A method for in vitro culture and isolation of nk cell exosomes loaded with pd-1 antibodies

Abstract

The application discloses an in-vitro culture and separation method of NK cell exosome loaded with PD-1 antibody, relates to the field of biomedical technology, and specifically comprises the following steps: (1) NK cell culture; (2) PD-1 antibody loading; (3) exosome extraction; (4) exosome purification; efficient loading of the PD-1 antibody is realized through electroporation, high expression of the antibody in the exosome is ensured, optimized NK cell culture conditions and exosome extraction procedures guarantee large-scale production of the exosome; by adding inorganic particles as microcarriers in cell culture, more attachment points are provided for cells, transfer of nutrients and removal of metabolic waste are optimized, the cell proliferation fold is improved, the cell survival rate is maintained, the overall loading efficiency and growth amount of the microcarriers are improved, thereby the growth and proliferation of the NK cells are promoted, and large-scale culture of the cells is realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for culturing and isolating NK cell exosomes loaded with PD-1 antibodies in vitro. Background Art

[0002] Exosomes are small vesicles with a diameter of 30-150 nm secreted by cells that participate in intercellular communication. In recent years, exosomes have shown great potential as drug carriers in tumor immunotherapy. NK cell (natural killer) exosomes have strong anti-tumor activity, and PD-1 antibodies are important drugs for inhibiting tumor immune escape. Therefore, it is of great significance to develop a method to load PD-1 antibodies into NK cell exosomes and achieve large-scale culture and isolation.

[0003] Large-scale cell culture is a fundamental technology in the biopharmaceutical industry. Large-scale culture of suspension cells is relatively simple; the cells can simply be placed in culture medium for suspension culture. However, not all cells can be cultured in suspension; in fact, most animal cells require adherent culture. Large-scale culture of adherent cells is more complex, requiring either the adherent cells to be tamed for suspension culture or a sufficiently large surface area to allow them to grow adherently. Microcarriers can provide a larger attachment area. Microcarriers are actually tiny microspheres that allow adherent cells to attach to the surface of the microspheres for adherent culture and growth. The microspheres themselves, acting as microcarriers, are suspended or dispersed in the bioreactor, achieving an effect similar to suspension culture, allowing them to be cultured in large-scale bioreactors.

[0004] For example, Chinese patent application number CN202211495737.6 discloses a microcarrier for cell culture and its preparation method. The application discloses a microcarrier for cell culture and its preparation method. The microcarrier used in the application is a natural or semi-natural inorganic particle; the natural or semi-natural inorganic particle is selected from at least one of the following materials: (1) fine sand; (2) particles ground from at least one of rocks, ores, diatomaceous earth, shells, eggshells, snail shells, starfish skeletons, and coral skeletons; (3) quartz sand or floating beads. The microcarrier used in the application for cell culture directly uses natural or semi-natural inorganic particles, which have a wide source of materials, low price, and low pollution; and can be applied to small-scale cell culture; providing a new solution and approach for cell adherent culture.

[0005] The loading efficiency of inorganic particles as microcarriers and the amount of cell growth on the microcarriers in the above scheme still need to be improved. Summary of the Invention

[0006] The embodiments of the present application provide an in vitro culture and isolation method for NK cell exosomes loaded with PD-1 antibodies, thereby solving the problems of low loading efficiency and cell growth of inorganic particles as microcarriers in the prior art, improving the loading efficiency and cell growth of inorganic particles, and realizing large-scale cell culture.

[0007] The present invention provides a method for culturing and isolating NK cell exosomes loaded with PD-1 antibodies in vitro, which specifically includes the following steps:

[0008] (1) NK cell culture:

[0009] NK cells were isolated from the blood of healthy donors and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2. NK cells were stimulated with IL-2 (50 IU / mL) and IL-15 (10 ng / mL).

[0010] (2) PD-1 antibody loading:

[0011] PD-1 antibodies were introduced into NK cells via electroporation, with the electroporation parameters set at 400 V / cm and a pulse time of 10 ms. Immunofluorescence was used to detect the expression of PD-1 antibodies in NK cells.

