Preparation method and application of super memory NK cell

Abstract

The invention provides a preparation method and application of super memory NK cells. The method comprises the following steps: S1, separating from peripheral blood or umbilical cord blood to obtain mononuclear cells; s2, culturing the mononuclear cells by using an NK activation culture medium; s3, separating CD3-CD56 + cells from the cultured mononuclear cells, so as to obtain purified NK (Natural Killer) cells; and S4, stimulating the NK cells by using a memory factor to induce the NK cells into memory-like NK cells, and culturing the memory-like NK cells by using an amplification culture medium to prepare the super-memory NK cells. Before the NK sorting kit is used for sorting and purifying the cells, the NK activation culture medium containing IL-2, IL-15 and IL-18 is used for conducting induction culture on the mononuclear cells, the NK cells can be activated, the total cell amount is greatly increased, the number of the NK cells obtained through follow-up sorting is increased, the problems that the number of the NK cells purified through PBMC in the early stage is small, and the culture difficulty is large are solved, and the cell quality is improved. The total cell quantity of the finally cultured super memory NK cells is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a preparation method and application of super memory NK cells. Background Art

[0002] Natural killer cells (NK cells) are a unique type of innate lymphocytes. Unlike immune cells such as B cells or T cells, NK cells can exhibit rapid and effective cytolytic activity against viruses and tumor cells without prior antigen sensitization. NK cells mainly exert their killing effects through two pathways: regulating immune responses by producing cytokines and directly killing target cells. NK cells also participate in complex interactions with other key immune cells (including dendritic cells, T cells, and B cells), and initiate adaptive immunity through cytokines and chemokines. Therefore, NK cells play an important role in the body's immune process against tumors, early antiviral or intracellular parasitic infections.

[0003] Existing studies have shown that under the conditions of hapten-specific, virus-specific and cytokine stimulation and induction, after the initial activation of NK cells, the response will be enhanced when restimulated, proving that NK cells have the function of immune memory.

[0004] The existing method for preparing memory NK cells is usually to separate peripheral blood mononuclear cells (PBMC) from peripheral blood and then directly sort and purify CD3 - CD56 + Cells, that is, NK cells are directly separated and purified from peripheral blood, and then the NK cells are induced and stimulated by memory factors, and then the NK cells stimulated by memory factors are cultured and amplified to prepare memory NK cells. The proportion of NK cells in peripheral blood mononuclear cells is between 5% and 15%, with an average proportion of about 11%. Only (3 to 8)×10 NK cells can be separated and purified from 50mL of peripheral blood. 6 This method for preparing memory NK cells has the problem that the number of NK cells separated and purified in the early stage is small, and the success rate of NK cell culture is low, which leads to the failure of the final culture or the total amount of memory NK cells obtained is small. Summary of the invention

[0005] In order to solve the problems of the existing preparation method of memory NK cells, such as the small number of NK cells separated and purified in the early stage, the low success rate of NK cell culture, and the small total amount of super memory NK cells finally obtained, the present invention provides a preparation method and application of super memory NK cells.

[0006] According to a first aspect of the present invention, a method for preparing super memory NK cells is provided, comprising the following steps:

[0007] S1. Mononuclear cells are isolated from peripheral blood or umbilical cord blood;

[0008] S2. Inducing and culturing mononuclear cells using NK activation medium;

[0009] The NK activation medium comprises the following components: 200-1200 IU / mL interleukin-2 (IL-2), 10-60 ng / mL interleukin-15 (IL-15), 10-60 ng / mL interleukin-18 (IL-18), 5-15 wt% plasma, and a first basal medium;

[0010] S3. Isolation of CD3 from induced activated NK cells - CD56 + cells, and obtain purified NK cells;

[0011] S4. Stimulating NK cells with memory factors to induce NK cells into memory-like NK cells, and culturing the memory-like NK cells with expansion medium to obtain super memory NK cells.

[0012] Different from the above-mentioned existing preparation methods of memory NK cells, in the preparation method of super memory NK cells provided by the present invention, the mononuclear cells separated from peripheral blood or umbilical cord blood are first cultured with NK activation medium, and then separated and purified by NK sorting technology to obtain purified NK cells, and then the NK cells are induced and stimulated with memory factors, and then the memory-like NK cells obtained after stimulation with memory factors are cultured with expansion medium, so as to prepare super memory NK cells. First, before separation and purification by NK sorting technology, the mononuclear cells are first induced and cultured with NK activation medium containing IL-2, IL-15 and IL-18, so that super memory NK cells can be amplified. The invention relates to a novel method for preparing NK cells for use in a clinic or clinic, which can greatly increase the number of NK cells obtained through subsequent sorting, solve the problem that the number of NK cells obtained after purification of mononuclear cells is small in the early stage and the culture is difficult, greatly improves the culture success rate of super memory NK cells, and increases the total number of cells that finally obtain super memory NK cells. Secondly, compared with the use of other culture media, the preparation method provided by the present invention can significantly increase the proliferation rate of mononuclear cells by regulating the components and contents of the NK activation culture medium used for culturing mononuclear cells, thereby obtaining more mononuclear cells, and obtaining more purified NK cells in the subsequent sorting process, thereby increasing the total number and yield of super memory NK cells.

[0013] Preferably, in S1, the mononuclear cells include at least one of peripheral blood mononuclear cells (PBMC) separated from peripheral blood, human umbilical cord blood mononuclear cells (CBMC) separated from umbilical cord blood, cryopreserved peripheral blood mononuclear cells (PBMC), and cryopreserved umbilical cord blood mononuclear cells (CBMC).

[0014] Preferably, in S4, the expansion medium comprises the following components: 200-1200 IU / mL IL-2, and a second basal medium.

