Separation and multiplication culture method of rat NK (Natural Killer) cells

Abstract

The invention relates to the technical field of immune cells, and discloses a rat NK cell separation and multiplication culture method which comprises the following steps: screening rat PBMC cells in a CD161 + positive screening mode to obtain rat NK cells; the rat NK cells and the rat PBMC cells are mixed and co-cultured, and the working concentration of the interleukin growth factor is adjusted in stages in the culture process. In the scheme of the application, the rat NK cells obtained by separation and amplification in the peripheral blood of the rat have the characteristics of large quantity, strong functional activity, no infectious pathogen pollution and the like, and can be used as an important source of non-autologous NK cells; abundant cell sources can be provided for clinically using non-autologous NK cells to treat diseases such as malignant tumors and virus infection. Meanwhile, efficient amplification of the rat NK cells can be realized, and the obtained rat NK cells have the characteristics of high purity and high activity.

Description

Technical Field

[0001] The present application relates to the field of immune cell technology, and mainly to a method for isolating, amplifying and culturing rat NK cells. Background Art

[0002] NK cells (natural killer cells) are among the most important effector cells in the animal immune system, comprising 3% to 10% of an individual's peripheral blood lymphocytes. They serve as the first line of defense for an animal's defenses. They are usually dormant, but once activated, they infiltrate most tissues and attack tumor cells and virus-infected cells. NK cells are a core component of an animal's innate immunity and the foundation of anti-tumor cellular immunity. Their cytotoxic activity is not restricted by the major histocompatibility complex (MHC) and is known as natural killer activity. Unlike T cells, which attack foreign cells, NK cells only kill tumor or virus-infected cells, demonstrating a distinctly targeted nature. NK cells exhibit an early killing effect on tumor cells, typically within one hour in vitro and four hours in vivo. They also compensate for the limitation of cytotoxic T lymphocytes, which must recognize MHC class I molecules to kill tumors or viral infections and are therefore unable to kill tumor cells that are negative for these molecules. Cancer patients often suffer from severe damage to their NK cells, making them unable to eliminate tumor cells on their own. This presents an opportunity for the use of NK cells in the treatment of tumors or viral infections. The so-called NK cell immunotherapy is to restore or rebuild the anti-tumor ability of NK cells through cell engineering, thereby improving the effect of NK cells in anti-tumor immunotherapy.

[0003] The killing ability of NK cells is mainly achieved through the following three mechanisms: (1) NK cells directly damage target cells by releasing perforins and granzymes; (2) NK cells bind to target cell membrane ligands through membrane TNF family molecules to induce target cell apoptosis; (3) NK cells bind to antibodies through the surface CD16 molecule, producing antibody-dependent cell-mediated cytotoxicity (ADCC).

[0004] Literature reports suggest that the combination of NK cells with ADCC activity and monoclonal antibody drugs can be more effective than single NK cells. Currently, there are many methods for expanding NK cells, but they still suffer from issues such as low cell purity, low expansion efficiency, and lack of ADCC activity. Therefore, further research is needed to obtain large quantities of NK cells with high purity and ADCC activity.

[0005] Because NK cells are present in very low concentrations in the peripheral blood of individual animals, accounting for only 6% to 12% of monocytes, their clinical application as adoptive immune cells has been greatly limited. For many years, researchers have been attempting to achieve large-scale expansion of NK cells in vitro. However, current conventional techniques primarily involve adding a combination of IL-2, IL-12, IL-15, and γ-IFN factors to the in vitro culture system to promote NK expansion. After a period of culture, NK cells can only expand several-fold, and the purity is not ideal, thus failing to meet the needs of practical applications. Furthermore, this method is costly, culture is unstable, and is unsuitable for large-scale application.

[0006] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0007] In view of the above-mentioned deficiencies in the prior art, the purpose of this application is to provide a method for isolating, amplifying and culturing rat NK cells. The isolated rat NK cells have the advantages of high activity, high purity and stable amplification multiples, aiming to solve the problems of low cell purity, low amplification efficiency and lack of ADCC effect in the existing NK cell amplification methods.

[0008] The technical solution of this application is as follows: A method for isolating, amplifying and culturing rat NK cells, comprising the following steps: Rat NK cells were obtained by CD161+ positive screening in rat PBMC cells; The rat NK cells and the rat PBMC cells are mixed and co-cultured, and the working concentration of the interleukin growth factor is adjusted in stages during the culture process.