[0012] (3) Exosome extraction:

[0013] The cultured NK cells were washed with PBS, resuspended in serum-free medium containing inorganic particles, and cultured for 48 hours to collect exosomes. Exosomes were extracted by ultracentrifugation.

[0014] (4) Exosome purification:

[0015] Exosomes were further purified using density gradient centrifugation. The exosome suspension was layered onto a 30% to 60% sucrose gradient solution and centrifuged at 100,000 g for 16 h to collect the exosome layer.

[0016] Sucrose was removed by dialysis to obtain purified exosomes.

[0017] Furthermore, the inorganic particles include sea sand.

[0018] Furthermore, the particle size range of the inorganic particles includes a small particle size of 10-50 μm, a medium particle size of 50-150 μm, and a large particle size of 150-500 μm.

[0019] Furthermore, in step (3), exosomes are extracted using ultracentrifugation, and the specific steps are as follows:

[0020] The cell culture supernatant was centrifuged at 300g for 10 min to remove cells and debris;

[0021] Cell debris and apoptotic bodies were removed by centrifugation at 2000 g for 20 min;

[0022] Large vesicles were removed by centrifugation at 10,000 g for 30 min;

[0023] Finally, the exosome pellet was collected by centrifugation at 100,000 g for 70 min and resuspended in PBS.

[0024] Furthermore, in step (3), when the NK cells are resuspended in the serum-free culture medium containing inorganic particles, intermittent stirring is performed.

[0025] Furthermore, the intermittent stirring includes stirring and settling, specifically, the initial stirring is carried out at a speed of 50-100 rpm, after stirring for 2-4 hours, stirring is stopped, and settling is carried out for 10-30 minutes as the first stirring cycle; after settling is completed, stirring is carried out at a speed of 40-80 rpm, after stirring for 2-4 hours, stirring is stopped, and settling is carried out for 10-30 minutes, which is the second stirring cycle; each subsequent stirring cycle is carried out at a speed of 30-60 rpm, after stirring for 2-4 hours, stirring is stopped, and settling is carried out for 10-30 minutes, and this cycle is repeated until the end of the culture.

[0026] Furthermore, the inorganic particles include a density of 1.2-3.0 g / cm 3 High-density sedimentation particles, density 1.0-1.2g / cm 3 of medium-density suspended particles.

[0027] Furthermore, the culture medium is also added with a density of <1.0 g / cm 3 Plastic microspheres are low-density floating particles.

[0028] Furthermore, the particle size of high-density sedimentation particles is 50-200 μm, the particle size of medium-density suspended particles is 10-50 μm, and the particle size of low-density floating particles is 1-10 μm.

[0029] Furthermore, the high-density settling particles account for 30%-40%; the medium-density suspended particles account for 40%-50%; and the low-density floating particles account for 10%-20%.

[0030] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0031] First, this scheme achieves efficient loading of PD-1 antibodies through electroporation, ensuring high expression of antibodies in exosomes, and optimizes NK cell culture conditions and exosome extraction processes to ensure large-scale production of exosomes; density gradient centrifugation and dialysis are used to obtain high-purity exosomes, reducing interference from impurities; this scheme can be extended to other antibodies and cell types, providing new ideas for the development of exosome drug carriers; this scheme provides an efficient, economical, and highly operational method for large-scale in vitro culture and separation of NK cell exosomes loaded with PD-1 antibodies, which has broad application prospects; in cell culture, inorganic particles, including sea sand, are added. Sea sand is easy to separate and has no effect on the purity of exosomes after centrifugation. While increasing the exosome yield, it does not reduce the purity of the exosomes. Low exosome purity; Inorganic particles of different sizes have different effects. Small-sized particles significantly increase cell attachment rate, while the addition of large-sized particles can maintain the overall attachment area. Therefore, the combination of particles of different sizes can provide more attachment points, optimize the delivery of nutrients and the removal of metabolic waste, increase cell proliferation multiples, maintain cell viability, and improve overall loading efficiency and growth, thereby promoting the growth and proliferation of NK cells. During the cell culture process, the combination ratio of inorganic particles is dynamically adjusted according to the growth status of NK cells and changes in the culture medium environment. Through particle size classification and optimized combination, the culture efficiency and quality of NK cells on inorganic particles can be further improved, and the adaptability and flexibility of cell culture can be improved, thereby realizing large-scale cell production.

