A factor kit for culturing γδ t cells, a culture medium and application thereof

Abstract

The present application relates to the technical field of culture medium preparation, in particular to a factor kit for culturing gamma delta T cells, a culture medium and application thereof, comprising the following raw materials by weight: 30-50 parts of a composite cytokine core, 20-30 parts of a composite anti-aggregation stabilizer, and 10-20 parts of a composite metabolic regulator; the present application processes interleukin-2, significantly improves its stability and anti-aggregation performance through electrostatic self-assembly, emulsification cross-linking and freeze-drying protection reaction; interleukin-15 realizes long-acting slow release and activity maintenance through electrostatic shielding, multiple emulsion embedding and skeleton support reaction; transforming growth factor-beta effectively solves the problem of easy adsorption and inactivation through affinity protection, biomimetic embedding and molecular inclusion reaction; through the synergistic effect of the three modified factors, the half-life of the cytokine in the culture system is prolonged, the adding frequency is reduced, and the gamma delta T cells obtained through culture have higher expansion fold and tumor killing activity.

Description

Technical Field

[0001] This invention relates to the field of culture medium preparation technology, specifically to a factor kit for culturing γδT cells, a culture medium, and their applications. Background Technology

[0002] As a bridge between the innate and adaptive immune systems, γδT cells have shown great promise in the fields of tumor immunotherapy and the treatment of infectious diseases due to their MHC-unrestricted tumor-killing ability and broad-spectrum antiviral activity. However, the content of γδT cells in human peripheral blood is extremely low, making it difficult to obtain a sufficient number of cells directly from patients for clinical treatment. Therefore, establishing an efficient and stable in vitro expansion and culture system is a key prerequisite for the clinical translation of γδT cells.

[0003] In existing technologies, interleukin-2 has a short half-life in culture medium, is prone to protein aggregation and loss of activity, leading to the need for frequent high-concentration additions, increasing culture costs and contamination risks. Interleukin-15, as a key proliferation factor, degrades rapidly in its natural form in in vitro culture systems, making it difficult to maintain a long-lasting stimulatory signal and limiting the continuous expansion capacity of γδT cells. Although transforming growth factor-β plays an important role in regulating γδT cell function, it readily adsorbs onto container walls and undergoes conformational changes in solution, resulting in an actual effective concentration far lower than the theoretical addition amount, making it difficult to exert the expected immunomodulatory function. Based on this, the present invention provides a factor kit, culture medium, and applications for culturing γδT cells. Summary of the Invention

[0004] The purpose of this invention is to provide a factor kit, culture medium, and application for culturing γδT cells. The factor kit and culture medium for culturing γδT cells prepared by this invention not only have a high γδT cell expansion rate and CD3+γδT cell purity, but also a high tumor cell killing rate.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a factor kit for culturing γδT cells, a culture medium and its application, characterized in that it is composed of the following raw materials in parts by weight: 30-50 parts of a compound cytokine core, 20-30 parts of a compound anti-agglomeration stabilizer, and 10-20 parts of a compound metabolic regulator. The core of the composite cytokine is composed of modified interleukin-2, modified interleukin-15, and modified transforming growth factor-β in a mass ratio of 3:2:1. The modified interleukin-2 was prepared by treating interleukin-2 with trehalose, chitosan quaternary ammonium salt, and polyvinyl alcohol. The modified interleukin-15 was prepared by treating interleukin-15 with arginine, mannitol, and PLGA. The modified transforming growth factor-β was prepared by treating transforming growth factor-β with sodium heparin, chondroitin sulfate, and hydroxypropyl-β-cyclodextrin.

[0006] Furthermore, the preparation method of the modified interleukin-2 is as follows: A 0.5-1.0 mg / mL interleukin-2 solution is mixed with a 0.5-1.5% (w / w) chitosan quaternary ammonium salt solution to form an IL-2 / chitosan complex solution through electrostatic self-assembly; the complex solution is mixed with a 2-4% (w / w) polyvinyl alcohol aqueous solution to undergo emulsification and cross-linking; the resulting emulsion is mixed with a 5-10% (w / w) trehalose solution for freeze-drying protection, followed by gradient sublimation drying to obtain a fluffy modified interleukin-2. The protein conformation is stabilized through electrostatic self-assembly with chitosan quaternary ammonium salt, a protective embedding structure is formed through polyvinyl alcohol emulsification and cross-linking, and the activity is solidified through trehalose freeze-drying protection. These three processes synergistically enhance the anti-aggregation ability and thermal stability of IL-2, effectively extending its half-life in the culture system and reducing the need for frequent additions.

