Immune cell culture expansion medium and preparation method thereof

Abstract

The invention belongs to the technical field of immune cell culture, and particularly relates to an immune cell culture expansion medium and a preparation method thereof. The culture medium consists of a matrix and active ingredients; the matrix comprises the following components in parts by weight: 10 to 15 parts of thiolated hyaluronic acid, 8 to 12 parts of mercapto polyethylene glycol, 0.5 to 1 part of thioglycolic acid, 0.01 to 0.05 parts of hydrogen peroxide, 1 to 2 parts of perfluorodecane, 0.5 to 1 part of soybean lecithin, 1 to 2 parts of cell protectants and 40 to 50 parts of PBS buffer; the active ingredients comprise the following components in parts by weight: 0.005 to 0.01 parts of IL-2, 0.001 to 0.005 parts of IL-15, 0.05 to 0.1 parts of α-galactosylceramide, 0.03 to 0.15 parts of glutathione, 0.5 to 1 parts of amino acids, 0.25 to 0.5 parts of L-glutamine, 0.05 to 0.1 parts of pyruvic acid and 0.1 to 0.5 parts of albumin. The present invention provides continuous oxygen support and controls the release of active ingredients through a dynamic three-dimensional network constructed by sulfhydryl hyaluronic acid and mercapto polyethylene glycol.

Description

Technical Field

[0001] The invention belongs to the technical field of immune cell culture, and particularly relates to an immune cell culture expansion medium and a preparation method thereof. Background Art

[0002] Immune cell culture expansion medium is a nutrient solution specifically used for in vitro culture and expansion of immune cells (such as T cells, NK cells, etc.). It is widely used in immune cell therapy, especially in cell immunotherapy (such as CAR-T cell therapy, TIL therapy) in cancer treatment. The core function of this culture medium is to provide immune cells with nutrients required for growth and expansion, such as basic components such as amino acids, vitamins, glucose, and inorganic salts, and also contains cytokines such as IL-2 and IL-15 to promote cell proliferation and maintain functional activity. In addition, serum or serum substitutes are usually added to the culture medium to provide additional growth factors and proteins, and a suitable pH value is maintained by adding a buffer system to ensure the stability of cells in an in vitro growth environment. In the process of culturing immune cells, precise optimization of the culture medium is crucial to ensure that the expanded cells can maintain their killing function and biological activity, especially when meeting the needs of large-scale expansion, it is necessary to maintain cell activity and prevent apoptosis. In recent years, with the development of immunotherapy, more and more research has been devoted to the development of serum-free or chemically defined culture media to reduce variation factors and improve safety, while exploring personalized culture media to adapt to the cell needs of different patients. The design of the culture medium also needs to be adapted to automated bioreactors to support efficient and large-scale expansion of immune cells, providing strong support in areas such as cancer treatment, viral infection control, and regulation of autoimmune diseases and transplant rejection reactions.

[0003] Although immune cell culture expansion medium plays a vital role in immune cell therapy, existing culture media still have some significant defects. First, traditional culture media often rely on serum or animal-derived components, such as fetal bovine serum (FBS), which may cause immune rejection reactions or safety hazards. Secondly, there is still room for improvement in the expansion efficiency and cell function maintenance of existing culture media. Although many culture media can promote cell proliferation, it is difficult to effectively maintain the functional activity of immune cells, especially in large-scale expansion, where the loss or weakening of cell function is more common. Therefore, based on the above problems, it is particularly necessary to develop a serum-free, safe and reliable expansion medium that can efficiently maintain the activity of immune cells. Summary of the invention

[0004] In view of the defects of the prior art, the object of the present invention is to provide an immune cell culture expansion medium and a preparation method thereof.

[0005] The technical effects of the present invention are achieved by the following technical solutions: an immune cell culture and expansion medium, comprising a matrix and active ingredients;

[0006] The matrix comprises the following components by weight: 10 to 15 parts of thiolated hyaluronic acid, 8 to 12 parts of mercapto polyethylene glycol, 0.5 to 1 part of thioglycolic acid, 0.01 to 0.05 parts of hydrogen peroxide, 1 to 2 parts of perfluorodecane, 0.5 to 1 part of soybean lecithin, 1 to 2 parts of cell protectants and 40 to 50 parts of PBS buffer;

[0007] The composition of the active ingredient includes the following components by weight: 0.005-0.01 parts of IL-2, 0.001-0.005 parts of IL-15, 0.05-0.1 parts of α-galactosylceramide, 0.03-0.15 parts of glutathione, 0.5-1 parts of amino acids, 0.25-0.5 parts of L-glutamine, 0.05-0.1 parts of pyruvic acid and 0.1-0.5 parts of albumin.

[0008] Preferably, the cell protectant is any one of trehalose, mannitol, sorbitol and betaine.

[0009] Preferably, the IL-2 is used at a concentration of 20 ng / mL; the IL-15 is used at a concentration of 10 ng / mL; the α-galactosylceramide is used at a concentration of 2 mmol / L; the glutathione is used at a concentration of 2 mmol / L; the amino acid is used at a concentration of 1 mmol / L; the L-glutamine is used at a concentration of 2 mmol / L; the pyruvate is used at a concentration of 1 mmol / L; and the albumin is used at a concentration of 0.5 mg / mL.

[0010] Preferably, the amino acid is any one of serine, glycine, aspartic acid and glutamic acid.