[0032] Secondly, stirring increases the mixing uniformity of particles and culture medium, while promoting the uniform distribution of cells on the surface of particles; by pausing the natural sedimentation process after stirring, particles of different particle sizes form a more optimized spatial distribution in the culture dish or reactor, thereby further improving the cell loading rate and growth rate; during the cell culture process, the combination of stirring and sedimentation can improve the uniform distribution of particles in the culture system, improve the mass transfer efficiency of nutrients and the discharge efficiency of metabolic waste; promote the uniform loading and efficient growth of cells on the surface of particles, increase the cell loading rate and growth rate, improve the cell culture environment, and improve the cell survival rate and growth quality, thereby meeting the needs of large-scale production of NK cells; by changing the stirring speed, stirring time and sedimentation time, the cell culture is adjusted and optimized, and the cell survival rate and growth quality are improved, thereby meeting the needs of large-scale production of NK cells;

[0033] Third, by adding three different densities of particles to the culture medium, a dynamic three-dimensional culture framework is formed, in which high-density particles settle to the bottom to form a support layer; medium-density particles are suspended in the culture medium to form a middle-layer framework; and low-density particles float on the upper layer to form an upper structure. The three-dimensional framework provides more attachment points and growth space for cells, significantly improving the cell loading rate. The combination of particles of different densities enables cells to be evenly distributed throughout the culture system, increasing the contact opportunities between cells and particles. The three-dimensional framework promotes uniform mixing of the culture medium and the supply of fresh oxygen, improves the mass transfer efficiency of nutrients and the discharge efficiency of metabolic waste, and promotes rapid cell growth and reproduction. The three-dimensional framework structure reduces the risk of cell damage due to excessive shear force, while providing a more stable and supportive growth environment, improving cell survival rate and maintaining good cell morphology and functional activity.

[0034] Fourthly, by precisely controlling the particle size of suspended particles and the combined ratio of sedimentation particles, suspended particles and floating particles, more attachment points and growth space are provided for cells, significantly improving the cell loading rate; the optimized three-dimensional mixed particle framework promotes the uniform distribution of nutrients and the effective discharge of metabolic waste, while reducing the competition effect between cells and promoting the rapid growth and reproduction of cells; the optimized three-dimensional mixed particle framework culture environment reduces the risk of cell damage due to environmental pressure, while providing more stable and supportive growth conditions, significantly improving cell viability and functional activity; this improved scheme is not only suitable for small-scale laboratory culture systems, but can also be further expanded to large-scale bioreactors, promoting the efficient, low-cost and large-scale production of NK cells. DETAILED DESCRIPTION

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains; the terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0036] Example 1: A method for loading PD-1 antibodies into NK cell exosomes and culturing and isolating them in large quantities in vitro, specifically comprising the following steps:

[0037] (1) NK cell culture:

[0038] NK cells were isolated from the blood of healthy donors and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2. NK cells were stimulated with IL-2 (50 IU / mL) and IL-15 (10 ng / mL).

[0039] (2) PD-1 antibody loading:

[0040] PD-1 antibodies were introduced into NK cells via electroporation, with the electroporation parameters set at 400 V / cm and a pulse time of 10 ms. Immunofluorescence was used to detect the expression of PD-1 antibodies in NK cells.