[0007] Further, the interleukin-2 and chitosan quaternary ammonium salt solution are mixed at a volume ratio of 1:2-4; the IL-2 / chitosan complex solution and polyvinyl alcohol aqueous solution are mixed at a volume ratio of 1:1-1.5; and the emulsion and trehalose solution are mixed at a volume ratio of 2-3:1.

[0008] Further, the preparation method of the modified interleukin-15 is as follows: A solution of interleukin-15 with a concentration of 0.8-1.2 mg / mL is mixed with a solution of arginine with a mass fraction of 2-4%, and electrostatic shielding and aggregation inhibition reactions are carried out to form an IL-15 / arginine complex; the complex is used as the inner aqueous phase and mixed with a PLGA oil phase with a mass fraction of 5-8%, emulsified to form a primary emulsion; the primary emulsion is then mixed with an outer aqueous phase containing 1-2% polyvinyl alcohol, emulsified to form a secondary emulsion, and subsequently subjected to rotary evaporation under reduced pressure to obtain a PLGA microcapsule suspension encapsulating IL-15. Finally, the obtained microencapsulated suspension was mixed with a 3-6% mannitol solution containing 0.5-1% nano-calcium carbonate, and subjected to scaffold support and freeze-drying. Then, gradient drying was performed to obtain modified interleukin-15. The protein aggregation was inhibited by arginine electrostatic shielding, and microencapsulation and long-term sustained release were achieved by PLGA encapsulation. The mannitol scaffold support ensured the stability of the dosage form. The three factors synergistically extended the action time of IL-15 in the culture system, achieving continuous release and maintaining high biological activity, effectively solving the problems of short half-life and frequent addition required by conventional IL-15.

[0009] Further, the interleukin-15 solution and arginine solution are mixed at a volume ratio of 1:1.5-2.5; the IL-15 / arginine complex is mixed with the PLGA oil phase at a volume ratio of 1:3-5; the colostrum is mixed with the external aqueous phase containing polyvinyl alcohol at a volume ratio of 1:5-10; and the microcapsule suspension is mixed with the mannitol solution at a volume ratio of 1:1-2.

[0010] Further, the preparation method of the modified transforming growth factor-β is as follows: a transforming growth factor-β solution with a concentration of 0.2-0.5 mg / mL is mixed with a heparin sodium solution with a mass fraction of 1-2%, and an affinity binding and conformational stabilization reaction is carried out to form a TGF-β / heparin sodium complex; the complex is mixed with a chondroitin sulfate solution with a mass fraction of 2-4% to form a mixture, and then added dropwise to a chitosan acetate solution with a mass fraction of 1-2%, and an ionic crosslinking and biomimetic encapsulation reaction is carried out to obtain chondroitin sulfate / chitosan water encapsulating TGF-β. Gel microsphere suspension: The suspension was mixed with a 10-15% (w / w) hydroxypropyl-β-cyclodextrin solution to carry out hydrophobic cavity inclusion and solubilization reactions. After centrifugation and freeze-drying, modified transforming growth factor-β was obtained. The active sites were protected by heparin sodium affinity binding to prevent adsorption to the container wall. A biomimetic microenvironment was constructed by chondroitin sulfate ion crosslinking to achieve hydrogel embedding. The hydrophobic region was stabilized by inclusion of hydroxypropyl-β-cyclodextrin molecules. The three factors synergistically enhanced the anti-adsorption capacity and conformational stability of TGF-β, solving its easy inactivation problem.

[0011] Further, the transforming growth factor-β solution and heparin sodium solution are mixed at a volume ratio of 1:1-1.5; the TGF-β / heparin sodium complex is mixed with chondroitin sulfate solution at a volume ratio of 1:2-3, and the final volume ratio of the mixture to the chitosan solution is 1:2-4; the chondroitin sulfate / chitosan hydrogel microsphere suspension is mixed with hydroxypropyl-β-cyclodextrin solution at a volume ratio of 1:1-2.

[0012] Furthermore, the composite anti-agglomeration stabilizer is composed of the following raw materials in parts by weight: 10-20 parts poloxamer 188, 5-15 parts human serum albumin, and 5-10 parts polyethylene glycol 4000; the composite metabolic regulator is composed of the following raw materials in parts by weight: 5-10 parts sodium pyruvate, 3-8 parts linoleic acid, and 2-5 parts glutathione.

[0013] A culture medium for culturing γδT cells, the culture medium comprising a basal medium and additives; the basal medium being a serum-free medium AIM-V or X-VIVO15; the additives comprising a factor kit, a complex anti-agglomeration stabilizer and a complex metabolic regulator, a 2-4 mmol / L glutamine substitute, 5-10 μg / mL transferrin and 5-10 nmol / L sodium selenite; the pH of the culture medium being 7.2-7.4.

