Method for improving lethality of umbilical cord blood-derived NK cells and application of method

Abstract

The invention provides a method for improving the killing power of umbilical cord blood-derived NK cells and application of the method. According to the method, immunomodulatory factors (such as IL-15 and IL-21) which are naturally rich in umbilical cord blood exosomes are used for promoting amplification and activation of the NK cells, and the killing power of the NK cells is enhanced; in addition, the exosome can improve the tumor microenvironment and reduce immunosuppression, so that the NK cells have stronger continuous killing ability in vivo and in vitro. Compared with single use of NK cells, the method can significantly improve the anti-tumor effect, and provides a safer, more efficient and more economical strategy for immune cell therapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for improving the killing power of NK cells derived from umbilical cord blood and an application thereof. Background Art

[0002] In recent years, immune cell therapy, as an emerging treatment strategy, has shown great application potential in many fields such as tumor immunotherapy, infectious diseases, and immune diseases. Compared with traditional treatments (such as chemotherapy and radiotherapy), immune cell therapy has obvious advantages. First, immune cell therapy can specifically identify and kill tumor cells, reduce damage to normal cells, and thus reduce side effects and toxic reactions. Secondly, immune cells can exert long-term anti-tumor effects through continuous proliferation and continuous immune surveillance during the treatment process. In addition, immune cells are highly plastic and adaptable, and can effectively respond to tumor immune escape mechanisms. With the continuous development of immune cell therapy technology, especially the further enhancement of immune cell function through cytokine engineering, gene editing and other means, the effect of immunotherapy has been significantly improved.

[0003] Natural killer cells (NK cells) in immune cells have become an important part of tumor immunotherapy due to their efficient targeted killing ability and low side effects. NK cells are part of the innate immune system. Unlike adaptive immune cells such as T cells and B cells, NK cells play an immune defense role by recognizing and directly killing mutant cells (such as tumor cells and virus-infected cells) without relying on antigen presentation or immune memory. This characteristic of NK cells makes them have great potential in the treatment of tumor immunotherapy, viral infection and other immune-related diseases. Compared with other immune cells, NK cells have the following unique advantages in immunotherapy: ① No need for antigen presentation: NK cells can directly recognize and kill abnormal cells through their activation receptors without relying on antigen presentation or specific antigen recognition; ② Breakthrough ability for tumor immune escape: NK cells can recognize cells lacking MHCI molecules and attack through other mechanisms; ③ Low immunogenicity and broad spectrum: NK cells have low immunogenicity, and their attack on allogeneic cells is not easy to induce a strong immune response. This allows NK cells to be used in allogeneic immunotherapy (such as NK cells from umbilical cord blood) and reduces the risk of immune rejection; ④ Self-renewal and rapid response ability: NK cells have strong self-renewal and proliferation capabilities and can quickly adapt to new immune challenges. Compared with T cells, NK cells can respond quickly without going through the antigen presentation process, reducing the delay time of treatment; ⑤ Lower side effects: Since the killing mechanism of NK cells is completed through receptor recognition and direct killing of target cells, it does not rely on large-scale immune amplification and cytokine secretion, so its side effects are lower, especially during the treatment process, it will not cause extensive damage to normal tissues like chemotherapy. The in vitro activation, amplification and improvement of the lethality of NK cells are important research directions for NK cells for immunotherapy.

[0004] Umbilical cord blood (UCB) is the blood that remains in the umbilical cord and placenta when the fetus is born. It is traditionally regarded as medical waste. However, with the deepening of research on immune cells, umbilical cord blood has gradually been found to be an important biomedical resource. Due to its unique cell composition, umbilical cord blood has been widely used in many medical fields, mainly including: hematopoietic stem cell transplantation, immunotherapy, tissue repair and autologous or allogeneic cell storage. At the same time, there are also a large number of primitive NK cells in umbilical cord blood, which are called umbilical cord blood NK cells (Umbilical Cord Blood Natural Killer Cell, UCB-NK).

[0005] Exosomes are small vesicles of nanometer size (30-150nm), which belong to a type of extracellular vesicles (EVs). They are formed by cells through endocytosis and released into the extracellular environment. Exosomes contain a variety of bioactive molecules, including proteins, lipids, mRNA, miRNA, etc., which can transmit signals between cells and regulate immune responses and cell behaviors. Umbilical cord blood plasma is rich in exosomes, which are derived from a variety of cell types, such as immune cells, hematopoietic stem cells, mesenchymal stem cells, etc. Due to the specificity of their source, exosomes in umbilical cord blood plasma show unique advantages in immunomodulation, anti-inflammatory effects and tissue repair.