[0011] Preferably, the specific preparation steps of the sulfonated hyaluronic acid are as follows:

[0012] A1: Dissolve hyaluronic acid in deionized water and stir to dissolve evenly to obtain a 0.5-1% hyaluronic acid solution; add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to the hyaluronic acid solution under ice bath conditions, and adjust the pH to 4.5-5. After stirring for 30 minutes, add N-hydroxysuccinimide and continue stirring for 60 minutes to obtain an activated solution;

[0013] A2: Add thioethylamine to the activation solution prepared in step A1, react for 4 to 6 hours in an ice bath, then add dithiothreitol and continue the reaction for 30 minutes. After the reaction is completed, keep the solution in a nitrogen environment, then dialyze the solution with a 3 to 6 kDa dialysis bag for 24 to 48 hours, and freeze-dry it at -60 vacuum for 12 hours to obtain thiolated hyaluronic acid.

[0014] Preferably, in step A1, the mass ratio of the hyaluronic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is 4:1.5-3:1.15-2.3;

[0015] Preferably, in step A2, the amount of thioethylamine used is 20-40% of the mass of hyaluronic acid; the amount of dithiothreitol used is 40% of the mass of hyaluronic acid.

[0016] Preferably, the specific preparation steps of the thiol polyethylene glycol sulfhydryl are as follows:

[0017] B1: dissolving polyethylene glycol in anhydrous dichloromethane, stirring and dissolving uniformly to obtain a polyethylene glycol solution with a concentration of 10%; placing the polyethylene glycol solution in an ice bath, adding potassium carbonate, stirring and mixing uniformly, slowly adding iodomethane, continuing to stir and react for 4 to 6 hours, filtering, washing the organic phase with distilled water, drying with anhydrous magnesium sulfate, filtering, and obtaining a dry organic phase;

[0018] B2: Add the dry organic phase prepared in step B1 to cold ether, collect the precipitate by centrifugation, and vacuum dry to constant weight to obtain iodinated polyethylene glycol; dissolve the iodinated polyethylene glycol in anhydrous tetrahydrofuran, stir until completely dissolved, add sodium hydrosulfide at room temperature, maintain a nitrogen atmosphere, and stir the reaction for 12 hours.

[0019] B3: After the reaction in step B2 is completed, 10 wt% hydrochloric acid solution is added to neutralize it, and then the organic phase is separated, dried over anhydrous magnesium sulfate, filtered, poured into cold ether, the precipitate is collected, and vacuum dried to constant weight to obtain mercapto polyethylene glycol.

[0020] Preferably, in step B1, the polyethylene glycol is PEG-2000; the ratio of the polyethylene glycol, potassium carbonate and iodomethane is 1 g: 0.4-0.45 g: 0.2-0.25 mL;

[0021] Preferably, in step B2, the volume of the cold ether used is the same as that of the anhydrous dichloromethane in step B1; the ratio of the iodinated polyethylene glycol, anhydrous tetrahydrofuran and sodium hydrosulfide used is 1g:8-10mL:0.15-0.2g;

[0022] Preferably, in step B3, the volume of cold ether used is the same as that of anhydrous tetrahydrofuran in step B2.

[0023] Preferably, another aspect of the present invention is to provide a method for preparing an immune cell culture expansion medium, and the specific preparation steps are as follows:

[0024] S1: Dissolve thiolated hyaluronic acid and mercapto polyethylene glycol in 80% by weight of PBS buffer, stir until completely dissolved, slowly add thioglycolic acid and hydrogen peroxide, control the pH to neutral, and stir and react at 37°C for 1 to 2 hours;

[0025] S2: adding soybean lecithin to 20% by weight of PBS buffer, heating to 40-50° C., stirring until completely dissolved, then slowly adding perfluorodecane, stirring at 800 rpm for 10-15 min, and then ultrasonically treating for 3-5 min to obtain a refined emulsion;

[0026] S3: After the reaction in step S1 is completed, the refined emulsion prepared in step S2 is added, and after being stirred at 1200 rpm and mixed evenly, a cell protective agent is added, and stirring is continued to dissolve and disperse evenly, and the mixture is allowed to stand at 4°C for 12 to 24 hours to obtain a matrix;

[0027] S4: Adjust the pH of the matrix prepared in step S3 to neutral and control the temperature at 4°C, then add the active ingredients IL-2, IL-15, α-galactosylceramide, glutathione, L-glutamine, pyruvate, amino acids and albumin in sequence, stir gently to ensure that the active ingredients are evenly dispersed, and obtain the expansion medium.

[0028] The beneficial effects of the present invention are as follows:

[0029] The hyaluronic acid used in the present invention is a natural polysaccharide, widely present in the extracellular matrix, has good biocompatibility and biodegradability, can simulate the natural extracellular environment, and provides suitable growth support for immune cells. The hydrophilicity and flexibility of hyaluronic acid help promote cell proliferation, migration and functional expression; the hydrogel network formed by thiolated hyaluronic acid and thiol polyethylene glycol (PEG) through disulfide cross-linking has a high degree of biocompatibility and dynamic regulation ability, and this flexible and stable three-dimensional scaffold can simulate the natural extracellular matrix, provide physical support for immune cells in three-dimensional space, ensure its normal proliferation, differentiation and migration, reduce the stress response and apoptosis of cells, so as to maintain a high survival rate. Based on the reversible cross-linked structure of disulfide bonds, the three-dimensional network not only has physical stability, but also has anti-enzyme degradation ability; proteases or matrix metalloproteinases (MMPs) secreted by immune cells during culture can degrade conventional matrix materials; however, through the dynamic characteristics of disulfide bonds, the matrix material can effectively resist enzymolysis and extend its functional life, thereby ensuring stability in long-term culture.