[0009] This application uses a combination of CD161+ positive screening and PBMC co-culture to produce rat NK cells with high purity (over 99%), a large expansion multiple, and strong activity. Furthermore, since the initial positive screening yields fewer rat NK cells, the subsequent required culture medium volume and growth factor dosage are significantly reduced, which not only reduces costs but also results in a higher number and purity of cells cultured.

[0010] The method for isolating, expanding and culturing rat NK cells, wherein, during the process of mixing and culturing the rat NK cells with the rat PBMC cells, the cell number ratio of the rat NK cells to the rat PBMC cells at the initial stage of culture is 1:2.

[0011] In the present application, the cell number ratio of rat NK cells to PBMC is 1:2. Culturing rat NK cells and PBMC at a ratio of 1:2 can meet the cell expansion multiples, and the proportion of rat NK cells in all the cells obtained reaches more than 99%, which is no less than pure NK cell culture.

[0012] The method for isolating, expanding and culturing rat NK cells, wherein the method for adjusting the working concentration of interleukin growth factor in stages during the culture process is as follows: At the initial culture, NK cell culture medium was used as the culture medium, and Rat IL-2 at a working concentration of 1000 IU / ml and Rat IL-21 at 20 ng / ml were added; On the third day of culture, an equal volume of the first supplement was added to the cell suspension. The first supplement was prepared by adding rat IL-15 with a working concentration of 50 ng / ml and rat IL-2 with a working concentration of 1000 IU / ml to the NK cell culture medium. On the sixth day of culture, a second supplement was added to adjust the cell concentration of the cell suspension to 1-1.5×10^6 cells / ml. The second supplement was prepared by adding 500 IU / ml of Rat IL-2 and 50 ng / ml of Rat IL-15 to the NK cell culture medium. On the 9th day of culture, a third supplement was added to adjust the cell concentration of the cell suspension to 1-1.5×10^6 cells / ml. The third supplement was prepared by adding 500 IU / ml of Rat IL-2, 50 ng / ml of Rat IL-15, and 50 ng / ml of Rat IL-18 to the NK cell culture medium. The NK cell culture medium was prepared by adding 450 ml of 1640 culture medium to every 50 ml of fetal bovine serum.

[0013] The method for isolating, expanding and culturing rat NK cells, wherein the initial culture specifically comprises the following steps: resuspending the rat NK cells in the NK cell culture medium to obtain rat NK cell fluid; resuspending the rat PBMC cells in the NK cell culture medium to obtain rat PBMC cell fluid; The rat NK cell solution was inoculated into a cell culture dish, and an equal volume of the rat PBMC cell solution was added, and Rat IL-2 with a working concentration of 1000 IU / ml and Rat IL-21 with a working concentration of 20 ng / ml were added, and the dish was placed in an incubator at 37° C. for culture; The cell concentration of the rat NK cell fluid is not less than 2×10^6 cells / ml; the cell concentration of the rat PBMC cell fluid is twice that of the rat NK cell fluid.

[0014] The method for isolating, amplifying and culturing rat NK cells, wherein the process of obtaining rat PBMC cells comprises the following steps: An equal volume of 1640 culture medium was added to the peripheral blood of rats to form a mixed solution. The mixed solution and lymph extract were added to a centrifuge tube at a volume ratio of 6:4-5, and the tube was centrifuged to obtain the rat PBMC cells.

[0015] The method for isolating, amplifying and culturing rat NK cells, wherein the process of centrifuging to obtain the rat PBMC cells comprises the following steps: Centrifuge at 1500 rpm for 30 minutes at room temperature; Pipette the cell fluid from the middle buffy coat layer into a container, add 1640 culture medium at a volume ratio of 1-1.5:5, mix well, and centrifuge at room temperature for 10 minutes at 1300 rpm. After centrifugation, the supernatant was discarded, the cells were resuspended in 1640 medium, washed, and centrifuged at room temperature for 10 min at 1000 rpm; After centrifugation, the supernatant was removed to obtain the rat PBMC cells.

[0016] The method for isolating, amplifying and culturing rat NK cells, wherein the process of screening rat NK cells from rat PBMC cells by CD161+ positive screening comprises the following steps: The rat PBMC cells were adjusted to a cell concentration of 4-5×10^6 cells / ml using 1640 culture medium, and Rat CD161-PE antibody was added at a volume ratio of 100-150:1, and incubated at room temperature in the dark for 30 minutes; Anti-PE magnetic beads were added at a volume ratio of 10-15:1, and the cells were incubated in the dark at room temperature for 25 to 30 minutes. CD161-positive cells were screened using a Miltenyi MACS system to obtain the rat NK cells.

