In-vitro culture and separation method of NK cell exosome loaded with PD-1 antibody

Abstract

The invention discloses an in-vitro culture and separation method of an NK cell exosome loaded with a PD-1 antibody, and relates to the technical field of biomedicine, and the in-vitro culture and separation method specifically comprises the following steps: (1) culturing an NK cell; (2) loading a PD-1 antibody; (3) exosome extraction; (4) purifying the exosome; efficient loading of the PD-1 antibody is realized through electroporation, high expression of the antibody in the exosome is ensured, and large-scale production of the exosome is ensured through optimized NK cell culture conditions and an exosome extraction process; inorganic particles are added in cell culture to serve as microcarriers, more attachment points are provided for cells, transmission of nutrient substances and removal of metabolic waste are optimized, the proliferation multiple of the cells is increased, the cell viability is kept, the overall loading efficiency and growth amount of the microcarriers are increased, and therefore growth and proliferation of the NK cells are promoted; the 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 an in vitro culture and separation method of NK cell exosomes loaded with PD-1 antibodies. Background Art

[0002] Exosomes are small vesicles with a diameter of 30-150nm secreted by cells, which are involved in the transmission of information between cells. In recent years, exosomes have shown great potential as drug carriers in tumor immunotherapy. NK cell (natural killer cell) 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 explore a method to load PD-1 antibodies into NK cell exosomes and achieve large-scale culture and separation.

[0003] Large-scale cell culture is a basic technology in the biopharmaceutical industry. Large-scale culture of suspension cells is relatively simple. Cells can be directly placed in culture medium for suspension culture. However, not all cells can be cultured in suspension; in fact, for most animal cells, they need to be cultured in an adherent manner. Large-scale culture of adherent cells is more complicated. Either the adherent cells are domesticated into suspension culture or a large enough surface area is provided for them to grow adherently. Microcarriers can provide a larger attachment area. The so-called 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, as microcarriers, are suspended or dispersed in the bioreactor, which has a similar effect to suspension culture, so that they can be cultured in large-scale bioreactors.

[0004] For example, the Chinese patent application number CN202211495737.6 discloses a microcarrier for cell culture and a method for preparing the same. The application discloses a microcarrier for cell culture and a method for preparing the same. 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 range of material sources, low prices, and little pollution; and can be applied to small-scale cell culture; it provides 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 separation 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 application embodiment provides a method for in vitro culture and separation of NK cell exosomes loaded with PD-1 antibodies, 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] The PD-1 antibody was introduced into NK cells by electroporation, with the electroporation parameters set at 400 V / cm and the pulse time at 10 ms. The expression of the PD-1 antibody in NK cells was detected by immunofluorescence.

[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 using ultracentrifugation;

[0014] (4) Exosome purification:

[0015] Exosomes were further purified using density gradient centrifugation, where 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), the exosomes are extracted by ultracentrifugation, and the specific steps are as follows:

[0020] The cell culture supernatant was centrifuged at 300 g 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 cells were centrifuged at 100,000 g for 70 min, and the exosome pellet was collected 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, the stirring is stopped, and the settling is carried out for 10-30 minutes as the first stirring cycle; after the settling is completed, the stirring is carried out at a speed of 40-80 rpm, after stirring for 2-4 hours, the stirring is stopped, and the 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, the stirring is stopped, and the settling is carried out for 10-30 minutes, and this cycle is repeated until the cultivation is completed.

