NK cell culture gel containing rhizoma atractylodis macrocephalae extract as well as preparation method and application of NK cell culture gel

Abstract

The invention relates to an NK cell culture gel containing a bighead atractylodes rhizome extract as well as a preparation method and application of the NK cell culture gel. According to the culture gel, a conventional NK cell basic culture medium is used as a matrix, self-made composite gel with the mass concentration of 0.5% is added, hyaluronic acid and chitosan are used as natural polysaccharide gel matrixes of the self-made composite gel, and the self-made composite gel is compounded with a rhizoma atractylodis macrocephalae extract, cell nutritional factors and immunopotentiation auxiliary components. The dual technical effects of huddling growth induction and culture period shortening of the NK cells are achieved by precisely regulating and controlling the pore structure of the gel and the slow release efficiency of the active ingredients. Experiments prove that by means of the culture gel, the huddling rate of the NK cells can be increased to 85% or above, the culture period is shortened by 28.6% compared with that of a traditional culture medium, the total number of the cultured NK cells can reach 245 times of the initial inoculation amount, and the killing rate of K562 tumor target cells is maintained to be 90% or above.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically relating to an NK cell culture gel containing Atractylodes macrocephala extract, its preparation method, and its application. Background Technology

[0002] Natural killer cells (NK cells) are a core component of the body's innate immune system. They possess the unique biological characteristic of rapidly recognizing and killing tumor cells, virus-infected cells, and abnormally proliferating cells without prior sensitization. Furthermore, they do not rely on antigen presentation during the killing process and do not cause graft-versus-host disease (GVHD). Therefore, they have irreplaceable advantages and broad application prospects in cell therapy fields such as tumor immunotherapy and antiviral therapy.

[0003] With the rapid development of cell therapy technology, the clinical demand for highly active and sufficient NK cells is increasing. However, NK cell in vitro culture technology still faces two major challenges that severely limit its large-scale clinical application:

[0004] First, traditional NK cell culture often uses liquid culture media, which cannot simulate the three-dimensional microenvironment of cell growth in vivo. This results in NK cells growing dispersedly during culture, lacking effective contact and signal transduction between cells, leading to problems such as slow proliferation, decreased activity, and increased apoptosis rate. Studies have shown that direct contact and interaction between cells are crucial for maintaining the proliferative activity and functional stability of immune cells. Dispersed NK cells not only have limited proliferation, but their cytotoxic activity also decreases significantly with prolonged culture time.

[0005] Secondly, current in vitro culture cycles for NK cells are relatively long, typically requiring 14-21 days to obtain a sufficient quantity of highly active NK cells. This excessively long culture period not only increases culture costs and the risk of contamination but also leads to functional exhaustion of cells during prolonged culture, affecting clinical treatment outcomes. Although existing technologies can promote NK cell proliferation to some extent by adding cytokines such as IL-2 and IL-15, they still cannot effectively solve the core problems of long culture cycles and dispersed cell growth.

[0006] Traditional Chinese medicine extracts have gradually become a research hotspot in the field of cell culture additives due to their advantages such as natural origin, good biocompatibility, clear immunomodulatory activity, and low cost. Atractylodes macrocephala, a commonly used traditional Chinese medicine, has a major active ingredient, Atractylodes macrocephala polysaccharide, which has significant immunomodulatory effects. It can promote the proliferation and differentiation of immune cells, inhibit apoptosis, and enhance the body's immune function. It also possesses antioxidant and anti-inflammatory biological activities, and exhibits good biocompatibility and no obvious toxic side effects. However, there are currently no reports on technologies that combine Atractylodes macrocephala extract with natural polysaccharide gels to construct NK cell culture gel systems, achieving the dual goals of inducing cell aggregation and shortening the culture cycle.

[0007] To address the shortcomings of existing technologies, this invention aims to provide an NK cell culture gel containing Atractylodes macrocephala extract, its preparation method, and its application. By constructing a hyaluronic acid-chitosan composite gel matrix, and compounding it with Atractylodes macrocephala extract and cell nutrient factors, the three-dimensional porous structure of the gel simulates the in vivo cell growth microenvironment. Simultaneously, the sustained-release effect of the active ingredients continuously stimulates NK cell proliferation, ultimately achieving the technical effects of increasing NK cell aggregation rate, shortening the culture cycle, and maintaining high cytotoxic activity. This provides a feasible technical solution for the large-scale preparation of clinical-grade NK cells. Summary of the Invention

[0008] The purpose of this invention is to overcome the technical defects of existing NK cell in vitro culture, such as cell dispersion, slow proliferation, and long culture cycle, and to provide an NK cell culture gel containing Atractylodes macrocephala extract, its preparation method, and its application. This culture gel can effectively induce NK cells to grow in clusters, significantly shorten the culture cycle, and maintain high cell killing activity, making it suitable for large-scale and efficient preparation of NK cells.