[0014] An application of culturing γδT cells, wherein the factor kit and culture medium are used in γδT cell drugs for treating solid tumors or viral infections.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention uses chitosan quaternary ammonium salt to electrostatically self-assemble interleukin-2, which stabilizes the natural protein conformation of IL-2 and reduces aggregation and inactivation caused by hydrophobic interactions in solution. The emulsification and cross-linking of polyvinyl alcohol can form a hydrophilic protective film on the surface of IL-2, further isolating it from adverse external environments. Trehalose maintains the active protein structure by replacing water molecules through hydrogen bonds during freeze-drying. The synergistic effect of these three factors enhances the anti-aggregation ability and thermal stability of the modified IL-2. Compared with the direct addition of natural IL-2 in the prior art, the modified IL-2 of this invention has a prolonged half-life in the culture system, reducing the need for frequent high-concentration additions due to factor inactivation during the culture process, thus reducing culture costs and pollution risks.

[0016] 2. This invention uses arginine to electrostatically shield interleukin-15, inhibiting the hydrophobic aggregation tendency between IL-15 molecules. PLGA double emulsion encapsulation technology encapsulates IL-15 within microcapsules to achieve slow release. Mannitol and nano-calcium carbonate construct a porous framework to support the microcapsule structure during freeze-drying. The synergistic effect of these three components enables the modified IL-15 to achieve a long-term continuous release effect. Compared with the existing technology where natural IL-15 rapidly degrades in culture medium and requires daily replenishment, the modified IL-15 of this invention prolongs the maintenance time of effective stimulation signals, enhances the continuous expansion capacity of γδT cells, and ensures the stability of protein bioactivity during release.

[0017] 3. This invention utilizes sodium heparin for affinity binding protection of transforming growth factor-β, specifically occupying adsorption sites on the TGF-β surface and effectively preventing its non-specific adsorption to the culture vessel wall. Chondroitin sulfate and chitosan construct a biomimetic extracellular matrix microenvironment through ionic cross-linking, embedding TGF-β in a hydrogel to simulate its in vivo storage state. Hydroxypropyl-β-cyclodextrin encapsulates the hydrophobic regions on the surface of the TGF-β molecule through a hydrophobic cavity, further stabilizing its conformation. The synergistic effect of these three components enhances the anti-adsorption capacity and conformational stability of the modified TGF-β. Compared with existing technologies where the actual active concentration of TGF-β is insufficient due to adsorption and conformational changes after addition to the culture vessel wall, the modified TGF-β of this invention exhibits a high activity retention rate, ensuring that its expected effect in the regulation of γδT cell function is fully realized. Detailed Implementation

[0018] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] It should be noted that the raw materials used in the following embodiments are all commercially available.

[0020] Example 1: A factor kit for culturing γδT cells, composed of the following raw materials in parts by weight: 30 parts of compound cytokine core, 20 parts of compound anti-agglomeration stabilizer, and 10 parts of compound metabolic regulator; The core of the compound cytokine is composed of modified interleukin-2, modified interleukin-15 and modified transforming growth factor-β in a mass ratio of 3:2:1; Modified interleukin-2 was prepared by treating interleukin-2 with trehalose, chitosan quaternary ammonium salt, and polyvinyl alcohol. Modified interleukin-15 is prepared by treating interleukin-15 with arginine, mannitol, and polylactic acid-glycolic acid copolymer; Modified transforming growth factor-β was prepared by treating transforming growth factor-β with sodium heparin, chondroitin sulfate, and hydroxypropyl-β-cyclodextrin.

[0021] The modified interleukin-2 was prepared as follows: A 0.5 mg / mL interleukin-2 solution was mixed with a 0.5% (w / w) chitosan quaternary ammonium salt solution at 2℃ and 200 rpm for 60 min of electrostatic self-assembly to form an IL-2 / chitosan complex solution. The complex solution was then mixed with a 2% (w / w) polyvinyl alcohol aqueous solution at room temperature and subjected to an emulsification and crosslinking reaction at 8000 rpm for 15 min. During emulsification, the system was kept cooled in an ice bath to achieve physical embedding and solidification of polyvinyl alcohol. The resulting emulsion was mixed with a 5% (w / w) trehalose solution and pre-frozen at -80℃ for 4 h under freeze-drying protection, followed by gradient sublimation drying under vacuum (maintained at -20℃ for 24 h, then at 25℃ for 6 h) to obtain modified interleukin-2.

[0022] Interleukin-2 and chitosan quaternary ammonium salt solution were mixed at a volume ratio of 1:2; IL-2 / chitosan complex solution and polyvinyl alcohol aqueous solution were mixed at a volume ratio of 1:1; emulsion and trehalose solution were mixed at a volume ratio of 2:1.

