CN116574679A - Preparation method and application of specific NK cells - Google Patents
Preparation method and application of specific NK cells Download PDFInfo
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Abstract
The invention discloses a preparation method and application of specific NK cells. The specificity of NK cells corresponds to the antigen loaded during preparation. Firstly, selecting and preparing corresponding antigens according to the specificity of the required NK cells; NK cells are prepared, activated by Stimulating Activation Complexes (SAC) such as IL-2, IL-12, IL-15, IL-18, IL-21 and the like, and continuously cultured under the action of amplification differentiation complexes (EDC) such as IL-2, IL-15, IL-21 and the like after antigen loading, so that NK cells are promoted to be amplified and differentiated, and the specificity aiming at the used antigen is obtained. The SNK cells prepared by the method aiming at the myeloid leukemia and the B cell lymphoma obviously enhance the activity of NK specificity to kill corresponding cancer cells.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method and application of specific NK cells.
Background
NK cells are increasingly used in basic research and clinical application in the wide fields of anti-cancer, anti-infection, anti-aging and the like, the research content is more and more, and the application effect is better. Such widespread use is also well reflected in the current understanding of NK cells, which are thought to be a component of the innate immune system that has a broad spectrum of effects; little research into the adaptive immunity of NK cells is seen.
Cytotoxic T Lymphocytes (CTLs) have a killing function similar to NK cells in the acquired immune system, but unlike NK cells, CTLs have antigen specificity. The antigen needs to be loaded in the process of preparing CTL, and the specificity of CTL depends on the difference in the antigen loaded. CTLs loaded with Cytomegalovirus (CMV) antigen only kill CMV-infected cells, but not adenovirus (Adv) -infected cells.
NK cells are generally considered to recognize and kill non-My cells, distinguishing self from non-self key molecules as MHC-class I molecules. The inhibitory receptor on NK cells is combined with MHC-I molecules on the surface of target cells to enable the NK cells to be in an inhibition state, so that killing effect can not occur. While the MHC-I molecules on the surface of the cells infected by viruses, cancerated and aged and dead are downwards regulated or disappeared, so that NK cells are released from inhibition, and the killing effect can be exerted. More interestingly, the action of CTLs requires MHC-I molecules, and antigen and MHC-I molecules are processed by antigen presenting cells to form immune complexes which are presented to the CTLs in order to achieve antigen-specific killing function. Thus, CTLs, unlike NK cells, are not lethal to cells lacking or down-regulated MHC class I molecules. NK cells with broad spectrum of actions are the first line of defense of organisms against serious diseases such as infection, cancer and the like, and specific CTLs are special soldiers who in vivo fight various diseases. It looks like NK cells and CTLs are a perfect combination of congenital and acquired properties, broad spectrum properties and specificity just like a group army, and the subordinate two armies have definite work, work and strength. However, this simple and crude understanding has in fact hampered the scientific development in this field and has also misled the industry so far that no better NK cell products have been developed. Since the actual situation is usually that no MHC-class I molecules are low, NK cell action is favored; the MHC-I molecules are not needed to be high, so that the CTL effect is facilitated; the case of both NK and CTL cell actions is not suitable for their best action. The novel immune cell which combines the broad spectrum of NK cells and the specificity of CTL is the specific NK (Specific natural killer, SNK) cell of the invention, which is more powerful as a synthetic army.
The physical basis of a specific immune response is memory immune cells. T cells and B cells are both postnatal training killers, and after antigen activation, a portion of the cells carry the memory of this antigen for life. Reinfection is when they are very physically present. When reinfection occurs, the memory cells hit rapidly and less than the potential of the Bell thief, often allowing control of viral replication at the early stages of infection. This is an important memory function of immune cells. As natural immune cells, NK cells have been widely known to have no memory function. However, as the research progresses, the research reports that NK cells have a 'memory-like response' in a cytomegalovirus infected mouse model, and later, the NK cells of human beings are further proved to have similar memory characteristics; its advantages are long service life and high activity. It was later discovered that similar ML-NK cells can also be produced by NK cells induced by cytokines; such cells are also known as cytokine-induced memory-like NK cells (CIML-NK) cells. In fact, ML-NK and CIML-NK cells are broad-spectrum nonspecific immune cells as common NK cells and CIK and DC-CIK cells which have been very popular in the Chinese immunocyte therapy community, because Specific NK (SNK) cells have not been seen so far. In fact, the current ML-NK and CIML-NK cells are more similar to common NK cells, and are still quite different from CIK cells and DC-CIK cells.
One difference is that the starting cells differ: peripheral blood lymphocytes (PBMC) are used as CIK cells and DC-CIK cells, and pure NK cells further purified from PBMC are used as ML-NK and CIML-NK cells; thus, CIK cells and DC-CIK cell products are mixtures of various lymphocytes, whereas ML-NK and CIML-NK cells are almost pure NK cells.
