A method for enhancing the killing ability of cord blood-derived NK cells and its application

Abstract

The present invention provides a method for enhancing the killing power of umbilical cord blood-derived NK cells and its application. This method utilizes immune regulatory factors (such as IL-15, IL-21) naturally rich in umbilical cord blood exosomes to promote the expansion 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 continuous 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 provides a safer, more efficient and more economical strategy for immunocyte therapy.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly to a method for enhancing the killing ability of cord blood-derived NK cells and its application. Background Art

[0002] In recent years, as an emerging treatment strategy, immunotherapy has shown great application potential in multiple fields such as cancer immunotherapy, infectious diseases, and immune diseases. Compared with traditional treatment methods (such as chemotherapy and radiotherapy), immunotherapy has obvious advantages. First of all, immunotherapy can specifically recognize and kill tumor cells, reducing the damage to normal cells, thereby reducing side effects and toxic reactions. Secondly, immune cells can play a long-term anti-tumor effect through continuous proliferation and continuous immune surveillance during the treatment process. In addition, immune cells have high plasticity and adaptability, and can effectively respond to the tumor immune escape mechanism. With the continuous development of immunotherapy technology, especially the further enhancement of immune cell functions through means such as cytokine engineering and gene editing, the effect of immunotherapy has been significantly improved.

[0003] Natural killer cells (NK cells) in immune cells have become an important part of cancer immunotherapy due to their efficient targeted killing ability and low side effects. NK cells are part of the innate immune system. Different from adaptive immune cells such as T cells and B cells, NK cells play an immune defense role by recognizing and directly killing abnormal cells (such as cancer cells and virus-infected cells) without relying on antigen presentation or immune memory. This characteristic of NK cells makes them have great potential in cancer immunotherapy, virus infection, and the treatment of other immune-related diseases. Compared with other immune cells, NK cells have the following unique advantages in immunotherapy: ① No antigen presentation required: NK cells can directly recognize and kill abnormal cells through their activating receptors without relying on antigen presentation or specific antigen recognition; ② Ability to break through tumor immune escape: NK cells can recognize cells lacking MHC I molecules and attack them through other mechanisms; ③ Low immunogenicity and broad spectrum: NK cells have low immunogenicity, and their attack on allogeneic cells is not likely to trigger a strong immune response. This enables NK cells to be used in allogeneic immunotherapy (such as umbilical cord blood-derived NK cells) and reduces the risk of immune rejection; ④ Self-renewal and rapid response ability: NK cells have strong self-renewal and proliferation abilities 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 treatment delay time; ⑤ Lower side effects: Since the killing mechanism of NK cells is completed by receptor recognition and direct killing of target cells without relying on large-scale immune amplification and cytokine secretion, their side effects are lower, especially during treatment, they do not cause extensive damage to normal tissues like chemotherapy. The in vitro activation, expansion, and improvement of the killing ability of NK cells are important research directions for the use of NK cells in immunotherapy.

[0004] Umbilical cord blood (UCB) is the blood remaining in the umbilical cord and placenta at the time of fetal birth and has traditionally been regarded as medical waste. However, with the in-depth study of 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 multiple 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, called umbilical cord blood NK cells (Umbilical Cord Blood Natural Killer Cell, UCB-NK).

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

[0006] Currently, most NK cells used in clinical applications are derived from peripheral blood. Due to the difficulty of acquisition and technical limitations, NK cells derived from umbilical cord blood have not been widely used. Even in a few applications, umbilical cord blood NK cells are usually used alone or in combination with specific antibody drugs for treatment. However, using umbilical cord blood NK cells alone often fails to achieve ideal therapeutic effects, and although using them in combination 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 application of umbilical cord blood NK cells and exosomes derived from their plasma to improve the killing ability of NK cells. Summary of the Invention

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

[0008] To achieve the above object, the technical solution of the present invention is realized as follows:

[0009] A method for improving the killing ability of umbilical cord blood-derived NK cells, the method comprising the following steps:

[0010] S1. Isolate and extract exosomes from umbilical cord blood plasma;

[0011] S2. Isolate mononuclear cells from umbilical cord blood plasma, then add IL-2, IL-15, IL-21, and every 48 hours, add the umbilical cord blood plasma exosomes obtained in S1 to the culture system according to the ratio of the number of cells: the number of exosome particles = 1:5 - 1:15 to promote the amplification efficiency of NK cells, and collect the obtained NK cells;

[0012] S3. Add the umbilical cord blood plasma exosomes obtained in S1 to the NK cells obtained in S2 at a ratio of the number of cells to the number of exosome particles of 1:5 - 1:15 and co-incubate them.