[0030] The present invention loads perfluorodecane into a hydrophilic network constructed by thiolated hyaluronic acid and mercapto polyethylene glycol by means of a nanoemulsion, and uses soybean lecithin as an emulsifier to evenly disperse hydrophobic perfluorodecane in a hydrophilic matrix to form a stable mixed system, thereby achieving effective load in a water-soluble network. Perfluorodecane, as an oxygen carrier in the network structure, can not only provide continuous oxygen support for immune cells, but also promote the redox reaction occurring in cells and materials, maintain the metabolic needs of cells and the stability of materials; perfluorodecane can maintain the normal metabolic activity of immune cells by providing continuous oxygen, and avoid stress reactions caused by hypoxia. This stable oxygen supply not only ensures the efficient proliferation and functional retention of immune cells, but also enhances the adaptability of the network to external mechanical stress and cell migration.

[0031] In addition, the disulfide-crosslinked network structure can control the release of active ingredients by adjusting the pore size and crosslinking density. For example, growth factors (IL-2, IL-15) can be released slowly and continuously through the network to avoid the risk of rapid consumption of active factors or cytotoxicity caused by excessive instantaneous release; for specific active ingredients such as α-galactosylceramide, the network structure can efficiently deliver them to target cells through selective transmission to exert their immunomodulatory function. Metabolites such as L-glutamine and pyruvate are crucial in the process of immune cell expansion. The disulfide bond network can prevent these small molecules from rapidly diffusing, thereby providing a stable energy supply, avoiding fluctuations in energy supply, and ensuring continuous cell proliferation. Glutathione has an antioxidant function, and its rapid oxidation can be effectively prevented by a crosslinked network. At the same time, the environment within the network can maintain its biological activity, thereby protecting immune cells from oxidative stress. The hydrophilicity and anti-protein adsorption properties of thiol polyethylene glycol further reduce the risk of nonspecific adsorption in the culture matrix and prevent adsorbed proteins or other macromolecules from affecting their functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 is a graph showing the percentage of cell activity of the expansion culture medium prepared in Examples 1 to 4 of the present invention and Comparative Examples 1 to 2;

[0034] Figure 2 is a flow cytometry image of the expansion medium prepared in Example 2 of the present invention;

[0035] Figure 3is a flow cytometry graph of the expansion medium prepared in Comparative Example 2 of the present invention;

[0036] Figure 4 It is a test chart of the anti-enzyme hydrolysis of the amplification culture medium prepared in Example 2 of the present invention and Comparative Examples 1-2;

[0037] Figure 5 It is a weight damage test diagram of the expansion culture medium prepared in Example 2 of the present invention and Comparative Examples 1-2;

[0038] Figure 6 It is a graph showing changes in the concentration of active factors in the amplification culture medium prepared in Example 2 of the present invention and Comparative Examples 1 to 2;

[0039] Figure 7 2 is a graph showing changes in oxygen concentration in the amplification culture medium prepared in Example 2 of the present invention and Comparative Example 1. DETAILED DESCRIPTION

[0040] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. It should be noted that, unless otherwise specified, the raw materials involved in the present invention are purchased through conventional commercial channels.

[0041] Example 1: An immune cell culture expansion medium, comprising a matrix and an active ingredient;

[0042] The matrix comprises the following components by weight: 10 parts of thiolated hyaluronic acid, 8 parts of mercapto polyethylene glycol, 0.5 parts of thioglycolic acid, 0.01 parts of hydrogen peroxide, 1 part of perfluorodecane, 0.5 parts of soybean lecithin, 1 part of cell protectant and 40 parts of PBS buffer;

[0043] The composition of the active ingredient includes the following components by weight: 0.005 parts of IL-2, 0.001 parts of IL-15, 0.05 parts of α-galactosylceramide, 0.03 parts of glutathione, 0.5 parts of amino acids, 0.25 parts of L-glutamine, 0.05 parts of pyruvic acid and 0.1 parts of albumin.

[0044] Preparation of sulfhydryl hyaluronic acid:

[0045] A1: Dissolve 20 g of hyaluronic acid in deionized water and stir to dissolve evenly to obtain a 1% hyaluronic acid solution; add 6 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to the hyaluronic acid solution under ice bath conditions, adjust the pH to 4.5, stir for 30 min, add 4.6 g of N-hydroxysuccinimide, and continue stirring for 60 min to obtain an activated solution;

[0046] A2: Add 4 g of thioethylamine to the activation solution prepared in step A1, react for 4 h in an ice bath, then add 8 g of dithiothreitol and continue to react for 30 min. After the reaction is completed, keep the solution in a nitrogen environment, then dialyze the solution with a 3 kDa dialysis bag for 24 h, and freeze-dry at -60 °C for 12 h to obtain thiolated hyaluronic acid;

[0047] Preparation of Thiol Polyethylene Glycol Sulfhydryl:

[0048] B1: Dissolve 10 g of polyethylene glycol in 100 mL of anhydrous dichloromethane, stir and dissolve evenly to obtain a polyethylene glycol solution with a concentration of 10%; place the polyethylene glycol solution in an ice bath, add 4 g of potassium carbonate, stir and mix evenly, slowly add 2 mL of iodomethane, continue stirring and reacting for 4 hours, filter, wash the organic phase with distilled water, dry with anhydrous magnesium sulfate, filter, and obtain a dry organic phase;

[0049] B2: Add the dried organic phase prepared in step B1 to 100 mL of cold ether, collect the precipitate by centrifugation, and vacuum dry to constant weight to obtain iodinated polyethylene glycol; dissolve 10 g of iodinated polyethylene glycol in 80 mL of anhydrous tetrahydrofuran, stir until completely dissolved, add 1.5 g of sodium hydrosulfide at room temperature, maintain a nitrogen atmosphere, and stir the reaction for 12 hours.