[0015] In the preparation method of super memory NK cells provided by the present invention, through the synergistic effect between the steps, the IL-2 in the expansion medium can promote the proliferation of mononuclear cells even at a lower concentration. At the same time, low concentration of IL-2 can improve the purity of the NK cells obtained subsequently and reduce the number of impurity cells.

[0016] If the concentration of IL-2 in the expansion medium is too high, although the cells grow quickly, the high concentration of IL-2 will stimulate (activate) T cell proliferation, thereby resulting in low purity of NK cells.

[0017] Preferably, the first basal culture medium includes at least one of KBM-581 culture medium, KBM-591 culture medium, and GT-T551 culture medium.

[0018] Preferably, the second basal culture medium is at least one of KBM-581 culture medium, KBM-591 culture medium, and GT-T551 culture medium.

[0019] Preferably, the first basal culture medium and the second culture medium are both GT-T551 culture medium.

[0020] Compared with other types of basal culture media, the use of KBM-591 medium as the basal culture medium does not require the addition of plasma or serum substitutes throughout the process and can achieve a culture effect similar to that of KBM581 medium. Moreover, based on the addition of plasma, the culture effect of KBM-591 medium is better than that of KBM-581 medium.

[0021] Preferably, S2 comprises the following operations: resuspending the mononuclear cells in NK activation medium and culturing the cells at a cell density of (1-5)×10 6 The cells were inoculated into NK activation medium and cultured at 5% CO2 and 37°C for 120-144h.

[0022] In the process of culturing mononuclear cells separated from peripheral blood or umbilical cord blood using NK activation medium, controlling the inoculated cell density and culture time within the above range is beneficial to increasing the growth rate of mononuclear cells, increasing the number of NK cells obtained through sorting, and further increasing the total number of super memory NK cells finally obtained.

[0023] If the inoculated cell density is too low, the cells will grow slowly; if the inoculated cell density is too high, the growth between cells will be inhibited, which will lead to a slowdown in the cell growth rate. Both of the above situations will lead to a significant reduction in the number of super memory NK cells produced.

[0024] If the culture time after mononuclear cells are inoculated into NK activation medium is too short, the number of NK cells obtained through culture will be too small, and the number of NK cells obtained through sorting will be too small, thereby increasing the difficulty of subsequent culture.

[0025] Preferably, in S2, the cell density used when the mononuclear cells are resuspended in the NK activation medium and inoculated into the NK activation medium is (2-3)×10 6 Pieces / mL.

[0026] The mononuclear cells were resuspended in NK activation medium and cultured at (2-3)×10 6 The induction effect can be further improved by inoculating cells at a density of 100 cells / mL into NK activation medium.

[0027] Preferably, in S2, during the process of resuspending the mononuclear cells in the NK activation medium and inoculating them into the NK activation medium for 120 to 144 hours, the following medium supplementation operation is also included: when the mononuclear cells are resuspended in the NK activation medium and inoculated into the NK activation medium for 72 hours, the NK activation medium and plasma are supplemented into the culture system to adjust the plasma concentration in the culture system to 5 to 15 wt % and the cell density to 1 to 1.5×10 6 After culturing at 5% CO2 and 37°C, NK activation medium and plasma were added to the culture system every 24 h to adjust the plasma concentration in the culture system to 5-15wt% and the cell density to 1-1.5×10 6 The cells were cultured at 37°C and 5% CO2.

[0028] Preferably, S3 includes the following operations: centrifuging the induced activated cultured mononuclear cells at 500g for 5 minutes, collecting the precipitate and resuspending the precipitate with PBS buffer, adding biotin-labeled NK cell antibody, mixing and incubating at 2-8°C for 5 minutes, adding buffer and NK cell sorting magnetic beads to the system, and sorting out the surface marker CD3 - CD56 + cells to obtain purified NK cells.

[0029] After induction culture, the proportion of NK cells in mononuclear cells increased greatly, and the total number of cells increased significantly. Then, non-NK cells were adsorbed through the sorting column, and the unadsorbed cells were collected. Their surface markers were CD3 - CD56 + NK cells can be obtained by final sorting from 50 mL of peripheral blood or umbilical cord blood. The number of NK cells obtained can reach (5-12)×10 7 This solves the problem that the number of NK cells after cell sorting is too small to be successfully cultured.

[0030] Preferably, in S4, the memory factor includes interleukin 15 (IL-15) and interleukin 18 (IL-18), and the memory factor further includes at least one of interleukin 12 (IL-12) and endotoxin.

[0031] The memory factors used in this scheme to stimulate NK cells include IL-15 and IL18. On this basis, IL-12 or endotoxin (LPS) is added. The memory factors containing the above components are used to stimulate and culture the isolated and purified NK cells, which can more efficiently induce NK cells into memory-like NK cells.

[0032] Preferably, S4 comprises the following steps: adding memory factors to activate the NK cells in culture, culturing for 20 to 30 hours under the conditions of 5% CO2, 5% O2, and 37°C, collecting the culture fluid, centrifuging at 500g for 5 minutes, collecting the memory-like NK cell precipitate, resuspending the memory-like NK cell precipitate with an expansion medium, and amplifying the NK cells at a cell density of (1 to 2)×10 6 / mL, 5wt% plasma was inoculated into the expansion medium, and 5wt% plasma was added to the system at the same time, and cultured at 5% CO2 and 37°C for 96 to 192h to obtain super memory NK cells.

[0033] In S4, by regulating the inoculation cell density and culture time, super memory NK cells with good cell status can be obtained. If the culture time is too long, the cell status will deteriorate, and the cell activity of the obtained super memory NK cells will decrease.