[0013] Furthermore, the specific method of S1 is as follows: Add anticoagulated umbilical cord blood to hydroxyethyl starch to precipitate red blood cells, and take the upper plasma layer after low-speed centrifugation; then, after high-speed centrifugation of the plasma layer, remove the impurity proteins contained in the plasma, and then after several ultrafiltration membrane filtrations to remove the immunoglobulins and albumin contained in the plasma, perform ultra-high-speed centrifugation and collect the plasma exosomes.

[0014] Even further, the specific steps of S1 are as follows:

[0015] S11. Add 6% hydroxyethyl starch with a mass fraction of 1 / 4 of the umbilical cord blood volume to the anticoagulated umbilical cord blood, mix well and let it stand for 15 - 20 min;

[0016] S12. Centrifuge at 750 rpm for 10 min to remove the red blood cell precipitate;

[0017] S13. Centrifuge the upper plasma at 4000 rpm for 30 min to remove impurity cells and cell debris;

[0018] S14. Balance the ultrafiltration cup and ultrafiltration tube;

[0019] S15. Filter and centrifuge the clarified plasma suspension with a 500 kDa ultrafiltration tube at 4000g for 30 min to remove immunoglobulin molecules, and collect the upper suspension;

[0020] S16. Filter and centrifuge with a 100 kDa ultrafiltration tube at 4000g for 30 min to remove most of the albumin molecules, and collect the upper suspension;

[0021] S17. Perform ultra-high-speed centrifugation of the upper suspension at 100000g for 1.5 hours and collect the lower exosomes.

[0022] 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.

[0023] Furthermore, in S2, the cell density is 1 - 1.5×10 6 cells / mL; and, count the cells every 48 hours and supplement the NK cell complete medium to maintain the cell density at 1 - 1.5×10 6 cells / mL.

[0024] Further, in S2, IL-2, IL-15, and IL-21 need to be replenished to the initial concentration every 48 hours, and umbilical cord blood plasma exosomes at 1 - 1.5×10 7 particles / mL are added to the culture system every 48 hours.

[0025] Further, in S2, the cells are cultured for 14 - 21 days until the cell growth enters the plateau phase.

[0026] Further, the specific steps of S2 are as follows:

[0027] S21: Centrifuge the anticoagulated umbilical cord blood at 2000 rpm for 20 min to remove the upper plasma;

[0028] S22: Add 0.9% saline by mass in a volume ratio of 1:1 to the lower cell suspension, mix well and then aliquot into centrifuge tubes;

[0029] S23: Slowly inject the cell suspension above the Ficoll separation solution in a volume ratio of cell suspension:Ficoll separation solution = 2:1, and then perform brake-free centrifugation at 2000 rpm for 20 min;

[0030] S24: Collect the mononuclear cell layer in the centrifuge tube and wash it three times with 0.9% saline;

[0031] S25: Culture the 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 at a ratio of cell number:exosome particle number = 1:10 every 48 hours to promote the expansion efficiency of NK cells;

[0032] S26: After a culture period of 14 - 21 days, collect the obtained NK cells.

[0033] Further, in S3, the co-incubation is not more than 24 hours.

[0034] The present invention also provides an application of NK cells obtained by the method for enhancing the killing ability of umbilical cord blood-derived NK cells as described above in killing tumor cells.

[0035] Compared with the prior art, the method for enhancing the killing ability of umbilical cord blood-derived NK cells and its application of the present invention have the following advantages:

[0036] (1) The method for enhancing the killing power of cord blood-derived NK cells in the present invention uses cord blood NK cells and has the following unique advantages: ① Stronger amplification ability: Cord blood NK cells have higher proliferation potential during in vitro amplification, and a large number of highly active NK cells can be obtained in a short time, improving the treatment efficiency; ② Lower immunogenicity: Cord blood NK cells have a lower HLA expression level and better histocompatibility with donors. Therefore, they are less likely 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 cord blood NK cells have stronger cytotoxicity compared to peripheral blood NK cells and can more effectively recognize and eliminate leukemia, lymphoma, and solid tumor cells; ④ Rich in cytokines: Cord blood contains relatively high levels of immunomodulatory factors such as IL-15 and IL-21, which help activate and maintain the function of NK cells, enabling them to have a more persistent anti-tumor and antiviral effect in vivo; ⑤ Less affected by the immunosuppressive microenvironment: Peripheral blood NK cells are easily immunosuppressed in the tumor microenvironment, while cord blood NK cells are relatively less inhibited by the tumor immune escape mechanism. Therefore, they may have more advantages in tumor immunotherapy.