[0050] B3: After the reaction in step B2 is completed, a 10 wt% hydrochloric acid solution is added to neutralize it, and then the organic phase is separated, the organic phase is dried over anhydrous magnesium sulfate, filtered, poured into cold ether, the precipitate is collected, and vacuum dried to constant weight to obtain mercapto polyethylene glycol;

[0051] Preparation of immune cell culture expansion medium:

[0052] S1: Dissolve thiolated hyaluronic acid and mercapto polyethylene glycol in 80% by weight of PBS buffer, stir until completely dissolved, slowly add thioglycolic acid and hydrogen peroxide, control the pH to neutral, and stir and react at 37°C for 1 hour;

[0053] S2: adding soybean lecithin to 20% by weight of PBS buffer, heating to 40°C, stirring until completely dissolved, then slowly adding perfluorodecane, stirring at 800 rpm for 15 min, and then ultrasonically treating for 3 min to obtain a refined emulsion;

[0054] S3: After the reaction in step S1 is completed, the refined emulsion prepared in step S2 is added, and after being stirred at 1200 rpm for uniform mixing, trehalose is added, and stirring is continued to dissolve and disperse uniformly, and the mixture is allowed to stand at 4°C for 12 hours to obtain a matrix;

[0055] S4: Adjust the pH of the matrix prepared in step S3 to neutral and control the temperature at 4°C, then add the active ingredients IL-2, IL-15, α-galactosylceramide, glutathione, L-glutamine, pyruvate, serine and albumin in sequence, stir gently to ensure that the active ingredients are evenly dispersed, and obtain the expansion medium.

[0056] Example 2: An immune cell culture expansion medium, comprising a matrix and an active ingredient;

[0057] The matrix comprises the following components in parts by weight: 13 parts of thiolated hyaluronic acid, 11 parts of mercapto polyethylene glycol, 1 part of thioglycolic acid, 0.04 parts of hydrogen peroxide, 2 parts of perfluorodecane, 1 part of soybean lecithin, 1.5 parts of cell protectant and 50 parts of PBS buffer;

[0058] The composition of the active ingredient includes the following components by weight: 0.01 parts of IL-2, 0.005 parts of IL-15, 0.08 parts of α-galactosylceramide, 0.1 parts of glutathione, 1 part of amino acids, 0.5 parts of L-glutamine, 0.1 parts of pyruvic acid and 0.5 parts of albumin.

[0059] Preparation of sulfhydryl hyaluronic acid:

[0060] A1: Dissolve 20 g of hyaluronic acid in deionized water and stir to dissolve evenly to obtain a 1% hyaluronic acid solution; add 10 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to the hyaluronic acid solution under ice bath conditions, adjust the pH to 5, stir for 30 min, add 8 g of N-hydroxysuccinimide, and continue stirring for 60 min to obtain an activated solution;

[0061] A2: Add 7 g of thioethylamine to the activation solution prepared in step A1, react for 6 h in an ice bath, then add 8 g of dithiothreitol and continue to react for 30 min. After the reaction is completed, keep the solution in a nitrogen environment, then dialyze the solution with a 3 kDa dialysis bag for 36 h, and freeze-dry at -60 °C for 12 h to obtain thiolated hyaluronic acid;

[0062] Preparation of Thiol Polyethylene Glycol Sulfhydryl:

[0063] B1: Dissolve 10 g of polyethylene glycol in 100 mL of anhydrous dichloromethane, stir and dissolve evenly to obtain a polyethylene glycol solution with a concentration of 10%; place the polyethylene glycol solution in an ice bath, add 4.2 g of potassium carbonate, stir and mix evenly, then slowly add 2.3 mL of iodomethane, continue stirring and reacting for 6 hours, filter, wash the organic phase with distilled water, dry with anhydrous magnesium sulfate, filter, and obtain a dry organic phase;

[0064] B2: Add the dried organic phase prepared in step B1 to 100 mL of cold ether, collect the precipitate by centrifugation, and vacuum dry to constant weight to obtain iodinated polyethylene glycol; dissolve 10 g of iodinated polyethylene glycol in 90 mL of anhydrous tetrahydrofuran, stir until completely dissolved, add 1.8 g of sodium hydrosulfide at room temperature, maintain a nitrogen atmosphere, and stir the reaction for 12 hours.

[0065] B3: After the reaction in step B2 is completed, a 10 wt% hydrochloric acid solution is added to neutralize it, and then the organic phase is separated, the organic phase is dried over anhydrous magnesium sulfate, filtered, poured into 90 mL of cold ether, the precipitate is collected, and vacuum dried to constant weight to obtain mercapto polyethylene glycol;

[0066] Preparation of immune cell culture expansion medium:

[0067] S1: Dissolve thiolated hyaluronic acid and mercapto polyethylene glycol in 80% by weight of PBS buffer, stir until completely dissolved, slowly add thioglycolic acid and hydrogen peroxide, control the pH to neutral, and stir and react at 37°C for 2h;

[0068] S2: adding soybean lecithin to 20% by weight of PBS buffer, heating to 45°C, stirring until completely dissolved, then slowly adding perfluorodecane, stirring at 800 rpm for 10 min, and then ultrasonically treating for 4 min to obtain a refined emulsion;

[0069] S3: After the reaction in step S1 is completed, add the refined emulsion prepared in step S2, stir and mix evenly at 1200 rpm, add mannitol, continue to stir and dissolve evenly, and stand at 4°C for 24 hours to obtain a matrix;

[0070] S4: Adjust the pH of the matrix prepared in step S3 to neutral and control the temperature at 4°C, then add the active ingredients IL-2, IL-15, α-galactosylceramide, glutathione, L-glutamine, pyruvate, glycine and albumin in sequence, gently stir to ensure that the active ingredients are evenly dispersed, and obtain the expansion medium.