[0034] Preferably, after the memory factor is mixed with the expansion medium, the concentration of IL-12 in the system is 5 to 15 ng / mL, the concentration of IL-15 is 40 to 60 ng / mL, and the concentration of IL-18 is 40 to 60 ng / mL.

[0035] By adding memory factors to the expansion medium and controlling the concentrations of the components in the system and the culture conditions within the above range to culture the memory-like NK cells for 20 to 30 hours, the above culture environment and conditions can effectively simulate the hypoxic and inflammatory microenvironment in the body. Stimulating NK cells with memory factors is more conducive to stimulating NK cells into memory NK cells. Then, the stimulated NK cells are resuspended in the expansion medium containing IL-2 and the second basal culture medium and inoculated into fresh expansion medium. At the same time, 5wt% plasma is added and the inoculated cell density is regulated. The synergistic effect of the above steps further increases the total cell amount of super memory NK cells.

[0036] Preferably, in S4, during the process of resuspending the collected memory-like NK cell precipitate and inoculating it into the NK expansion medium for 96 to 192 hours, the following fluid replenishment operation is also included: fresh expansion medium is added to the culture system every 48 hours to adjust the cell density in the culture system to (0.5 to 1.5)×10 6 / mL, cultured at 5% CO2, 37°C to obtain super memory NK cells.

[0037] This protocol utilizes expansion and activation culture medium to culture the resuspended memory-like NK cell pellet for 96 to 192 hours, and also includes replenishing the culture medium and regulating the cell density in the culture system to continue culturing the memory-like NK cells, which can further increase the total number of super memory NK cells ultimately obtained.

[0038] Preferably, the plasma comprises at least one of fresh umbilical cord blood, frozen umbilical cord blood, fresh peripheral blood, and frozen peripheral blood.

[0039] The existing technology usually directly separates and purifies NK cells from peripheral blood, then uses cytokines to induce and stimulate NK cells, and then cultivates and amplifies the cytokine-stimulated NK cells to prepare memory NK cells. In this case, due to the small number of NK cells separated and purified in the early stage, only fresh peripheral blood or mononuclear cells separated from umbilical cord blood can be used for successful cultivation. Cryopreserved peripheral blood PBMC or umbilical cord blood CBMC are more difficult to culture successfully due to their low activity.

[0040] The method for preparing super memory NK cells provided by the present invention is to culture the PBMC separated from peripheral blood with NK activation medium, then sort and purify to obtain NK cells, then use memory factors to induce and stimulate the NK cells, and then use expansion medium to culture the NK cells stimulated by cytokines, so as to prepare super memory NK cells. In this method, even if frozen PBMC or CBMC are used, the above-mentioned difficulty in culturing NK cells will not occur, resulting in a small number of super memory NK cells. That is, even if frozen PBMC and CBMC are used in the process of preparing super memory NK cells, a sufficient number of memory NK cells can still be obtained. In addition, the amount of PBMC and CBMC required by the method provided by the present invention only needs to be greater than 3×10 7 A large number of super memory NK cells can be successfully cultured.

[0041] According to a second aspect of the present invention, a super memory NK cell is provided. The super memory NK cell is prepared by the above-mentioned super memory NK cell preparation method.

[0042] According to the third aspect of the present invention, there is provided the use of the above-mentioned super memory NK cells in the preparation of anti-tumor drugs.

[0043] Preferably, the tumor includes at least one of non-small cell lung cancer, chronic myeloid leukemia, colorectal cancer, lymphoma, multiple myeloma, gastric cancer, liver cancer, breast cancer, and prostate cancer.

[0044] The super memory NK cells prepared by the method provided by the present invention are applied to the preparation of anti-tumor drugs, and the drugs have good anti-tumor effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a graph showing the results of detecting lymphocyte subsets in common NK cells using flow cytometry.

[0046] Figure 2 This is a graph showing the results of detecting lymphocyte subsets in the super memory NK cells prepared in Example 1 using flow cytometry.

[0047] Figure 3 This is a graph showing the results of detecting the CD107a expression rate on the cell membrane before and after stimulation of ordinary NK cells using flow cytometry.

[0048] Figure 4 This is a graph showing the results of detecting the CD107a expression rate on the cell membrane before and after stimulation of the super memory NK cells prepared in Example 1 using flow cytometry. DETAILED DESCRIPTION

[0049] The following is a further clear and complete description of the technical features in the technical solution provided by the present invention in conjunction with the specific implementation methods. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0050] Example 1

[0051] A method for preparing super memory NK cells, characterized in that it comprises the following steps:

[0052] S1. Collect 40 mL of peripheral blood using a green vacuum blood collection tube containing sodium heparin, gently invert and mix to obtain anticoagulated blood; add 15 mL of Ficoll lymphocyte separation solution to a 50 mL sterile lymphocyte separation tube, centrifuge at 300 g for 5 min for later use; take the anticoagulated blood and add it to the lymphocyte separation tube, centrifuge horizontally at 800 g for 15 min, set the ascending speed to 5 and the descending speed to 4, and after centrifugation, use a pipette to aspirate the upper autologous plasma, inactivate it at 56°C for 30 min, place it at 4°C for 10 min, and then centrifuge it at 1000 g for 10 min, aspirate the upper autologous plasma into a new sterile 50 mL centrifuge tube for later use; aspirate the mononuclear cells in the remaining cloud layer in the separation tube into a new 50 mL centrifuge tube, and wash it twice with PBS buffer. The specific operation of PBS washing is: add PBS buffer to the centrifuge tube to resuspend, add PBS buffer to 40 mL and mix, centrifuge at 500 g for 5 min, discard the supernatant, and obtain peripheral blood mononuclear cells.

