of NK cells.

[0030] A third aspect of the present invention provides the application of the immune cell culture kit as described above in the preparation of immune cells and / or supernatant exosomes.

[0031] The fourth aspect of the present invention provides the use of NK cells as described above in the preparation of drugs for treating and / or preventing tumors.

[0032] Beneficial Effects: This invention provides an immune cell culture kit, culture method, and application. The immune cell culture kit includes a basal culture medium, recombinant antibodies, and cytokines. The basal culture medium includes RPMI-1640, thioglycerol, L-ascorbic acid, dexamethasone, and β-mercaptoethanol. The recombinant antibodies include two or more of CD16, CD56, NKp30, NKp44, NKp46, NKG2D, and 2B4 antibodies. The cytokines include IL-2, IL-15, IL-18, IL-21, and FLT3L. The immune cell culture kit of this invention is low-cost, and the cultured NK cells have high purity and viability, and good tumor-killing activity. The NK cells cultured by this method exhibit high killing activity against both A549 and K562 cells. Attached Figure Description

[0033] Figure 1 Cell morphology diagrams for each embodiment and comparative example;

[0034] Figure 2 The NK cell expansion curves for each embodiment and comparative example are shown.

[0035] Figure 3 Flow cytometry images of NK cells at day 7 and day 14 of culture in each of the embodiments and comparative examples;

[0036] Figure 4 Flow cytometry analysis of NK cell expression of the lung-targeting marker chemokine CD3-CD56+CCR5+;

[0037] Figure 5 Figure showing the cytotoxic activity of NK cells against A549 cells;

[0038] Figure 6 The graph shows the cytotoxic activity of NK cells against K562 cells. Detailed Implementation

[0039] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0040] As a first aspect of the present invention, the immune cell kit of the present invention is composed of component A, component B and component C:

[0041] Component A is the basal culture medium, 2×1L, including RPMI-1640, 1-10 μg / mL thioglycerol, 10-100 μg / mL L-ascorbic acid, 0.1-1 μM dexamethasone, and 0.1-0.6 μM β-mercaptoethanol;

[0042] Component B consists of recombinant antibodies, 1 mL, including two or more of the following: 10–100 μg / mL anti-human CD16 antibody, 10–100 μg / mL anti-human CD56 antibody, 10–100 μg / mL anti-human NKp30 antibody, 10–100 μg / mL anti-human NKp44 antibody, 10–100 μg / mL anti-human NKp46 antibody, 10–100 μg / mL anti-human NKG2D antibody, and 10–100 μg / mL anti-human 2B4 antibody.

[0043] Component C consists of cytokines, 1 mL, including: 50,000–500,000 U / mL IL-2, 10–100 μg / mL IL-15, 25–75 μg / mL IL-18, 1–10 μg / mL IL-21, and 10–100 μg / mL FLT3L.

[0044] The immune cell culture kit of the present invention uses component A, which is RPMI-1640 with added thioglycerol, L-ascorbic acid, dexamethasone and β-mercaptoethanol. This combination is beneficial to the survival rate and expansion rate of NK cells, and can reduce the corresponding cost compared with other basic culture media.

[0045] The immune cells in this invention can also be T cells, etc.

[0046] A second aspect of the present invention claims protection for the application of the immune cell culture kit in the in vitro preparation of NK cells, specifically the use of the immune cell culture kit to culture NK cells in PBMCs, resulting in NK cells with high survival and expansion rates.

[0047] In a second aspect, the invention seeks protection for the application of the above-mentioned immune cell culture kit in immune cell amplification and immune cell exosome preparation. The immune cell culture kit can improve the amplification rate of immune cells in vitro, and the cost of the immune cell culture kit is relatively low, which helps enterprises save costs.

[0048] In a fourth aspect of the present invention, the NK cells obtained in the second aspect of the present invention are used in the preparation of some tumor drugs. The positive rate of NK cells expressing the lung marker chemokine CCR5 is as high as 99.6%, and they have high killing activity against both A549 cells and K562 cells.

[0049] To better illustrate the technical effects of the present invention, the present invention has constructed the following embodiments and comparative examples for testing.