Another difference is the cytokine used: ML-NK cells are prepared by using cytokines such as IL-12 and IL-18. Cytokine such as IL-2 and IFN-gamma is used for preparing CIK cells, and IL-4 and GM-CSF is used for preparing DC cells; DC-CIK cells are the products of separate culture of DC and CIK cells and then combined together for culture. DC cells are in fact an antigen presenting cell, and at the same time have the effect of stimulating lymphocyte expansion. However, DC cells are used only to stimulate CIK cell expansion during the preparation of DC-CIK. Both CIK cells and DC-CIK cells belong to the broad spectrum of action immune cells. Although most of these two cells are T cells, there should be a large number of cd8+ T, i.e., CTL cells, but not antigen-stimulated CTL cells are activated and cannot exert a killing effect. NK cells play a broad-spectrum killing role in the two types of cells, but the proportion of NK cells in the DC-CIK cell product of the upgrade version of the NK cells is low and cannot exceed 50%; the NK cell proportion in the NK cell product is at least 70-80%, and often is more than 95%. Although CTL in CIK and DC-CIK cells is not easy to kill, NK cells have low proportion, so that killing power is weak, but other T cells belong to helper T (Th) cells and have certain immunity promoting effect, and are also useful.
The memory of ML-NK and CIML-NK cells refers to the fact that they secrete large amounts of IFN-gamma when they are again stimulated by cytokines such as IL-12 and IL-18, which are more potent, but still broad-spectrum, and no concept, product, or application has been presented to date for Specific NK (SNK) cells.
The invention discloses a preparation method and application of myeloid leukemia (Acute myeloid leukemia) SNK (AML-SNK) cells; meanwhile, the preparation method and the application of the SNK cell of the B cell lymphoma are also disclosed, so that the universality of the preparation method of the SNK cell is verified.
Although the present inventors have worked on NK cells and CTL cells and TIL cells for decades, various cells have been prepared basically according to the old concept. Although NK cells are expected to be more specific, no suitable preparation method has been found. Until recently we have found that we have used specific NK cells for a long time, but not to be aware. The preparation method of the high-activity NK (HANK) cells for more than ten years uses K562 engineering cells expressing IL-15, IL-21 and other several immune activating factors, and has good curative effects on various leukemia, solid tumors and AIDS. In the past we have thought that K562 cells only provide a solid support for several cytokines that is suitable for the biological activity of the K562 cells. In the past work, the killing activity of the HANK cells on K562 cells is found to be very high, but the killing activity of the HANK cells on other tumor cells is relatively low; the HANK cells have better curative effects on various leukemia and cancers, but have the best curative effects on myeloid leukemia. However, these phenomena are considered to be a cause of the fact that, in particular, the killing activity against K562 cells is high, and the lack of MHC class I molecules on K562 cells results in no inhibition of HANK cells, which is expected to be high. The significant fact that K562 engineered cells were used in the HANK cell preparation process was neglected. In the face of the results of the excellent curative effect of HANK cells on the myeloid leukemia and the excellent curative effect on the lymphoma, the countermeasure of multipurpose myeloid leukemia and less lymphoma is adopted. However, the present inventors believe that the high killing rate of the HANK cells against the K562 cells and the high cure rate of the HANK cells against the myeloid leukemia should be related to the K562 cells used in the preparation process. That is, the K562 cells used in the preparation process have a specific effect on the K562 cells and the myeloid leukemia; this also demonstrates the antigen specificity of NK cells. However, the inventors believe that rather than their time spent demonstrating the new function of an older product that has been used for many years, a completely new method of preparing SNK cells for AML has not been developed as inferred above. We have prepared SNK cells of B cell lymphoma at the same time, further verify the universality of the invention for preparing SNK cells.
Disclosure of Invention
The main object of the present invention is to provide a method for preparing specific NK (specific nature killer, SNK) cells and applications thereof. The specificity of NK cells depends on the antigen used; thus, SNK cells against any antigen can be prepared. SNK cells for the preparation of AML and B cell lymphomas are exemplified herein for purposes of illustration, but are not intended to limit the scope of applicability of the invention.
In the above-mentioned method for preparing HANK cells, several cytokines are expressed on the surface of K562 cells, and the effect of K562 cells with several cytokines is the whole effect of the recombinant cells, possibly masking the effect of K562 cells alone. Therefore, in the specific NK cell preparation method disclosed in the present invention, we used unmodified cancer cells as antigens and stimulated NK cell activation with free Stimulating and Activating Complexes (SAC) such as IL-2, IL-12, IL-15, IL18, IL-21, etc. in order to illustrate the respective actions of each component.
In order to achieve the above purpose, the preparation method of SNK cells provided by the invention comprises the steps of loading the activated NK cells with corresponding antigens, and continuously culturing and differentiating under the action of an Expansion and Differentiation Complex (EDC) to obtain the required SNK cells.
The material as the corresponding antigen is not limited, and may be any substance having antigenicity, including a polypeptide, a protein, a nucleic acid, and the like; any cell, including normal cells, cancer cells, primary cells, passaged cell lines; the method of treating the cells or cell lines is not limited, and may be whole cells, or lysates or extracts. In the present specification, the 100Gy gamma ray irradiation treated subculture cancer cell line is used. The cancer cells after irradiation treatment are in a metabolically dead state and have the form of a primitive cell line, but lose their growth ability, and are lysed and vanished about 7 days under culture conditions.