[0053] S2. Resuspend the peripheral blood mononuclear cells in activation medium and set the cell density at 2×10 6 The cells were inoculated into activation medium and cultured at 5% w CO2 and 37°C for 72 h. The activation medium was added to the culture system and the cell density was adjusted to 2×10 6 / mL, and continue to culture at 5% CO2 and 37℃ for 48h;

[0054] NK activation medium includes the following components: 1000 IU / mL IL-2, 50 ng / mL IL-15, 50 ng / mL IL-18, 10 wt% inactivated plasma, KBM-581 medium;

[0055] S3. Centrifuge the induced activated cultured mononuclear cells at 500g for 5 minutes, collect the precipitate and resuspend it with buffer, add biotin-labeled NK cell antibody, mix and incubate at 2-8℃ for 5 minutes, then add buffer and NK cell sorting magnetic bead mixture, mix and incubate at 2-8℃ for 10 minutes, adsorb non-NK cells with sorting column, collect the unadsorbed cells, whose surface marker is CD3 - CD56 + , obtain purified NK cells;

[0056] S4. The memory factor was mixed with the expansion medium and used to culture the NK cells at 5% CO2, 5% O2, and 37°C for 25 h. The culture medium was collected and centrifuged to collect the cell pellet. The cell pellet was resuspended in the expansion medium and the cell density was set at 2.0×10 6 1×10 / mL were inoculated into the expansion medium, and 5 wt% plasma was added to the system. The culture was incubated at 5% CO2 and 37°C. Fresh expansion medium was added to the culture system every 48 h to adjust the cell density in the culture system to 1×10 6 / mL, cultured at 5% CO2, 37℃ for 168h to obtain super memory NK cells;

[0057] Memory factors include IL-12, IL-15, and IL-18;

[0058] The expansion medium included the following components: 1000 IU / mL IL-2, KBM-581 medium;

[0059] After the memory factor was mixed with the expansion medium, the concentration of IL-12 in the system was 10 ng / mL, the concentration of IL-15 was 50 ng / mL, and the concentration of IL-18 was 50 ng / mL.

[0060] Example 2

[0061] This embodiment provides a method for preparing super memory NK cells. Compared with Example 1, the difference in structure is that in the NK activation medium and expansion medium used in the whole culture process, an equal amount of KBM-591 medium is used to replace KBM-581 medium, and no inactivated plasma is added.

[0062] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0063] Example 3

[0064] This embodiment provides a method for preparing super memory NK cells. Compared with Example 1, the difference in structure is that an equal amount of GT-T551 medium is used to replace KBM-581 medium in the NK activation medium and expansion medium used in the whole culture process.

[0065] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0066] Example 4

[0067] This embodiment provides a method for preparing super memory NK cells. Compared with the embodiment 1, the difference in structure is that endotoxin (LPS) is used to replace IL-12 in the memory factor used in step S4. After adding endotoxin, the endotoxin concentration in the culture medium is 5000 ng / mL.

[0068] The endotoxin (LPS) used was purchased from ThermoFisher Company, with the catalog number 00-4976-03, which was a 500X (500 times working concentration) LPS aqueous solution with a working concentration of 5000 ng / mL;

[0069] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0070] Example 5

[0071] This embodiment provides a method for preparing super memory NK cells. Compared with Embodiment 1, the difference in structure is that in step S1, the peripheral blood mononuclear cells used in step S1 are peripheral blood mononuclear cells that have been frozen and resuscitated instead of peripheral blood mononuclear cells separated from fresh peripheral blood.

[0072] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0073] Example 6

[0074] This embodiment provides a method for preparing super memory NK cells. Compared with Example 1, the difference in structure is: in step S4, the culture system does not control O2 when NK cells are stimulated with a combination of factors, that is, the memory factors and the expansion culture medium are mixed and used to culture the NK cells under 5% CO2 and 37°C for 25 hours, and then the culture fluid is collected for subsequent experiments.

[0075] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0076] Example 7

[0077] This comparative example provides a method for preparing super memory NK cells. Compared with Example 1, the difference in structure is: in step S4, after the memory factor is mixed with the expansion medium, the concentration of IL-12 in the system is 3 ng / mL, the concentration of IL-15 is 62 ng / mL, and the concentration of IL-18 is 35 ng / mL.

[0078] Except for the above differences, the materials, formula ratios and preparation operations used in this comparative example are strictly consistent with those in Example 1.

[0079] Example 8

[0080] This comparative example provides a method for preparing super memory NK cells. Compared with Example 1, the difference in structure is: in step S4, after the memory factor is mixed with the expansion medium, the concentration of IL-12 in the system is 18 ng / mL, the concentration of IL-15 is 35 ng / mL, and the concentration of IL-18 is 62 ng / mL.

[0081] Except for the above differences, the materials, formula ratios and preparation operations used in this comparative example are strictly consistent with those in Example 1.

[0082] Comparative Example 1

[0083] A method for preparing super memory NK cells comprises the following steps:

[0084] (1) Coating culture: coating the culture flask with coating solution, and keeping the culture flask at 4°C for 24 hours during the coating culture;

[0085] (2) collecting peripheral blood, separating and collecting human peripheral blood mononuclear cells using a lymphocyte separation solution, and washing to obtain human peripheral blood mononuclear cells;

[0086] (3) Purifying NK cells from PBMC cells using an NK cell isolation kit;

[0087] (4) Inoculation and culture: the purified NK cells are mixed with activation medium and inoculated into the culture bottle after the coating culture, and then plasma is added and placed in an incubator for culture. The parameters of the incubator are set as follows: temperature 37°C, 5% CO2;

[0088] (5) After 72 h of culture, activation medium was added to the culture flask, and then the culture was continued in the incubator;

[0089] (6) After 96 hours of culture, add activation medium to the culture flask, add memory factor at 120 hours, and then continue to culture in the incubator;

[0090] (7) After culturing for 16 h, centrifuge and discard the supernatant. Resuspend the obtained cells in expansion medium and culture them in a new culture bottle. Then add plasma and continue to culture in the incubator.