[0050] Example 1: The immune cell culture kit used consists of components A, B, and C.

[0051] Component A is the basal culture medium, 2×1L, including RPMI-1640, 3μg / mL thioglycerol, 25μg / mL L-ascorbic acid, 0.8μM dexamethasone, and 0.2μM β-mercaptoethanol;

[0052] Component B consists of recombinant antibodies, 1 mL, including 20 μg / mL anti-human CD56 antibody and 100 μg / mL anti-human NKG2D antibody;

[0053] Component C consists of cytokines, 1 mL, including: 200,000 U / mL IL-2, 20 μg / mL IL-15, 30 μg / mL IL-18, 8 μg / mL IL-21, and 50 μg / mL FLT3L.

[0054] The NK cell culture steps are as follows:

[0055] S1: Coat a T25 or T75 culture flask with a mixture of 1 mL of component B and 9 mL of D-PBS, and incubate at room temperature for 1 hour or at 4°C overnight;

[0056] S2: Isolation of human peripheral blood mononuclear cells (PBMCs);

[0057] S3: Inoculate PBMCs into coated T25 or T75 culture flasks at a density of 3–8 × 10⁶. 6 The cells / mL were added to the first mixed culture medium (the first mixed culture medium was obtained by mixing component C: component A = 1:1000) for incubation;

[0058] S4: From day 0 to 12 of culture, add an appropriate amount of the first mixed medium and continue culturing. During this period, replenish the first mixed medium as needed based on cell density and medium color, maintaining the cell concentration at 2.0–3.5 × 10⁻⁶ cells / day. 6 Following the principle of cells / mL, cells were sequentially transferred to T175 culture flasks, T225 culture flasks, and 1L culture bags according to the volume of the culture medium. During the first 9 days of culture, 1-10% autologous plasma was added, with 10% being the most preferred. After 9 days, no further addition of autologous plasma was required.

[0059] S5: On day 12, add 1L of basal medium (containing 500U / mL IL-2) and continue culturing until day 14.

[0060] S6: After culture, collect cells and perform tests on NK cell purity, expansion fold, and cytotoxic activity.

[0061] Controlled test experiment 1

[0062] The effect of thioglycerol addition on NK cell culture was determined using the controlled variable method. Specifically, the immune cell culture kit composition of control test 1 was the same as that in Example 1, while keeping the contents of other components constant. Different concentrations of thioglycerol were set, and the culture method was the same as in Example 1. The total cell viability was then detected after 14 days of culture.

[0063] In the control test experiment 1, three test groups were set up, with the concentrations of thioglycerol added being 0, 1 μg / mL, 3 μg / mL, 5 μg / mL, 7 μg / mL, and 10 μg / mL. The total cell viability after 14 days of culture was recorded and the average value was calculated. The results are shown in Table 1.

[0064] Table 1. Cell viability at different concentrations of thioglycerol

[0065] Thioglycerol (μg / mL) 0 1 3 5 7 10 Cell viability (%) in test group 1 89.35 90.17 94.32 95.01 92.37 91.28 Cell survival rate (%) in test group 2 88.47 92.34 95.14 94.12 94.25 93.24 Cell survival rate (%) in test group 3 87.62 92.88 94.01 93.27 93.14 92.77 Average cell viability (%) 88.48 91.80 94.49 94.13 93.25 92.43

[0066] Table 1 shows that increasing the concentration of thioglycerol within the range of this regimen can increase cell survival.

[0067] Controlled test experiment 2

[0068] The effect of L-ascorbic acid addition on NK cell culture was determined using the controlled variable method. Specifically, the immune cell culture kit composition of control test 2 was the same as that in Example 1, while keeping the contents of other components constant. Different concentrations of L-ascorbic acid were set, and the culture method was the same as in Example 1. The total cell viability was then detected after 14 days of culture.

[0069] In the control test 2, three test groups were set up, with L-ascorbic acid added at concentrations of 0, 10 μg / mL, 25 μg / mL, 40 μg / mL, 55 μg / mL, 70 μg / mL, and 100 μg / mL. The total cell viability after 14 days of culture was recorded and the average value was calculated. The results are shown in Table 2.