[0071] Example 3: An immune cell culture expansion medium, comprising a matrix and an active ingredient;

[0072] The matrix comprises the following components by weight: 12 parts of thiolated hyaluronic acid, 10 parts of mercapto polyethylene glycol, 0.6 parts of thioglycolic acid, 0.02 parts of hydrogen peroxide, 1.5 parts of perfluorodecane, 0.8 parts of soybean lecithin, 1.5 parts of cell protectant and 45 parts of PBS buffer;

[0073] The composition of the active ingredient includes the following components by weight: 0.008 parts of IL-2, 0.002 parts of IL-15, 0.06 parts of α-galactosylceramide, 0.12 parts of glutathione, 0.6 parts of amino acids, 0.4 parts of L-glutamine, 0.08 parts of pyruvic acid and 0.3 parts of albumin.

[0074] Preparation of sulfhydryl hyaluronic acid:

[0075] A1: Dissolve 20 g of hyaluronic acid in deionized water and stir to dissolve evenly to obtain a 0.6% hyaluronic acid solution; add 9 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to the hyaluronic acid solution under ice bath conditions, adjust the pH to 4.8, stir for 30 min, add 7 g of N-hydroxysuccinimide, and continue stirring for 60 min to obtain an activated solution;

[0076] A2: Add 6 g of thioethylamine to the activation solution prepared in step A1, react for 5 h in an ice bath, then add 8 g of dithiothreitol and continue to react for 30 min. After the reaction is completed, keep the solution in a nitrogen environment, then dialyze the solution with a 5 kDa dialysis bag for 36 h, and freeze-dry at -60 °C for 12 h to obtain thiolated hyaluronic acid;

[0077] Preparation of Thiol Polyethylene Glycol Sulfhydryl:

[0078] B1: Dissolve 10 g of polyethylene glycol in 100 mL of anhydrous dichloromethane, stir and dissolve evenly to obtain a polyethylene glycol solution with a concentration of 10%; place the polyethylene glycol solution in an ice bath, add 4.4 g of potassium carbonate, stir and mix evenly, then slowly add 2.4 mL of iodomethane, continue stirring and reacting for 5 hours, filter, wash the organic phase with distilled water, dry with anhydrous magnesium sulfate, and filter to obtain a dry organic phase;

[0079] B2: Add the dried organic phase prepared in step B1 to 100 mL of cold ether, collect the precipitate by centrifugation, and vacuum dry to constant weight to obtain iodinated polyethylene glycol; dissolve 10 g of iodinated polyethylene glycol in 85 mL of anhydrous tetrahydrofuran, stir until completely dissolved, add 1.9 g of sodium hydrosulfide at room temperature, maintain a nitrogen atmosphere, and stir the reaction for 12 hours.

[0080] B3: After the reaction in step B2 is completed, a 10 wt% hydrochloric acid solution is added to neutralize it, and then the organic phase is separated, the organic phase is dried over anhydrous magnesium sulfate, filtered, poured into 85 mL of cold ether, the precipitate is collected, and vacuum dried to constant weight to obtain mercapto polyethylene glycol;

[0081] Preparation of immune cell culture expansion medium:

[0082] S1: Dissolve thiolated hyaluronic acid and mercapto polyethylene glycol in 80% by weight of PBS buffer, stir until completely dissolved, slowly add thioglycolic acid and hydrogen peroxide, control the pH to neutral, and stir the reaction at 37°C for 1.5h;

[0083] S2: adding soybean lecithin to 20% by weight of PBS buffer, heating to 48°C, stirring until completely dissolved, then slowly adding perfluorodecane, stirring at 800 rpm for 12 minutes, and then ultrasonically treating for 4 minutes to obtain a refined emulsion;

[0084] S3: After the reaction in step S1 is completed, the refined emulsion prepared in step S2 is added, and after being stirred at 1200 rpm for uniform mixing, sorbitol is added, and stirring is continued to dissolve and disperse uniformly, and the mixture is allowed to stand at 4° C. for 18 hours to obtain a matrix;

[0085] S4: The pH of the matrix prepared in step S3 is adjusted to neutral, and the temperature is controlled at 4°C, and then the active ingredients IL-2, IL-15, α-galactosylceramide, glutathione, L-glutamine, pyruvate, aspartic acid and albumin are added in sequence, and gently stirred to ensure that the active ingredients are evenly dispersed to obtain an expansion medium.

[0086] Example 4: An immune cell culture expansion medium, comprising a matrix and an active ingredient;

[0087] The matrix comprises the following components by weight: 15 parts of thiolated hyaluronic acid, 12 parts of mercapto polyethylene glycol, 1 part of thioglycolic acid, 0.05 parts of hydrogen peroxide, 2 parts of perfluorodecane, 1 part of soybean lecithin, 2 parts of cell protectants and 50 parts of PBS buffer;

[0088] The composition of the active ingredient includes the following components by weight: 0.01 parts of IL-2, 0.005 parts of IL-15, 0.1 parts of α-galactosylceramide, 0.15 parts of glutathione, 1 part of amino acids, 0.5 parts of L-glutamine, 0.1 parts of pyruvic acid and 0.5 parts of albumin.