[0091] (8) replenishing the expansion medium and then continuing to culture in the incubator;

[0092] (9) Transferring the cell suspension in the culture flask into a culture bag, supplementing the expansion medium, and then continuing to culture in the incubator;

[0093] (10) replenishing the expansion medium and then continuing to culture in the incubator;

[0094] (11) Use a cell counter to measure the cell concentration in the culture medium and collect the cells when the measurement results meet the standard.

[0095] The coating solution included the following components: 50 ng CD3 antibody, 500 ng CD16 antibody, 10 mL PBS;

[0096] The activation medium included the following components: 1000 IU / mL IL-2, 50 ng / mL IL-15, 50 ng / mL IL-18, 10 wt% plasma, KBM-581;

[0097] The expansion medium included the following components: 1000 IU / mL IL-2, KBM-581 medium;

[0098] Memory factors include the following components: IL-12, IL-15, and IL-18;

[0099] After the memory factor was mixed with the expansion medium, the concentration of IL-12 in the system was 10 ng / mL, the concentration of IL-15 was 50 ng / mL, and the concentration of IL-18 was 50 ng / mL.

[0100] Comparative Example 2

[0101] A method for preparing super memory NK cells comprises the following steps:

[0102] (1) collecting peripheral blood, separating and collecting human peripheral blood mononuclear cells using a lymphocyte separation solution, and washing to obtain human peripheral blood mononuclear cells;

[0103] (2) Purifying NK cells from PBMC cells using an NK cell isolation kit;

[0104] (3) Inoculation and culture: the purified NK cells were mixed with activation medium and inoculated into a culture bottle, and then placed in an incubator for culture. The parameters of the incubator were set as follows: temperature 37° C., 5% CO2;

[0105] (4) After 72 h of culture, activation medium was added to the culture flask, and then the culture was continued in the incubator;

[0106] (5) After 96 hours of culture, add activation medium to the culture flask, add memory factor at 120 hours, and then continue to culture in the incubator;

[0107] (6) After culturing for 16 hours, the memory factor and activation medium are washed away, and then the cells are separated and purified using a centrifuge. The purified cells are resuspended in expansion medium and cultured in a new culture bottle, and then plasma is added and cultured in an incubator;

[0108] (7) replenishing the expansion medium and then continuing to culture in the incubator;

[0109] (8) Transferring the cell suspension in the culture flask into a culture bag, supplementing the expansion medium, and then continuing to culture in the incubator;

[0110] (9) replenishing the expansion medium and then continuing to culture in the incubator;

[0111] (10) Using a cell counter to measure the cell concentration of the culture medium, and collecting the cells after the measurement results meet the standard;

[0112] The activation medium included the following components: 1000 IU / mL IL-2, 50 ng / mL IL-15, 50 ng / mL IL-18, 10 wt% plasma, KBM-581;

[0113] The expansion medium included the following components: 1000 IU / mL IL-2, KBM-581 medium;

[0114] Memory factors include the following components: IL-12, IL-15, and IL-18;

[0115] After the memory factor was mixed with the expansion medium, the concentration of IL-12 in the system was 10 ng / mL, the concentration of IL-15 was 50 ng / mL, and the concentration of IL-18 was 50 ng / mL.

[0116] Comparative Example 3

[0117] (1) Coating culture: coating the culture flask with coating solution, and the culture flask is kept at 4 degrees Celsius for 24 hours during the coating culture;

[0118] (2) collecting peripheral blood, separating and collecting human peripheral blood mononuclear cells using a lymphocyte separation solution, and washing to obtain human peripheral blood mononuclear cells;

[0119] (3) Inoculation and culture: the purified NK cells are mixed with expansion medium and inoculated into the culture bottle after the coating culture, and then plasma is added and placed in an incubator for culture. The parameters of the incubator are set as follows: temperature 37°C, 5% CO2;

[0120] (4) adding the expansion medium and plasma to the culture bottle after culturing for 72 hours, and then continuing to culture in the incubator;

[0121] (5) After 96 hours of culture, add activation medium to the culture flask, add memory factor at 120 hours, and then continue to culture in the incubator;

[0122] (6) After culturing for 16 h, centrifuge and discard the supernatant. Resuspend the obtained cells in expansion medium and culture them in a new culture bottle. Then add plasma and continue to culture in the incubator.

[0123] (7) replenishing the expansion medium and adding plasma, and then continuing to culture in the incubator;

[0124] (8) Transferring the cell suspension in the culture flask into a culture bag, supplementing the expansion medium, and then continuing to culture in the incubator;

[0125] (9) replenishing the expansion medium and then continuing to culture in the incubator;

[0126] (10) Using a cell counter to measure the cell concentration of the culture medium, and collecting the cells after the measurement results meet the standard;

[0127] The coating solution included the following components: 50 ng CD3 antibody, 500 ng CD16 antibody, 10 ml PBS;

[0128] The expansion medium included the following components: 1000 IU / mL IL-2, KBM-581 medium;

[0129] Memory factors include the following components: IL-12, IL-15, and IL-18;

[0130] After the memory factor was mixed with the expansion medium, the concentration of IL-12 in the system was 10 ng / mL, the concentration of IL-15 was 50 ng / mL, and the concentration of IL-18 was 50 ng / mL.