[0041] (3) Exosome extraction:

[0042] The cultured NK cells were washed with PBS, resuspended in serum-free medium containing inorganic particles, and cultured for 48 h to collect exosomes;

[0043] The volume ratio of the inorganic particles to the serum-free culture medium is 2%; the inorganic particles include sea sand, and the particle size range of the inorganic particles includes a small particle size of 10-50 μm, a medium particle size of 50-150 μm, and a large particle size of 150-500 μm;

[0044] The specific preparation steps of serum-free culture medium containing inorganic particles are as follows:

[0045] Cleaning and high-temperature dry heat sterilization of inorganic particles;

[0046] The inorganic particles are combined in particle size, including all small particle size, all medium particle size, all large particle size, small particle size: medium particle size mass ratio of 1:1, medium particle size: large particle size mass ratio of 1:1, small particle size: medium particle size: large particle size mass ratio of 1:1:1;

[0047] Inorganic particles of different size combinations were added to serum-free culture medium;

[0048] Exosomes were extracted using ultracentrifugation. The specific steps are as follows:

[0049] The cell culture supernatant was centrifuged at 300g for 10 min to remove cells and debris;

[0050] Cell debris and apoptotic bodies were removed by centrifugation at 2000 g for 20 min;

[0051] Large vesicles were removed by centrifugation at 10,000 g for 30 min;

[0052] Finally, the exosome pellet was collected by centrifugation at 100,000 g for 70 min and resuspended in PBS;

[0053] (4) Exosome purification:

[0054] Density gradient centrifugation was used to further purify the exosomes. The exosome suspension was layered onto a 30% to 60% sucrose gradient solution and centrifuged at 100,000 g for 16 hours to collect the exosome layer. The sucrose was removed by dialysis to obtain purified exosomes.

[0055] The cell attachment, growth status and proliferation were regularly observed; the cell viability was detected using trypan blue staining; the cell count was performed to evaluate the proliferation multiple; the cell culture results are shown in Table 1:

[0056]

[0057] Table 1

[0058] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0059] This protocol achieves efficient loading of PD-1 antibodies through electroporation, ensuring high antibody expression in exosomes. Optimized NK cell culture conditions and exosome extraction processes ensure large-scale exosome production. Density gradient centrifugation and dialysis are used to obtain highly pure exosomes, reducing impurity interference. This protocol can be extended to other antibodies and cell types, providing new insights into the development of exosome drug carriers. This protocol provides an efficient, economical, and highly operational method for the large-scale in vitro culture and isolation of NK cell exosomes loaded with PD-1 antibodies, which has broad application prospects.

[0060] Inorganic particles, including sea sand, are added to cell culture. Sea sand is easy to separate and has no effect on the purity of exosomes after centrifugation. It increases the yield of exosomes without reducing the purity of exosomes. Inorganic particles of different sizes have different effects. Small particles have a larger specific surface area due to their smaller size, which can provide more attachment points for cells, thereby improving the efficiency of cell attachment, allowing cells to anchor and expand better, and promoting their proliferation and activation. In the cell culture system, the gaps between small particles are smaller, allowing nutrients and growth factors to diffuse rapidly, allowing NK cells to obtain nutrients and growth factors immediately, maintaining NK cell activity and improving their proliferation capacity. Culture medium containing small particles allows cells to be closer together, which can promote direct contact and interaction between cells.

[0061] Large particles increase the overall cell attachment area. Although large particles have a relatively small specific surface area, they can provide more overall attachment area within the same volume due to their larger volume. The larger gaps between large particles promote the entry of oxygen from the culture medium into the cell culture system and the discharge of metabolic waste, supporting the three-dimensional growth of cells. Large particles provide cells with a growth space similar to a three-dimensional microenvironment, which is closer to the natural growth state of cells in the body and promotes cell differentiation and functional expression.