[0089] Preparation of sulfhydryl hyaluronic acid:

[0090] A1: Dissolve 20 g of hyaluronic acid in deionized water and stir to dissolve evenly to obtain a 0.5% hyaluronic acid solution; add 12 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to the hyaluronic acid solution under ice bath conditions, adjust the pH to 5, stir for 30 min, add 9.2 g of N-hydroxysuccinimide, and continue stirring for 60 min to obtain an activated solution;

[0091] A2: Add 8 g of thioethylamine to the activation solution prepared in step A1, react for 6 h in an ice bath, then add 8 g of dithiothreitol and continue to react for 30 min. After the reaction is completed, keep the solution in a nitrogen environment, then dialyze the solution with a 6 kDa dialysis bag for 48 h, and freeze-dry at -60 °C for 12 h to obtain thiolated hyaluronic acid;

[0092] Preparation of Thiol Polyethylene Glycol Sulfhydryl:

[0093] B1: Dissolve 10 g of polyethylene glycol in 100 mL of anhydrous dichloromethane, stir and dissolve evenly to obtain a polyethylene glycol solution with a concentration of 10%; place the polyethylene glycol solution in an ice bath, add 4.5 g of potassium carbonate, stir and mix evenly, then slowly add 2.5 mL of iodomethane, continue stirring and reacting for 6 hours, filter, wash the organic phase with distilled water, dry with anhydrous magnesium sulfate, filter, and obtain a dry organic phase;

[0094] B2: Add the dried organic phase prepared in step B1 to 100 mL of cold ether, collect the precipitate by centrifugation, and vacuum dry to constant weight to obtain iodinated polyethylene glycol; dissolve 10 g of iodinated polyethylene glycol in 100 mL of anhydrous tetrahydrofuran, stir until completely dissolved, add 2 g of sodium hydrosulfide at room temperature, maintain a nitrogen atmosphere, and stir the reaction for 12 hours.

[0095] B3: After the reaction in step B2 is completed, a 10 wt% hydrochloric acid solution is added to neutralize it, and then the organic phase is separated, the organic phase is dried over anhydrous magnesium sulfate, filtered, poured into 100 mL of cold ether, the precipitate is collected, and vacuum dried to constant weight to obtain mercapto polyethylene glycol;

[0096] Preparation of immune cell culture expansion medium:

[0097] S1: Dissolve thiolated hyaluronic acid and mercapto polyethylene glycol in 80% by weight of PBS buffer, stir until completely dissolved, slowly add thioglycolic acid and hydrogen peroxide, control the pH to neutral, and stir and react at 37°C for 2h;

[0098] S2: adding soybean lecithin to 20% by weight of PBS buffer, heating to 50°C, stirring until completely dissolved, then slowly adding perfluorodecane, stirring at 800 rpm for 15 minutes, and then ultrasonically treating for 5 minutes to obtain a refined emulsion;

[0099] S3: After the reaction in step S1 is completed, add the refined emulsion prepared in step S2, stir and mix evenly at 1200 rpm, add betaine, continue to stir and dissolve evenly, and stand at 4°C for 24 hours to obtain a matrix;

[0100] S4: Adjust the pH of the matrix prepared in step S3 to neutral, and control the temperature at 4°C, then add the active ingredients IL-2, IL-15, α-galactosylceramide, glutathione, L-glutamine, pyruvate, glutamate and albumin in sequence, and gently stir to ensure that the active ingredients are evenly dispersed to obtain an expansion medium.

[0101] Comparative Example 1: The operation of Comparative Example 1 is basically the same as that of Example 2, except that no perfluorodecane is added in Comparative Example 1, and soybean lecithin is added normally.

[0102] Comparative Example 2: The operation of Comparative Example 2 is basically the same as that of Example 2, except that in Comparative Example 2, hyaluronic acid and gelatin are used to construct a network structure through glutaraldehyde, replacing the network structure constructed by thioglycolic acid with thiolated hyaluronic acid and thiopolyethylene glycol.

[0103] Performance Test:

[0104] Cell activity test: The expansion culture medium samples prepared in Examples 1 to 4 and Comparative Examples 1 to 2 were placed at the bottom of the culture plate, and 8 wells per group of samples were evenly distributed in a 96-well plate. 50 μL of DMEM culture medium was added to each well, and then the NKT cells were diluted to 1×10 4 After culturing for 24 h, 48 h, and 72 h, 5 μL of 5 mg / mL MTT solution was added. After culturing for 4 h, the culture medium in the wells was aspirated, and then 50 μL of dimethyl sulfoxide was added to the wells. The absorbance (OD value) was measured at 490 nm using an ELISA reader, and the activity percentage was measured [activity percentage (%) = (OD value of the embodiment - OD value of the negative control group) / (OD value of the positive control group - OD value of the negative control group) × 100%]. The blank control group contained only culture medium and MTT solution. Each sample was processed and analyzed three times, and the results were averaged. The cell survival rate results are shown in the figure. Figure 1 shown.