[0131] Comparative Example 4

[0132] A method for preparing super memory NK cells comprises the following steps:

[0133] (1) collecting peripheral blood, separating and collecting human peripheral blood mononuclear cells using a lymphocyte separation solution, and washing to obtain human peripheral blood mononuclear cells;

[0134] (2) Inoculation and culture: the purified NK cells are mixed with expansion medium and inoculated into the culture bottle after the coating culture, and then plasma is added and placed in an incubator for culture. The parameters of the incubator are set as follows: temperature 37°C, 5% CO2;

[0135] (3) After 72 h of culture, add expansion medium to the culture flask and continue culturing in the incubator;

[0136] (4) After 96 hours of culture, add expansion medium to the culture flask, add memory factor at 120 hours, and then continue to culture in the incubator;

[0137] (5) After culturing for 16 h, centrifuge and discard the supernatant. Resuspend the obtained cells in expansion medium and culture them in a new culture bottle. Then add plasma and continue to culture in the incubator.

[0138] (6) replenishing expansion medium and adding plasma, and then continuing to culture in the incubator;

[0139] (7) Transferring the cell suspension in the culture flask into a culture bag, supplementing the expansion medium, and then continuing to culture in the incubator;

[0140] (8) replenishing the expansion medium and then continuing to culture in the incubator;

[0141] (9) Using a cell counter to measure the cell concentration of the culture medium, and collecting the cells after the measurement results meet the standard;

[0142] The coating solution included the following components: 50 ng CD3 antibody, 500 ng CD16 antibody;

[0143] The expansion medium included the following components: 1000 IU / mL IL-2, KBM-581 medium;

[0144] Memory factors include the following components: IL-12, IL-15, and IL-18;

[0145] After the memory factor was mixed with the expansion medium, the concentration of IL-12 in the system was 10 ng / mL, the concentration of IL-15 was 50 ng / mL, and the concentration of IL-18 was 50 ng / mL.

[0146] Test Case

[0147] 1. Participants

[0148] In this test example, the super memory NK cells prepared in Examples 1 to 8 and Comparative Examples 1 to 4 were used as test objects to conduct relevant performance tests.

[0149] 2. Test content

[0150] (1) Cell quantity and purity (lymphocyte subset detection)

[0151] After super memory NK cells are collected, they are counted and samples are taken to detect lymphocyte subsets, including CD3 - CD56 + Cells and CD3 - CD56 + CD16 + The ratio of cells is used to indicate the purity of super memory NK cells.

[0152] (2) Cytokines

[0153] TNF-α (tumor necrosis factor-α) is cytotoxic to various tumor cells; INF-γ (interferon-γ) can directly inhibit viral replication, induce virus-infected cells to express viral antigens, increase the immune system's ability to recognize and kill infected cells, and overcome tumor progression by inhibiting angiogenesis in tumor tissues, inducing apoptosis of regulatory T cells, and / or stimulating the activity of M1 pro-inflammatory macrophages.

[0154] The expression levels of cytokines TNF-α and INF-γ in super memory NK cells were detected.

[0155] The cytokines TNF-α and INF-γ characterize the toxicity of super memory NK cells. The higher the concentration of TNF-α and INF-γ, the stronger the cytotoxicity of super memory NK cells and the stronger the killing effect on tumor cells.

[0156] (3) CD107a expression rate

[0157] As a sensitive marker of NK cell degranulation, CD107a molecule is directly related to cytotoxic activity and can reflect the level of cytotoxic cell killing activity.

[0158] Super memory NK cells were cultured with RPMI 1640 medium, stimulated by the addition of PMA (phorbol ester, final concentration 2.5 μg / mL) and ionomycin (final concentration 0.5 μg / mL), and incubated for 6 h. The expression rate of CD107a on the cell membrane before and after stimulation was detected by flow cytometry, where △CD107a (%) = CD107a positive cell ratio after stimulation - CD107a positive cell ratio before stimulation.

[0159] In the above tests, ordinary NK cells were used as controls, and the preparation method thereof was as follows:

[0160] A. Coating culture: Coating the culture flask with coating solution. During the coating culture, the culture flask is kept at 4°C for 24 hours.

[0161] B. collecting peripheral blood, separating and collecting human peripheral blood mononuclear cells using lymphocyte separation solution, and washing to obtain human peripheral blood mononuclear cells;

[0162] C. Peripheral blood mononuclear cells were cultured with NK activation medium for 6 days;

[0163] D. Common NK cells were obtained by culturing peripheral blood mononuclear cells with NK expansion medium for 8 days.

[0164] The coating solution included the following components: 50 ng CD3 antibody, 500 ng CD16 antibody, 10 mL PBS buffer;

[0165] The activation medium includes the following components: 1000 IU / mL IL-2, 50 ng / mL IL-15, 50 ng / mL IL-18, 10 wt% inactivated plasma, KBM-581 medium;

[0166] The expansion medium includes the following components: 1000 IU / mL IL-2, KBM-581 medium.

[0167] 3. Experimental results

[0168] Table 1 The cell number and purity of super memory NK cells finally obtained in each embodiment and comparative example

[0169]

[0170] The cell number and purity of the super memory NK cells finally obtained by the methods provided in Examples 1 to 8 and Comparative Examples 1 to 4 are shown in Table 1. In addition, the results of detecting the lymphocyte subsets of ordinary NK cells by flow cytometry are shown in Table 1. Figure 1 As shown, the results of detecting the lymphocyte subsets of the super memory NK cells prepared in Example 1 by flow cytometry are as follows Figure 2 As shown.