[0062] Table 1 shows that among the particle size combinations, the combination with a mass ratio of small particle size: medium particle size: large particle size of 1:1:1 exhibits the best cell attachment rate, proliferation multiple and cell viability. Small particle size particles significantly improve the cell attachment rate, while the addition of large particle size particles has little effect on the attachment rate, but can maintain the overall attachment area. Therefore, the combination of particles of different particle sizes can provide more attachment points, optimize the delivery of nutrients and the removal of metabolic waste, increase the cell proliferation multiple, maintain cell viability, and improve the overall loading efficiency and growth amount, thereby promoting the growth and proliferation of NK cells. During the cell culture process, the combination ratio of inorganic particles is dynamically adjusted according to the growth status of NK cells and changes in the culture medium environment. Through particle size classification and optimized combination, the culture efficiency and quality of NK cells on inorganic particles can be further improved, the adaptability and flexibility of cell culture can be improved, and the large-scale production of cells can be achieved.

[0063] Example 2: The above Example 1 improves the cell attachment rate, proliferation multiples and cell viability by adding inorganic particles of different particle size combinations to the culture medium, thereby promoting the growth and proliferation of NK cells. In order to improve the growth efficiency, survival rate and quality of NK cells on inorganic particles, further improvements are made on the basis of Example 1.

[0064] When the NK cells are resuspended in the serum-free culture medium containing inorganic particles in step (3), intermittent stirring is performed, including stirring and sedimentation. Specifically, the initial stirring is performed at a speed of 50-100 rpm. After stirring for 2-4 hours, the stirring is stopped and the sedimentation is performed for 10-30 minutes as the first stirring cycle; after the sedimentation is completed, the stirring is performed at a speed of 40-80 rpm. After stirring for 2-4 hours, the stirring is stopped and the sedimentation is performed for 10-30 minutes. This is the second stirring cycle; each subsequent stirring cycle is stirred at a speed of 30-60 rpm. After stirring for 2-4 hours, the stirring is stopped and the sedimentation is performed for 10-30 minutes. This cycle is repeated until the culture is completed.

[0065] The stirring strategies of the comparative experiment of this embodiment include no stirring and continuous stirring. The other technical features are the same as those of this embodiment. The cell growth, loading rate and survival rate are continuously monitored during the culture process. The cell culture results are shown in Table 2:

[0066] Stirring strategy Cell loading rate (%) Cell doubling time (days) Cell viability (%) No stirring 60 4.5 85 Continuous stirring 75 3.8 90 Intermittent stirring 90 3.0 95

[0067] Table 2

[0068] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0069] Stirring increases the mixing uniformity of particles and culture medium, while promoting the uniform distribution of cells on the particle surface. The natural sedimentation process after pausing stirring allows particles of different sizes to form a more optimized spatial distribution in the culture dish or reactor, thereby further improving the cell loading rate and growth rate.

[0070] During the cell culture process, the combination of stirring and sedimentation can improve the uniform distribution of particles in the culture system, improve the mass transfer efficiency of nutrients and the discharge efficiency of metabolic waste; promote the uniform loading and efficient growth of cells on the particle surface, increase the cell loading rate and growth rate; improve the cell culture environment, and enhance the cell survival rate and growth quality, thereby meeting the needs of large-scale production of NK cells; by changing the stirring speed, stirring time and sedimentation time, the cell culture can be adjusted and optimized to improve the cell survival rate and growth quality, thereby meeting the needs of large-scale production of NK cells;

[0071] The cell culture results in Table 2 show that the combination of stirring and sedimentation enables particles of different particle sizes to form a more optimized spatial distribution in the culture system, increasing the contact opportunities between cells and particles, thereby significantly improving the cell loading rate; due to the improved efficiency of nutrient mass transfer and metabolic waste discharge, the cell growth environment is optimized and the growth rate is significantly accelerated; the stirring process promotes the uniform mixing of the culture medium and the supply of fresh oxygen, while the sedimentation process reduces the risk of cell damage due to excessive shear force. Under the combined action of stirring and sedimentation, the cell survival rate is significantly improved, and the cells have good morphology and high functional activity.

[0072] Example 3: The above-mentioned Example 2 improves the growth efficiency, survival rate and quality of NK cells on inorganic particles by combining stirring and sedimentation during the culture process. In order to improve the loading rate, growth rate and cell survival rate of NK cells on inorganic particles, further improvements are made on the basis of Example 2.