[0070] Table 2. Cell viability at different concentrations of L-ascorbic acid

[0071]

[0072] As shown in Table 2, increasing the concentration of L-ascorbic acid within the range of this regimen can increase cell survival.

[0073] Controlled test experiment 3

[0074] The effect of dexamethasone addition on NK cell culture was determined using the controlled variable method. Specifically, the immune cell culture kit composition of control test 3 was the same as that in Example 1, with the contents of other components kept constant. Different concentrations of dexamethasone were set, and the culture method was the same as in Example 1. The total cell viability was then detected after 14 days of culture.

[0075] In the control test experiment 3, three test groups were set up, with dexamethasone added at concentrations of 0, 0.1 μg / mL, 0.2 μg / mL, 0.4 μg / mL, 0.8 μg / mL, 1 μg / mL, and 2 μg / mL. The total cell viability after 14 days of culture was recorded and the average value was calculated. The results are shown in Table 3.

[0076] Table 3. Cell viability at different concentrations of dexamethasone

[0077]

[0078] Table 3 shows that increasing the concentration of dexamethasone within the range specified in this study can increase cell survival.

[0079] Controlled test experiment 4

[0080] The effect of β-mercaptoethanol addition on NK cell culture was determined using the controlled variable method. Specifically, the immune cell culture kit composition of control test 4 was the same as that in Example 1, with the contents of other components kept constant. Different concentrations of β-mercaptoethanol were set, and the culture method was the same as in Example 1. The total cell viability was then detected after 14 years of culture.

[0081] In the control test experiment 4, three test groups were set up, with β-mercaptoethanol added at concentrations of 0, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, and 0.6 μg / mL. The total cell viability after 14 days of culture was recorded and the average value was calculated. The results are shown in Table 4.

[0082] Table 4. Cell viability at different concentrations of β-mercaptoethanol

[0083]

[0084] Table 4 shows that increasing the concentration of β-mercaptoethanol within the range of this regimen can increase cell survival.

[0085] In addition, comparative example 1 and comparative example 2 have been added to this invention. The details of comparative example 1 and comparative example 2 are as follows.

[0086] Comparative Example 1: The immune cell culture kit used consisted of components A, B, and C.

[0087] Component A is the basal culture medium, 2 × 1 L RPMI-1640;

[0088] Component B consists of recombinant antibodies, 1 mL, including 20 μg / mL anti-human CD56 antibody and 100 μg / mL anti-human NKG2D antibody;

[0089] Component C consists of cytokines, 1 mL, including: 200,000 U / mL IL-2, 20 μg / mL IL-15, 30 μg / mL IL-18, 8 μg / mL IL-21, and 50 μg / mL FLT3L.

[0090] The NK cell culture steps for Comparative Example 1 were the same as those for Example 1.

[0091] Comparative Example 2: The immune cell culture kit used consisted of components A, B, and C.

[0092] Component A is the basal culture medium, 2 × 1 L AIM-V (Gibco);

[0093] Component B consists of recombinant antibodies, 1 mL, including 20 μg / mL anti-human CD56 antibody and 100 μg / mL anti-human NKG2D antibody;

[0094] Component C consists of cytokines, 1 mL, including: 200,000 U / mL IL-2, 20 μg / mL IL-15, 30 μg / mL IL-18, 8 μg / mL IL-21, and 50 μg / mL FLT3L.

[0095] The NK cell culture steps for Comparative Example 2 were the same as those for Example 1.

[0096] Tests and trials

[0097] Examples of the embodiments, Comparative Example 1 and Comparative Example 2 were tested, including NK cell morphology, purity, total cell viability, expansion fold, and killing activity against non-small cell lung cancer A549 cells and leukemia cells K562 cells.

[0098] Specifically, peripheral blood mononuclear cells (PBMCs) were extracted from peripheral blood using the Ficoll method (density gradient centrifugation), and the PBMC viability was detected. NK cells were then cultured, and all cells were collected after 14 days of culture.