[0009] To achieve the above objectives, the present invention provides the following technical solution: an NK cell culture gel containing Atractylodes macrocephala extract, using 581 basal culture medium as the matrix and adding a self-made composite gel with a mass concentration of 0.5%; the self-made composite gel, by weight, consists of 20 parts hyaluronic acid, 10 parts chitosan, 3 parts Atractylodes macrocephala extract, 5 parts fetal bovine serum substitute, 2 parts L-glutamine, 60 parts PBS buffer, and 0.5 parts genipin; the Atractylodes macrocephala extract contains ≥32% Atractylodes macrocephala polysaccharide and is obtained by ethanol reflux extraction, macroporous resin purification, and freeze-drying of Atractylodes macrocephala slices.

[0010] As a preferred embodiment of the present invention, the preparation process of Atractylodes macrocephala extract is as follows: Atractylodes macrocephala slices are pulverized to 40 mesh, 10 times the amount of 70% ethanol is added, and the mixture is refluxed and extracted twice, 1.5 hours each time. The extracts are combined, concentrated under reduced pressure to a relative density of 1.15 at 60°C, loaded onto a D101 macroporous adsorption resin column, and after washing away impurities with water, the mixture is eluted with 30% ethanol. The eluent is collected and freeze-dried to obtain Atractylodes macrocephala extract.

[0011] As a preferred embodiment of the present invention, the self-made composite gel has a pore diameter of 50-200 μm, a porosity of 75%-85%, and a sustained-release period of 8-12 days for the active ingredients.

[0012] A method for preparing NK cell culture gel containing Atractylodes macrocephala extract includes the following steps:

[0013] (1) Raw material preparation: Weigh out 20 parts of hyaluronic acid, 10 parts of chitosan, 3 parts of Atractylodes macrocephala extract, 5 parts of fetal bovine serum substitute, 2 parts of L-glutamine, 60 parts of PBS buffer, and 0.5 parts of genipin by weight.

[0014] (2) Gel matrix preparation: Hyaluronic acid and chitosan were added to 30 portions of PBS buffer preheated to 45°C and magnetically stirred for 30 min until completely dissolved;

[0015] (3) Preparation of active ingredient solution: Add Atractylodes macrocephala extract, fetal bovine serum substitute and L-glutamine to the remaining 30 parts of PBS buffer and sonicate for 12 min to dissolve;

[0016] (4) Preparation of composite gel: The active ingredient solution was added dropwise to 581 basic culture medium, stirred evenly, and then genipin was added to adjust the pH to 7.0. Crosslinking was carried out at room temperature for 2.5 h.

[0017] (5) Sterilization treatment: The cross-linked gel system was autoclaved at 121°C for 20 min and cooled to room temperature to obtain NK cell culture gel containing Atractylodes macrocephala extract.

[0018] As a preferred embodiment of the present invention, the ultrasonic power in step (3) is 150-200W and the ultrasonic temperature is controlled at 25-30℃.

[0019] As a preferred embodiment of the present invention, in step (4), the dropping rate is 1-2 mL / min and the stirring speed is 100-150 r / min.

[0020] The application of NK cell culture gel containing Atractylodes macrocephala extract in large-scale NK cell culture includes the following culture steps:

[0021] (1) Mononuclear cell acquisition: Mononuclear cells are obtained from peripheral blood or umbilical cord blood by centrifugation with lymphocyte separation solution;

[0022] (2) Cell seeding: Mononuclear cells were seeded at a density of 1.06 × 10⁶ cells / mL in culture flasks containing the culture gel;

[0023] (3) Culture conditions control: Incubate at 37℃ in a 5% CO2 incubator, and replace the culture gel medium with fresh medium once a day;

[0024] (4) Cell density maintenance: During culture, maintain the cell density at 8×10⁵-1.2×10⁶ cells / mL;

[0025] (5) Cell harvesting: NK cells were harvested after 10 days of culture.

[0026] As a preferred embodiment of the present invention, the centrifugation conditions in step (1) are 2000 r / min, centrifugation time 15-20 min, and centrifugation temperature 4℃.

[0027] As a preferred embodiment of the present invention, during the culture process, NK cells form a stable cell cluster consisting of 80-100 cells on the 3rd day of culture, and the cell cluster survival rate is ≥85%.

[0028] As a preferred embodiment of the present invention, the harvested NK cells have a killing rate of ≥90% against K562 tumor target cells, and the total number of cells is more than 245 times the initial inoculation amount.

[0029] Compared with the prior art, the present invention has the following significant advantages:

[0030] (1) For the first time, the composite application of Atractylodes macrocephala extract and natural polysaccharide gel was realized, and a microenvironment for NK cell culture with a three-dimensional porous structure was constructed. This can effectively induce NK cells to grow in clusters. On the third day of culture, a stable cell cluster consisting of 80-100 cells can be formed, and the cell cluster survival rate is over 85%. This solves the technical problem of cell dispersion and slow proliferation in traditional liquid culture medium. The close contact and signal transduction between cells significantly promote the proliferation of NK cells. After 10 days of culture, the total number of cells can reach 245 times the initial inoculum, and the proliferation multiple is 34.5% higher than that of traditional culture.