Methods for preparing SNK cells include stimulating NK cells with a stimulating activator complex (stimulating and activating complexes, SAC) composed of cytokines such as IL-2, IL-12, IL-15, IL-18, IL-21, and the like, and expanding differentiated SNK cells by expanding a differentiation complex (expanding and differentiating complexes, EDC). The amount of these cytokines to be used is not limited, and may be increased or decreased within an appropriate range; the combination of these cytokines is not limited, and may be used in total, or a combination of several of them; the combination form (dosage form) of these cytokines is not limited, and may be free, may be expressed on cells as an antigen by a genetic recombination technique, may be of a encapsulated or liposome nature, or the like.
The source of the initial cells for preparing SNK cells is not limited, and can be an NK cell line or a primary NK cell; can be autologous or allogenic; the variant can be any healthy adult or umbilical cord blood, and the adult can be relative or unrelated; it may also be iPSC-NK cells from iPSC.
The purity of the starting cells for preparing SNK cells is not limited, and may be mixed cell lymphocytes after blood is separated only by lymphocyte separation liquid, or NK cells further purified by various methods. Purified NK cells were used in the following examples in order to demonstrate the reliability of the results produced. In practical application, purified NK cells are not needed, because mixed lymphotenia with the purity of about 10% separated by lymphocyte separation liquid is adopted, and SNK cells with the purity as high as 95% and even higher can be finally obtained by the culture differentiation technology provided by the method without any further separation and purification.
The method of culturing SNK cells is not limited, and can be a feeder cell method or a cytokine method, and the type and concentration of the cytokine used are not limited.
The method for producing SNK cells is not limited in the length of cell culture, and may be 1 week or 2 weeks or longer.
The method for preparing SNK cells is not limited in the point of time of loading antigen during cell culture and specific loading method, and can be loaded at the time of initial culture or at any point of time from the time of initial culture to the time of harvesting cells.
The invention also discloses application of the specific NK cells.
The invention discloses SNK cells prepared by a preparation method of specific NK cells, which have specific killing effects on myeloid leukemia and B cell lymphoma cells respectively, and remarkably improve the overall function.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to the structures shown in these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flowchart of a preparation method of SNK cells according to an embodiment of the present invention;
FIG. 2 is a graph showing comparison of killing activity of two NK cells against DB target cells in example one of the present invention;
FIG. 3 is a graph showing the comparison of the killing activity of two NK cells against KG-1 target cells in the first embodiment of the present invention;
FIG. 4 is a graph showing comparison of the killing activity of two NK cells against DB target cells in the second embodiment of the present invention;
FIG. 5 is a graph showing the comparison of the killing activity of two NK cells against KG-1 target cells in the second embodiment of the invention.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Furthermore, the description of "first," "second," etc. in this disclosure is for descriptive purposes only and is not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be considered to be absent and not within the scope of protection claimed in the present invention.
In the preparation method of SNK cells disclosed in the embodiment of the invention, referring to FIG. 1, a cancer cell line killed by radiation is used as an antigen, SAC composed of cytokines such as IL-2, IL-12, IL-15, IL18, IL-21 and the like is used for stimulating the activation of NK cells, and EDC is used for promoting the expansion and differentiation of NK cells, so that SNK cells aiming at the antigen are obtained.
Embodiment one: preparation of SNK cells for B cell lymphoma
1. Preparation of B cell lymphoma antigen
DB cells are a DB cell line isolated and cultured from ascites of patients with Diffuse Large B Cell Lymphoma (DLBCL) and cultured and amplified in RPMI1640 culture solution containing 10% fetal bovine serum and 1% green streptomycin.
DB cell concentration was adjusted to 5X10 with the above culture solution 6 Each of the cells was packed in 15ml tip centrifuge tubes.
Treating with 100Gy radiation, centrifuging with physiological saline, washing for 3 times, suspending in X-VIVO15 serum-free culture solution, and preserving with liquid nitrogen. The DB cell code DBC after irradiation treatment was used as B cell lymphoma antigen in subsequent studies. DBC belongs to metabolically dead cells, and although it has the form of a primitive cell line, it loses its ability to grow and is cleaved and disappeared about 7 days under culture conditions.
2. Preparation of B cell lymphoma specific NK (BCL-SNK) cells
(1) Isolation of PBMC
1) Transferring 10ml of whole blood into a 50ml centrifuge tube, adding 10ml of PBS solution for dilution, and gently mixing;
2) Two 15ml centrifuge tubes were taken and 5ml Ficoll solution was added first. Then lightly adding diluted blood to the upper layers of ficolls of two centrifuge tubes, wherein the upper layers are required to be gentle, so that the two solutions are prevented from being mixed together, and 10ml of diluted blood is respectively added into each centrifuge tube;
3) 2,000rpm,20min, note that the no break, or only 1-2 brake, must be set in the deceleration setting. The cell layer where PBMC is located is white.
4) The layer of cells can then be pipetted into another clean 15ml centrifuge tube.