[0023] The modified interleukin-15 was prepared as follows: A 0.8 mg / mL interleukin-15 solution was mixed with a 2% arginine solution under oscillation conditions at 8℃ and 100 rpm, and subjected to electrostatic shielding and aggregation inhibition reaction for 40 min to form an IL-15 / arginine complex. This complex was then used as the inner aqueous phase and mixed with a 5% PLGA oil phase, emulsified at 10000 rpm for 3 min to form a primary emulsion. The primary emulsion was then mixed with an outer aqueous phase containing 1% polyvinyl alcohol. The mixture was emulsified at 8000 rpm for 5 min to form a double emulsion. Subsequently, the double emulsion was subjected to rotary evaporation under reduced pressure for 2 h at 35 °C through solvent evaporation reaction to obtain a PLGA microcapsule suspension encapsulating IL-15. Finally, the obtained microcapsule suspension was mixed with a 3% mannitol solution containing 0.5% nano-calcium carbonate, pre-frozen at -75 °C for 3 h through framework support and freeze-drying, and then subjected to gradient drying under vacuum conditions (maintained at -15 °C for 20 h, and at 20 °C for 8 h) to obtain modified interleukin-15.

[0024] Interleukin-15 solution and arginine solution were mixed at a volume ratio of 1:1.5; IL-15 / arginine complex was mixed with PLGA oil phase at a volume ratio of 1:3; colostrum was mixed with external aqueous phase containing polyvinyl alcohol at a volume ratio of 1:5; microcapsule suspension was mixed with mannitol solution at a volume ratio of 1:1.

[0025] The modified transforming growth factor-β was prepared as follows: A 0.2 mg / mL solution of transforming growth factor-β was mixed with a 1% sodium heparin solution under oscillation conditions at 2℃ and 50 rpm for 30 min to form a TGF-β / sodium heparin complex. The complex was then mixed with a 2% chondroitin sulfate solution and added dropwise to a 1% chitosan acetate solution. The mixture was then subjected to ionic crosslinking and biomimetic encapsulation reactions at 23℃ and 300 rpm for 60 min to obtain chondroitin sulfate / chitosan hydrogel microspheres encapsulating TGF-β. The hydrogel suspension was mixed with a 10% hydroxypropyl-β-cyclodextrin solution and subjected to hydrophobic cavity inclusion and solubilization reactions at 23℃ and 150 rpm. After being protected from light for 12 h, the precipitate was collected by centrifugation at 4℃ and 1000 rpm for 20 min and then freeze-dried to obtain the modified transforming growth factor-β.

[0026] Transforming growth factor-β solution and heparin sodium solution were mixed at a volume ratio of 1:1; TGF-β / heparin sodium complex and chondroitin sulfate solution were mixed at a volume ratio of 1:2, and the final volume ratio of the mixture to chitosan solution was 1:2; chondroitin sulfate / chitosan hydrogel microsphere suspension and hydroxypropyl-β-cyclodextrin solution were mixed at a volume ratio of 1:1.

[0027] The compound anti-agglomeration stabilizer is composed of the following raw materials in parts by weight: 10 parts poloxamer 188, 5 parts human serum albumin, and 5 parts polyethylene glycol 4000; the compound metabolic regulator is composed of the following raw materials in parts by weight: 5 parts sodium pyruvate, 3 parts linoleic acid, and 2 parts glutathione.

[0028] A culture medium for culturing γδT cells, comprising a basal medium and additives; the basal medium is serum-free medium AIM-V or X-VIVO15; the additives include a factor kit, a complex anti-agglomeration stabilizer and a complex metabolic regulator, a 2 mmol / L glutamine substitute, 5 μg / mL transferrin and 5 nmol / L sodium selenite; the pH of the culture medium is 7.2.

[0029] An application of culturing γδT cells, a factor kit and culture medium for use in γδT cell drugs for treating solid tumors or viral infections.

[0030] Example 2: A factor kit for culturing γδT cells, composed of the following raw materials in parts by weight: 40 parts of compound cytokine core, 25 parts of compound anti-agglomeration stabilizer, and 15 parts of compound metabolic regulator; The core of the compound cytokine is composed of modified interleukin-2, modified interleukin-15 and modified transforming growth factor-β in a mass ratio of 3:2:1; Modified interleukin-2 was prepared by treating interleukin-2 with trehalose, chitosan quaternary ammonium salt, and polyvinyl alcohol. Modified interleukin-15 is prepared by treating interleukin-15 with arginine, mannitol, and polylactic acid-glycolic acid copolymer; Modified transforming growth factor-β was prepared by treating transforming growth factor-β with sodium heparin, chondroitin sulfate, and hydroxypropyl-β-cyclodextrin.