[0031] (2) Significantly shortens the culture cycle. Traditional NK cell culture requires 14 days to obtain a sufficient number of highly active cells. This invention continuously releases Atractylodes macrocephala polysaccharides and nutrients through a gel sustained-release system, continuously stimulating NK cell proliferation and shortening the culture cycle to 10 days, a reduction of 28.6%. This significantly reduces culture costs and pollution risks, providing time assurance for the large-scale clinical application of NK cells.

[0032] (3) The cultured NK cells maintain high killing activity. According to the test, the killing rate of the NK cells cultured in this invention against K562 tumor target cells is maintained at more than 90%, which is not significantly different from the traditional culture method, thus ensuring the clinical therapeutic effect of the cells. At the same time, the cell survival rate is more than 95%, and the phenotypic purity (CD3⁻CD56⁺) is ≥90%, which meets the clinical grade cell standard.

[0033] (4) The culture gel has good biocompatibility and no cytotoxicity. The gel matrix uses natural polysaccharides such as hyaluronic acid and chitosan. The active ingredient, Atractylodes macrocephala extract, is derived from natural sources. All raw materials meet the cell culture grade standards. The cytotoxicity test verified that the culture gel has a toxicity rate of <5% to NK cells. It has excellent biocompatibility, low raw material cost, and simple and controllable preparation process, making it suitable for large-scale industrial production.

[0034] (5) The preparation process is stable and reliable. In the preparation process of the culture gel of the present invention, the parameters of each step are clear. For example, the dissolution temperature of the gel matrix, stirring speed, ultrasonic power, cross-linking time, etc. have been optimized to ensure the stability and repeatability of product quality. At the same time, the parameters such as cell seeding density, culture conditions, and density maintenance range in the culture process are clear, which are highly operable and convenient for clinical laboratories and manufacturing enterprises to promote and apply. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification and claims of this application are intended to cover non-exclusive inclusion.

[0037] The term "embodiment" as used herein means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of the phrase "embodiment" in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0038] A NK cell culture gel containing Atractylodes macrocephala extract is prepared using 581 basal medium as the matrix and a self-made composite gel with a mass concentration of 0.5%. The self-made composite gel consists of 20 parts hyaluronic acid, 10 parts chitosan, 3 parts Atractylodes macrocephala extract, 5 parts fetal bovine serum substitute, 2 parts L-glutamine, 60 parts PBS buffer, and 0.5 parts genipin by weight. The Atractylodes macrocephala extract contains ≥32% Atractylodes macrocephala polysaccharide.

[0039] Atractylodes macrocephala extract was prepared by the following process: Atractylodes macrocephala slices were pulverized to 40 mesh, 10 times the amount of 70% ethanol was added, and the mixture was refluxed twice for 1.5 hours each time. The extracts were combined, concentrated under reduced pressure to a relative density of 1.15 at 60℃, and loaded onto a D101 macroporous adsorption resin column. Impurities were first eluted with deionized water, and then the target components were eluted with 30% ethanol. The eluent was collected, freeze-dried, and the Atractylodes macrocephala extract was obtained. The content of Atractylodes macrocephala polysaccharides was found to be ≥32%.

[0040] The self-made composite gel has a pore diameter of 50-200 μm and a porosity of 75%-85%. This pore structure can provide a suitable growth space for NK cells, promote intercellular contact and signal transduction, and achieve the slow release of active ingredients. The sustained release period is 8-12 days, which can continuously provide proliferation signals to NK cells throughout the entire culture period.

[0041] The preparation method of NK cell culture gel containing Atractylodes macrocephala extract includes the following steps:

[0042] Raw material preparation: Accurately weigh 20 parts by weight of hyaluronic acid, 10 parts by weight of chitosan, 3 parts by weight of Atractylodes macrocephala extract, 5 parts by weight of fetal bovine serum substitute, 2 parts by weight of L-glutamine, 60 parts by weight of PBS buffer, and 0.5 parts by weight of genipin; all raw materials meet cell culture grade standards, and the molecular weight of hyaluronic acid is 1.5 × 10⁻⁶. 6 -2.0×10 6 Da, the degree of deacetylation of chitosan is ≥85%.

[0043] Gel matrix preparation: Weigh out hyaluronic acid and chitosan and add them to 30 portions of PBS buffer preheated to 45°C. Place the mixture on a magnetic stirrer and stir at 100-150 r / min for 30 min until the hyaluronic acid and chitosan are completely dissolved to form a uniform gel matrix solution.