[0017] The method for isolating, amplifying and culturing rat NK cells further comprises the following steps: On the 11th to 12th day of culture, cells were collected; The process of collecting cells is to centrifuge the cell suspension at room temperature, 1000 rpm, for 10 minutes, discard the supernatant, and repeat twice.

[0018] The rat NK cell isolation, expansion and culture method, wherein the rat NK cells and the rat PBMC cells are derived from the same rat.

[0019] The method for isolating, expanding and culturing rat NK cells, wherein the rat peripheral blood is rat peripheral blood taken within 5 minutes of death of an adult rat.

[0020] Beneficial Effects: In this application, rat NK cells isolated and amplified from rat peripheral blood have the characteristics of large number, strong functional activity, and no contamination by infectious pathogens. They can be used as an important source of non-autologous NK cells, providing a rich cell source for the clinical use of non-autologous NK cells to treat diseases such as malignant tumors and viral infections. At the same time, this application can achieve efficient amplification of rat NK cells without the need for any light radiation device, and the obtained rat NK cells have the characteristics of high purity and high activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a statistical diagram of the NK cell culture days and cell numbers in the experimental group and control group 1 in Example 1 of the present application.

[0022] Figure 2 These are microscopic images of the cell fluid of the experimental group in Example 1 of the present application after being cultured for different days.

[0023] Figure 3 These are microscope images of the cell fluid of Control Example 1 in Example 1 of the present application cultured for different days.

[0024] Figure 4 These are microscopic images of the cell fluid of Control Example 2 in Example 1 of the present application cultured for different days.

[0025] Figure 5 These are flow cytometric images of the cell fluids of the experimental group in Example 2 of the present application cultured for different days.

[0026] Figure 6 This is a schematic diagram of the NK cell purity of the cell fluid of the experimental group in Example 2 of the present application after culture for different days. DETAILED DESCRIPTION

[0027] The present application provides a method for isolating, amplifying, and culturing rat NK cells. To make the purpose, technical solutions, and effects of the present application clearer and more specific, the present application is further described below. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present application and are not intended to limit the present application.

[0028] The present application provides a method for isolating, expanding and culturing rat NK cells, comprising the following steps: (1) Collection and transportation of rat peripheral blood; (2) Storage of rat peripheral blood; (3) Isolation of rat peripheral blood mononuclear cells (PBMCs) from rat peripheral blood; (4) Screening and obtaining rat NK cells; (5) Expansion and culture of rat NK cells; (6) The process of releasing rat NK cells.

[0029] Specifically, in step (1), the rat peripheral blood is rat peripheral blood (fresh rat peripheral blood) obtained within 5 minutes of death of an adult rat. The collection process can be to add the rat peripheral blood into a blood tube containing EDTA-anticoagulant using a negative pressure tube. The transportation process can be to place the blood tube in a transport thermostat with a 4°C ice box, and maintain a cold chain at 4°C throughout the entire process for rapid transportation to the cell bank.

[0030] In step (2), the storage process of rat peripheral blood includes: A. Identity information of the adult rats registered for blood draw: The identity information includes the basic information of the adult rats, family DNA pedigree, virus test results, etc. B. Register the tracking information of the adult rat: The tracking information includes the blood test report of the adult rat 2 weeks before and after collection, the normal eating and sleeping habits, etc. C. Count the cells in the collected peripheral blood of the rats and record the statistical data.

[0031] In step (3), the process of isolating rat PBMC cells from rat peripheral blood includes the following steps: An equal volume of 1640 medium is added to rat peripheral blood to form a mixture. This mixture and lymphatic extract are added to a centrifuge tube at a volume ratio of 6:4-5. Centrifuge to obtain rat PBMCs and count the cells. This method is limited to obtaining rat PBMCs.

[0032] Specifically, the process of centrifuging, obtaining rat PBMC cells, and counting the number includes the following steps: Centrifuge at 1500 rpm for 30 minutes at room temperature; Slowly and carefully pipette the middle buffy coat cell solution into a tube without damaging the middle interface, add 1640 medium at a volume ratio of 1-1.5:5, mix well, and centrifuge at room temperature for 10 minutes at 1300 rpm. After centrifugation, the supernatant was removed, and the cells were resuspended in 1640 medium, washed, and centrifuged at room temperature for 10 minutes at 1000 rpm. After centrifugation, the supernatant was removed to obtain rat PBMC cells; Resuspend the cells in 1640 medium and count the cells.