[0006] At present, most of the NK cells used in clinical applications are derived from peripheral blood, while NK cells derived from umbilical cord blood have not been widely used due to the difficulty of obtaining and technical limitations. Even in a few applications, umbilical cord blood NK cells are usually used alone or in combination with specific antibody drugs. However, it is often difficult to achieve the desired therapeutic effect when using umbilical cord blood NK cells alone, and although the combined use with antibody drugs can enhance the efficacy, it will significantly increase the economic burden on patients and may cause additional side effects. Therefore, the present invention will propose a method for the combined use of umbilical cord blood NK cells and their plasma-derived exosomes to improve the lethality of NK cells. Summary of the invention

[0007] In view of this, the present invention aims to propose a method for improving the killing power of NK cells derived from umbilical cord blood and its application. This method uses the immunomodulatory factors (such as IL-15 and IL-21) naturally rich in exosomes to promote the proliferation and activation of NK cells and enhance their killing power; in addition, exosomes can improve the tumor microenvironment, reduce immunosuppression, and enable NK cells to have stronger sustained killing ability in vivo and in vitro. Compared with the use of NK cells alone, this method can significantly improve the anti-tumor effect and provide a safer, more efficient and economical strategy for immune cell therapy.

[0008] To achieve the above object, the technical solution of the present invention is achieved as follows: A method for improving the killing ability of NK cells derived from umbilical cord blood, the method comprising the following steps: S1. Isolation and extraction of exosomes from umbilical cord blood plasma; S2, separating mononuclear cells from umbilical cord blood plasma, then adding IL-2, IL-15, and IL-21, and adding umbilical cord blood plasma exosomes obtained in S1 to the culture system every 48 hours at a ratio of cell number: exosome particle number = 1:5-1:15 to promote NK cell expansion efficiency, and collecting the obtained NK cells; S3. The NK cells obtained in S2 were added with the cord blood plasma exosomes obtained in S1 at a ratio of 1:5-1:15 in terms of the number of cells: the number of exosome particles.

[0009] Furthermore, the specific method of S1 is: adding hydroxyethyl starch to anticoagulated umbilical cord blood to precipitate red blood cells, and taking the upper plasma layer after low-speed centrifugation; then the plasma layer is subjected to high-speed centrifugation to remove impurity proteins contained in the plasma, and then the plasma is filtered through an ultrafiltration membrane several times to remove immunoglobulins and albumin contained in the plasma, followed by ultracentrifugation, and then the plasma exosomes are collected.

[0010] Furthermore, the specific steps of S1 are: S11, adding 6% hydroxyethyl starch by mass fraction to 1 / 4 of the volume of the anticoagulated umbilical cord blood, mixing thoroughly and letting stand for 15-20 minutes; S12, low-speed centrifugation at 750 rpm for 10 min to remove red blood cell pellet; S13, the upper plasma was centrifuged at 4000 rpm for 30 min to remove impurities and cell debris; S14, balance ultrafiltration cup and ultrafiltration tube; S15, the clarified plasma suspension was filtered and centrifuged using a 500 kDa ultrafiltration tube at 4000 g for 30 min to remove immunoglobulin molecules, and the upper suspension was collected; S16, filter and centrifuge at 4000g for 30 min using a 100 kDa ultrafiltration tube to remove most of the albumin molecules, and collect the upper suspension; S17. Ultracentrifuge the upper suspension at 100,000 g for 1.5 h and collect the exosomes in the lower layer.

[0011] Furthermore, in S2, the concentration of IL-2 is 50-300 U / mL, the concentration of IL-15 is 5-20 ng / mL, and the concentration of IL-21 is 5-20 ng / mL.

[0012] Furthermore, in S2, the cell density was 1-1.5×10 6 / mL; and, the cells were counted every 48 hours, and NK cell complete medium was added to maintain the cell density at 1-1.5×10 6 Pieces / mL.

[0013] Furthermore, in S2, IL-2, IL-15, and IL-21 were added to the initial concentration every 48 hours, and 1-1.5×10 umbilical cord blood plasma exosomes were added to the culture system every 48 hours. 7 Particles / mL.

[0014] Furthermore, in S2, the cells are cultured for 14-21 days until the cell growth enters a stationary phase.

[0015] Further, the specific steps of S2 are: S21, centrifuging the anticoagulated umbilical cord blood at 2000 rpm for 20 min to remove the upper plasma; S22, add 0.9% saline solution by volume at a ratio of 1:1 to the lower cell suspension, mix thoroughly and dispense into centrifuge tubes; S23, slowly injecting the cell suspension onto the top of the Ficoll separation solution according to the volume ratio of cell suspension to Ficoll separation solution = 2:1, and then centrifuging at 2000 rpm for 20 min without brake; S24, collect the mononuclear cell layer in the centrifuge tube and wash it three times with 0.9% saline; S25, culture mononuclear cells, add interleukin 2 (IL-2), interleukin 15 (IL-15), and interleukin 21 (IL-21) to the mononuclear cells, and add umbilical cord blood plasma exosomes to the culture system every 48 hours at a ratio of cell number: exosome particle number = 1:10 to promote NK cell expansion efficiency; S26. After a culture period of 14-21 days, the obtained NK cells are collected.