[0031] The modified interleukin-2 was prepared as follows: A 0.7 mg / mL interleukin-2 solution was mixed with a 1% (w / w) chitosan quaternary ammonium salt solution at 4℃ and 300 rpm for 75 min of electrostatic self-assembly to form an IL-2 / chitosan complex solution. The complex solution was then mixed with a 3% (w / w) polyvinyl alcohol aqueous solution at room temperature and subjected to an emulsification and crosslinking reaction at 10000 rpm for 22 min. During emulsification, the system was kept cooled in an ice bath to achieve physical embedding and solidification of polyvinyl alcohol. The resulting emulsion was mixed with a 5-10% (w / w) trehalose solution, pre-frozen at -60℃ for 5 h under freeze-drying protection, and then subjected to gradient sublimation drying under vacuum (maintained at -15℃ for 30 h, then at 27℃ for 9 h) to obtain modified interleukin-2.

[0032] Interleukin-2 and chitosan quaternary ammonium salt solution were mixed at a volume ratio of 1:3; IL-2 / chitosan complex solution and polyvinyl alcohol aqueous solution were mixed at a volume ratio of 1:1.2; emulsion and trehalose solution were mixed at a volume ratio of 2.5:1.

[0033] The modified interleukin-15 was prepared as follows: A 1 mg / mL interleukin-15 solution was mixed with a 3% arginine solution under oscillation conditions at 10℃ and 125 rpm, and subjected to electrostatic shielding and aggregation inhibition reaction for 50 min to form an IL-15 / arginine complex. This complex was then used as the inner aqueous phase and mixed with a 6.5% PLGA oil phase, emulsified at 12500 rpm for 4 min to form a primary emulsion. The primary emulsion was then mixed with an outer aqueous phase containing 1.5% polyvinyl alcohol. The mixture was emulsified at 9000 rpm for 6 min to form a double emulsion. Subsequently, the double emulsion was subjected to rotary evaporation under reduced pressure at 37 °C for 3 h via solvent evaporation reaction to obtain a PLGA microcapsule suspension encapsulating IL-15. Finally, the obtained microcapsule suspension was mixed with a 4.5% mannitol solution containing 0.7% nano-calcium carbonate, and pre-frozen at -65 °C for 4 h via framework support and freeze-drying. Then, it was subjected to gradient drying under vacuum conditions (maintained at -10 °C for 25 h, and at 25 °C for 10 h) to obtain modified interleukin-15.

[0034] Interleukin-15 solution and arginine solution were mixed at a volume ratio of 1:2; IL-15 / arginine complex was mixed with PLGA oil phase at a volume ratio of 1:4; colostrum was mixed with external aqueous phase containing polyvinyl alcohol at a volume ratio of 1:7.5; microcapsule suspension was mixed with mannitol solution at a volume ratio of 1:1.5.

[0035] The modified transforming growth factor-β was prepared as follows: A 0.3 mg / mL solution of transforming growth factor-β was mixed with a 1.5% sodium heparin solution under oscillation conditions at 4℃ and 65 rpm for 37 min to form a TGF-β / sodium heparin complex. The complex was then mixed with a 3% chondroitin sulfate solution and added dropwise to a 1.5% chitosan acetate solution. The mixture was then subjected to ionic crosslinking and biomimetic encapsulation reactions at 25℃ and 400 rpm for 75 min to obtain chondroitin sulfate / chitosan hydrogel microspheres encapsulating TGF-β. The hydrogel suspension was mixed with a 12.5% ​​hydroxypropyl-β-cyclodextrin solution and subjected to hydrophobic cavity inclusion and solubilization reactions at 25℃ and 175 rpm. After being protected from light for 18 hours, the precipitate was collected by centrifugation at 4℃ and 1500 rpm for 25 min and then freeze-dried to obtain the modified transforming growth factor-β.

[0036] Transforming growth factor-β solution and heparin sodium solution were mixed at a volume ratio of 1:1.2; TGF-β / heparin sodium complex and chondroitin sulfate solution were mixed at a volume ratio of 1:2.5, and the final volume ratio of the mixture to chitosan solution was 1:3; chondroitin sulfate / chitosan hydrogel microsphere suspension and hydroxypropyl-β-cyclodextrin solution were mixed at a volume ratio of 1:1.5.

[0037] The compound anti-agglomeration stabilizer is composed of the following raw materials in parts by weight: 15 parts poloxamer 188, 10 parts human serum albumin, and 7.5 parts polyethylene glycol 4000; the compound metabolic regulator is composed of the following raw materials in parts by weight: 7.5 parts sodium pyruvate, 5.5 parts linoleic acid, and 3.5 parts glutathione.