[0037] (2) The method for enhancing the killing power of cord blood-derived NK cells in the present invention combines cord blood NK cells with exosomes derived from their plasma, which can play a synergistic role, enhance the immunotherapy effect, and improve the killing ability of NK cells. Its advantages are reflected in the following aspects: ① Improving the amplification efficiency of NK cells: During in vitro amplification, cord blood exosomes can act as natural growth-promoting factors to increase the proliferation rate of NK cells, rapidly increasing their number and improving the feasibility of clinical application; ② Enhancing the killing ability of NK cells: By providing immune activation factors, cord blood exosomes can promote the activation of NK cells, making their effect stronger in anti-tumor treatment; ③ Improving the tumor microenvironment and enhancing treatment tolerance: The tumor microenvironment usually inhibits the activity of NK cells, while cord blood exosomes can improve the survival and persistence of NK cells in vivo by regulating immune factors, increasing the success rate of immunotherapy. Cord blood plasma exosomes are a natural immunomodulator that can promote the proliferation, activation of NK cells, and enhance their anti-tumor ability. When used in combination with cord blood-derived NK cells, it can significantly enhance the immunotherapy effect, improve the tumor microenvironment, and enhance the safety and effectiveness of cell therapy. In the future, the cell therapy strategy based on the combination of cord blood NK cells and exosomes is expected to become a new direction for tumor immunotherapy and cell therapy.

[0038] (3)The method for enhancing the killing power of umbilical cord blood-derived NK cells in the present invention utilizes the immunomodulatory factors (such as IL-15, IL-21) naturally rich in exosomes to promote the expansion 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 continuous killing ability in vivo and in vitro. Compared with the sole use of NK cells, this method can significantly improve the anti-tumor effect and provide a safer, more efficient, and more economical strategy for immunocyte therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic 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 drawings:

[0040] Figure 1 is the electron microscopy image of umbilical cord blood plasma exosomes;

[0041] Figure 2 is the particle size diagram of umbilical cord blood plasma exosomes;

[0042] Figure 3 is the graph of the surface protein expression of exosome particles detected by flow cytometry; (a) is the positive expression rate of CD9, and (b) is the positive expression rate of CD81;

[0043] Figure 4 is the diagram of NK cell culture. (a) are the NK cells cultured in the patent group (Group A) on the 14th day, (b) are the NK cells cultured in Control Group B on the 14th day; (c) are the NK cells cultured in Control Group C on the 14th day;

[0044] Figure 5 is the comparison graph of the amplification efficiency of NK cells in three groups;

[0045] Figure 6 is the flow cytometry detection graph of NK cells in three groups. (a) is the flow cytometry phenotype of NK cells in the patent group (Group A) on the 14th day; (b) is the flow cytometry phenotype of NK cells in Control Group B on the 14th day, and (c) is the flow cytometry phenotype of NK cells in Control Group C on the 14th day;

[0046] Figure 7 is the comparison of the lysis rate of K562 cells after inoculating NK cells and K562 cells according to different protocols in the E+T group for 6 hours. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0047] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0048] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0049] Example 1 Isolation and Extraction of Cord Blood Plasma Exosomes

[0050] Take 100 mL of 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 of the plasma layer, a large amount of impurity proteins such as fibrin in the plasma are removed, and then after several ultrafiltration membrane filtrations to remove immunoglobulins and albumin contained in the plasma, ultracentrifugation is performed to collect plasma exosomes.