[0016] Further, in S3, the co-incubation does not exceed 24 hours.

[0017] The present invention also provides an application of NK cells obtained by the above-mentioned method for improving the killing ability of NK cells derived from umbilical cord blood in killing tumor cells.

[0018] Compared with the prior art, the method for improving the killing ability of NK cells derived from umbilical cord blood and its application in the present invention have the following advantages: (1) The method for improving the killing power of umbilical cord blood-derived NK cells described in the present invention uses umbilical cord blood NK cells, which have the following unique advantages: ① Stronger expansion ability: umbilical cord blood NK cells have a higher proliferation potential during in vitro expansion, and a large number of highly active NK cells can be obtained in a short time, thereby improving the treatment efficiency; ② Lower immunogenicity: umbilical cord blood NK cells have a lower HLA expression level and better tissue compatibility with donors. Therefore, they are not prone to cause severe immune rejection reactions during allogeneic transplantation or cell therapy, and are suitable for "universal" immunotherapy; ③ Stronger killing activity: Studies have found that umbilical cord blood NK cells have stronger cytotoxicity than peripheral blood NK cells, and can more effectively identify and eliminate leukemia, lymphoma and solid tumor cells; ④ Rich in cytokines: Umbilical cord blood contains higher levels of immunomodulatory factors, such as IL-15 and IL-21, which help activate and maintain the function of NK cells, giving them a more lasting anti-tumor and antiviral effect in the body; ⑤ Less affected by the immunosuppressive microenvironment: Peripheral blood NK cells are susceptible to immunosuppression in the tumor microenvironment, while umbilical cord blood NK cells are relatively less susceptible to inhibition by tumor immune escape mechanisms, and therefore may have more advantages in tumor immunotherapy.

[0019] (2) The method of improving the killing power of NK cells derived from umbilical cord blood described in the present invention combines umbilical cord blood NK cells with exosomes derived from their plasma, which can play a synergistic role, improve the immunotherapy effect, and improve the killing ability of NK cells. Its advantages are reflected in the following aspects: ① Improve the expansion efficiency of NK cells: During in vitro expansion, umbilical cord blood exosomes can serve as natural growth promoting factors to increase the proliferation rate of NK cells, increase their number rapidly, and improve the feasibility of clinical application; ② Enhance the killing ability of NK cells: By providing immune activation factors, umbilical cord blood exosomes can promote the activation of NK cells, making them more effective in anti-tumor treatment; ③ Improve the tumor microenvironment and improve treatment tolerance: The tumor microenvironment usually inhibits the activity of NK cells, while umbilical cord blood exosomes can improve the survival and persistence of NK cells in the body by regulating immune factors, thereby improving the success rate of immunotherapy. Umbilical cord blood plasma exosomes are a natural immunomodulator that can promote the proliferation and activation of NK cells and improve their anti-tumor ability. Combined use with NK cells from umbilical cord blood can significantly enhance the effect of immunotherapy, improve the tumor microenvironment, and increase the safety and effectiveness of cell therapy. In the future, cell therapy strategies based on the combination of umbilical cord blood NK cells and exosomes are expected to become a new direction for tumor immunotherapy and cell therapy.

[0020] (3) The method of improving the killing power of NK cells derived from umbilical cord blood described in the present invention utilizes the immunomodulatory factors (such as IL-15 and IL-21) naturally rich in exosomes to promote the proliferation and activation of NK cells and enhance their killing power; in addition, exosomes can improve the tumor microenvironment, reduce immunosuppression, and enable NK cells to have stronger sustained killing ability in vivo and in vitro. Compared with the use of NK cells alone, this method can significantly improve the anti-tumor effect and provide a safer, more efficient and economical strategy for immune cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings: Figure 1 This is an electron microscopic image of cord blood plasma exosomes; Figure 2 This is the particle size diagram of exosomes in umbilical cord blood plasma; Figure 3 The expression of surface proteins of exosome particles detected by flow cytometry; (a) is the positive expression rate of CD9, and (b) is the positive expression rate of CD81; Figure 4 Figure 1 is a picture of NK cell culture, (a) is the NK cells cultured in the patent group (group A) on the 14th day, (b) is the NK cells cultured in the control group B on the 14th day; (c) is the NK cells cultured in the control group C on the 14th day; Figure 5 This is a comparison chart of the expansion efficiency of three groups of NK cells; Figure 6 The flow cytometry images of three groups of NK cells are shown in FIG. (a) is the flow cytometry phenotype of NK cells of the patent group (group A) on the 14th day; (b) is the flow cytometry phenotype of NK cells of the control group B on the 14th day; and (c) is the flow cytometry phenotype of NK cells of the control group C on the 14th day. Figure 7 The lysis rate of K562 cells was compared 6 hours after NK cells and K562 cells were inoculated in the E+T group according to different schemes. DETAILED DESCRIPTION