[0044] Preparation of active ingredient solution: Add Atractylodes macrocephala extract, fetal bovine serum substitute, and L-glutamine to the remaining 30 portions of PBS buffer, place in an ultrasonic cleaner, and sonicate for 12 minutes at 150-200W power and 25-30℃ to fully dissolve each component and obtain the active ingredient solution.

[0045] Preparation of composite gel: The active ingredient solution was slowly added dropwise to 581 basal culture medium at a dropping rate of 1-2 mL / min, while stirring continuously at a speed of 100-150 r / min to ensure uniform mixing; after the addition was completed, genipin was added as a cross-linking agent, the pH of the system was adjusted to 7.0, and the mixture was placed at room temperature for 2.5 h for cross-linking to form a preliminary composite gel system.

[0046] Sterilization treatment: The cross-linked composite gel system was placed in a high-pressure steam sterilizer and sterilized at 121℃ and 0.1MPa for 20 minutes. After sterilization, it was taken out and allowed to cool naturally to room temperature to obtain NK cell culture gel containing Atractylodes macrocephala extract.

[0047] (III) Application of NK cell culture gel containing Atractylodes macrocephala extract

[0048] The application of this culture gel in large-scale NK cell culture includes the following steps:

[0049] Mononuclear cell acquisition: Peripheral blood or umbilical cord blood from healthy donors was collected, diluted with an equal volume of physiological saline, and then slowly added to centrifuge tubes pre-coated with lymphocyte separation solution. The mixture was centrifuged at 4°C and 2000 rpm for 15-20 minutes. The intermediate mononuclear cell layer was aspirated and washed twice with PBS buffer to obtain purified mononuclear cells.

[0050] Cell seeding: The purified mononuclear cells were resuspended in the NK cell culture gel containing Atractylodes macrocephala extract as described in this invention, and the cell density was adjusted to 1.06 × 10⁶ cells / year. 6 Cells per mL were inoculated into sterile culture flasks. The inoculation volume was adjusted according to the culture flask specifications to ensure uniform cell distribution.

[0051] Culture conditions control: After inoculation, the culture flasks are placed in a constant temperature incubator at 37°C, 5% CO2, and 95% humidity. Fresh culture gel medium of the present invention is replaced once a day, with the replacement volume being 100% of the original culture medium volume, to ensure sufficient nutrition and removal of metabolic waste.

[0052] Cell density maintenance: Cell density was monitored daily using a cell counter during culture, and the culture medium volume was adjusted accordingly to maintain a cell density of 8 × 10⁶ cells / day. 5 -1.2×10 6 Cells / mL should be kept low to avoid affecting proliferation efficiency, or too high a cell density leading to insufficient nutrition and decreased activity.

[0053] Cell harvesting and detection: After 10 days of culture, NK cells were collected from the culture flasks, and the cell phenotype (CD3⁻CD56⁺) was detected by flow cytometry. The cell killing activity against K562 tumor target cells was detected by CCK-8 assay, and the cell viability was also detected.

[0054] Example 1

[0055] Preparation of NK cell culture gel containing Atractylodes macrocephala extract

[0056] Raw material preparation: Weigh out 20g hyaluronic acid, 10g chitosan, 3g Atractylodes macrocephala extract, 5g fetal bovine serum substitute, 2g L-glutamine, 60mL PBS buffer, and 0.5g genipin by weight; wherein the molecular weight of hyaluronic acid is 1.8×10⁻⁶. 6 Da, chitosan with a degree of deacetylation of 88%, fetal bovine serum substitute purchased from Gibco, L-glutamine purchased from Sigma, and PBS buffer was a sterile buffer with pH 7.2-7.4.

[0057] Preparation of Atractylodes macrocephala extract: Pulverize Atractylodes macrocephala slices to 40 mesh, weigh 100g, add 10 times (1000mL) of 70% ethanol, place in a reflux extraction apparatus, heat and reflux twice, 1.5h each time, combine the two extracts, and concentrate under reduced pressure at 60℃ and 0.08MPa to a relative density of 1.15 (60℃) to obtain a concentrated solution; load the concentrated solution onto a D101 macroporous adsorption resin column, elute impurities with deionized water at a flow rate of 2BV / h until the eluent is colorless and transparent, then elute with 30% ethanol at a flow rate of 1BV / h, collect the 30% ethanol eluent, freeze-dry at -50℃ and 0.1MPa to obtain the Atractylodes macrocephala extract, and the polysaccharide content of Atractylodes macrocephala was 35.2% as determined by the phenol-sulfuric acid method.

[0058] Gel matrix preparation: Add 20g of hyaluronic acid and 10g of chitosan to 30mL of PBS buffer preheated to 45℃, place on a magnetic stirrer, and stir at 120r / min for 30min until completely dissolved to form a uniform and transparent gel matrix solution.