[0033] The process of this step (3) has been optimized by the inventors. By optimizing the ratio of the mixed solution to the lymph extract and the centrifugation parameters, rat PBMC cells can be better and more completely extracted from rat peripheral blood. If the ratio of the mixed solution to the lymph extract is too high, the whole blood extraction will be incomplete; if the ratio is too low, the white coat layer will be sparse and not conducive to operation. If the centrifugation speed is too high or the time is too long, the white coat layer cells will be reduced; if the centrifugation speed is too low or the time is too short, the separation will be incomplete.

[0034] 80% of the peripheral blood mononuclear cells obtained in this step (3) can be used for rat NK cell screening, and 20% can be used for co-culture with rat NK cells.

[0035] In step (4), the process of screening and obtaining rat NK cells is to screen out rat NK cells through CD161+ positive screening, which specifically includes the following steps: The obtained rat PBMC cells were adjusted to a cell concentration of 4~5×10^6 cells / ml with 1640 culture medium. Rat CD161-PE antibody was added at a volume ratio of 100-150:1 to label mature NK cells. The cells were incubated at room temperature in the dark for 30 minutes. Anti-PE magnetic beads were added at a volume ratio of 10-15:1 to capture the PE-labeled mature NK cells. The cells were incubated at room temperature in the dark for 25-30 minutes. CD161-positive cells were screened using the Miltenyi MACS system, which are rat NK cells. At this time, rat NK cells include NK cells and NKT cells.

[0036] The process of this step (4) has been optimized by the inventors. By controlling the addition ratio of RatCD161-PE antibody and anti-PE magnetic beads, more rat NK cells can be screened more accurately. Adding too much RatCD161-PE antibody will result in false positives; adding too little RatCD161-PE antibody will result in incomplete NK cell labeling. Adding too much anti-PE magnetic beads will result in excessive costs and blockage of the screening channel. Adding too little anti-PE magnetic beads will result in incomplete capture of mature NK cells labeled with PE.

[0037] In step (5), during the amplification and culture of rat NK cells, autologous PBMC cells are added and mixed and cultured to simulate the in vivo microenvironment stimulation and activation, and interleukin growth factor is added to the cell culture medium in stages for culture. In this step (5), the incubator is preferably a carbon dioxide incubator.

[0038] In step (5), the culture medium used for culturing rat NK cells is NK cell culture medium, which is prepared by adding 50 ml of fetal bovine serum to 450 ml of 1640 culture medium.

[0039] Specifically, step (5) includes the following steps: A. Initial culture: Rat NK cells were resuspended in NK cell culture medium at a concentration of 2×10^6 cells / ml to obtain rat NK cell fluid; The rat PBMC cells obtained in step (3) were resuspended in NK cell culture medium at a concentration of 4×10^6 cells / ml to obtain rat PBMC cell fluid; Rat NK cell fluid was inoculated into a cell culture dish, and an equal volume of rat PBMC cell fluid was added. Then, Rat IL-2 at a working concentration of 1000 IU / ml and Rat IL-21 at 20 ng / ml were added, and the cells were cultured in a 37°C incubator (the working concentration refers to the addition of Rat IL-2 and Rat IL-21 so that the concentration of Rat IL-2 in the cell suspension is 1000 IU / ml and the concentration of Rat IL-21 is 20 ng / ml).

[0040] In the present application, the cell number ratio of rat NK cells and rat PBMC cells is 1:2. Culturing rat NK cells and rat PBMC cells in a ratio of 1:2 can meet the cell expansion multiples, and the proportion of rat NK cells in all the cells obtained reaches more than 99%, which is no less than pure NK cell culture.

[0041] B. On the third day of culture, an equal volume of the first supplement was added. The first supplement was prepared by adding rat IL-15 at a working concentration of 50 ng / ml and rat IL-2 at 1000 IU / ml to the NK cell culture medium.

[0042] C. On the sixth day of culture, a second supplement was added to adjust the cell concentration of the cell suspension to 1-1.5×10^6 cells / ml. The second supplement was prepared by adding a working concentration of 500 IU / ml of RatIL-2 and 50 ng / ml of RatIL-15 to the NK cell culture medium.

[0043] D. On the ninth day of culture, add the third supplement to adjust the cell concentration of the cell suspension to 1-1.5×10^6 cells / ml. The third supplement is prepared by adding 500 IU / ml of Rat IL-2, 50 ng / ml of Rat IL-15, and 50 ng / ml of Rat IL-18 to NK cell culture medium.