[0099] Cell morphology was recorded on days 3, 7, and 14 of culture. Cells collected at different culture times were AO / PI stained, and cell counts were performed using a cell counter to calculate cell viability and cell number, and cell expansion curves were plotted. Cell morphology images are shown below. Figure 1 As shown. NK cell expansion curve as shown. Figure 2 As shown.

[0100] The purity of NK cells in the collected cells was detected by flow cytometry. The antibodies used were: PerCP-Cy5.5-CD3 antibody, Brilliant Violet 421 anti-human CD56 antibody, and PE-CCR5 antibody. Among them, the NK cells were CD3-CD56+ cell population, and the NK cells with lung targeting potential were CD3-CD56+CCR5+ cell population.

[0101] Specifically, the flow cytometry detection plots for Example 1, Comparative Example 1, and Comparative Example 2 are as follows: Figure 3 As shown in Table 5, the purity results of NK cells are as follows.

[0102] Table 5. Purity of NK cells in each experimental group

[0103] Group Day0 Day 7 Day 14 Example 1 6.90％ 54.0％ 90.1％ Comparative Example 1 6.90％ 13.2％ 24.0％ Comparative Example 2 6.90％ 30.0％ 51.2％

[0104] from Figure 1 As can be seen from the comparison between Example 1 and Comparative Example 1, when RPMI-1640 was used as the basal culture medium for the kit alone (Comparative Example 1), it was insufficient to meet the normal proliferation requirements of cells, and no significant cell expansion was observed. However, the immune cell culture kit of Example 1, which incorporates thioglycerol, L-ascorbic acid, dexamethasone, and β-mercaptoethanol into RPMI-1640 as the basal culture medium (i.e., component A), effectively increases the survival and expansion rate of NK cells, thus meeting the growth and proliferation requirements of NK cells.

[0105] Combination Figure 1 , Figure 2 and Figure 3 By comparing Example 1 and Comparative Example 2, it can be seen that the basal medium in Example 1 is superior to AIM-V basal medium in culturing NK cells. It can achieve rapid proliferation of NK cells and the purity of NK cells in the obtained total cells is high. The purity of NK cells cultured in Example 1 can reach 90.1% on day 14, while it is only 51.2% in Comparative Example 2. At the same time, the culture medium cost of Example 1 is much lower than that of AIM-V basal medium used in Comparative Example 2, which is beneficial for saving production costs in large-scale production.

[0106] Furthermore, the NK cells with lung-targeting potential were identified as CD3-CD56+CCR5+ cell populations, which were detected using flow cytometry. The results are as follows: Figure 4 As shown.

[0107] Combined with appendix Figure 4 In Example 1, the positive rate of NK cells expressing the lung marker chemokine CCR5 was as high as 99.6%, suggesting that the NK cells cultured using this example have lung-targeting potential and are expected to provide an effective cell therapy in the treatment of lung cancer.

[0108] Lethality test:

[0109] The killing activity of collected NK cells against non-small cell lung cancer A549 cells and leukemia cells K562 cells was tested using an LDH release assay. The specific experimental steps are as follows:

[0110] Log-phase non-small cell lung cancer A549 cells or leukemia cells K562 cells, as well as NK cells obtained from each experimental group after 14 days of culture, were collected. A549 or K562 cells were used as target cells, and NK cells as effector cells. A549 cells in log-phase growth were digested with 0.25% trypsin (K562 cells did not require digestion) to prepare single-cell suspensions. Trypan blue staining and cell counting were performed, and the cell density was adjusted to 1×10⁻⁶. 5 cells / mL;

[0111] Add 50 μL of A549 or K562 cell suspension to each well of a 96-well plate. Add effector cells (NK cells) at different effector / target ratios (1:1, 5:1, 10:1), also in 50 μL per well.

[0112] Simultaneously, effector cells and target cells were set up with natural release wells, target cells maximum release wells and culture medium natural release wells, and volume calibration control wells. Each well had a volume of 100uL and three replicates were set up.

[0113] Incubate at 37°C and 5% CO2 for 12 hours. Add 10 μL of lysis buffer to each well of the target cell maximum release well 45 minutes before the end of the reaction.