[0073] Inorganic particles including density 1.2-3.0g / cm 3 High-density sedimentation particles, density 1.0-1.2g / cm 3 The medium density suspended particles with a density of <1.0 g / cm 3 Plastic microspheres are low-density floating particles.

[0074] The comparative experiment of this embodiment uses traditional two-dimensional culture and a plastic-free three-dimensional inorganic particle framework. The difference between the traditional two-dimensional culture and this embodiment is that no inorganic particles are added; the difference between the plastic-free three-dimensional inorganic particle framework and this solution is that the added inorganic particles do not contain plastic microspheres.

[0075] During the culture process, the cell growth, loading rate and survival rate were continuously monitored. The cell culture results are shown in Table 3:

[0076]

[0077] Table 3

[0078] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0079] Three different densities of particles are added to the culture medium to form a dynamic three-dimensional culture framework. The high-density particles settle to the bottom, forming a support layer; the medium-density particles are suspended in the culture medium, forming a middle-layer framework; and the low-density particles float in the upper layer, forming the upper structure.

[0080] The cell culture results in Table 3 show that the 3D particle framework improves cell culture efficiency, with the cell culture efficiency in the 3D mixed particle framework increasing by 60%. The 3D framework provides more attachment points and growth space for cells, significantly increasing the cell loading rate. The combination of particles of different densities enables cells to be evenly distributed throughout the culture system, increasing the contact opportunities between cells and particles. The 3D framework promotes uniform mixing of the culture medium and the supply of fresh oxygen, improving the mass transfer efficiency of nutrients and the discharge efficiency of metabolic waste, and promoting rapid cell growth and reproduction. The 3D framework structure reduces the risk of cell damage due to excessive shear force, while providing a more stable and supportive growth environment, improving cell survival rate and maintaining good cell morphology and functional activity.

[0081] The three-dimensional dynamic culture framework is not only suitable for small-scale laboratory culture systems, but can also be further expanded to large-scale bioreactors, thereby improving the scale production efficiency and quality consistency of NK cells;

[0082] Natural inorganic particles are widely available and low-cost, and the use of plastic particles is also relatively small, which reduces the overall culture cost; reduces dependence on organic synthetic materials and reduces the risk of environmental pollution; and can adjust the density, particle size and combination ratio of particles according to specific needs to adapt to the needs of different types of cell culture.

[0083] Example 4: The above-mentioned Example 3 constructs a dynamic three-dimensional culture framework by combining inorganic particles of different densities and materials with a small amount of floating plastic particles, thereby improving the loading rate, growth rate, survival rate and culture quality of NK cells in the culture system. In order to improve the loading rate, growth rate and cell survival rate of NK cells on inorganic particles, further improvements are made on the basis of Example 3.

[0084] The particle size of high-density settling particles is 50-200μm, the particle size of medium-density suspended particles is 10-50μm; the particle size of low-density floating particles is 1-10μm; among them, high-density settling particles account for 30%-40%; medium-density suspended particles account for 40%-50%; and low-density floating particles account for 10%-20%.

[0085] The comparative experiments of this embodiment used traditional two-dimensional culture and a three-dimensional mixed particle framework without particle size combination. The only difference between the traditional two-dimensional culture and this embodiment is that no inorganic particles are added. The difference between the three-dimensional mixed particle framework without particle size combination and this solution is that only 10-50 μm particles are added.

[0086] During the culture process, the cell growth, loading rate and survival rate were continuously monitored. The cell culture results are shown in Table 4:

[0087]

[0088] Table 4

[0089] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0090] The cell culture results in Table 4 show that compared with traditional two-dimensional culture, the optimized three-dimensional hybrid particle framework significantly increased the NK cell loading rate, reaching over 98%. The cell doubling time was shortened to approximately 2.8 days, an increase of approximately 35% compared with the traditional method. The cell viability remained above 99%, demonstrating extremely high cell survival and good growth status.