[0171] By comparing the data of Comparative Examples 1 to 4 and Examples 1 to 8, it can be seen that compared with Comparative Examples 1 to 4, the number of super memory NK cells obtained by the method provided in Examples 1 to 8 is greater than that in Comparative Examples 1 to 4, and CD3 - CD56 + Cells and CD3 - CD56 + CD16 +The proportion of cells is higher, indicating that the purity of the obtained super memory NK cells is higher. The reason for the above results is that in the preparation method of super memory NK cells provided by the present invention, the mononuclear cells separated from peripheral blood or umbilical cord blood are first cultured using NK activation medium, and then the purified NK cells are separated and purified using NK sorting technology, and then the NK cells are induced and stimulated using memory factors, and then the memory-like NK cells obtained after stimulation with memory factors are cultured using amplification medium, thereby preparing super memory NK cells. First, before separation and purification using NK sorting technology, the mononuclear cells are first induced and cultured using NK activation medium containing IL-2, IL-15 and IL-18, which can amplify a large number of NK cells, greatly increasing the number of NK cells obtained after subsequent sorting, solving the problem that the number of NK cells obtained after purification of mononuclear cells is small in the early stage and the difficulty of culture is high, greatly improving the success rate of super memory NK cell culture, and increasing the total amount of cells that finally obtain super memory NK cells. Second, compared with Compared with the use of other culture media, the preparation method provided by the present invention can significantly increase the proliferation rate of mononuclear cells by regulating the components and contents of the NK activation culture medium used to culture mononuclear cells, thereby obtaining more mononuclear cells, and obtaining more purified NK cells in the subsequent sorting process, thereby increasing the total number of cells and the yield of super memory NK cells; while Comparative Example 1 mainly prepares super memory NK cells through the steps of coating, purification, activation, memory stimulation and amplification, and Comparative Example 2 prepares super memory NK cells through the steps of purification, activation, memory stimulation and amplification, without coating, and Comparative Example 3 prepares super memory NK cells through the steps of coating, purification, amplification culture and memory stimulation, and activation culture is not involved in the amplification culture process, and Comparative Example 4 prepares super memory NK cells through the steps of purification, amplification and memory stimulation, and the amplification culture process does not involve activation culture, and Comparative Example 4 prepares super memory NK cells through the steps of purification, amplification and memory stimulation, and the amplification is not coated and activated, and the super memory NNK cells provided in Comparative Examples 1 to 4 may result in fewer cells being obtained in the end

[0172] Compared with Example 1, the IL-2, IL-15 and IL-18 in the system after the memory factor used in the preparation step S4 of the super memory NK cells in Examples 7 to 8 is mixed with the expansion medium do not meet the requirements of "IL-12 concentration is 5 to 15 ng / mL, IL-15 concentration is 40 to 60 ng / mL, and IL-18 concentration is 40 to 60 ng / mL". The test results show that the number of super memory NK cells obtained by the methods provided in Examples 7 to 8 is lower than that in Example 1, and CD3 - CD56 + Cells and CD3 - CD56 + CD16 + The proportion of cells is also lower than that in Example 1.

[0173] By comparing the data of Example 1, Example 2 and Example 3, it can be seen that the type of basal culture medium in the NK activation medium and the expansion medium used in the preparation process of super memory NK cells will affect the cell number and purity of the super memory NK cells finally obtained.

[0174] By comparing the data of Example 1 and Example 4, it can be seen that compared with Example 1, the memory factors used in the preparation process of super memory NK cells in Example 4 include LPS, IL-15 and IL-18, and the cell number and purity of the super memory NK cells finally obtained are lower than those in Example 1.

[0175] By comparing the data of Example 1 and Example 5, it can be seen that in the process of preparing super memory NK cells using the method provided by the present invention, even if peripheral blood mononuclear cells that have been revived after freezing are used, a sufficient number of super memory NK cells can still be obtained.

[0176] By comparing the data of Example 1 and Example 6, it can be seen that by adding memory factors to the expansion medium and controlling the concentrations of the components in the system and the culture conditions within the above range to culture the memory-like NK cells for 20 to 30 hours, the above culture environment and conditions can effectively simulate the hypoxia and inflammatory microenvironment in vivo, and stimulate NK cells with memory factors, which is more conducive to stimulating NK cells into memory NK cells. Then, the stimulated NK cells are resuspended in the expansion medium containing IL-2 and the second basal medium and inoculated into fresh expansion medium. At the same time, the inoculated cell density is regulated. Through the synergistic effect of the above steps, the total cell amount and purity of super memory NK cells can be further improved.

[0177] Table 2 Cytokine detection results

[0178]

[0179] The cytokine expression levels in the super memory NK cells finally obtained by the methods provided in Examples 1 to 8 and Comparative Examples 1 to 4 were detected, and the results are shown in Table 2.

[0180] Through the data of comparative examples 1 to 4 and embodiments 1 to 8, it can be seen that compared with comparative examples 1 to 4, the concentrations of cytokines TNF-α and INF-γ in the super memory NK cells obtained by the method provided in embodiments 1 to 8 are higher than those in comparative examples 1 to 4, indicating that the killing effect of the super memory NK cells obtained by the method provided in embodiments 1 to 8 is better than that of comparative examples 1 to 4.