[0051] The specific implementation method is as follows:

[0052] 1. Aspirate 100 mL of cord blood anticoagulated with sodium citrate into a 200 mL centrifuge bottle, add 25 mL of 6% hydroxyethyl starch (Braun, Germany, product number: L6511) to the culture bottle, mix well and let stand for 15 - 20 min;

[0053] 2. Centrifuge the centrifuge bottle at 750 rpm for 10 min;

[0054] 3. Take out the centrifuge bottle, carefully aspirate the upper plasma layer to about 2 mm from the red blood cell layer, and place the upper plasma layer in a new centrifuge bottle;

[0055] 4. Centrifuge the centrifuge bottle at 4000 rpm for 30 min to remove residual cells, cell debris, fibrinogen and other substances;

[0056] 5. Add 80 mL of 0.9% saline solution to an Amicon® Stirred Ultrafiltration Cell - 500 kDa (Millipore, product number: UFSC20001), place it in a centrifuge and centrifuge at 4000 g for 30 min to balance the ultrafiltration cell, and discard the lower layer liquid after centrifugation;

[0057] 6. Aspirate the upper plasma layer after centrifugation in step 4, inhale the plasma layer into the balanced Amicon® Stirred Ultrafiltration Cell - 500 kDa, centrifuge at 4000 g for 30 min, aspirate the suspension above the filter membrane into a new centrifuge tube, and repeatedly rinse the filter membrane with 10 mL of 0.9% saline solution, and mix the rinsed solution with the solution above the filter membrane;

[0058] 7. Add 50 mL of 0.9% saline solution to a Centricon Plus 70 ultrafiltration tube - 100 kDa (Millipore, catalog number: UFC710008), place it in a centrifuge and centrifuge at 4000 g for 30 min to balance the ultrafiltration cup. Discard the lower liquid after centrifugation.

[0059] 8. Aspirate the solution in Step 6 into a Centricon Plus 70 ultrafiltration tube - 100 kDa, centrifuge at 4000 g for 30 min, aspirate the suspension above the filter membrane into a new centrifuge tube, and repeatedly rinse the filter membrane with 5 mL of 0.9% saline solution. Mix the rinsed solution with the solution above the filter membrane.

[0060] 9. Place the solution in several ultracentrifuge tubes (Beckman Coulter, catalog number: 361625), balance them and place them in an ultracentrifuge (Beckman Coulter Optimal XE - 90, catalog number A99833) and centrifuge at 100000 g for 90 min.

[0061] 10. After centrifugation, discard the upper solution in the ultracentrifuge tube, resuspend the lower precipitate with an appropriate amount of 0.9% saline solution, then make up the solution to the ultracentrifuge tube, and repeat Step 9.

[0062] 11. Discard the upper solution after centrifugation. The lower precipitate is the umbilical cord blood plasma exosomes. Resuspend the exosome particles with an appropriate amount of 0.9% saline for subsequent detection and preservation.

[0063] Example 2 Cultivation and Expansion of Umbilical Cord Blood NK Cells

[0064] 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 and mix well, then perform ficoll density gradient centrifugation to obtain the middle mononuclear cell layer. Spread it evenly in a culture flask, add IL - 2, IL - 15, and IL - 21 cytokines to the culture flask, and add the exosome particles separated from the plasma. At the same time, set a control group (the control group is a culture protocol without adding exosome particles), and culture in a serum - free culture system for 14 - 21 days to harvest NK cells.

[0065] The specific implementation method is as follows:

[0066] 1. Take 100 mL of umbilical cord blood anticoagulated with sodium citrate, dispense it into 4 50 - ml centrifuge tubes, 25 ml per tube. Place the centrifuge tubes in a centrifuge and centrifuge at 2000 rpm for 20 min. After centrifugation, take out the centrifuge tubes and aspirate and discard the upper plasma with a pipette.

[0067] 2. Add 0.9% normal saline injection in equal volume to the lower-layer cell suspension containing red blood cells, and mix well.

[0068] 3. Take 4 50-mL centrifuge tubes, add 15 mL of Ficoll lymphocyte separation solution (Tianjin Haoyang, product number: LDS1075CB) to the centrifuge tubes, slowly add 30 mL of the cell suspension to the upper layer of the lymphocyte separation solution without breaking the separation layer.

[0069] 4. Place the centrifuge tubes in a centrifuge for density gradient centrifugation at 2000 rpm for 20 min, and set the braking deceleration to 0.

[0070] 5. After centrifugation, take out the centrifuge tubes and aspirate the upper-layer solution. Carefully aspirate the white film layer cells in the middle, that is, the umbilical cord blood mononuclear cell layer, with a sterile Pasteur pipette and place them in a new 50-mL centrifuge tube.