5) Adding PBS to 10-15ml, centrifuging at 1,500rpm for 10min, removing supernatant, adding culture medium, and cleaning with the same operation;
6) 5-10ml of culture medium is added to resuspend the cells, and the cells are counted for later use.
3. Removal of monocytes and B cells: after removing monocytes therein by adhesion, B cells therein were removed by nylon cotton column method.
(1) The concentration of mononuclear cells was adjusted to 4X10 with RPMI1640 medium containing 10% fetal bovine serum 6 Per ml, in sterile plastic dishes, 37℃and 7.5% CO 2 After 2h of incubation in the environment, monocytes and B cells adhering to the plastic were removed;
(2) Pre-incubating non-adherent cells with RPMI1640 solution containing 10% fetal bovine serum for 1h, passing through nylon Long Mianzhu, adhering B cells and residual mononuclear cells on a nylon cotton column, washing the column with a culture solution, and collecting washed T cells and NK cells;
(3) Separation of T cells and NK cells with magnetic field: the remaining T cells and NK cells were isolated with a magnetized cell separator (MACS). The test uses a negative selection method to label and isolate cells according to the sterility requirement.
1) The freshly isolated T cells and NK cells were labeled in a water bath, and the cells were incubated with anti-surface antigen monoclonal antibodies for 10min (10 7 Individual cells were treated with 100ul of anti-CD 3 mab);
2) Washing the cells, and incubating with biotin-labeled 100ul goat anti-mouse antiserum for 10min;
3) Washing, adding 25ul of FITC-labeled streptavidin, and reacting for 8min;
4) After washing, the reaction was carried out for 8min with biotin-labeled magnetic particles (anti-CD 3 mab plus 100ul magnetic particles).
After each of the above reactions, the reaction was terminated by washing with 10 volumes of PBS containing 1% bovine serum albumin and centrifuging at 3000r/min for 8min.
5) Immunomagnetic separation using magnetized cell separator (MACS) the separation column was autoclaved prior to use.
(1) The cell suspension marked with the magnetic compound is resuspended in 2-5 ml PBS containing 1% bovine serum albumin, and the cell concentration is adjusted to 5X10 7 ～1×10 8 Cells/ml;
(2) pre-incubating the separation column with PBS containing 1% bovine serum albumin for 30min, pre-cooling at 4 ℃, placing the pre-incubated separation column in a magnetic field of a permanent magnet, and adding 80-100 ml of labeled cell suspension;
(3) sucking out floating cells without magnet, centrifuging to precipitate cells, namely purified NK cells, and suspending with RPMI1640 culture solution for later use; and counted by fluorescent microscopy.
4. NK cell culture
(1) Stimulation and activation: suspending purified NK cells in RPMI1640 culture solution containing 10% fetal bovine serum and 1% penicillin streptomycin at a cell concentration of 1-2X10 6 The cells/ml are added with a stimulus-activated complex (SAC) composed of IL-12 of 1-80ng/ml, IL-15 of 1-100ng/ml, IL-18 of 2-90ng/ml, 37 ℃ and 5% CO 2 Culturing in incubator for 12-16 hr, and centrifuging with RPMI1640 culture solution for 3 times;
(2) Loading antigen: suspending the NK cells in RPMI1640 culture solution containing 10% fetal bovine serum and 1% green streptomycin at a cell concentration of 0.8-1.2X10 6 Cells/ml, adding DB cells (DBC) after irradiation treatment to a concentration 1-2 times that of NK cells;
(3) Amplification and differentiation: adding 100-1200IU/ml IL-2,1-100ng/ml IL-15,2-100ng/ml IL-21 amplification differentiation complex (EDC), culturing for 7 days, and half-changing every 3 days; adding proper amount of new culture solution to maintain cell concentration at 0.8-1.2X10 6 Individual cells/ml.
Another set of untreated DB cells (DBC) was used as a control for normal NK cells.
(4) Collecting cells: one group of the collected DBC-loaded NK+DBC, namely B cell lymphoma specific NK (BCL-SNK) cells, and the control group of the collected DBC-unloaded NK-DBC cells, namely common NK cells.
5. In vitro killing test
Detection of cytotoxic function by Calcein-AM/Cell Tracker Deep Red viable cell dye double-label flow method
(1) Labeling target cells (T): 2 different target cells were used in the assay, one being a DB cell line (T1) for antigen preparation for testing the antigen specificity of NK cells; the other is KG-1 cell (T2), which belongs to the acute myelogenous leukemia cell line as a control; the culture and labeling conditions were identical for both cells. Target cells (T1 and T2) were adjusted to 1X10, respectively, using PMRI 1640 medium containing 10% fetal bovine serum and 1% penicillin 6 Double staining with Calcein-AM (CAM) added to a final concentration of 2 μmol/L and Cell Tracker Deep Red Dye (DRD) to a final concentration of 0.1 μmol/L; and 1 tube Undyed (US), 1 tube CAM single dyed, 1 tube DRD single dyed as control; after incubation at 37deg.C for 30min in the absence of light, PBS was used to wash for 2 times and the cell density was adjusted to 2×10 with medium 5 Adding 96-well U-shaped bottom plates per mL, and adding 100 mu L of the bottom plates per well; together, test groups 8 pairs (multiple wells, 4 pairs of effector cells in each group, corresponding to E: T ratios of 0.5:1, 1:1, 2:1, 5:1, respectively) were provided; control wells were set: the number of Undyed (US) pairs was 1, CAM single-stained pairs was 1, DRD single-stained pairs was 1, and target cells (T and T2) pairs were 1, respectively.