[0022] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Example 1 Isolation and extraction of exosomes from umbilical cord blood plasma Take 100 mL of umbilical cord blood anticoagulated with sodium citrate, add hydroxyethyl starch to precipitate red blood cells, and take the upper plasma layer after low-speed centrifugation. After high-speed centrifugation, remove a large amount of impurity proteins such as fibrin contained in the plasma, and then remove the immunoglobulin and albumin contained in the plasma through several ultrafiltration membrane filtrations, and then collect plasma exosomes after ultracentrifugation.

[0025] The specific implementation is as follows: 1. Pipette 100 mL of umbilical cord blood anticoagulated with sodium citrate into a 200 mL centrifuge bottle, add 25 mL of 6% hydroxyethyl starch (B. Braun, Germany, item number: L6511) into the culture bottle, mix thoroughly and let stand for 15-20 minutes; 2. Centrifuge the centrifuge bottle at 750 rpm for 10 min; 3. Take out the centrifuge bottle, carefully aspirate the upper plasma layer to about 2 mm away from the red blood cell layer, and place the upper plasma layer in a new centrifuge bottle; 4. Centrifuge the centrifuge bottle at 4000 rpm for 30 min to remove residual cells, cell debris, fibrinogen and other substances; 5. Add 80 mL of 0.9% saline solution to the Amicon® stirred ultrafiltration cup-500 kDa (Millipore, catalog number: UFSC20001), place in a centrifuge and centrifuge at 4000 g for 30 min, balance the ultrafiltration cup, and discard the lower layer of liquid after centrifugation; 6. Take the upper plasma layer after centrifugation in step 4, and aspirate the plasma layer into the balanced Amicon® stirred ultrafiltration cup-500kDa, centrifuge at 4000g for 30min, aspirate the suspension on the upper layer of the filter membrane into a new centrifuge tube, and repeatedly rinse the filter membrane with 10mL of 0.9% saline solution, and mix the rinsed solution with the upper layer solution of the filter membrane; 7. Add 50 mL of 0.9% saline solution to a Centricon Plus 70 ultrafiltration tube -100 kDa (Millipore, catalog number: UFC710008), place in a centrifuge and centrifuge at 4000 g for 30 min, balance the ultrafiltration cup, and discard the lower layer of liquid after centrifugation; 8. Pipette the solution in step 6 into a Centricon Plus 70 ultrafiltration tube -100 kDa, centrifuge at 4000 g for 30 min, pipette the suspension on the upper layer of the filter membrane into a new centrifuge tube, and repeatedly rinse the filter membrane with 5 mL of 0.9% saline solution, and mix the rinsed solution with the upper layer solution of the filter membrane; 9. Place the solution in several ultracentrifuge tubes (Beckman Coulter, Catalog No.: 361625), balance them and place them in an ultracentrifuge (Beckman Coulter Optimal XE-90, Catalog No. A99833) at 100,000 g for 90 min. 10. After centrifugation, discard the upper solution of the ultracentrifuge tube, resuspend the lower precipitate with an appropriate amount of 0.9% saline solution, then fill the solution into the ultracentrifuge tube and repeat step 9; 11. Discard the upper solution after centrifugation, and the lower sediment is the cord blood plasma exosomes. Resuspend the exosome particles with an appropriate amount of 0.9% saline for subsequent detection and preservation.

[0026] Example 2 Cultivation and Expansion of Umbilical Cord Blood NK Cells Centrifuge 100 mL of umbilical cord blood anticoagulated with sodium citrate to remove the upper plasma, add an equal volume of 0.9% saline to the lower cell suspension, mix well, and perform ficoll density gradient centrifugation to obtain the middle mononuclear cell layer. Spread it in a culture bottle, add IL-2, IL-15, and IL-21 cytokines to the culture bottle, and add exosome particles separated from plasma. At the same time, set up a control group (the control group is a culture program without adding exosome particles), and culture it in a serum-free culture system for 14-21 days to harvest NK cells.