[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 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 the present application have at least the following technical effects or advantages:

[0031] First, this scheme achieves efficient loading of PD-1 antibodies through electroporation to ensure high expression of antibodies in exosomes, optimizes NK cell culture conditions and exosome extraction process, and ensures large-scale production of exosomes; uses density gradient centrifugation and dialysis to obtain high-purity exosomes and reduce 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. On the premise of increasing the exosome yield, it does not reduce the purity of the exosomes. Low purity of exosomes; Inorganic particles of different sizes have different effects. Small-sized particles significantly increase the 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 the proliferation multiples of cells, maintain cell viability, and improve the 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 state 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 production of cells;

[0032] Second, stirring can increase the mixing uniformity of particles and culture solution, while promoting the uniform distribution of cells on the surface of particles; by pausing the natural sedimentation process after stirring, particles of different sizes can form a more optimized spatial distribution in the culture dish or reactor, thereby further improving the cell loading rate and growth rate; in 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 into 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; low-density particles float on the upper layer to form an upper structure; the three-dimensional framework provides cells with more attachment points and growth space, 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 growth and reproduction of cells; the three-dimensional framework structure reduces the risk of cells being damaged by excessive shear force, while providing a more stable and supportive growth environment; improves the survival rate of cells and maintains the good morphology and functional activity of cells;

[0034] Fourth, by precisely controlling the particle size of suspended particles, as well as the combined 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 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 realization of 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 technicians in the technical field to which the present invention belongs; the terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the term "and / or" used herein includes any and all combinations of one or more related 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% CO. 2NK cells were cultured under the conditions of IL-2 (50 IU / mL) and IL-15 (10 ng / mL) were used to stimulate NK cells;

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

[0040] The PD-1 antibody was introduced into NK cells by electroporation, with the electroporation parameters set at 400 V / cm and the pulse time at 10 ms. The expression of the PD-1 antibody in NK cells was detected by immunofluorescence.

[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 serum-free culture medium containing inorganic particles is prepared in the following specific steps:

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

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

[0047] Inorganic particles of different particle 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 300 g 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 into 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 observed regularly; the cell viability was detected by 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 scheme achieves efficient loading of PD-1 antibodies through electroporation, ensuring high expression of antibodies in exosomes. The optimized NK cell culture conditions and exosome extraction process ensure the large-scale production of exosomes. Density gradient centrifugation and dialysis are used to obtain high-purity exosomes and reduce 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 operable method for large-scale in vitro culture and separation of NK cell exosomes loaded with PD-1 antibodies, which has broad application prospects.

[0060] In the 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. It does not reduce the purity of exosomes while increasing the yield of exosomes. Inorganic particles of different sizes have different functions. 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 small, and nutrients and growth factors can diffuse quickly, so that NK cells can obtain nutrients and growth factors immediately, maintain the activity of NK cells, and improve their proliferation ability. The culture medium containing small particles has a closer distance between cells, 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 large volume. The gaps between large particles are larger, which promotes the entry of oxygen in 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 in the combination of particle sizes, when the mass ratio of small particle size: medium particle size: large particle size is 1:1:1, the best cell attachment rate, proliferation multiple and cell viability are shown. 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 proliferation multiple of cells, maintain cell viability, and improve the overall loading efficiency and growth amount, thereby promoting the growth and proliferation of NK cells; in the cell culture process, according to the growth state of NK cells and changes in the culture medium environment, the combination ratio of inorganic particles is dynamically adjusted. 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 production of cells.

[0063] Example 2: The above Example 1 improves the cell attachment rate, proliferation multiple and cell viability by adding inorganic particles of different particle size combinations into 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 in step (3) are resuspended in the serum-free culture medium containing inorganic particles, 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 growth, loading rate and survival rate of the cells 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; through the natural sedimentation process after pausing 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;

[0070] In 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, and increase the cell loading rate and growth rate; improve the cell culture environment, improve the cell survival rate and growth quality, so as to meet 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, so as to meet 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, which increases the contact opportunity between cells and particles, thereby significantly improving the cell loading rate; due to the improvement of nutrient mass transfer efficiency and metabolic waste discharge efficiency, the cell growth environment is optimized and the growth rate is significantly accelerated; the stirring process promotes the uniform mixing of the culture solution 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 survival rate of cells 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 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 adopts the traditional two-dimensional culture and the 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] The cell growth, loading rate and survival rate were continuously monitored during the culture process. 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, 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.