(2) Preparation of effector cells (E): effector cells are DBC+NK cells, and DBC-NK cell group 2, respectively. Each group of target cells was diluted 1X10 with medium 5 Individual/mL (0.5:1), 2X10 5 Individual/mL (1:1), 4X10 5 /mL(2:1)、1X10 6 4 concentrations of/mL (5:1), 100. Mu.L was added to each corresponding target cell well; and 100 μl was added to each of US well, CAM well, DRD well, T1 and T2 well.
(3) Incubation of effective target: the culture plate is placed in a culture box at 37 ℃ for standing for 4 hours for killing after centrifugation for 1min at 1000 r/min.
(4) Flow cytometer detection: after incubation time, cells were homogenized, loaded with flow cytometry, and note that the same volume of the sub-set of samples was sampled. The double positive cell population is the remaining viable target cells.
(5) Calculation of effector cytotoxicity
Effector cytotoxicity (%) = (target control value-experimental group value)/target control value) ×100
(6) Results: the NK-DBC cells, namely common NK cells, are prepared without loading DBC antigen; NK+DBC cells, which are BCL-SNK cells, are prepared after DBC antigen is loaded. As can be seen from table 1, fig. 2 and fig. 3, the average number of living cells and the killing rate of both groups of cells are in good dose-effect relationship, and in the case of fixing the number of target cells (T1), as the number of effector cells (E) increases, the average number of living cells decreases, cytotoxicity increases, and the number of target cells (T1) killed increases; the test design is reasonable and the operation is standard. Only 1 pair of single DB target cell composite holes (T1) and KG-1 target cells (T2) of the shared unadditized effector cells (E) are arranged in the killing test, the average number of T1 living cells is 2096, and the average number of T2 living cells is 2324.
The killing activity of normal NK cells of NK-DBC group against related DB target cells by BCL-SNK cells of unrelated DB target cells and NK+DBC was analyzed by taking a group in which E: T1 is 10:1 as an example. The average number of the survival target cells of the DBC-NK group is 953, and the killing rate is 54.53%; the number of the target cells for the survival of the NK+DBC group B cell lymphoma specific NK (BCL-SNK) cells is 325, and the killing rate is up to 84.49%; the result shows that the killing activity of the B cell lymphoma specific NK (BCL-SNK) cells prepared after loading the DBC antigen to the corresponding target cells is obviously improved, and the increased killing rate of about 30 percent belongs to the contribution of antigen specificity.
Taking a group in which E: T2 is 10:1 as an example, the killing activity of normal NK cells of NK-DBC group against irrelevant KG-1 target cells and BCL-SNK cells of NK+DBC group against irrelevant KG-1 target cells was analyzed. The survival average number of the irrelevant target cells of the common NK-DBC group is 982, and the killing rate is 57.75%; the number of the survival of irrelevant target cells of the B cell lymphoma specific NK (BCL-SNK) cells of the NK+DBC group is 874, and the killing rate is up to 62.39%; the method shows that the killing activity of the B cell lymphoma specific NK (BCL-SNK) cells prepared after loading the DBC antigen on KG-1 target cells which are not related with the B cell lymphoma specific NK (BCL-SNK) cells is not different from that of common NK cells, and the B cell lymphoma specific NK cells belong to non-specific broad-spectrum killing effects.
The killing activity of NK cells prepared by the two methods on two different target cells fully verifies the specificity of the B cell lymphoma specific NK (BCL-SNK) cells.
Example two preparation of acute myeloid leukemia specific NK (AML-SNK) cells
1. Preparation of Acute Myeloid Leukemia (AML) antigen
KG-1 cells are an AML cell line and are amplified by culture in RPMI1640 medium containing 10% fetal bovine serum and 1% penicillin.
KG-1 cell concentration was adjusted to 5X10 with the above culture solution 6 Each of the cells was packed in 15ml tip centrifuge tubes.
Treating with 100Gy radiation, centrifuging with physiological saline, washing for 3 times, suspending in X-VIVO15 serum-free culture solution, and preserving with liquid nitrogen. KGC, the KG-1 cell code after irradiation, was used as AML antigen in the subsequent studies. KGC belongs to a metabolically dead cell, and although it has the form of a primitive cell line, it loses its ability to grow, and it is cleaved and disappears about 7 days under culture conditions.
2. Preparation of AML-SNK cells
(1) Isolation of PBMC
1) Transferring 10ml of whole blood into a 50ml centrifuge tube, adding 10ml of PBS solution for dilution, and gently mixing;
2) Two 15ml centrifuge tubes were taken and 5ml Ficoll solution was added first. Then lightly adding diluted blood to the upper layers of ficolls of two centrifuge tubes, wherein the upper layers are required to be gentle, so that the two solutions are prevented from being mixed together, and 10ml of diluted blood is respectively added into each centrifuge tube;
3) 2,000rpm,20min, note that the no break, or only 1-2 brake, must be set in the deceleration setting. The cell layer where PBMC is located is white.