[0071] 6. Add 0.9% normal saline to the cell suspension to make it 50 mL, and centrifuge at 1500 rpm for 5 min for rinsing; after rinsing, discard the upper-layer solution, and continue to rinse the cell precipitate with normal saline, repeating three times.

[0072] 7. Resuspend the cell precipitate with NK cell complete medium and adjust the cell density to 1 - 1.5×10 6 / mL. The preparation method of the complete medium is to add 1% culture supplement (NK MACS® supplement, human, product number: 130 - 114 - 429) to the NK cell basal medium (NK MACS® Medium, human, product number: 130 - 114 - 429), and add 5% human AB serum (GeminiBio, product number: 100 - 912 - 100), and place it in a culture flask for culture.

[0073] 8. Add 100 U / mL IL-2 (Tongli Haiyuan, product number: GMP-TL906), 10 ng / mL IL-15 (Tongli Haiyuan, product number: GMP-TL202 - 0050), and 10 ng / mL IL-21 (Tongli Haiyuan, product number: GMP-TL509 - 0050) to the cell suspension.

[0074] 9. Count the cells every 48 hours, supplement NK cell complete medium to maintain the cell density at 1 - 1.5×10 6 cells / mL, supplement IL-2, IL-15, and IL-21 growth factors every 48 hours, and add 1 - 1.5×10 7 particles / mL of umbilical cord blood plasma exosomes to the culture system every 48 hours. After the cells grow vigorously, transfer them to a culture bag for continued culture.

[0075] 10. Culture NK cells for 21 days according to the above culture method, draw a growth curve until the cell growth enters the plateau phase;

[0076] 11. Take out the cultured cells from the culture bag, aspirate them into several 200 mL centrifuge bottles, centrifuge at 1500 rpm for 15 min, then aspirate and discard the supernatant, and resuspend the cell pellet with the complete NK cell medium to obtain cord blood-derived NK cells, denoted as group A.

[0077] Control group B (hereinafter referred to as group B):

[0078] The difference from the above NK cell culture and expansion method is that exosome particles are not added in step 9.

[0079] Control group C (hereinafter referred to as group C):

[0080] The difference from the above NK cell culture and expansion method is that cord blood plasma exosomes are replaced with exosomes derived from mesenchymal stem cells (MSCs), and the extraction of the exosomes can be carried out by a conventional extraction method.

[0081] Experiment 1 Transmission electron microscopy observation of exosome particles

[0082] The specific operation method is as follows:

[0083] 1. Take 50 μl of the isolated and purified exosome precipitate, add an equal volume of 2.5% glutaraldehyde by mass fraction, and place it in a refrigerator at 4°C for fixation for 1 hr;

[0084] 2. Drop 20 μl of the fixed exosome suspension onto the front of the electron microscopy copper grid and let it stand for 20 min;

[0085] 3. Carefully suck off the excess solution with a filter paper; then wash the copper grid 5 times with ultrapure water, 30 seconds each time, and dry it with a filter paper;

[0086] 4. Drop 1 drop of 2% uranyl acetate staining solution onto the front of the copper grid, stain for 1 min, and then suck off the excess staining solution along the edge of the copper grid with a filter paper;

[0087] 5. Place the copper grid in the air at room temperature to dry naturally, and wait until it is dry before putting it on the machine for observation.

[0088] The results are as Figure 1 shown. Under the electron microscope, cord blood plasma exosomes have a typical bilayer membrane structure under the microscope, with the unique "teacup-like" structure of exosomes and a complete envelope. This shows that the method for extracting cord blood plasma exosomes described in this patent is feasible, simple, and has good effects.

[0089] At the same time, analyze the particle size of cord blood plasma exosomes, and the results are as Figure 2As shown, after NTA detection, the exosome particle size has a single peak at less than 200 nm, with a peak value of approximately 103 nm, and the average length of most particle sizes is between 30 - 200 nm. This conforms to the particle size characteristics of exosomes.

[0090] Experiment 2 Detection of the expression of surface proteins on exosome particles by flow cytometry

[0091] The specific operation method is as follows:

[0092] 1. Invert and mix the components of the Thermofisher CD63 magnetic beads (product number 10606D) for 10 min; aspirate 20 μl of the magnetic bead suspension into a 1.5 mL round-bottom EP tube.