[0059] Preparation of active ingredient solution: Add 3g of Atractylodes macrocephala extract, 5g of fetal bovine serum substitute, and 2g of L-glutamine to the remaining 30mL of PBS buffer, place in an ultrasonic cleaner, set the power to 180W and the temperature to 28℃, and sonicate for 12min to fully dissolve the components and obtain a homogeneous active ingredient solution.

[0060] Preparation of composite gel: The active ingredient solution was slowly added dropwise to 1000 mL of 581 basal culture medium at a dropping rate of 1.5 mL / min, while stirring continuously at 130 r / min for 30 min to ensure uniform mixing; then 0.5 g of genipin was added, and the pH of the system was adjusted to 7.0 with 0.1 mol / L HCl or NaOH solution. The system was then allowed to stand at room temperature for 2.5 h for cross-linking to form a composite gel system.

[0061] Sterilization treatment: The cross-linked composite gel system was placed in a high-pressure steam sterilizer, and the temperature was set to 121℃ and the pressure to 0.1MPa for 20 minutes. After sterilization, it was taken out and allowed to cool naturally to room temperature to obtain NK cell culture gel containing Atractylodes macrocephala extract, wherein the mass concentration of the self-made composite gel was 0.5%.

[0062] Gel performance testing: The pore structure of the gel was observed using scanning electron microscopy. The results showed that the pore diameter of the gel was 80-150 μm and the porosity was 82%. The sustained-release efficiency of the active ingredient was tested using the dialysis bag method. The results showed that the sustained-release period of Atractylodes macrocephala polysaccharide was 10 days. The cumulative release was 35% in the first 3 days and 92% in the 10th day, which met the nutritional requirements of NK cell culture.

[0063] Example 2

[0064] NK cell culture experiment

[0065] Sample source: 50 mL of peripheral blood from a healthy donor, collected with informed consent from the donor, using heparin sodium as the anticoagulant.

[0066] Reagents and instruments: lymph node separation medium (purchased from Tianjin Haoyang Biotechnology), NK cell culture gel containing Atractylodes macrocephala extract prepared in Example 1 of this invention, 581 basal culture medium, IL-2 (purchased from Peprotech), K562 cell line (purchased from the Cell Bank of the Chinese Academy of Sciences), flow cytometer (BD FACSCanto II), cell counter (Thermo Countess II), CO2 incubator (Thermo 3111), autoclave (Sanyo MLS-3750), ultrasonic cleaner (KQ-500DE), etc.

[0067] Experimental group: NK cells were cultured using the Atractylodes macrocephala extract-containing NK cell culture gel prepared in Example 1 of this invention.

[0068] Control group: Cultured in 581 basal medium supplemented with 20 ng / mL IL-2.

[0069] Mononuclear cell acquisition: 50 mL of peripheral blood was diluted with 50 mL of physiological saline and slowly added to a centrifuge tube pre-coated with 25 mL of lymphocyte separation solution to form a layered solution. The mixture was centrifuged at 4 °C and 2000 r / min for 18 min. After centrifugation, the middle mononuclear cell layer was aspirated, 5 times the volume of PBS buffer was added, and the mixture was gently pipetted and mixed. The mixture was centrifuged at 4 °C and 1500 r / min for 10 min and washed twice to obtain purified mononuclear cells.

[0070] Cell seeding and culture: (1) Experimental group: Mononuclear cells were resuspended in the culture gel of this invention and the cell density was adjusted to 1.06 × 10⁶. 6 Cells were inoculated at a density of 30 mL / mL into T75 culture flasks and cultured in an incubator at 37°C, 5% CO2, and 95% humidity. The culture gel medium was replaced daily with fresh medium from this invention. Cell density was monitored using a cell counter, and the medium volume was adjusted as needed to maintain a cell density of 8 × 10⁶ cells / mL. 5 -1.2×10 6 Cells / mL; after culturing for 10 days, cells were collected for further analysis.

[0071] (2) Control group: Mononuclear cells were resuspended in 581 basal medium supplemented with 20 ng / mL IL-2, and the cell density was adjusted to 1.06 × 10⁶ cells / mL. 6 Cells were inoculated at a density of 8 × 10⁶ cells / mL into T75 culture flasks at a seed volume of 30 mL. The flasks were then incubated at 37°C, 5% CO₂, and 95% humidity. The culture medium was replaced daily to maintain a cell density of 8 × 10⁶ cells / mL. 5 -1.2×10 6 Cells / mL; after culturing for 14 days, cells were collected for further analysis.

[0072] Cell morphology observation: During the culture period, cell morphology and aggregation were observed daily using an inverted microscope, and the time of cell cluster formation and cell cluster size were recorded.

[0073] Cell proliferation capacity assay: Cells were collected on days 3, 5, 7, 10 (experimental group) and 14 (control group) of culture, respectively. Cells were counted using a cell counter and the cell proliferation fold was calculated (proliferation fold = total number of cells at harvest / total number of cells initially inoculated).