4) The layer of cells can then be pipetted into another clean 15ml centrifuge tube.
5) Adding PBS to 10-15ml, centrifuging at 1,500rpm for 10min, removing supernatant, adding culture medium, and cleaning with the same operation;
6) 5-10ml of culture medium is added to resuspend the cells, and the cells are counted for later use.
(2) Removal of monocytes and B cells: after removing monocytes therein by adhesion, B cells therein were removed by nylon cotton column method.
1) The concentration of mononuclear cells was adjusted to 4X10 with RPMI1640 medium containing 10% fetal bovine serum 6 Per ml, in sterile plastic dishes, 37℃and 7.5% CO 2 After 2h of incubation in the environment, monocytes and B cells adhering to the plastic were removed;
2) Non-adherent cells were preincubated with RPMI1640 solution containing 10% fetal bovine serum for 1h and then passed through nylon Long Mianzhu, B cells and remaining monocytes were adhered to nylon cotton column, the column was washed with culture solution, and T cells and NK cells were collected.
(3) Separation of T cells and NK cells with magnetic field: the remaining T cells and NK cells were isolated with a magnetized cell separator (MACS). The test uses a negative selection method to label and isolate cells according to the sterility requirement.
1) The freshly isolated T cells and NK cells were labeled in a water bath, and the cells were incubated with anti-surface antigen monoclonal antibodies for 10min (10 7 Individual cells were treated with 100ul of anti-CD 3 mab);
2) Washing the cells, and incubating with biotin-labeled 100ul goat anti-mouse antiserum for 10min;
3) Washing, adding 25ul of FITC-labeled streptavidin, and reacting for 8min;
4) After washing, the reaction was carried out for 8min with biotin-labeled magnetic particles (anti-CD 3 mab plus 100ul magnetic particles).
After each of the above reactions, the reaction was terminated by washing with 10 volumes of PBS containing 1% bovine serum albumin and centrifuging at 3000r/min for 8min.
5) Immunomagnetic separation using magnetized cell separator (MACS) the separation column was autoclaved prior to use.
(1) The cell suspension marked with the magnetic compound is resuspended in 2-5 ml PBS containing 1% bovine serum albumin, and the cell concentration is adjusted to 5X10 7 ～1×10 8 Cells/ml;
(2) pre-incubating the separation column with PBS containing 1% bovine serum albumin for 30min, pre-cooling at 4 ℃, placing the pre-incubated separation column in a magnetic field of a permanent magnet, and adding 80-100 ml of labeled cell suspension;
(3) sucking out floating cells without magnet, centrifuging to precipitate cells, namely purified NK cells, and suspending with RPMI1640 culture solution for later use; and counted by fluorescent microscopy.
(4) NK cell culture
1) Stimulation and activation: suspending purified NK cells in RPMI1640 culture solution containing 10% fetal bovine serum and 1% penicillin streptomycin at a cell concentration of 1-2X10 6 The cells/ml are added with a stimulus-activated complex (SAC) composed of IL-12 of 1-80ng/ml, IL-15 of 1-100ng/ml, IL-18 of 2-90ng/ml, 37 ℃ and 5% CO 2 Culturing in incubator for 12-16 hr, and centrifuging with RPMI1640 culture solution for 3 times;
2) Loading antigen: suspending the NK cells in RPMI1640 culture solution containing 10% fetal bovine serum and 1% green streptomycin at a cell concentration of 0.8-1.2X10 6 The KG-1 cells (KGC) after irradiation treatment are added to the concentration which is 1 to 2 times that of NK cells per ml;
3) Amplification and differentiation: adding 100-1200IU/ml IL-2,1-100ng/ml IL-15,2-100ng/ml IL-21 amplification differentiation complex (EDC), culturing for 7 days, and half-changing every 3 days; adding proper amount of new culture solution to maintain cell concentration at 0.8-1.2X10 6 Individual cells/ml.
A further set of KG-1 cells (KGC) without irradiation was used as a control.
4) Collecting cells: collecting a group of NK cells loaded with KGC as NK+KGC, namely AML-SNK cells; one group of NK cells that were not loaded with KGC were collected as NK-KGC, which were common NK cells.