[0093] 2. Add 200 μl of the magnetic bead washing solution to the EP tube and mix well with a pipette tip.

[0094] 3. Place the EP tube on the magnetic stand for 1 min; then aspirate and discard the supernatant.

[0095] 4. Take 50 μl of the extracted plasma exosome suspension, add 50 μl of the washing solution to a final volume of 100 μl, and mix well.

[0096] 5. Place the exosome-washing solution mixture on a rotary mixer, set the rotation speed to 10 rpm, and incubate at 2 - 8 °C overnight with rotation.

[0097] 6. Centrifuge the sample quickly for 3 - 5 seconds the next day to collect the precipitate.

[0098] 7. Add 300 μl of the washing solution to the sample and mix well with a pipette tip for 30 seconds.

[0099] 8. Place the sample on the magnetic stand for about 1 min, aspirate and discard the supernatant.

[0100] 9. Add 400 μl of the washing solution to the sample and mix well with a pipette tip for 30 seconds.

[0101] 10. Place the sample on the magnetic stand for about 1 min, aspirate and discard the supernatant, and resuspend with 300 μl of the washing solution.

[0102] 11. Take 100 μl of each sample, add the CD9-PE and CD81-FITC flow antibodies to the samples respectively, and incubate at 4 °C in the dark for 30 min.

[0103] 12. Place the incubated sample on the magnetic stand for about 1 min, aspirate and discard the supernatant, and wash with 300 μl of the washing solution.

[0104] 13. After repeating step 12 once, resuspend the selected samples with 300 μl of PB and then perform on-machine detection.

[0105] The results are as follows Figure 3 As shown, the positive expression rates of CD9 and CD81 detected by flow cytometry for the exosome particles obtained by the method of the present invention are: 94.37% for CD9 and 97.71% for CD81, both meeting the surface protein expression requirements of exosomes.

[0106] Experiment 3: Detection of NK cell phenotype

[0107] 1. Adjust the density of the amplified NK cell suspension obtained in Example 2 to 1×10 6 / mL. Take a flow cytometry tube, add 5 μl of CD3-FITC antibody (Invitrogen) and 5 μl of CD16CD56-PE antibody (Invitrogen) to the tube. Add 200 μl of the cell suspension into the flow cytometry tube, shake well, incubate in the dark at 4°C for 30 min. After incubation, add 1 ml of PBS to the tube, centrifuge at 300 g for 5 min at 4°C; after centrifugation, discard the supernatant, resuspend the precipitate with 500 μl of PBS, mix well and then detect by machine.

[0108] 2. Turn on the BD FACSCantoII flow cytometer, turn on the FACSDiva analysis software after preheating, create an FSC-SSC scatter plot, set the gate P1 for lymphocytes and monocytes, create another four-quadrant scatter plot FL1-FL2, use CD3 as the abscissa and CD56 as the ordinate to observe the phenotype of the amplified NK cells, and calculate the amplification efficiency and phenotypic differences of the NK cells in groups A-C. The results are as follows Figures 4 - 6 as shown.

[0109] It can be seen from Figure 4 that in group A of this patent, that is, adding exosomes derived from umbilical cord blood plasma during the culture of NK cells, observing the growth state of NK cells on the 14th day, the cells grow in clusters, the growth state is good, and there are more cells within the visual field. Control group B only uses the conventional cytokine culture method, that is, without adding exosomes. Observing on the 14th day of culture, the cells are significantly not aggregated and grow scattered, the cell state is good, and the number of cells within the visual field is less than that of group A of this patent. Control group C, that is, adding exosomes derived from mesenchymal stem cells during the culture of NK cells, observing on the 14th day of culture, the cells grow slightly in clusters, the growth state is good, the cells within the visual field are denser than those in control group B, but the cell density within the visual field is slightly lower than that of group A of this patent.