[0044] During this culture process, the initial cell concentration cannot be lower than 2×10^6 cells / ml, otherwise the cells cannot be successfully inoculated; during the culture process, the cell concentration cannot exceed the range of 1-1.5×10^6 cells / ml. If the concentration is too low, the cells will die, and if it is too high, the cells cannot grow normally.

[0045] E. Cells were collected on day 11-12.

[0046] The process of collecting cells is to centrifuge the cultured cell suspension at room temperature, 1000 rpm, for 10 minutes, discard the supernatant, and repeat twice.

[0047] In the embodiment of the present application, it is best to collect cells on days 11 to 12 because the cells are in the best condition at this time.

[0048] In step (5), the following steps may also be included: Detection of rat NK cells.

[0049] After the 11th day of rat NK cell expansion culture, the obtained rat NK cells can be subjected to mycoplasma, endotoxin, flow cytometry, cell viability, and other tests to determine the quality and efficiency of rat NK cell expansion. The obtained rat NK cells can also be tested on days 3, 6, 9, and 11 (or at a time determined by experimental requirements) to observe the quality and efficiency of rat NK cell expansion during the culture process.

[0050] In step (6), the process for releasing the rat NK cells is to resuspend the amplified rat NK cells with physiological saline. For example, the cells can be resuspended with physiological saline at a concentration of 4×10^7 cells / ml. In a specific embodiment of the present application, the cells are resuspended with physiological saline at a concentration of 4×10^7 cells / ml, placed in a 1.5ml EP tube, and sealed; the EP tubes are systematically numbered, and the EP tubes containing rat NK cells from the same adult rat are grouped together. The corresponding storage information and system number of the adult rat are uniformly input into a computer and uploaded to the cell information archive for management, archiving, and release.

[0051] The method for isolating, expanding and culturing rat NK cells provided in this application has the following advantages: (1) Compared with the traditional method of directly inducing NK cells with PBMC, this application uses a combination of CD161+ positive screening and autologous PBMC mixed co-culture to make the final product rat NK cells have the characteristics of high purity (more than 99%), large expansion multiples, and strong activity. In addition, the traditional PBMC induction method uses a large amount of PBMC, which leads to an increase in the volume of culture medium and the amount of growth factors used, resulting in a high cost of the required growth factors and a low purity of the final product NK cells. In the present application scheme, since the rat NK cells obtained in the initial positive screening are relatively small, the volume of culture medium and the amount of growth factors required in the subsequent culture are greatly reduced, and the number and purity of the cells finally cultured are both high.

[0052] (2) Compared with the traditional PMBC-induced NK cell addition method of cell growth factors, the present application adopts the segmented addition of different growth factors and concentrations. After repeated experimental comparisons, it was found that Rat IL-12 would temporarily accelerate cell proliferation, but would destroy cell activity, so it was not adopted; adding Rat IL-18 would accelerate cell proliferation without destroying cell activity, but adding Rat IL-18 in the early stage of culture would not affect NK cells. Only when added in the later stage of culture would it lead to accelerated cell proliferation. The method of adding cell growth factors in the present application can effectively reduce the loss of cell culture activity while also ensuring the expansion multiple of the cell number.

[0053] (3) Compared with the method of using NK92 cells as the trophoblast, the mixed culture method of the present application can best mimic the in vivo microenvironment and maintain the activity of rat NK cells at its best; PBMCs will die on the 7th to 8th day of culture, and a small part of them will be transformed into rat NK cells, which has no effect on the subsequent purity of rat NK cells.

[0054] (4) Compared with the method of forming a trophoblast layer by irradiating PBMC with light, the culture method of this application does not require light irradiation. Light irradiation will cause irreversible damage to cells and cause unpredictable changes in cells, resulting in greater randomness in the experiment. In addition, the cost of radiation equipment is high and the experimental process is time-consuming and labor-intensive, which most laboratories cannot support.

[0055] In the present application, the rat NK cells obtained by separation and amplification in rat peripheral blood have the characteristics of large number, strong functional activity, and no contamination by infectious pathogens. They can be used as an important source of non-autologous NK cells, and can provide a rich source of cells for the clinical use of non-autologous NK cells to treat diseases such as malignant tumors and viral infections. At the same time, the present application can achieve efficient amplification of rat NK cells, and the obtained rat NK cells have the characteristics of high purity and high activity. It enhances the application of NK cell therapy to medical technology treatments and provides a new cell culture method for the treatment and prevention of tumors and immune system diseases caused by viral infections. At the same time, the separation, amplification and culture method of rat NK cells provided in the present application has the advantages of simple operation, low cost and wide application.