[0027] The specific implementation is as follows: 1. Take 100 mL of umbilical cord blood anticoagulated with sodium citrate and divide it into 4 50 mL centrifuge tubes, 25 mL in each tube. Place the centrifuge tubes in a centrifuge and centrifuge at 2000 rpm for 20 minutes. After centrifugation, take out the centrifuge tubes and discard the upper plasma with a pipette. 2. Add equal volume of 0.9% saline injection to the red blood cell suspension in the lower layer and mix thoroughly; 3. Take 4 50mL centrifuge tubes, add 15mL Ficoll lymphocyte separation solution (Tianjin Haoyang, product number: LDS1075CB) into the centrifuge tubes, and slowly add 30mL of cell suspension to the upper layer of lymphocyte separation solution without breaking the separation layer; 4. Place the centrifuge tube in a centrifuge and perform density gradient centrifugation at 2000 rpm for 20 min, with the brake speed set to 0; 5. After centrifugation, take out the centrifuge tube and discard the upper solution. Use a sterile Pasteur pipette to carefully aspirate the middle white film layer cells, i.e. the umbilical cord blood mononuclear cell layer, and place it in a new 50mL centrifuge tube; 6. Add 0.9% saline to the cell suspension to 50 mL, centrifuge at 1500 rpm for 5 min to rinse; discard the upper solution after rinsing, and continue to rinse the cell pellet with saline, repeating three times; 7. Resuspend the cell pellet in NK cell complete medium and adjust the cell density to 1-1.5×10 6 / mL. The complete medium is prepared by adding 1% culture supplement (NK MACS® supplement, human, catalog number: 130-114-429) to NK cell basal medium (NK MACS® Medium, human, catalog number: 130-114-429), and adding 5% human AB serum (GeminiBio, catalog number: 100-912-100) and placing in a culture flask for culture; 8. Add 100U / mL IL-2 (Tongli Haiyuan, catalog number: GMP-TL906), 10ng / mL IL-15 (Tongli Haiyuan, catalog number: GMP-TL202-0050), and 10ng / mL IL-21 (Tongli Haiyuan, catalog number: GMP-TL509-0050) to the cell suspension; 9. Count the cells every 48 hours and add NK cell complete medium to maintain the cell density at 1-1.5×10 6 IL-2, IL-15, and IL-21 growth factors were added every 48 hours, and 1-1.5×10 umbilical cord blood plasma exosomes were added to the culture system every 48 hours. 7 Particles / mL, after the cells grow vigorously, transfer them into culture bags for further culture; 10. Cultivate NK cells for 21 days according to the above culture method and draw a growth curve until the cell growth enters the plateau phase; 11. The cultured cells were taken out from the culture bag and aspirated into several 200 mL centrifuge bottles. After centrifugation at 1500 rpm for 15 min, the supernatant was discarded and the cell pellet was resuspended in NK cell complete medium to obtain umbilical cord blood-derived NK cells, which were recorded as Group A.

[0028] Control group B (hereinafter referred to as group B): The difference from the above NK cell culture and expansion method is that exosome particles are not added in step 9.

[0029] Control group C (hereinafter referred to as group C): The difference from the above-mentioned NK cell culture and expansion method is that the umbilical cord blood plasma exosomes are replaced with exosomes derived from mesenchymal stem cells (MSC), and the extraction of the exosomes can adopt conventional extraction methods.

[0030] Experiment 1 Transmission electron microscopy observation of exosome particles The specific operation method is as follows: 1. Take 50 μml of the isolated and purified exosome precipitate, add an equal volume of 2.5% glutaraldehyde, and place it in a 4°C refrigerator for fixation for 1 hour; 2. Add 20 μl of the fixed exosome suspension onto the front of the electron microscope copper grid and let it stand for 20 minutes; 3. Carefully absorb the excess solution with absorbent filter paper; then wash the copper mesh with ultrapure water 5 times, each time for 30 seconds, and dry it with filter paper; 4. Add 1 drop of 2% uranyl acetate dye solution to the front of the copper mesh, dye for 1 minute, and then use filter paper to absorb the excess dye along the edge of the copper mesh; 5. Place the copper mesh in air at room temperature to dry naturally, and then observe it on the machine after it is dry.

[0031] The results are as follows Figure 1 As shown, under an electron microscope, the umbilical cord blood plasma exosomes have a typical double-layer membrane structure, a unique "tray-like" structure of exosomes, and a complete capsule. This shows that the umbilical cord blood plasma exosome extraction method described in this patent is feasible, simple, and has good effects.

[0032] At the same time, the particle size of cord blood plasma exosomes was analyzed, and the results were as follows Figure 2 As shown in the figure, after NTA detection, the exosome particle size has a single peak at less than 200nm, with a peak value of about 103nm, and the average length of most particles is between 30-200nm, which is consistent with the particle size characteristics of exosomes.