[0110] It can be seen from Figure 5 that the total inoculation amounts of the three groups of NK cells at the initial stage of culture on the 0th day are about 0.7×10 8 cells. On the 7th day, the number of NK cells in group A of this patent is (3.80 ± 0.294)×10 8cells, the number of NK cells in control group B was (4.53 ± 0.368) × 10 8 cells, the number of NK cells in control group C was (4.17 ± 0.116) × 10 8 cells; on the 14th day, the number of NK cells in Patent Group A of this patent was (38.22 ± 3.21) × 10 8 cells, the number of NK cells in control group B was (30.61 ± 0.697) × 10 8 cells, the number of NK cells in control group C was (33.27 ± 1.970) × 10 8 cells; on the 21st day, the number of NK cells in Patent Group A of this patent was (113.50 ± 5.97) × 10 8 cells, the number of NK cells in control group B was (80.83 ± 5.69) × 10 8 cells, the number of NK cells in control group C was (92.45 ± 4.89) × 10 8 cells; the final amplification multiple of NK cells in Patent Group A of this patent after 21 days of culture was approximately 162.1 - fold, the final amplification multiple of NK cells in control group B after 21 days of culture was approximately 115.5 - fold, and the final amplification multiple of NK cells in control group C after 21 days of culture was approximately 132.1 - fold.

[0111] It can be seen that on the 14th day of culture, flow cytometry was performed on NK cells from three groups respectively. The proportion of the NK cell surface marker CD3 - CD16 / 56+ detected in Patent Group A of this patent was 91.8%, which was better than the surface marker proportion of 80.3% in control group B and better than the surface marker proportion of 86.5% in control group C. Figure 6

[0112] It can be found through the above that adding exosomes derived from umbilical cord blood plasma during the culture of NK cells can more effectively improve the cell amplification efficiency than not adding exosomes and adding exosomes derived from mesenchymal stem cells.

[0113] Experiment 4: In vitro killing experiment of NK cells against K562 cells

[0114] The LDH release method (lactate dehydrogenase release method) is an experimental method commonly used for 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 enzyme activity of lactate dehydrogenase (LDH). When cells are stimulated (such as by drugs, radiation, damage), the cell membrane may be damaged, resulting in the release of intracellular substances (such as LDH) into the extracellular fluid. By detecting the LDH activity released in the culture medium, the degree of cell damage or death can be indirectly reflected, and thus the killing ability of effector cells can be calculated.

[0115] The specific implementation method is as follows:

[0116] 1. Take out K562 cells from liquid nitrogen and resuscitate the cells in a 37°C water bath. Resuspend the cells with RPMI1640 medium containing 10% FBS, and centrifuge at 1500 rpm for 5 min to remove the cryopreservation solution. Adjust the seeding density to 0.5 - 1×10 6 cells / mL, seed in a culture flask, and maintain in RPMI - 1640 medium (containing 10% FBS) in an incubator at 37°C and 5% CO2. Change the medium or passage every 2 - 3 days to ensure good cell status.

[0117] 2. Take K562 cells in the logarithmic growth phase, wash them twice with PBS, and adjust the concentration to 1×10 5 cells / mL. Use trypan blue staining to ensure that the cell viability is greater than 95%.

[0118] 3. Take a 24 - well plate and group as follows (3 replicates per well, the control group NK cells are the same as the NK cells in this patent method):

[0119] (1) Experimental group (E + T group): Seed the NK cells and K562 cells obtained in Example 2 according to different effector - to - target ratios. The effector - to - target ratio (E:T ratio, that is, NK cell concentration:K562 cell concentration) is 0.25:1, 0.5:1, 1:1, 2:1; that is, adjust the concentration of NK cells 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:

[0120] This patent group: First, add exosome particles to the NK cells in group A from umbilical cord blood plasma according to the ratio of cell:exosome particle number equal to 1:10, and co - incubate for no more than 24 hours; then add K562 cells according to the effector - to - target ratio.

[0121] Simple exosome amplification group: The NK cells in group A are directly added with K562 cells according to the effector - to - target ratio without prior co - incubation with exosomes.

[0122] Simple exosome incubation group: First, add exosome particles to the NK cells in group B from umbilical cord blood plasma according to the ratio of cell:exosome particle number equal to 1:10, and co - incubate for no more than 24 hours; then add K562 cells according to the effector - to - target ratio.

[0123] Control group (without exosomes): The NK cells in group B were directly added with K562 cells according to the effector-to-target ratio without prior co-incubation with exosomes.

[0124] (2) Spontaneous release control group of target cells (T group): Only K562 cells were inoculated without inoculating NK cells.

[0125] (3) Spontaneous release control group of effector cells (E group): Only the NK cells of each group were inoculated without inoculating K562 cells.