[0038] A culture medium for culturing γδT cells, comprising a basal medium and additives; the basal medium is serum-free medium AIM-V or X-VIVO15; the additives include a factor kit, a complex anti-agglomeration stabilizer and a complex metabolic regulator, a 3 mmol / L glutamine substitute, 7.5 μg / mL transferrin and 7.5 nmol / L sodium selenite; the pH of the culture medium is 7.3.

[0039] An application of culturing γδT cells, a factor kit and culture medium for use in γδT cell drugs for treating solid tumors or viral infections.

[0040] Example 3: A factor kit for culturing γδT cells, composed of the following raw materials in parts by weight: 50 parts of compound cytokine core, 30 parts of compound anti-agglomeration stabilizer, and 20 parts of compound metabolic regulator; The core of the compound cytokine is composed of modified interleukin-2, modified interleukin-15 and modified transforming growth factor-β in a mass ratio of 3:2:1; Modified interleukin-2 was prepared by treating interleukin-2 with trehalose, chitosan quaternary ammonium salt, and polyvinyl alcohol. Modified interleukin-15 is prepared by treating interleukin-15 with arginine, mannitol, and polylactic acid-glycolic acid copolymer; Modified transforming growth factor-β was prepared by treating transforming growth factor-β with sodium heparin, chondroitin sulfate, and hydroxypropyl-β-cyclodextrin.

[0041] The modified interleukin-2 was prepared as follows: A 1.0 mg / mL interleukin-2 solution was mixed with a 1.5% (w / w) chitosan quaternary ammonium salt solution at 6℃ and 400 rpm for 90 min of electrostatic self-assembly to form an IL-2 / chitosan complex solution. The complex solution was then mixed with a 4% (w / w) polyvinyl alcohol aqueous solution at room temperature and subjected to an emulsification and crosslinking reaction at 12000 rpm for 30 min. During emulsification, the system was kept cooled in an ice bath to achieve physical embedding and solidification of polyvinyl alcohol. The resulting emulsion was mixed with a 10% (w / w) trehalose solution and pre-frozen at -40℃ for 6 h under freeze-drying protection, followed by gradient sublimation drying under vacuum (maintained at -10℃ for 36 h and 30℃ for 12 h) to obtain modified interleukin-2.

[0042] Interleukin-2 and chitosan quaternary ammonium salt solution were mixed at a volume ratio of 1:4; IL-2 / chitosan complex solution and polyvinyl alcohol aqueous solution were mixed at a volume ratio of 1:1.5; emulsion and trehalose solution were mixed at a volume ratio of 3:1.

[0043] The modified interleukin-15 was prepared as follows: A 1.2 mg / mL interleukin-15 solution was mixed with a 4% arginine solution under oscillation conditions at 12℃ and 150 rpm, and subjected to electrostatic shielding and aggregation inhibition reaction for 60 min to form an IL-15 / arginine complex. This complex was then used as the inner aqueous phase and mixed with an 8% PLGA oil phase, emulsified at 15000 rpm for 5 min to form a primary emulsion. The primary emulsion was then mixed with an outer aqueous phase containing 2% polyvinyl alcohol. The mixture was emulsified at 10,000 rpm for 8 min to form a double emulsion. Subsequently, the double emulsion was subjected to rotary evaporation under reduced pressure for 4 h at 40 °C through solvent evaporation reaction to obtain a PLGA microcapsule suspension encapsulating IL-15. Finally, the obtained microcapsule suspension was mixed with a 6% mannitol solution containing 1% nano-calcium carbonate, and pre-frozen at -50 °C for 5 h through framework support and freeze-drying. Then, it was subjected to gradient drying under vacuum conditions (maintained at -5 °C for 30 h and at 25 °C for 12 h) to obtain modified interleukin-15.

[0044] Interleukin-15 solution and arginine solution were mixed at a volume ratio of 1:2.5; IL-15 / arginine complex was mixed with PLGA oil phase at a volume ratio of 1:5; colostrum was mixed with external aqueous phase containing polyvinyl alcohol at a volume ratio of 1:10; microcapsule suspension was mixed with mannitol solution at a volume ratio of 1:2.

[0045] The modified transforming growth factor-β was prepared as follows: A 0.5 mg / mL solution of transforming growth factor-β was mixed with a 2% sodium heparin solution under oscillation conditions at 6℃ and 80 rpm for 45 min to form a TGF-β / sodium heparin complex. The complex was then mixed with a 4% chondroitin sulfate solution and added dropwise to a 2% chitosan acetate solution. The mixture was then subjected to ionic crosslinking and biomimetic encapsulation reactions at 27℃ and 500 rpm for 90 min to obtain chondroitin sulfate / chitosan hydrogel microspheres encapsulating TGF-β. The hydrogel suspension was mixed with a 15% hydroxypropyl-β-cyclodextrin solution and subjected to hydrophobic cavity inclusion and solubilization reactions at 27℃ and 200 rpm. After being protected from light for 24 h, the precipitate was collected by centrifugation at 4℃ and 2000 rpm for 30 min and then freeze-dried to obtain the modified transforming growth factor-β.