3. In vitro killing test
Detection of cytotoxic function by Calcein-AM/Cell Tracker Deep Red viable cell dye double-label flow method
Labeling target cells (T): 2 different target cells were used in the assay, one being KG-1 cell line (T1) for antigen preparation for testing the antigen specificity of NK cells; the other is DB cell (T2), belonging to B cell lymphoma cell line as control; the culture and labeling conditions were identical for both cells. With 10% foetal calf serum and 1% green streptomycinPMRI 1640 medium adjusts target cells (T1 and T2) to 1X10, respectively 6 Double staining with Calcein-AM (CAM) added to a final concentration of 2 μmol/L and Cell Tracker Deep Red Dye (DRD) to a final concentration of 0.1 μmol/L; and 1 tube Undyed (US), 1 tube CAM single dyed, 1 tube DRD single dyed as control; after incubation at 37deg.C for 30min in the absence of light, PBS was used to wash for 2 times and the cell density was adjusted to 2×10 with medium 5 Adding 96-well U-shaped bottom plates per mL, and adding 100 mu L of the bottom plates per well; together, test groups 8 pairs (multiple wells, 4 pairs of effector cells in each group, corresponding to E: T ratios of 0.5:1, 1:1, 2:1, 5:1, respectively) were provided; control wells were set: the number of Undyed (US) pairs was 1, CAM single-stained pairs was 1, DRD single-stained pairs was 1, and target cells (T and T2) pairs were 1, respectively.
(1) Preparation of effector cells (E): effector cells are KGC+NK cells, and KGC-NK cells, group 2, respectively. Each group of target cells was diluted 1X10 with medium 5 Individual/mL (0.5:1), 2X10 5 Individual/mL (1:1), 4X10 5 /mL(2:1)、1X10 6 4 concentrations of/mL (5:1), 100. Mu.L was added to each corresponding target cell well; and 100 μl was added to each of US well, CAM well, DRD well, T1 and T2 well.
(2) Incubation of effective target: the culture plate is placed in a culture box at 37 ℃ for standing for 4 hours for killing after centrifugation for 1min at 1000 r/min.
(3) Flow cytometer detection: after incubation time, cells were homogenized, loaded with flow cytometry, and note that the same volume of the sub-set of samples was sampled. The double positive cell population is the remaining viable target cells.
(4) Calculation of effector cytotoxicity
(5) Effector cytotoxicity (%) = (target control value-experimental group value)/target control value) ×100
(6) Results: the non-loaded KGC antigen is prepared into NK-KGC group cells, which belong to common NK cells; the NK+KGC cells are prepared after KGC antigen is loaded, and the KGC cells are AML-SNK cells. As can be seen from table 2, fig. 4 and fig. 5, the average number of living cells and the killing rate of both groups of cells are in good dose-effect relationship, and in the case of fixing the number of target cells (T1), as the number of effector cells (E) increases, the average number of living cells decreases, cytotoxicity increases, and the number of target cells (T1) killed increases; the test design is reasonable and the operation is standard. Only 1 pair of single KG-1 target cells (T1) and DB target cells (T2) of the shared unadditized effector cells (E) are arranged in the killing test, the average number of T1 living cells is 2145, and the average number of T2 living cells is 2027.
Taking a group of 10:1 as an example, the killing activity of normal NK cells of the NK-KGC group on the related KG-1 target cells by the acute myeloid leukemia specific NK (AML-SNK) cells of the unrelated KG-1 target cells and the NK+KGC group was analyzed. The average number of the survival target cells of the NK-KGC group is 853, and the killing rate is 60.23%; the number of the survival target cells of the acute myelogenous leukemia specific NK (AML-SNK) cells of the NK+KGC group is 315, and the killing rate is up to 85.31%; the acute myelogenous leukemia specific NK (AML-SNK) cells prepared after the KGC antigen is loaded are obviously improved in killing activity on target cells corresponding to the acute myelogenous leukemia specific NK (AML-SNK) cells, and the increased killing rate of about 25% belongs to the contribution of antigen specificity.
Taking a group of 10:1 as an example, the killing activity of normal NK cells of NK-KGC group on irrelevant DB target cells and acute myeloid leukemia specific NK (AML-SNK) cells of NK+KGC group on irrelevant DB target cells was analyzed. The average number of the survival target cells of the common NK group is 776, and the killing rate is 61.72%; the number of surviving target cells of the specific NK (AML-SNK) cells of the acute myeloid leukemia is 781, and the killing rate is up to 61.47%; the result shows that the killing activity of the acute myelogenous leukemia specific NK (AML-SNK) cells prepared after the KGC antigen is loaded on DB target cells irrelevant to the acute myelogenous leukemia specific NK (AML-SNK) cells is not greatly different from that of common NK cells, and the acute myelogenous leukemia specific NK cells belong to nonspecific broad-spectrum killing effects.
The killing activity of NK cells prepared by the two methods on two different target cells fully verifies the specificity of the acute myeloid leukemia specific NK (AML-SNK) cells.
Of course, the SNK cell preparation method of the present invention can be used to prepare not only SNK cells against cancer cells such as myeloid leukemia and B-cell lymphoma, but also SNK cells against any other antigen. With the increasing basic research and clinical application of SNK cells in the wide fields of anti-infection, anti-aging and the like, the research content is deeper and deeper, and the application effect is better.
The foregoing description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the invention, and all equivalent structural changes made by the description of the present invention and the accompanying drawings or direct/indirect application in other related technical fields are included in the scope of the invention.