[0126] (4) Complete lysis positive control group (Max LDH group): Cell lysate was added to K562 cells (providing the maximum LDH release).

[0127] (5) Medium blank control group (Blank group): Only medium was added without adding any cells.

[0128] 5. The cells of each group were added to a 24-well plate, and the total cell volume per well was adjusted to 200 μL, as follows:

[0129] Table 1 Experimental conditions of each group

[0130]

[0131] 6. After gently shaking and mixing, the 24-well plate was placed in an incubator at 37 °C and 5% CO2 for 6 hours;

[0132] 7. After incubation, centrifuge at 400 g for 5 min, and collect 100 μl of the supernatant into a 96-well plate;

[0133] 8. According to the instructions of the CyQUANT LDH kit (Thermofisher, catalog number: C20300), add 100 μL of the LDH reaction substrate and incubate in the dark at room temperature for 30 min;

[0134] 9. After terminating the reaction, measure the absorbance at 490 nm (OD: 490 nm);

[0135] 10. Calculate the killing ability of the NK cells of each group against the target cells through the following formula:

[0136] Cell killing efficiency % = [(OD value of the experimental group - OD value of the spontaneous release control group of target cells - OD value of the spontaneous release control group of effector cells) / (OD value of the complete lysis positive control group - OD value of the spontaneous release control group of target cells - OD value of the spontaneous release control group of effector cells)] × 100%;

[0137] 11. Plot the killing efficiency curves of each group and calculate the feasibility of the method of this patent for improving the in vitro killing efficiency of NK cells.

[0138] Figure 7 It is the lysis rate (i.e., cell killing efficacy) of K562 cells after the E+T group inoculated NK cells and K562 cells according to different protocols for 6 hours. In the figure, the control group is the culture medium blank control group. It can be seen from the figure that among the four groups of protocols, the killing ability of NK cells against K562 target cells at 6 hours is different at different effector-to-target ratios. When the effector-to-target ratio is 0.25:1, the killing rates of the patent group and other groups are 32.8%, 21.5%, 27.2% and 19.6% respectively; when the effector-to-target ratio is 0.5:1, the killing rates of the patent group and other groups are 58.6%, 42.9%, 40.3% and 34.7% respectively; when the effector-to-target ratio is 1:1, the killing rates of the patent group and other groups are 85.9%, 74.1%, 80.7% and 73.5% respectively; when the effector-to-target ratio is 2:1, the killing rates of the patent group and others are 99.9%, 98.7%, 98.9% and 96.3% respectively. It shows that at the same effector-to-target ratio, the killing efficiency of NK cells prepared by the patent group is better than that of other control groups. Especially in the case of a lower effector-to-target ratio (≤1), the killing ability of NK cells in the patent group against target cells is significantly better than that of NK cells in other control groups.

[0139] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for enhancing the killing ability of umbilical cord blood-derived NK cells, characterized in that: The method includes the following steps: S1. Isolate and extract exosomes from umbilical cord blood plasma; S2. Isolate mononuclear cells from umbilical cord blood, then add IL-2, IL-15, and IL-21, and add the umbilical cord blood plasma exosomes obtained in S1 to the culture system at a ratio of cell number: exosome particle number = 1:5 - 1:15 every 48 hours to promote the NK cell amplification efficiency, and collect the obtained NK cells; 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; culture the cells for 14 - 21 days until the cell growth enters the plateau phase; S3. Incubate the NK cells obtained in S2 with the umbilical cord blood plasma exosomes obtained in S1 at a ratio of cell number: exosome particle number equal to 1:5 - 1:15, and the co-incubation time does not exceed 24 hours.

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

3. The method for enhancing the killing ability of cord blood-derived NK cells according to claim 1, characterized in that: In S2, the cell density is 1 - 1.5×10 6 cells / mL; and, the cells are counted every 48 hours, and NK cell complete medium is supplemented to maintain the cell density at 1 - 1.5×10 6 cells / mL.

4. The method for enhancing the killing ability of umbilical cord blood-derived NK cells according to claim 3, wherein: In S2, IL-2, IL-15, and IL-21 should be replenished to the initial concentration every 48 hours, and umbilical cord blood plasma exosomes at 1 - 1.5×10 7 particles / mL should be added to the culture system every 48 hours.

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