[0091] By precisely controlling the particle size of suspended particles and the ratio of settled particles, suspended particles, and floating particles, more attachment points and growth space are provided for cells, significantly improving the cell loading rate. The optimized three-dimensional mixed particle framework promotes the uniform distribution of nutrients and the effective discharge of metabolic waste, while reducing the competition effect between cells and promoting rapid cell growth and reproduction.

[0092] The optimized three-dimensional hybrid particle framework culture environment reduces the risk of cell damage due to environmental stress, while providing more stable and supportive growth conditions, significantly improving cell viability and functional activity. This improved solution is not only suitable for small-scale laboratory culture systems, but can also be further expanded to large-scale bioreactors, promoting the efficient, low-cost and large-scale production of NK cells.

[0093] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Various modifications and variations are readily apparent to those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for culturing and isolating NK cell exosomes loaded with PD-1 antibodies in vitro, characterized in that: The specific steps include: (1) NK cell culture: NK cells were isolated from the blood of healthy donors and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2. NK cells were stimulated with 50 IU / mL IL-2 and 10 ng / mL IL-15. (2) PD-1 antibody loading: PD-1 antibody was introduced into NK cells by electroporation, with the electroporation parameters set to 400 V / cm and a pulse time of 10 ms; the expression of PD-1 antibody in NK cells was detected by immunofluorescence. (3) Exosome extraction: The cultured NK cells were washed with PBS and resuspended in serum-free medium containing inorganic particles. The volume ratio of inorganic particles to serum-free medium was 2%. The inorganic particles include particles with a small particle size of 10-50 μm, a medium particle size of 50-150 μm and a large particle size of 150-500 μm, and the mass ratio of the small particle size particles, the medium particle size particles and the large particle size particles is 1:1:1; Perform intermittent stirring, which includes stirring and settling, specifically: The initial stirring is carried out at a speed of 50-100 rpm. After stirring for 2-4 hours, the stirring is stopped and the sedimentation is carried out for 10-30 minutes as the first stirring cycle; After the sedimentation is completed, stir at a speed of 40-80 rpm for 2-4 hours, then stop stirring and let it settle for 10-30 minutes for the second stirring cycle; Each subsequent cycle of stirring is performed at a speed of 30-60 rpm. After stirring for 2-4 hours, stirring is stopped and the mixture is allowed to settle for 10-30 minutes. This cycle is repeated until the culture is completed. The exosomes were collected after culturing for 48 hours and extracted by ultracentrifugation. Wherein, the inorganic particles also include those with a density of 1.2-3.0 g / cm 3 High-density sedimentation particles with a density of 1.0-1.2g / cm 3 Medium density suspended particles and density less than 1.0g / cm 3 Low-density floating particles of plastic microspheres; The particle size of the high-density sedimentation particles is 50-200 μm, the particle size of the medium-density suspended particles is 10-50 μm, and the particle size of the low-density floating particles is 1-10 μm; The high-density settling particles account for 30%-40% of the total amount of inorganic particles, the medium-density suspended particles account for 40%-50%, and the low-density floating particles account for 10%-20%; (4) Exosome purification: Density gradient centrifugation was used to further purify the exosomes. The exosome suspension was layered onto a 30% to 60% sucrose gradient solution and centrifuged at 100,000 g for 16 h to collect the exosome layer. The sucrose was removed by dialysis to obtain purified exosomes.

2. The in vitro culture and isolation method of NK cell exosomes loaded with PD-1 antibodies according to claim 1, characterized in that: The inorganic particles include sea sand.

3. The in vitro culture and isolation method of NK cell exosomes loaded with PD-1 antibodies according to claim 1, characterized in that: In step (3), exosomes are extracted using ultracentrifugation, and the specific steps are as follows: The cell culture supernatant was centrifuged at 300g for 10 min to remove cells and debris; Cell debris and apoptotic bodies were removed by centrifugation at 2000 g for 20 min; Large vesicles were removed by centrifugation at 10,000 g for 30 min; Finally, the exosome pellet was collected by centrifugation at 100,000 g for 70 min and resuspended in PBS.

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