[0105] Depend on Figure 1The results show that the expansion medium prepared by the present invention shows a higher cell survival rate. From the results of Comparative Example 1 and Example 2, it can be seen that in the absence of perfluorodecane, the cell survival rate showed a significant downward trend at 48 hours. From the results of Comparative Example 2 and Example 2, it can be seen that due to the lack of dynamic reversibility of the glutaraldehyde cross-linked network, it is difficult to respond to changes in the cell microenvironment. In addition, gelatin is more easily degraded by matrix metalloproteinases secreted by cells, resulting in an unstable three-dimensional network structure, which may lead to a significant decrease in cell activity after 72 hours.

[0106] Flow cytometry analysis test: After culturing NKT cells in the expansion medium prepared in Example 2 and Comparative Example 2 for 14 days, they were stained with fluorescent labeled antibodies and detected by flow cytometry. The results of Example 2 are as follows: Figure 2 As shown, the results of comparative example 3 are as follows Figure 3 shown.

[0107] Depend on Figure 2 and Figure 3 The results show that the expansion culture medium prepared by the present invention has a significant effect on the expansion culture of immune cells.

[0108] Anti-enzyme hydrolysis ability test: The expansion culture medium prepared in Example 2 and Comparative Examples 1 to 2 was immersed in a PBS buffer containing 1 μg / mL of MMP-2 metalloprotease, and a blank control group without adding MMP-2 metalloprotease was set up. The culture medium was cultured at 37°C and 5% CO2. At the same time, the MMP-2 metalloprotease solution was replaced every 2 days. The samples were removed at the 1st, 3rd and 7th day time points and the surface liquid was absorbed and weighed. The weight loss was calculated as (weight before the test - weight after the test) / weight before the test × 100%. The results are as follows Figure 4 shown.

[0109] Depend on Figure 4 As a result, the expansion medium prepared by the present invention has excellent resistance to enzymolysis and very little weight loss; from the results of Comparative Example 1 and Example 2, it can be seen that the antioxidant capacity of the network structure is affected after the oxygen carrier support of perfluorodecane is lacking, and the stability of the disulfide bond may be more affected by the degradation of the enzyme, which may lead to the obvious weight loss change of the culture medium starting from the 3rd day; from the results of Comparative Example 2 and Example 2, it can be seen that Comparative Example 2 uses hyaluronic acid and gelatin network cross-linked by glutaraldehyde, and the cross-linked structure formed by glutaraldehyde is relatively rigid and has good initial resistance to enzymolysis, but this irreversible cross-linked structure is susceptible to enzyme attack in long-term exposure to MMP-2, and the gelatin component is particularly susceptible to degradation by metalloproteinases. Therefore, in the 14th day culture test, the weight damage of the network structure dropped drastically.

[0110] Long-term stability test: 1 mL of 1×10 5 NKT cells / mL were cultured at 37°C and 5% CO2 for 14 days. The surface liquid of the culture medium was removed on the 1st, 3rd, 7th and 14th days to measure the weight change of the culture medium. The results are as follows: Figure 5 As shown; on the 1st day, 3rd day, 7th day and 14th day, the percentage changes in the concentrations of active factors IL-2 and IL-15 were measured, and the results are shown Figure 6 As shown; At the same time, the oxygen concentration changes of the culture medium of Example 2 and Comparative Example 1 were measured, and the results are as follows Figure 7 shown.

[0111] Depend on Figure 5 As a result, it can be seen that the expansion medium prepared by the present invention has good long-term stability; from the results of Comparative Example 1 and Example 2, it can be seen that due to the lack of continuous oxygen support, cell metabolism gradually weakens, and the cells may enter an oxygen-deficient state, resulting in a decrease in metabolic levels, resulting in a relative decrease in weight loss starting on the 7th day, which may be due to the inhibition of cell activity and the impact of enzyme activity. On the 14th day, the weight change of the culture medium fluctuates, which may be due to the impact of cell activity and waste accumulation; from the results of Comparative Example 2 and Example 2, it can be seen that although the network structure cross-linked by glutaraldehyde is relatively stable, gelatin is more easily degraded by matrix metalloproteinases, and the enzymes secreted by the cells have caused a certain degree of damage to the network structure on the 3rd day, resulting in a relatively obvious change in weight damage starting on the 3rd day, and the cell activity began to decrease on the 7th day, and the weight loss began to gradually decrease and stabilize.

[0112] Depend on Figure 6 As a result, it can be seen that the expansion medium prepared by the present invention has good long-term stability; from the results of comparative example 1 and example 2, it can be seen that due to the absence of perfluorodecane, although the initial release of cytokines is relatively stable, as the proliferation and metabolism of cells begin to be affected by hypoxia, the demand for cytokines increases, while the efficiency of cell proliferation decreases, which leads to a slight decrease in the concentration of IL-2 and IL-15 starting from the 3rd day, and the release of cell activity factors becomes unstable from the 7th day; from the results of comparative example 2 and example 2, it can be seen that in the early stage, due to the relatively rapid degradation of gelatin under the action of enzymes, the release of IL-2 and IL-15 may be relatively rapid in the early stage, which leads to a rapid change in their concentration on the 3rd day, and on the 7th day, the cell activity and enzyme activity begin to decrease, the concentrations of IL-2 and IL-15 are low and the release tends to be stable, and the lack of an effective sustained-release mechanism leads to the rapid consumption of active factors in the early stage, which has a significant impact on cell survival.

[0113] Depend on Figure 7The results show that the expansion medium prepared by the present invention has good long-term stability, and the oxygen concentration changes are relatively stable. From the results of Comparative Example 1 and Example 2, it can be seen that due to the absence of perfluorodecane, the oxygen mainly comes from the dissolved oxygen in the culture medium. Starting from the 7th day, the proliferation of cells is restricted, the metabolic activity of the cells is weakened, the oxygen consumption is also reduced, and the overall oxygen concentration is maintained at a low level.