[0056] The present application is further described below through specific examples.

[0057] Experimental Example 1: Experiment on the culture of rat NK cells Experimental group: (1) Preparation of NK cell culture medium: Add 450 ml of 1640 culture medium to every 50 ml of fetal bovine serum to prepare RPMI 1640 cell culture medium containing 10% fetal bovine serum.

[0058] (2) PBMC extraction: Add an equal volume of 1640 culture medium to fresh peripheral blood extracted from adult rats to form a mixed solution. The mixed solution and lymph extract are added to a centrifuge tube at a volume ratio of 6:4. Centrifuge at room temperature for 30 minutes at 1500 rpm. After centrifugation, slowly and carefully aspirate the cell fluid of the middle buffy coat layer into a tube without destroying the middle interface. Add 1640 culture medium at a volume ratio of 1:5 and mix evenly. Centrifuge at room temperature for 10 minutes at 1300 rpm. After centrifugation, remove the supernatant, resuspend in 1640 culture medium, rinse, centrifuge at room temperature for 10 minutes at 1000 rpm. After centrifugation, remove the supernatant, resuspend in 6 ml of 1640 culture medium, and count the cells. 80% of the rat PBMC cells obtained in this step are used for screening rat NK cells, and 20% are used for co-culture with rat NK cells.

[0059] (3) Screening of rat NK cells: Resuspend the rat PBMC cells obtained in step (2) in 1640 medium and adjust the cell concentration to 4×10^6 cells / ml using 1640 medium. Take 5 ml of the rat PBMC cell resuspension and add Rat CD161-PE antibody at a volume ratio of 100:1. Incubate at room temperature in the dark for 30 minutes. Add anti-PE magnetic beads at a volume ratio of 10:1 and incubate for 25-30 minutes. Use the Miltenyi MACS system to screen CD161-positive cells. At this time, the rat NK cells are a mixture of rat NK and NKT cells.

[0060] (4) Initial culture: The rat PBMC cells obtained in step (2) were resuspended in NK cell culture medium at a cell concentration of 4×10^6 cells / ml; the rat NK cells obtained in step (3) were resuspended in NK cell culture medium at a cell concentration of 2×10^6 cells / ml and inoculated into a 24-well cell culture plate. An equal volume of autologous rat PBMC cell fluid was added, followed by addition of Rat IL-2 at a working concentration of 1000 IU / ml and Rat IL-21 at 20 ng / ml, and the cells were cultured in a 37°C incubator.

[0061] (5) On the third day of culture, slowly add an equal volume of the first supplement from the well wall. The first supplement is prepared by adding 1000 IU / ml Rat IL-2 and 50 ng / ml Rat IL-15 to NK cell culture medium. Do not flush the cells.

[0062] (6) On the sixth day of culture, observe whether the cell suspension turns yellow. After pipetting the cells to mix, count them and adjust the cell concentration to 1-1.5×10^6 cells / ml with the second supplement. The second supplement is prepared by adding 500 IU / ml Rat IL-2 and 50 ng / ml Rat IL-15 to the NK cell culture medium. If the volume of the cell suspension is greater than 5 ml, transfer it to a 6-well plate and culture it in a 37°C incubator.

[0063] (7) On the 9th day of culture, observe whether the cell suspension turns yellow, pipette the cells to mix, and then count them. Use the third supplement to adjust the cell concentration to 1-1.5×10^6 cells / ml. The third supplement is prepared by adding 500 IU / ml of Rat IL-2, 50 ng / ml of Rat IL-15, and 50 ng / ml of Rat IL-18 to the NK cell culture medium. If the volume of the cell suspension is greater than 10 ml, transfer it to a T25 culture flask and culture it in a 37°C incubator. (8) On the 12th day of culture, observe whether the cell suspension turns yellow, blow the cells to mix and count, take a small portion for detection, take a small portion and continue to culture until the 15th day to observe the cell growth status, collect the remaining cell suspension, centrifuge at room temperature for 10 minutes at 1000 rpm, discard the supernatant after centrifugation, repeat twice; resuspend the cells with physiological saline at 4×10^7 cells / ml, place them in a 1.5ml centrifuge tube, seal it, register the information, and ship it out.

[0064] Control group 1 The culture process of control group 1 was the same as that of the experimental group, with the only difference being that in step (4), autologous rat PBMC cells were not added, but rat NK cell fluid with a cell concentration of 2×10^6 cells / ml and an equal volume of NK cell culture medium (pure NK cell culture) were added.