[0080] The cell culture results in Table 3 show that the three-dimensional particle framework improves the cell culture efficiency, and the cell culture efficiency in the three-dimensional mixed particle framework is increased by 60%; 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 the uniform mixing of culture fluid and the supply of fresh oxygen, improves the mass transfer efficiency of nutrients and the discharge efficiency of metabolic waste, and promotes the rapid growth and reproduction of cells; 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; improves the survival rate of cells and maintains the good morphology and functional activity of cells;

[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-up production efficiency and quality consistency of NK cells;

[0082] Natural inorganic particles are widely available and low-cost, and the amount of plastic particles used 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 the 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 experiment of this embodiment adopts the traditional two-dimensional culture and the three-dimensional mixed particle framework without particle size combination; the 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] The cell growth, loading rate and survival rate were continuously monitored during the culture process. 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 the traditional two-dimensional culture, the optimized three-dimensional hybrid particle framework significantly increased the NK cell loading rate, reaching more than 98%; the cell doubling time was shortened to about 2.8 days, which was about 35% higher than the traditional method; the cell viability was maintained above 99%, showing extremely high cell survival rate and good growth state;

[0091] By precisely controlling the particle size of suspended particles and the combined 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 the rapid growth and reproduction of cells;

[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 realization of efficient, low-cost and large-scale production of NK cells.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for in vitro culture and separation of NK cell exosomes loaded with PD-1 antibodies, 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 IL-2 (50 IU / mL) and IL-15 (10 ng / mL). (2) PD-1 antibody loading: The PD-1 antibody was introduced into NK cells by electroporation, with the electroporation parameters set at 400 V / cm and the pulse time at 10 ms. The expression of the PD-1 antibody in NK cells was detected by immunofluorescence. (3) Exosome extraction: 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 using ultracentrifugation; The volume ratio of inorganic particles to serum-free medium was 2%; (4) Exosome purification: Exosomes were further purified using density gradient centrifugation, where 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; Sucrose was removed by dialysis to obtain purified exosomes.

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

3. The method for in vitro culture and separation of NK cell exosomes loaded with PD-1 antibodies according to claim 1, characterized in that: 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.

4. The method for in vitro culture and separation of NK cell exosomes loaded with PD-1 antibodies according to claim 1, characterized in that: In step (3), the exosomes are extracted by ultracentrifugation, and the specific steps are as follows: The cell culture supernatant was centrifuged at 300 g 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 cells were centrifuged at 100,000 g for 70 min, and the exosome pellet was collected and resuspended in PBS.

5. The method for in vitro culture and separation of NK cell exosomes loaded with PD-1 antibodies according to claim 1, characterized in that: In step (3), when the NK cells are resuspended in the serum-free culture medium containing inorganic particles, intermittent stirring is performed.

6. The method for in vitro culture and separation of NK cell exosomes loaded with PD-1 antibodies according to claim 5, characterized in that: 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, the stirring is stopped and the settling is carried out for 10-30 minutes as the first stirring cycle; after the settling is completed, the stirring is carried out at a speed of 40-80 rpm. After stirring for 2-4 hours, the stirring is stopped and the 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, the stirring is stopped and the settling is carried out for 10-30 minutes, and this cycle is repeated until the cultivation is completed.

7. The method for in vitro culture and separation of NK cell exosomes loaded with PD-1 antibodies according to claim 5, characterized in that: 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 Medium density suspended particles.

8. The method for in vitro culture and separation of NK cell exosomes loaded with PD-1 antibodies according to claim 7, characterized in that: The culture medium also contains a density of <1.0 g / cm 3 Plastic microspheres are low-density floating particles.

9. The method for in vitro culture and separation of NK cell exosomes loaded with PD-1 antibodies according to claim 8, characterized in that: 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.

10. The method for in vitro culture and separation of NK cell exosomes loaded with PD-1 antibodies according to claim 9, characterized in that: 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%.

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