[0033] Experiment 2 Detection of exosome surface protein expression by flow cytometry The specific operation method is as follows: 1. Mix the components of Thermofisher CD63 magnetic beads (Cat. No. 10606D) by inversion for 10 minutes; pipette 20 μl of the magnetic bead suspension into a 1.5 mL round-bottom EP tube; 2. Add 200 μl of magnetic bead cleaning solution to the EP tube and mix thoroughly with a pipette tip; 3. Place the EP tube on the magnetic rack for 1 minute, then discard the supernatant; 4. Take 50 μl of the extracted plasma exosome suspension, add 50 μl of washing solution to a final volume of 100 μl, and mix thoroughly; 5. Place the exosome-washing solution mixture on a rotary mixer, set the speed to 10 rpm, and incubate overnight at 2-8°C; 6. The next day, centrifuge the sample for 3 to 5 seconds to collect the precipitate; 7. Add 300 μl of cleaning solution to the sample and mix thoroughly with a pipette tip for 30 seconds; 8. Place the sample on the magnetic rack for about 1 minute and discard the supernatant; 9. Add 400 μl of cleaning solution to the sample and mix thoroughly with a pipette tip for 30 seconds; 10. Place the sample on the magnetic rack for about 1 minute, discard the supernatant, and resuspend with 300 μl of washing solution; 11. Take 100 μl of each sample, add CD9-PE and CD81-FITC flow cytometry antibodies to the samples, and incubate at 4°C in the dark for 30 minutes; 12. Place the incubated sample on the magnetic rack for about 1 minute, discard the supernatant, and add 300 μl of cleaning solution for washing; 13. Repeat step 12 once, resuspend the selected sample with 300 μl PB and then test on the instrument.

[0034] The results are as follows Figure 3 As shown, the flow cytometry detection of the exosome particles obtained by the method of the present invention showed that the positive expression rate of CD9 was 94.37%, and the positive expression rate of CD81 was 97.71%, both of which met the surface protein expression requirements of exosomes.

[0035] Experiment 3 NK cell phenotype detection 1. The density of the amplified NK cell suspension obtained in Example 2 was adjusted to 1×10 6 / mL, take a flow tube, add 5μl of CD3-FITC antibody (Invitrogen) and 5μl of CD16CD56-PE antibody (Invitrogen) to the tube, add 200μl of cell suspension to the flow tube, shake and mix thoroughly, incubate at 4°C in the dark for 30min, after incubation, add 1ml of PBS to the tube, centrifuge at 300g, 5min, 4°C; after centrifugation, discard the supernatant, resuspend the precipitate with 500μl PBS, mix well and test on the flow tube; 2. Turn on the BD FACSCantoII flow cytometer, open the FACSDiva analysis software after preheating, create an FSC-SSC scatter plot, set gate P1 for lymphocytes and monocytes, and create a four-quadrant scatter plot FL1-FL2, with CD3 as the horizontal axis and CD56 as the vertical axis, observe the phenotype of the amplified NK cells, and calculate the amplification efficiency and phenotype differences of the NK cells in groups AC. The results are as follows: Figure 4-6 shown.

[0036] from Figure 4It can be seen that in Group A of this patent, i.e., exosomes derived from umbilical cord blood plasma were added during the culture of NK cells. The growth state of NK cells was observed on the 14th day. The cells grew in clusters and were in good growth state. There were more cells in the field of view. Control group B only used conventional cytokine culture method, i.e., no exosomes were added. On the 14th day of culture, the cells were obviously not aggregated, but grew scattered. The cells were in good condition, and there were fewer cells in the field of view than in this patent group. Control group C, i.e., exosomes derived from mesenchymal stem cells were added during the culture of NK cells. On the 14th day of culture, the cells grew slightly in clusters and were in good growth state. The cells in the field of view grew denser than those in control group B, but the density of cells in the field of view was slightly lower than that in Group A of this patent.

[0037] from Figure 5 It can be seen that the total number of NK cells in the three groups at the initial day 0 of culture was about 0.7×10 8 cells. On the 7th day, the number of NK cells in group A of this patent was (3.80±0.294)×10 8 cells, and the number of NK cells in the control group B was (4.53±0.368)×10 8 cells, and the number of NK cells in the control group C was (4.17±0.116)×10 8 cells; On the 14th day, the number of NK cells in group A of this patent was (38.22±3.21)×10 8 cells, and the number of NK cells in the control group B was (30.61±0.697)×10 8 cells, and the number of NK cells in the control group C was (33.27±1.970)×10 8 cells; On the 21st day, the number of NK cells in group A of this patent was (113.50±5.97)×10 8 cells, and the number of NK cells in the control group B was (80.83±5.69)×10 8 cells, and the number of NK cells in the control group C was (92.45±4.89)×10 8 cells; the final expansion multiple of the NK cells in group A of the present patent after 21 days of culture is about 162.1 times, the final expansion multiple of the NK cells in control group B after 21 days of culture is about 115.5 times, and the final expansion multiple of the NK cells in control group C after 21 days of culture is about 132.1 times.