[0114] After the reaction is complete, 50 μL of LDH enzyme reaction solution and 50 μL of supernatant are aspirated from each well and added to another new 96-well plate. The plate is incubated at room temperature in the dark for 30 min. Then, 50 μL of reaction stop solution is added, and the OD value is measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0115] The killing activity was calculated using the following formula: Kill rate (%) = (OD value of the assay tube - OD value of the target cell spontaneous release tube - OD value of the effector cell spontaneous release tube) / (OD value of the target cell maximum release tube - OD value of the target cell spontaneous release tube) × 100%. The calculation results are as follows: Figure 5-6 As shown.

[0116] Depend on Figure 5-6 It can be seen that the NK cells amplified in Example 1 of the present invention have high killing activity against both A549 cells and K562 cells.

[0117] In summary, the basal culture medium in Example 1 is superior to the AIM-V basal culture medium when culturing NK cells. Using the NK cells of the present invention is beneficial for saving costs in large-scale production and ensuring the production of NK cell products with better tumor killing activity.

[0118] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An immune cell culture kit, characterized in that, The immune cells are NK cells. The immune cell culture kit includes: basal culture medium, recombinant antibody, and cytokines. The basal culture medium includes RPMI-1640 medium, thioglycerol, L-ascorbic acid, dexamethasone, and β-mercaptoethanol. The cytokines include IL-2, IL-15, IL-18, IL-21, and FLT3L. In the basal culture medium, the concentration of thioglycerol is 1-10 μg / mL, the concentration of L-ascorbic acid is 10-100 μg / mL, the concentration of dexamethasone is 0.1-1 μM, and the concentration of β-mercaptoethanol is 0.1-0.6 μM. Among the cytokines, the concentration of IL-2 is 50,000-500,000 μg / mL. The recombinant antibody is a combination of CD56 antibody and NKG2D antibody, with the concentrations of IL-15 (10-100 μg / mL), IL-18 (25-75 μg / mL), IL-21 (1-10 μg / mL), and FLT3L (10-100 μg / mL).

2. The immune cell culture kit according to claim 1, characterized in that, The immune cell culture kit includes 3 μg / mL thioglycerol, 25 μg / mL L-ascorbic acid, 0.8 μM dexamethasone, 0.2 μM β-mercaptoethanol, 20 μg / mL anti-human CD56 antibody, 100 μg / mL anti-human NKG2D antibody, 200,000 U / mL IL-2, 20 μg / mL IL-15, 30 μg / mL IL-18, 8 μg / mL IL-21, and 50 μg / mL FLT3L.

3. A method for culturing NK cells, characterized in that, include: NK cells were cultured using the immune cell culture kit as described in any one of claims 1-2; The culture vessel was coated with a mixture of recombinant antibody and buffer. The first mixed culture medium was obtained by mixing the basal culture medium and cytokines; PBMCs were isolated from human peripheral blood. PBMCs were seeded into coated culture containers, and cultured in the first mixed medium for a predetermined time. During the culture process, the first mixed medium was supplemented to maintain a cell concentration of 2.0–3.5 × 10⁻⁶ cells / year. 6 cells / mL; After the first predetermined time of incubation, a second mixed culture medium is added to the culture container, wherein the second mixed culture medium consists of the basal culture medium and 500 U / mL IL-2, and the culture is carried out for a second predetermined time. After the second predetermined incubation period, the cultured NK cells are collected from the culture container.

4. The method for culturing NK cells according to claim 3, characterized in that, The first reservation period is 12 days, and the second reservation period is 2 days.

5. The method for culturing NK cells according to claim 4, characterized in that, The first predetermined time period of culture also includes adding autologous plasma to the culture container at the initial stage of culture, wherein the concentration of the autologous plasma is 1-10%, and the initial stage of culture is 0-9 days of the first predetermined time period.

6. The method for culturing NK cells according to claim 5, characterized in that, The first mixed culture medium was obtained by mixing the basal culture medium with cytokines at a ratio of 1000:

1.

7. The application of an immune cell culture kit as described in any one of claims 1-2 in the preparation of NK cells.

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