[0065] Control group 2 The culture process of control group 2 was the same as that of the experimental group, with the only difference being that in step (4), instead of adding the mixture of rat NK and NKT cells, autologous rat PBMC cell fluid with a cell concentration of 4×10^6 cells / ml and an equal volume of NK cell culture medium (pure PBMC culture) were added.

[0066] The cell count and cell activity of the cell fluids of the experimental group and the control group 1 on the 0th, 6th, 9th and 12th day of culture were detected respectively. The activity detection was carried out by the traditional trypan blue cell activity detection method. The results are shown in Table 1 and Figure 1 As shown in the figure, "1NK:2PBMC" represents the experimental group, and "NK" represents the control group 1.

[0067] Table 1 Statistics of NK cell culture days and cell numbers

[0068] Figure 2 These are microscopic images of cell fluids cultured for different days in the experimental group, where a is a microscopic image of the cell fluid on the 3rd day of culture; b is a microscopic image of the cell fluid on the 6th day of culture; c is a microscopic image of the cell fluid on the 9th day of culture; d is a microscopic image of the cell fluid on the 12th day of culture; and e is a microscopic image of the cell fluid on the 15th day of culture (all cells were dead on a large scale).

[0069] Figure 3 These are microscopic images of cell fluids cultured for different days in control group 1, where a is a microscopic image of cell fluid on the 3rd day of culture; b is a microscopic image of cell fluid on the 6th day of culture; c is a microscopic image of cell fluid on the 9th day of culture; d is a microscopic image of cell fluid on the 12th day of culture; and e is a microscopic image of cell fluid on the 15th day of culture (all cells were dead on a large scale).

[0070] Figure 4 These are microscopic images of cell fluids cultured for different days in the control group 2, where a is a microscopic image of the cell fluid on the first day of culture; b is a microscopic image of the cell fluid on the third day of culture; c is a microscopic image of the cell fluid on the fifth day of culture; d is a microscopic image of the cell fluid on the sixth day of culture; and e is a microscopic image of the cell fluid on the eighth day of culture (PBMC cell death).

[0071] Example 2: Detection of cell types by flow cytometry A small amount of NK cell culture medium from the experimental group in Example 1 on days 3, 6, 9, and 11 was collected and centrifuged at 400 rpm for 10 minutes at room temperature. The supernatant was discarded and the cells were resuspended in 100 μl of PBS. Percp-CD45, PE-CD3, FITC-CD16, and FITC-CD56 were added at a volume ratio of 500:1, respectively. The cells were shaken and incubated at room temperature in the dark for 20 minutes. The ratio of CD3- / CD16+CD56+ positive cells was analyzed by flow cytometry. The results are shown in Figure 2. Figure 5 As shown, a is a flow cytometric graph of the cell fluid on the 3rd day of culture, b is a flow cytometric graph of the cell fluid on the 6th day of culture, c is a flow cytometric graph of the cell fluid on the 9th day of culture, and d is a flow cytometric graph of the cell fluid on the 12th day of culture.

[0072] The changes in the proportion of NK cells over time are shown in Tables 2 and Figure 6 shown.

[0073] Table 2 Statistics of culture days and NK%

[0074] Based on the above examples, the following conclusions can be drawn: (1) Rat PBMC cells cultured alone will die on the 8th day, and rat NK cells cultured alone will not have enough cell proliferation and low activity on the 11th day, which is not enough to meet the experimental requirements.

[0075] (2) Rat NK cells and rat PBMC cells were cultured at a ratio of 1:2, which satisfied the multiples of cell expansion, and the proportion of rat NK cells in all cells reached more than 99%, which was no less than that of pure NK culture. Therefore, the method provided in this application not only solved the problem of insufficient NK cell expansion, but also greatly improved the purity and activity of NK cell products. According to the observation of the culture cycle, 12 days was the optimal time.

[0076] It should be understood that the application of this application is not limited to the above examples. For ordinary technicians in this field, they can make improvements or changes based on the above description, and all these improvements and changes should fall within the scope of protection of this application.

Claims

1. A method for isolating, amplifying and culturing rat NK cells, characterized in that: The following steps are involved: Rat NK cells were obtained by CD161+ positive screening in rat PBMC cells; The rat NK cells and the rat PBMC cells are mixed and co-cultured, and the working concentration of the interleukin growth factor is adjusted in stages during the culture process.

2. The method for separating, amplifying and culturing rat NK cells according to claim 1, wherein During the co-culturing of the rat NK cells and the rat PBMC cells, the ratio of the number of rat NK cells to the number of rat PBMC cells is 1:2 at the beginning of the culture.