[0038] from Figure 6 It can be seen that on the 14th day of culture, three groups of NK cells were taken for flow cytometry detection. The proportion of CD3-CD16 / 56+ in the NK cell surface marker detection of the patent group was 91.8%, which was better than the proportion of 80.3% of the surface marker of the control group B and the proportion of 86.5% of the surface marker of the control group C.

[0039] From the above, it can be found that adding exosomes derived from umbilical cord blood plasma during the culture of NK cells can more effectively improve the cell expansion efficiency than not adding exosomes and adding exosomes derived from mesenchymal stem cells.

[0040] Experiment 4 In vitro killing experiment of NK cells on K562 cells LDH release method (lactate dehydrogenase release method) is an experimental method commonly used in cytotoxicity detection, mainly used to evaluate the degree of cell damage or cell death. Its principle is based on the relationship between the integrity of the cell membrane and the activity of lactate dehydrogenase (LDH) enzyme. When cells are stimulated (such as drugs, radiation, damage), the cell membrane may be damaged, causing substances inside the cell (such as LDH) to be released into the extracellular fluid. By detecting the activity of LDH released in the culture medium, the degree of cell damage or death can be indirectly reflected, thereby calculating the killing ability of effector cells.

[0041] The specific implementation is as follows: 1. Take out K562 cells from liquid nitrogen and revive them in a 37°C water bath. Resuspend the cells in RPMI1640 medium containing 10% FBS and centrifuge at 1500 rpm for 5 minutes to remove the freezing solution. Adjust the inoculation density to 0.5-1×10 6 cells / mL, inoculated into culture flasks, maintained in RPMI-1640 medium (containing 10% FBS) in a 37°C, 5% CO2 incubator, and replaced the medium or subcultured every 2-3 days to ensure that the cells were in good condition.

[0042] 2. Take K562 cells in the logarithmic growth phase, wash them twice with PBS, and adjust the concentration to 1×10 5 cells / mL, and trypan blue staining was used to ensure that the cell viability was greater than 95%.

[0043] 3. Take a 24-well plate and divide it into groups as follows (three replicates per well, the NK cells in the control group are the same as those in this patent method): (1) Experimental group (E+T group): The NK cells and K562 cells of group A and group B obtained in Example 2 were inoculated according to different effector-target ratios (E:T ratio, i.e., NK cell concentration: K562 cell concentration), and the effector-target ratios were 0.25:1, 0.5:1, 1:1, and 2:1; that is, the concentration of NK cells was adjusted to 0.125×10 5 cells / mL、0.25×10 5 cells / mL、0.5×10 5 cells / mL、1×10 5 cells / mL, the specific scheme is as follows: This patent group: first add exosome particles to the NK cells of group A and the exosomes derived from umbilical cord blood plasma at a ratio of cells: exosome particles equal to 1:10, and incubate for no more than 24 hours; then add K562 cells according to the effector-target ratio.

[0044] Exosome-only expansion group: NK cells in group A were directly added to K562 cells according to the effector-target ratio without being co-incubated with exosomes first.

[0045] Simple exosome incubation group: first add exosome particles to the NK cells of group B and the exosomes derived from umbilical cord blood plasma at a ratio of cells: exosome particles equal to 1:10, and incubate for no more than 24 hours; then add K562 cells according to the effector-target ratio.

[0046] Control group (without exosomes): NK cells in group B were directly added to K562 cells according to the effector-target ratio without prior co-incubation with exosomes.

[0047] (2) Target cell spontaneous release control group (Group T): Only K562 cells were inoculated without NK cells.

[0048] (3) Effector cell spontaneous release control group (Group E): Only NK cells of each group were inoculated without K562 cells.

[0049] (4) Complete lysis positive control group (Max LDH group): Add cell lysis buffer (providing maximum LDH release) to K562 cells.

[0050] (5) Blank control group: only culture medium was added without any cells.