Claims (6)
1. The preparation method of the specific NK cells is characterized by comprising the following steps of:
s101, preparing corresponding antigens;
s102, preparing SNK cells, which comprise the following steps:
s1021, separating PBMC;
s1022, removing mononuclear cells and B cells, removing the mononuclear cells by an adhesion method, and removing the B cells by a nylon cotton column method;
s1023, separating NK cells by using a magnetic field;
s103.nk cell culture, comprising:
s1031, stimulation and activation: suspending purified NK cells in RPMI1640 culture solution containing 10% fetal bovine serum and 1% penicillin streptomycin at a cell concentration of 1-2X10 6 1-80ng/ml IL-12,1-100ng/ml IL-15,2-90ng/ml IL-18 stimulated activation complex (stimulating and activating complexes, SAC), 37 ℃ C., 5% CO 2 Culturing in incubator for 12-16 hr, and centrifuging with RPMI1640 culture solution for 3 times;
s1032. loading antigen: suspending the NK cells in RPMI1640 culture solution containing 10% of fetal calf serum and 1% of green streptomycin, and adding proper amount of corresponding antigen;
s1033, amplification and differentiation: adding 100-1200IU/ml IL-2,1-100ng/ml IL-15,2-100ng/ml IL-21 amplification differentiation complex (expanding and differentiating complexes, EDC), culturing for 7-16 days, and half-changing every 3 days; adding proper amount of new culture solution to maintain cell concentration at 0.8-1.2X10 6 Individual cells/ml;
a group of non-antigen is additionally arranged and used as a common NK cell control;
s1034, collecting SNK cells.
2. The method for producing an SNK cell according to claim 1, wherein the step S101 comprises:
s1011, selecting a corresponding cancer cell line as an antigen;
s1012, regulating the concentration of cancer cells to 5X10 by using the culture solution 6 Split charging into 15ml sharp-bottomed centrifuge tubes;
s1013, performing irradiation treatment by using 100Gy gamma rays, then performing centrifugal washing by using normal saline for 3 times, suspending in X-VIVO15 serum-free culture solution, and preserving by using liquid nitrogen.
3. The method for producing an SNK cell according to claim 1, wherein the step S1021 includes:
s10211, transferring 10ml of whole blood into a 50ml centrifuge tube, adding 10ml of PBS solution for dilution, and lightly mixing;
s10212, taking two 15ml centrifuge tubes, adding 5ml of Ficoll solution, and then lightly adding diluted blood to the upper layers of the Ficoll of the two centrifuge tubes, wherein the two solutions are required to be gently prevented from being mixed together, and each centrifuge tube is respectively 10ml of diluted blood;
s10213.2,000rpm,20min, note that the deceleration setting must be set to nobreak, or only 1-2 brake, the cell layer where PBMC is located is white;
s10214, sucking the layer of cells into another clean 15ml centrifuge tube by using a suction tube;
s10215, adding PBS to 10-15ml, centrifuging at 1,500rpm for 10min, removing supernatant, and adding culture medium for cleaning in the same operation;
s10216, adding 5-10ml of culture medium to re-suspend the cells, and counting for later use.
4. The method of preparing specific NK cells according to claim 1, wherein in S1022:
s10221 the concentration of mononuclear cells is adjusted to 4×10 with RPMI1640 medium containing 10% fetal bovine serum 6 Per ml, in sterile plastic dishes, 37℃and 7.5% CO 2 After 2h of incubation in the environment, monocytes and B cells adhering to the plastic were removed;
s10222 pre-incubating non-adherent cells with RPMI1640 solution containing 10% fetal bovine serum for 1h, passing through nylon Long Mianzhu, adhering B cells and residual mononuclear cells on nylon cotton column, washing the column with culture solution, and collecting washed T cells and NK cells.
5. The method of preparing specific NK cells according to claim 1, wherein in S1023, comprising:
s10231 labeling the newly separated T cells and NK cells in a water bath, incubating the cells with an anti-surface antigen monoclonal antibody for 10min (10 7 Individual cells were treated with 100ul of anti-CD 3 mab);
s10232, washing the cells, and incubating with biotin-labeled 100ul goat anti-mouse antiserum for 10min;
s10233, adding 25ul of FITC labeled streptavidin after washing, and reacting for 8min;
s10234, adding biotin-labeled magnetic particles (100 ul magnetic particles are added for the anti-CD 3 monoclonal antibody) after washing, and reacting for 8min;
after each reaction step, washing with 10 times of PBS containing 1% bovine serum albumin, and centrifuging at 3000r/min for 8min to terminate the reaction;
s10235 immunomagnetic separation Using magnetized cell separator (MACS) separation column was autoclaved before use
S102351 suspending the cell suspension marked with the magnetic complex in 2-5 ml PBS containing 1% bovine serum albumin to adjust the cell concentration to 5×10 7 ～1×10 8 Cells/ml;
s102352 pre-incubating the separation column with PBS containing 1% bovine serum albumin for 30min, pre-cooling at 4 ℃, placing in a magnetic field of a permanent magnet, and adding 80-100 ml of labeled cell suspension;
s102353 sucking out floating cells without magnet, centrifuging to precipitate cells, namely purified NK cells, and suspending with RPMI1640 culture solution for later use; and counted by fluorescent microscopy.
6. Use of specific NK cells, characterized in that the SNK cells according to any of the preceding claims 1 to 5 are used to kill the abnormal cells corresponding thereto.
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