[0046] Transforming growth factor-β solution and heparin sodium solution were mixed at a volume ratio of 1:1.5; TGF-β / heparin sodium complex and chondroitin sulfate solution were mixed at a volume ratio of 1:3, and the final volume ratio of the mixture to chitosan solution was 1:4; chondroitin sulfate / chitosan hydrogel microsphere suspension and hydroxypropyl-β-cyclodextrin solution were mixed at a volume ratio of 1:2.

[0047] The compound anti-agglomeration stabilizer is composed of the following raw materials in parts by weight: 20 parts poloxamer 188, 15 parts human serum albumin, and 10 parts polyethylene glycol 4000; the compound metabolic regulator is composed of the following raw materials in parts by weight: 10 parts sodium pyruvate, 8 parts linoleic acid, and 5 parts glutathione.

[0048] A culture medium for culturing γδT cells, comprising a basal medium and additives; the basal medium is serum-free medium AIM-V or X-VIVO15; the additives include a factor kit, a complex anti-agglomeration stabilizer and a complex metabolic regulator, 4 mmol / L glutamine substitute, 10 μg / mL transferrin and 10 nmol / L sodium selenite; the pH of the culture medium is 7.4.

[0049] An application of culturing γδT cells, a factor kit and culture medium for use in γδT cell drugs for treating solid tumors or viral infections.

[0050] Comparative Example 1: The difference between this comparative example and Example 1 is that the modified interleukin-2 in this comparative example is replaced with untreated interleukin-2.

[0051] Comparative Example 2 differs from Example 1 in that the modified interleukin-15 is replaced with untreated interleukin-15.

[0052] Comparative Example 3 differs from Example 1 in that the modified transforming growth factor-β is replaced with transforming growth factor-β.

[0053] Performance testing: The relevant performance of the factor kit, culture medium, and preparation method for culturing γδT cells provided in Examples 1 to 3 and Comparative Examples 1 to 3 were tested respectively. The test data are recorded in Table 1 below: Table 1: Performance Test Table of Factor Kits and Culture Media for Culturing γδT Cells Among them, the factor kits for culturing γδT cells and the γδT cell expansion fold test of the culture medium prepared using the test methods in GB / T30441-2013 in Examples 1, 2, 3, Comparative Examples 1, 2, and 3; The purity of the γδT cell culture factor kits and culture medium CD3+γδT cells prepared using the test methods in YY / T1792-2021 in Examples 1, 2, 3, Comparative Examples 1, 2, and 3 was tested. The tumor cell killing rate of the factor kits and culture media prepared for culturing γδT cells were tested using the test methods in ISO 10993-5:2009 in Examples 1, 2, 3, Comparative Examples 1, 2, and 3.

[0054] By comparing and analyzing the relevant data in the table, it can be seen that the factor kit and culture medium for culturing γδT cells prepared in this invention not only increase the γδT cell expansion fold but also improve CD3 count. + The γδT cells exhibit high purity and a high tumor cell killing rate. This indicates that the γδT cell culture factor kit, culture medium, and preparation method provided by this invention have broader market prospects and are more suitable for widespread application.

[0055] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0056] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A factor kit for culturing γδT cells, characterized in that, It is composed of the following raw materials in parts by weight: 30-50 parts of compound cytokine core, 20-30 parts of compound anti-agglomeration stabilizer, and 10-20 parts of compound metabolic regulator; The core of the composite cytokine is composed of modified interleukin-2, modified interleukin-15, and modified transforming growth factor-β in a mass ratio of 3:2:

1. The modified interleukin-2 was prepared by treating interleukin-2 with trehalose, chitosan quaternary ammonium salt, and polyvinyl alcohol. The modified interleukin-15 was prepared by treating interleukin-15 with arginine, mannitol, and PLGA. The modified transforming growth factor-β was prepared by treating transforming growth factor-β with sodium heparin, chondroitin sulfate, and hydroxypropyl-β-cyclodextrin.

2. The factor kit for culturing γδT cells according to claim 1, characterized in that, The modified interleukin-2 is prepared as follows: a 0.5-1.0 mg / mL interleukin-2 solution is mixed with a 0.5-1.5% (w / w) chitosan quaternary ammonium salt solution to form an IL-2 / chitosan complex solution through electrostatic self-assembly; the complex solution is mixed with a 2-4% (w / w) polyvinyl alcohol aqueous solution to undergo emulsification and cross-linking reaction; the resulting emulsion is mixed with a 5-10% (w / w) trehalose solution to undergo lyophilization protection reaction, followed by gradient sublimation drying to obtain a fluffy modified interleukin-2.