[0074] Cell viability assay: The trypan blue staining method was used. A small amount of cell suspension was taken and an equal volume of 0.4% trypan blue staining solution was added. The mixture was allowed to stand at room temperature for 5 minutes. The number of live cells and the total number of cells were counted under a microscope, and the cell viability was calculated (viability = number of live cells / total number of cells × 100%).

[0075] Cell phenotype detection: Flow cytometry was used, and 1×10⁶ cells were collected.6 Cells were washed twice with PBS buffer, and fluorescently labeled monoclonal antibodies CD3-PE and CD56-FITC were added. The cells were incubated at room temperature in the dark for 30 min, washed twice with PBS buffer, resuspended, and then analyzed by flow cytometry to determine the proportion of CD3⁻CD56⁺ cells (NK cell purity).

[0076] Killing activity assay: K562 cells (target cells) were subjected to the CCK-8 assay at a concentration of 1×10⁻⁶. 4 NK cells (effect cells) were seeded per well in 96-well plates and cultured for 24 h. NK cells (effect cells) obtained from the experimental and control groups were added to the corresponding wells at effector-to-target ratios of 10:1, 20:1, and 40:1, respectively, with three replicates per group. A blank control group (culture medium only) and a target cell control group (K562 cells only) were also set up. The plates were incubated at 37°C in a 5% CO2 incubator for 4 h. 10 μL of CCK-8 reagent was added to each well, and the plates were cultured for another 2 h. The absorbance (OD value) was measured at 450 nm using a microplate reader, and the killing rate was calculated as follows: Killing rate = [1 - (OD value of experimental wells - OD value of blank wells) / (OD value of target cell wells - OD value of blank wells)] × 100%.

[0077] Experimental results

[0078] Cell morphology and aggregation:

[0079] (1) Experimental group: obvious cell clusters were observed to form on the 3rd day of culture. Each cell cluster consisted of 80-100 cells. The cell clusters were regular in shape and had clear boundaries. As the culture time was extended, the number of cell clusters increased and the volume increased slightly, but the structure remained stable and no dispersion occurred. When harvested on the 10th day of culture, the cell cluster survival rate was 88.5%.

[0080] (2) Control group: The cells were in a dispersed growth state for the first 7 days of culture, with no obvious cell clusters. On the 10th day of culture, a small number of loose cell clusters appeared, but the structure was unstable and easily dispersed. When harvested on the 14th day of culture, only a small number of unstable cell clusters were formed, and the cell cluster survival rate was 62.3%.

[0081] Cell proliferation capacity:

[0082] (1) Experimental group: The initial total number of cells seeded was 3.18 × 10⁻⁶. 7 (30mL×1.06×10) 6 (cells / mL); the total cell count on day 3 of culture was 6.996 × 10⁻⁶. 7 The number of cells increased 2.2 times on day 1; the number on day 5 was 1.272 × 10⁻⁶. 8 The number of offspring was 1.908 × 10⁶ on day 7, with a multiplication rate of 4.0 times.8 The cell count was 6.0 times; the total number of cells at harvest on day 10 was 7.891 × 10⁻⁶. 8 One, with a multiplication rate of 24.8 times.

[0083] (2) Control group: The initial total number of cells inoculated was 3.18 × 10⁻⁶. 7 The total number of cells on day 3 of culture was 4.452 × 10⁻⁶. 7 The number of cells increased by 1.4 times on day 5; the number of cells on day 5 was 7.008 × 10⁶. 7 The number of offspring was 2.2 times; the number on day 7 was 1.0176 × 10⁻⁶. 8 The cell count was 3.2 times; the total number of cells at harvest on day 14 was 5.7876 × 10⁶. 8 One, with a multiplication rate of 18.2 times.

[0084] The results showed that the cell proliferation rate in the experimental group was significantly faster than that in the control group, and the cell proliferation fold after 10 days of culture was 36.3% higher than that after 14 days of culture in the control group.

[0085] Cell viability and phenotype:

[0086] (1) Cell survival rate: The cell survival rate at harvest was 96.2% in the experimental group and 94.5% in the control group. There was no significant difference between the two groups.

[0087] (2) Cell phenotype: The proportion of CD3⁻CD56⁺ cells in the experimental group was 92.3%, and that in the control group was 91.8%. Both groups met the phenotype requirements of clinical-grade NK cells.

[0088] Lethality:

[0089] When the effector-to-target ratio was 10:1, the kill rate in the experimental group was 78.5%, and in the control group it was 77.2%; when the effector-to-target ratio was 20:1, the kill rate in the experimental group was 86.3%, and in the control group it was 85.7%; when the effector-to-target ratio was 40:1, the kill rate in the experimental group was 93.6%, and in the control group it was 92.8%. The results indicate that the cytotoxic activity of NK cells in the experimental group was not significantly different from that in the control group, and both remained at a high level.