[0051] 5. Add cells from each group to a 24-well plate and adjust the total volume of cells per well to 200 µL as follows: Table 1 Experimental conditions of each group

[0052] 6. After gently shaking to mix, place the 24-well plate in a 37°C, 5% CO2 incubator and incubate for 6 hours; 7. After incubation, centrifuge at 400g for 5 min and collect 100 μl of supernatant into a 96-well plate; 8. According to the instructions of the CyQUANT LDH kit (Thermofisher, Cat. No. C20300), add 100 µL of LDH reaction substrate and incubate at room temperature for 30 min in the dark; 9. After terminating the reaction, measure the absorbance at 490nm (OD: 490nm); 10. Calculate the killing power of NK cells in each group against target cells using the following formula: Cell killing efficacy %=[(OD value of experimental group - OD value of target cell spontaneous release control group - OD value of effector cell spontaneous release control group) / (OD value of complete lysis positive control group - OD value of target cell spontaneous release control group - OD value of effector cell spontaneous release control group)]×100%; 11. Draw the killing efficacy curve of each group and calculate the feasibility of this patented method in improving the killing efficacy of NK cells in vitro.

[0053] Figure 7 It is the lysis rate (i.e., cell killing efficacy) of K562 cells after 6 hours of inoculation of NK cells and K562 cells in the E+T group according to different schemes. The control group in the figure is a blank control group of culture medium. It can be seen from the figure that the killing ability of NK cells in the four groups of schemes for 6 hours against K562 target cells has different killing abilities under different effect-target ratios. When the effect-target ratio is 0.25:1, the killing power of this patent group and other groups is 32.8%, 21.5%, 27.2% and 19.6% respectively; when the effect-target ratio is 0.5:1, the killing power of this patent group and other groups is 58.6%, 42.9%, 40.3% and 34.7% respectively; when the effect-target ratio is 1:1, the killing power of this patent group and other groups is 85.9%, 74.1%, 80.7% and 73.5% respectively; when the effect-target ratio is 2:1, the killing power of this patent group and other groups is 99.9%, 98.7%, 98.9% and 96.3% respectively. This indicates that under the same effector-target ratio, the killing efficiency of NK cells prepared by this patent group is better than that of other control groups. In particular, under the condition of a lower effector-target ratio (≤1), the killing ability of NK cells of this patent group on target cells is significantly better than that of NK cells of other control groups.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for improving the killing ability of NK cells derived from umbilical cord blood, characterized in that: The method comprises the following steps: S1. Isolation and extraction of exosomes from umbilical cord blood plasma; S2, separating mononuclear cells from umbilical cord blood plasma, then adding IL-2, IL-15, and IL-21, and adding umbilical cord blood plasma exosomes obtained in S1 to the culture system every 48 hours at a ratio of cell number: exosome particle number = 1:5-1:15 to promote NK cell expansion efficiency, and collecting the obtained NK cells; S3. The NK cells obtained in S2 were added with the cord blood plasma exosomes obtained in S1 at a ratio of 1:5-1:15 in terms of the number of cells: the number of exosome particles.

2. The method for improving the killing ability of NK cells derived from umbilical cord blood according to claim 1, characterized in that: The specific method of S1 is: add hydroxyethyl starch to the anticoagulated umbilical cord blood to precipitate the red blood cells, and take the upper plasma layer after low-speed centrifugation; then the plasma layer is subjected to high-speed centrifugation to remove the impurity proteins contained in the plasma, and then filtered through an ultrafiltration membrane several times to remove the immunoglobulins and albumin contained in the plasma, and then ultracentrifuged to collect the plasma exosomes.

3. The method for improving the killing ability of NK cells derived from umbilical cord blood according to claim 1, characterized in that: In S2, the concentration of IL-2 is 50-300 U / mL, the concentration of IL-15 is 5-20 ng / mL, and the concentration of IL-21 is 5-20 ng / mL.

4. The method for improving the killing ability of NK cells derived from umbilical cord blood according to claim 1, characterized in that: S2, the cell density is 1-1.5×10 6 / mL; and, the cells were counted every 48 hours, and NK cell complete medium was added to maintain the cell density at 1-1.5×10 6 Pieces / mL.

5. The method for improving the killing ability of NK cells derived from umbilical cord blood according to claim 4, characterized in that: In S2, IL-2, IL-15, and IL-21 were added to the initial concentration every 48 hours, and 1-1.5×10 umbilical cord blood plasma exosomes were added to the culture system every 48 hours. 7 Particles / mL.

6. The method for improving the killing ability of NK cells derived from umbilical cord blood according to claim 1, characterized in that: In S2, the cells are cultured for 14-21 days until the cell growth enters a stationary phase.

7. The method for improving the killing ability of NK cells derived from umbilical cord blood according to claim 1, characterized in that: Incubate for no longer than 24 hours in S3.

8. Use of NK cells obtained by the method for improving the killing ability of umbilical cord blood-derived NK cells according to any one of claims 1 to 7 in killing tumor cells.

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