3. The method for separating, amplifying and culturing rat NK cells according to claim 1, wherein The method for adjusting the working concentration of interleukin growth factor in stages during the culture process is as follows: At the initial culture, NK cell culture medium was used as the culture medium, and Rat IL-2 at a working concentration of 1000 IU / ml and Rat IL-21 at 20 ng / ml were added; On the third day of culture, an equal volume of the first supplement was added to the cell suspension. The first supplement was prepared by adding rat IL-15 with a working concentration of 50 ng / ml and rat IL-2 with a working concentration of 1000 IU / ml to the NK cell culture medium. On the sixth day of culture, a second supplement was added to adjust the cell concentration of the cell suspension to 1-1.5×10^6 cells / ml. The second supplement was prepared by adding 500 IU / ml of Rat IL-2 and 50 ng / ml of Rat IL-15 to the NK cell culture medium. On the 9th day of culture, a third supplement was added to adjust the cell concentration of the cell suspension to 1-1.5×10^6 cells / ml. The third supplement was prepared by adding 500 IU / ml of Rat IL-2, 50 ng / ml of Rat IL-15, and 50 ng / ml of Rat IL-18 to the NK cell culture medium. The NK cell culture medium was prepared by adding 450 ml of 1640 culture medium to every 50 ml of fetal bovine serum.

4. The method for separating, amplifying and culturing rat NK cells according to claim 3, wherein The initial culture specifically comprises the following steps: resuspending the rat NK cells in the NK cell culture medium to obtain rat NK cell fluid; resuspending the rat PBMC cells in the NK cell culture medium to obtain rat PBMC cell fluid; The rat NK cell solution was inoculated into a cell culture dish, and an equal volume of the rat PBMC cell solution was added, and Rat IL-2 with a working concentration of 1000 IU / ml and Rat IL-21 with a working concentration of 20 ng / ml were added, and the dish was placed in an incubator at 37° C. for culture; The cell concentration of the rat NK cell fluid is not less than 2×10^6 cells / ml; the cell concentration of the rat PBMC cell fluid is twice that of the rat NK cell fluid.

5. The method for separating, amplifying and culturing rat NK cells according to claim 1, wherein The process of obtaining the rat PBMC cells comprises the following steps: An equal volume of 1640 culture medium was added to the peripheral blood of rats to form a mixed solution. The mixed solution and lymph extract were added to a centrifuge tube at a volume ratio of 6:4-5, and the tube was centrifuged to obtain the rat PBMC cells.

6. The method for isolating, amplifying and culturing rat NK cells according to claim 5, wherein The process of centrifuging and obtaining the rat PBMC cells The following steps are involved: Centrifuge at 1500 rpm for 30 minutes at room temperature; Pipette the cell fluid from the middle buffy coat layer into a container, add 1640 culture medium at a volume ratio of 1-1.5:5, mix well, and centrifuge at room temperature for 10 minutes at 1300 rpm. After centrifugation, the supernatant was discarded, the cells were resuspended in 1640 medium, washed, and centrifuged at room temperature for 10 min at 1000 rpm; After centrifugation, the supernatant was removed to obtain the rat PBMC cells.

7. The method for isolating, amplifying and culturing rat NK cells according to claim 1, wherein The process of obtaining rat NK cells by screening rat PBMC cells through CD161+ positive screening comprises the following steps: The rat PBMC cells were adjusted to a cell concentration of 4-5×10^6 cells / ml using 1640 culture medium, and Rat CD161-PE antibody was added at a volume ratio of 100-150:1, and incubated at room temperature in the dark for 30 minutes; Anti-PE magnetic beads were added at a volume ratio of 10-15:1, and the cells were incubated in the dark at room temperature for 25 to 30 minutes. CD161-positive cells were screened using a Miltenyi MACS system to obtain the rat NK cells.

8. The method for isolating, amplifying and culturing rat NK cells according to claim 1, wherein The method for separating, amplifying and culturing rat NK cells further comprises the following steps: On the 11th to 12th day of culture, cells were collected; The process of collecting cells is to centrifuge the cell suspension at room temperature, 1000 rpm, for 10 minutes, discard the supernatant, and repeat twice.

9. The method for isolating, amplifying and culturing rat NK cells according to claim 1, wherein The rat NK cells and the rat PBMC cells are derived from the same rat.

10. The method for isolating, amplifying and culturing rat NK cells according to claim 5, wherein: The rat peripheral blood is the rat peripheral blood taken from an adult rat within 5 minutes after death.

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