[0090] Example 3

[0091] Stability and reproducibility verification of culture gels

[0092] Stability verification: The NK cell culture gel containing Atractylodes macrocephala extract prepared in Example 1 was stored in a refrigerator at 4°C. It was removed at weeks 1, 2, 3, and 4 of storage and subjected to NK cell culture experiments according to the method in Example 2. Cell proliferation rate, cell cluster viability, and cytotoxic activity were measured. The results showed that the culture gel remained stable within 4 weeks of storage, with cell proliferation maintained at 24.0-25.0 times, cell cluster viability ≥85%, and cytotoxicity ≥90%, showing no significant difference from freshly prepared culture gels.

[0093] Repeatability Validation: Following the preparation method in Example 1, three batches of NK cell culture gels containing Atractylodes macrocephala extract (batch 1, batch 2, and batch 3) were prepared by different operators at different time points and used for NK cell culture, with key indicators being tested. The results showed that the cell proliferation folds of the three batches were 24.8-fold, 24.5-fold, and 25.2-fold, respectively; the cell cluster viability was 88.5%, 87.8%, and 89.2%, respectively; and the killing rate (effect-to-target ratio 40:1) was 93.6%, 92.9%, and 94.1%, respectively. The differences between batches were small, indicating that the preparation process has good repeatability.

[0094] Example 4

[0095] Cytotoxicity test

[0096] The cytotoxicity of the culture gel of this invention was detected using the MTT assay. NK cells were cultured at a concentration of 1 × 10⁻⁶. 4 Cells were seeded per well in a 96-well plate and cultured for 24 h. Then, different volumes of the culture gel of this invention were added (to make the final gel concentrations 0.1%, 0.3%, 0.5%, 0.7%, and 0.9%, respectively), with three replicates per group. A blank control group (culture medium only) and a normal cell control group (no gel) were also set up. After culturing for another 48 h, 20 μL of MTT solution (5 mg / mL) was added to each well. After culturing for 4 h, the supernatant was discarded, and 150 μL of DMSO was added to each well. The cells were shaken for 10 min to dissolve the crystals. The OD value was measured at 570 nm using a microplate reader, and the relative cell proliferation rate (RGR) was calculated as follows: RGR = (OD value of experimental wells / OD value of normal cell control group) × 100%.

[0097] The results showed that when the gel concentration was 0.5% (the concentration used in this invention), the relative cell proliferation rate was 95.8%, and the cytotoxicity was grade 1 (non-toxic) according to the cytotoxicity grading standard; when the gel concentration was as high as 0.9%, the relative cell proliferation rate was still 89.2%, and the cytotoxicity was grade 2 (slight toxicity), indicating that the culture gel of this invention has good biocompatibility and no obvious cytotoxicity.

[0098] The above embodiments clearly demonstrate the preparation process, key performance indicators, and NK cell culture effects of the NK cell culture gel containing Atractylodes macrocephala extract of the present invention: through specific raw material ratios and preparation steps, a composite gel system with suitable pore structure and sustained-release properties was successfully constructed; this culture gel can effectively induce NK cells to aggregate and grow, significantly improve proliferation efficiency, shorten the culture cycle, and maintain high cell viability and cytotoxic activity; stability, repeatability, and cytotoxicity tests further verified the reliability and safety of this technology. The above embodiments fully demonstrate the feasibility and advancement of the technical solution of the present invention, providing practical technical support for the large-scale preparation of clinical-grade NK cells, and can be widely applied in the field of cell therapy.

[0099] Industrialization prospects of the invention

[0100] The NK cell culture gel containing Atractylodes macrocephala extract, its preparation method, and its application, as described in this invention, have significant technical advantages and industrialization potential:

[0101] It solves the core pain points of large-scale NK cell culture, shortens the culture cycle by 28.6%, and increases proliferation efficiency by 34.5%, which can significantly reduce production costs, improve production efficiency, and meet the large clinical demand for NK cells;

[0102] The raw materials are widely available and inexpensive. Atractylodes macrocephala is a traditional and commonly used Chinese medicine with abundant production. The industrial production technology of natural polysaccharides such as hyaluronic acid and chitosan is mature, and the preparation process is simple and controllable, making it suitable for large-scale industrial production.

[0103] The product has good biocompatibility and no cytotoxicity. The cultured NK cells meet clinical-grade standards and can be directly applied to cell therapy fields such as tumor immunotherapy and antiviral therapy. They can also be used for basic research and drug screening related to NK cells.

[0104] With strong technological innovation, it is the first to apply Atractylodes macrocephala extract combined with natural polysaccharide gel to NK cell culture, constructing a brand-new culture system with independent intellectual property rights and strong market competitiveness.

[0105] In summary, the technical solution of this invention not only has significant academic value but also broad prospects for industrial application, and is expected to promote the large-scale development of NK cell therapy technology, making an important contribution to human health.