[0114] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An immune cell culture expansion medium, characterized in that: The invention is composed of a matrix and active ingredients; the matrix comprises the following components in parts by weight: 10 to 15 parts of thiolated hyaluronic acid, 8 to 12 parts of mercapto polyethylene glycol, 0.5 to 1 part of thioglycolic acid, 0.01 to 0.05 parts of hydrogen peroxide, 1 to 2 parts of perfluorodecane, 0.5 to 1 part of soybean lecithin, 1 to 2 parts of cell protectants and 40 to 50 parts of PBS buffer; the active ingredients comprise the following components in parts by weight: 0.005 to 0.01 parts of IL-2, 0.001 to 0.005 parts of IL-15, 0.05 to 0.1 parts of α-galactosylceramide, 0.03 to 0.15 parts of glutathione, 0.5 to 1 parts of amino acids, 0.25 to 0.5 parts of L-glutamine, 0.05 to 0.1 parts of pyruvic acid and 0.1 to 0.5 parts of albumin; the immune cells are NKT immune cells; The cell protectant is any one of trehalose, mannitol, sorbitol and betaine; The amino acid is any one of serine, glycine, aspartic acid and glutamic acid.

2. The immune cell culture and expansion medium according to claim 1, characterized in that: The specific preparation steps of the sulfonated hyaluronic acid are as follows: A1: dissolving hyaluronic acid in deionized water, stirring and dissolving uniformly to obtain a hyaluronic acid solution; adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to the hyaluronic acid solution under ice bath conditions, adjusting the pH, stirring, adding N-hydroxysuccinimide, and continuing to stir to obtain an activated solution; A2: Add thioethylamine to the activation solution prepared in step A1, react under ice bath conditions, then add dithiothreitol and continue the reaction. After the reaction is completed, keep the solution in a nitrogen environment, dialyze with a dialysis bag, and vacuum freeze-dry to obtain thiolated hyaluronic acid.

3. An immune cell culture expansion medium according to claim 2, characterized in that: In step A1, the mass ratio of the hyaluronic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is 4:1.5-3:1.15-2.3; in step A2, the amount of the thioethylamine is 20-40% of the mass of the hyaluronic acid; and the amount of dithiothreitol is 40% of the mass of the hyaluronic acid.

4. The immune cell culture and expansion medium according to claim 3, characterized in that: The specific preparation steps of the mercapto polyethylene glycol are as follows: B1: dissolving polyethylene glycol in anhydrous dichloromethane, stirring and dissolving uniformly to obtain a polyethylene glycol solution; placing the polyethylene glycol solution in an ice bath, adding potassium carbonate, stirring and mixing uniformly, slowly adding iodomethane, continuing to stir and react, filtering, washing the organic phase with distilled water, drying with anhydrous magnesium sulfate, filtering, and obtaining a dry organic phase; B2: Add the dried organic phase prepared in step B1 to cold ether, collect the precipitate by centrifugation, and vacuum dry to constant weight to obtain iodinated polyethylene glycol; dissolve the iodinated polyethylene glycol in anhydrous tetrahydrofuran, stir until completely dissolved, add sodium hydrosulfide at room temperature, maintain a nitrogen atmosphere, and stir to react; B3: After the reaction in step B2 is completed, hydrochloric acid solution is added to neutralize it, and then the organic phase is separated, the organic phase is dried over anhydrous magnesium sulfate, filtered, poured into cold ether, the precipitate is collected, and vacuum dried to constant weight to obtain mercapto polyethylene glycol.

5. An immune cell culture expansion medium according to claim 4, characterized in that: In step B1, the polyethylene glycol is PEG-2000; the ratio of the polyethylene glycol, potassium carbonate and methyl iodide is 1g:0.4-0.45g:0.2-0.25mL; in step B2, the volume of the cold ether is the same as that of the anhydrous dichloromethane in step B1; the ratio of the iodinated polyethylene glycol, anhydrous tetrahydrofuran and sodium hydrosulfide is 1g:8-10mL:0.15-0.2g; in step B3, the volume of the cold ether is the same as that of the anhydrous tetrahydrofuran in step B2.

6. A method for preparing the immune cell culture and expansion medium according to any one of claims 1 to 5, characterized in that: The specific preparation steps are as follows: S1: Dissolve the sulfhydryl hyaluronic acid and mercapto polyethylene glycol in 80% by weight of PBS buffer, stir until completely dissolved, slowly add thioglycolic acid and hydrogen peroxide, control the pH to neutral, and stir to react; S2: adding soybean lecithin to 20% by weight of PBS buffer, heating, stirring until completely dissolved, then slowly adding perfluorodecane, stirring, and then ultrasonically treating to obtain a refined emulsion; S3: After the reaction in step S1 is completed, the refined emulsion prepared in step S2 is added, and after stirring and mixing evenly, a cell protective agent is added, and stirring is continued to dissolve and disperse evenly, and the mixture is allowed to stand to obtain a matrix; S4: Adjust the pH of the matrix prepared in step S3 to neutral, and control the temperature, then add the active ingredients IL-2, IL-15, α-galactosylceramide, glutathione, L-glutamine, pyruvate, amino acids and albumin in sequence, and gently stir to ensure that the active ingredients are evenly dispersed to obtain an expansion medium.

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