[0181] By comparing the data of Example 1 and Example 6, it can be seen that the concentrations of cytokines TNF-α and INF-γ in the super memory NK cells obtained by the method provided in Example 6 are slightly higher than those in Example 1. This is mainly because: by adding memory factors to the amplification culture medium and controlling the concentrations of each component in the system and the culture conditions within the above range to culture the memory-like NK cells for 20 to 30 hours, although the human body environment can be simulated so that the microenvironment during memory factor stimulation is close to the tumor microenvironment, the resulting cells can remember the tumor microenvironment to enhance the cell's homing ability and survival ability in the tumor microenvironment, but in such an environment, the growth of memory NK cells will be inhibited, the cell amount will be reduced, and the cytokine secretion will also be reduced. Therefore, if O2 is not controlled, that is, if hypoxia treatment is not performed, the secreted cytokines will increase. When evaluating the cell quality and effect of super memory NK cells, we not only look at the number of cells and cytokines, but also the survival time of super memory NK cells in the tumor microenvironment and the response speed when encountering antigens. If O2 is not controlled, it will be difficult to simulate the hypoxia and inflammatory microenvironment in the body. The survival time of the super memory NK cells in the tumor microenvironment will be reduced and the response speed when encountering antigens will be slower.

[0182] Table 3 CD107a expression rate detection results

[0183]

[0184]

[0185] The results of detecting the CD107a expression rate of the super memory NK cells prepared in Examples 1 to 8 and Comparative Examples 1 to 4 before and after stimulation by flow cytometry are shown in Table 3. The results of detecting the CD107a expression rate on the cell membrane of ordinary NK cells before and after stimulation by flow cytometry are shown in Table 3. Figure 3 As shown, the results of detecting the CD107a expression rate on the cell membrane of the super memory NK cells prepared in Example 1 before and after stimulation by flow cytometry are as follows: Figure 4 shown.

[0186] Compared with Comparative Examples 1 to 4, the CD107a expression rate of the super memory NK cells prepared by the method provided in Examples 1 to 8 after stimulation is higher than that of Comparative Examples 1 to 4, indicating that the killing activity of the super memory NK cells prepared by the method provided in Examples 1 to 8 is higher than that of Comparative Examples 1 to 4.

[0187] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the technical solutions of the present invention can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. A method for preparing super memory NK cells, characterized in that: The following steps are involved: S1. Mononuclear cells are isolated from peripheral blood or umbilical cord blood; S2. inducing and culturing the mononuclear cells using NK activation medium; The NK activation medium comprises the following components: 200-1200 IU / mL IL-2, 10-60 ng / mL IL-15, 10-60 ng / mL IL-18, 5-15 wt% plasma, and a first basal medium; S3. Isolation of CD3 from the mononuclear cells induced and cultured - CD56 + cells, and obtain purified NK cells; S4. Stimulating the NK cells with memory factors to induce the NK cells into memory-like NK cells, and culturing the memory-like NK cells with expansion medium to obtain the super memory NK cells.

2. The method for preparing super memory NK cells according to claim 1, characterized in that: In S4, the expansion medium includes the following components: 200-1200 IU / mL IL-2 and a second basal medium.

3. The method for preparing super memory NK cells according to claim 2, characterized in that: The first basal culture medium includes at least one of KBM-581 culture medium, KBM-591 culture medium, and GT-T551 culture medium, and / or the second basal culture medium includes at least one of KBM-581 culture medium, KBM-591 culture medium, and GT-T551 culture medium.

4. The method for preparing super memory NK cells according to claim 1, characterized in that: The S2 comprises the following operations: resuspending the mononuclear cells in NK activation medium and culturing the cells at a cell density of (1-5)×10 6 The cells / mL were inoculated into the NK activation medium and cultured at 5% CO2 and 37°C for 120 to 144 h.

5. The method for preparing super memory NK cells according to claim 1, characterized in that: The S3 comprises the following operations: centrifuging the induced cultured mononuclear cells at 500 g for 5 min, collecting the precipitate and resuspending the precipitate with PBS buffer, adding biotin-labeled NK cell antibody or CD3 magnetic beads, mixing and incubating at 2-8° C. for 5 min, adding PBS buffer and NK cell sorting magnetic beads to the system, mixing and incubating at 2-8° C. for 10 min, and sorting out the cells with surface markers of CD3 using a sorting column. - CD56 + cells to obtain purified NK cells.

6. The method for preparing super memory NK cells according to claim 1, characterized in that: In S4, the memory factors include IL-15 and IL-18, and the memory factors also include at least one of IL-12 and endotoxin.

7. The method for preparing super memory NK cells according to claim 6, characterized in that: The S4 comprises the following steps: mixing the memory factor with the expansion medium and using it to culture the NK cells under the conditions of 5% CO2, 5% O2, and 37°C for 20 to 30 hours, collecting the culture fluid, centrifuging, collecting the memory-like NK cell precipitate, resuspending the memory-like NK cell precipitate with the expansion medium, and performing a 30-well plate filtration at a cell density of (1 to 2)×10 6 The super memory NK cells were obtained by inoculating cells / mL into the amplification medium, adding 5wt% plasma to the system, and culturing them at 5% CO2 and 37°C for 96 to 192 hours.

8. The method for preparing super memory NK cells according to claim 7, characterized in that: In S4, during the process of resuspending the collected memory-like NK cell precipitate and inoculating it into the expansion medium for 96 to 192 hours, the following fluid replenishment operation is also included: fresh expansion medium is added to the culture system every 48 hours to adjust the cell density in the culture system to (0.5 to 1.5)×10 6 / mL, and cultured at 5% CO2 and 37°C to obtain the super memory NK cells.

9. A super memory NK cell, characterized in that: The super memory NK cells are prepared by the super memory NK cell preparation method according to any one of claims 1 to 8.

10. The use of super memory NK cells in the preparation of anti-tumor drugs as claimed in claim 9, characterized in that: The tumor includes at least one of non-small cell lung cancer, chronic myeloid leukemia, colorectal cancer, lymphoma, multiple myeloma, gastric cancer, liver cancer, breast cancer, and prostate cancer.

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