3. The factor kit for culturing γδT cells according to claim 2, characterized in that: The interleukin-2 and chitosan quaternary ammonium salt solution are mixed at a volume ratio of 1:2-4; the IL-2 / chitosan complex solution and polyvinyl alcohol aqueous solution are mixed at a volume ratio of 1:1-1.5; the emulsion and trehalose solution are mixed at a volume ratio of 2-3:

1.

4. The factor kit for culturing γδT cells according to claim 1, characterized in that, The modified interleukin-15 is prepared as follows: A 0.8-1.2 mg / mL interleukin-15 solution is mixed with a 2-4% arginine solution to perform electrostatic shielding and aggregation inhibition reactions, forming an IL-15 / arginine complex. This complex is then mixed with a 5-8% PLGA oil phase as the inner aqueous phase and emulsified to form a primary emulsion. The primary emulsion is then mixed with an outer aqueous phase containing 1-2% polyvinyl alcohol and emulsified to form a secondary emulsion. Subsequently, a PLGA microcapsule suspension encapsulating IL-15 is obtained by rotary evaporation under reduced pressure. Finally, the obtained microcapsule suspension is mixed with a 3-6% mannitol solution containing 0.5-1% nano-calcium carbonate, and subjected to skeletal support and freeze-drying reactions, followed by gradient drying to obtain the modified interleukin-15.

5. The factor kit for culturing γδT cells according to claim 4, characterized in that: The interleukin-15 solution and arginine solution are mixed at a volume ratio of 1:1.5-2.5; the IL-15 / arginine complex is mixed with the PLGA oil phase at a volume ratio of 1:3-5; the colostrum is mixed with the external aqueous phase containing polyvinyl alcohol at a volume ratio of 1:5-10; and the microcapsule suspension is mixed with the mannitol solution at a volume ratio of 1:1-2.

6. The factor kit for culturing γδT cells according to claim 1, characterized in that, The modified transforming growth factor-β is prepared as follows: a transforming growth factor-β solution with a concentration of 0.2-0.5 mg / mL is mixed with a heparin sodium solution with a mass fraction of 1-2% to form a TGF-β / heparin sodium complex through affinity binding and conformational stabilization reaction; the complex is mixed with a chondroitin sulfate solution with a mass fraction of 2-4% to form a mixed solution, which is then added dropwise to a chitosan acetate solution with a mass fraction of 1-2% to conduct ionic crosslinking and biomimetic encapsulation reaction to obtain a chondroitin sulfate / chitosan hydrogel microsphere suspension encapsulating TGF-β; the suspension is mixed with a hydroxypropyl-β-cyclodextrin solution with a mass fraction of 10-15% to conduct hydrophobic cavity inclusion and solubilization reaction, and after centrifugation and freeze-drying, the modified transforming growth factor-β is obtained.

7. The factor kit for culturing γδT cells according to claim 6, characterized in that: The transforming growth factor-β solution and heparin sodium solution are mixed at a volume ratio of 1:1-1.5; the TGF-β / heparin sodium complex is mixed with chondroitin sulfate solution at a volume ratio of 1:2-3, and the final volume ratio of the mixture to chitosan solution is 1:2-4; the chondroitin sulfate / chitosan hydrogel microsphere suspension is mixed with hydroxypropyl-β-cyclodextrin solution at a volume ratio of 1:1-2.

8. The factor kit for culturing γδT cells according to claim 1, characterized in that: The composite anti-agglomeration stabilizer is composed of the following raw materials in parts by weight: 10-20 parts poloxamer 188, 5-15 parts human serum albumin, and 5-10 parts polyethylene glycol 4000; the composite metabolic regulator is composed of the following raw materials in parts by weight: 5-10 parts sodium pyruvate, 3-8 parts linoleic acid, and 2-5 parts glutathione.

9. A culture medium for culturing γδT cells, comprising a factor kit for culturing γδT cells according to any one of claims 1-8, characterized in that: The culture medium consists of a basal medium and additives; the basal medium is serum-free medium AIM-V or X-VIVO15; the additives include a factor kit, a compound anti-agglomeration stabilizer and a compound metabolic regulator, 2-4 mmol / L glutamine substitute, 5-10 μg / mL transferrin and 5-10 nmol / L sodium selenite; the pH of the culture medium is 7.2-7.

4.

10. An application for culturing γδT cells, comprising a factor kit for culturing γδT cells according to any one of claims 1-8, characterized in that: The factor kit and culture medium are used in γδT cell drugs for treating solid tumors or viral infections.

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