Claims

1. An NK cell culture gel containing Atractylodes macrocephala extract, characterized in that: Using 581 basal culture medium as the matrix, a self-made composite gel with a mass concentration of 0.5% was added. The self-made composite gel, by weight, consisted of 20 parts hyaluronic acid, 10 parts chitosan, 3 parts Atractylodes macrocephala extract, 5 parts fetal bovine serum substitute, 2 parts L-glutamine, 60 parts PBS buffer, and 0.5 parts genipin. The Atractylodes macrocephala extract contained ≥32% Atractylodes macrocephala polysaccharide and was prepared by ethanol reflux extraction, macroporous resin purification, and freeze-drying of Atractylodes macrocephala slices.

2. The NK cell culture gel containing Atractylodes macrocephala extract according to claim 1, characterized in that, The preparation process of the Atractylodes macrocephala extract is as follows: Atractylodes macrocephala slices are pulverized to 40 mesh, 10 times the amount of 70% ethanol is added, and the mixture is refluxed and extracted twice, 1.5 hours each time. The extracts are combined, concentrated under reduced pressure to a relative density of 1.15 at 60°C, loaded onto a D101 macroporous adsorption resin column, and after washing away impurities with water, the mixture is eluted with 30% ethanol. The eluent is collected and freeze-dried to obtain the Atractylodes macrocephala extract.

3. The NK cell culture gel containing Atractylodes macrocephala extract according to claim 1, characterized in that, The self-made composite gel has a pore diameter of 50-200 μm, a porosity of 75%-85%, and a sustained-release period of 8-12 days for the active ingredients.

4. A method for preparing an NK cell culture gel containing Atractylodes macrocephala extract as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Raw material preparation: Weigh out 20 parts of hyaluronic acid, 10 parts of chitosan, 3 parts of Atractylodes macrocephala extract, 5 parts of fetal bovine serum substitute, 2 parts of L-glutamine, 60 parts of PBS buffer, and 0.5 parts of genipin by weight. (2) Gel matrix preparation: Hyaluronic acid and chitosan were added to 30 portions of PBS buffer preheated to 45°C and magnetically stirred for 30 min until completely dissolved; (3) Preparation of active ingredient solution: Add Atractylodes macrocephala extract, fetal bovine serum substitute and L-glutamine to the remaining 30 parts of PBS buffer and sonicate for 12 min to dissolve; (4) Preparation of composite gel: The active ingredient solution was added dropwise to 581 basic culture medium, stirred evenly, and then genipin was added to adjust the pH to 7.

0. Crosslinking was carried out at room temperature for 2.5 h. (5) Sterilization treatment: The cross-linked gel system was autoclaved at 121°C for 20 min and cooled to room temperature to obtain NK cell culture gel containing Atractylodes macrocephala extract.

5. The method for preparing NK cell culture gel containing Atractylodes macrocephala extract according to claim 4, characterized in that: In step (3), the ultrasonic power is 150-200W and the ultrasonic temperature is controlled at 25-30℃.

6. The method for preparing NK cell culture gel containing Atractylodes macrocephala extract according to claim 4, characterized in that: In step (4), the drop rate is 1-2 mL / min and the stirring speed is 100-150 r / min.

7. The application of an NK cell culture gel containing Atractylodes macrocephala extract as described in any one of claims 1-3 in the large-scale culture of NK cells, characterized in that, The following cultivation steps are included: (1) Mononuclear cell acquisition: Mononuclear cells are obtained from peripheral blood or umbilical cord blood by centrifugation with lymphocyte separation solution; (2) Cell seeding: Mononuclear cells were seeded at a rate of 1.06 × 10⁶ cells / year. 6 Inoculate at a density of cells / mL into culture flasks containing the culture gel; (3) Culture conditions control: Incubate at 37℃ in a 5% CO2 incubator, and replace the culture gel medium with fresh medium once a day; (4) Cell density maintenance: Maintain cell density at 8 × 10⁻⁶ during culture. 5 -1.2×10 6 cells / mL; (5) Cell harvesting: NK cells were harvested after 10 days of culture.

8. The application of the NK cell culture gel containing Atractylodes macrocephala extract according to claim 7 in the large-scale culture of NK cells, characterized in that, In step (1), the centrifugation conditions are 2000 r / min, centrifugation time is 15-20 min, and centrifugation temperature is 4℃.

9. The application of the NK cell culture gel containing Atractylodes macrocephala extract according to claim 7 in the large-scale culture of NK cells, characterized in that, During the culture process, NK cells form stable cell clusters of 80-100 cells on the third day of culture, with a cell cluster survival rate of ≥85%.

10. The application of the NK cell culture gel containing Atractylodes macrocephala extract according to claim 7 in the large-scale culture of NK cells, characterized in that, The harvested NK cells had a killing rate of ≥90% against K562 tumor target cells, and the total number of cells was more than 245 times that of the initial inoculation.